Specific depletion of the B-cell population induced by aberrant expression of human interferon regulatory factor 1 gene in transgenic mice . 
Interferons ( IFNs ) are well known both as antiviral proteins and as potent regulators of cell growth and differentiation . 
In fact , IFNs inhibit growth of various normal and transformed cell types . 
Previously , a nuclear factor , IRF-1 ( interferon regulatory factor 1 ) , which binds to type I IFN and some IFN-inducible gene promoters , was identified and cloned . 
Since the IRF-1 gene is both virus and IFN inducible , an intriguing issue is raised as to whether the IRF-1 gene is functioning in IFN-mediated regulation of cell growth and differentiation . 
In this study , we generated transgenic mice carrying the human IRF-1 gene linked to the human immunoglobulin heavy-chain enhancer . 
In the transgenic mice , all the lymphoid tissues examined showed a dramatic reduction in the number of B lymphocytes ( B cells ) . 
Preparation and analysis of bone marrow cells from the chimeric mice indicated that the bone marrow is the effective site for specific depletion of the B-cell population . 
In fact , transgenic bone marrow cells cocultured with a bone marrow-derived stromal cell line revealed an altered B-cell maturation pattern . 
Identification and cloning of TCF-1 , a T lymphocyte-specific transcription factor containing a sequence-specific HMG box . 
CD3-epsilon expression is controlled by a downstream T lymphocyte-specific enhancer element . 
We report the identification of a T cell-specific transcription factor , TCF-1 , binding to this element . 
The multimerized recognition motif of TCF-1 constituted a T cell-specific enhancer . 
Subsequent cloning of TCF-1 identified three splice alternatives . 
TCF-1 contained a single DNA-binding HMG box most closely related to similar boxes in the putative mammalian sex-determining gene SRY and in the Schizosaccharomyces pombe Mc mating type gene . 
TCF-1 mRNA was expressed uniquely in T lymphocytes . 
Upon cotransfection into non-T cells , TCF-1 could transactivate through its cognate motif . 
These results identify TCF-1 as a T cell-specific transcription factor , which might play a role in the establishment of the mature T cell phenotype . 
Expression of c-jun , jun B and jun D proto-oncogenes in human peripheral-blood granulocytes . 
We have found that purified human peripheral-blood granulocytes express constitutively significant levels of proto-oncogenes c-jun , jun B and jun D mRNA . 
Upon functional activation of granulocytes by 4 beta-phorbol 12-myristate 13-acetate ( PMA ) , the levels of c-jun , jun B and jun D transcripts were increased . 
The three jun genes showed a similar time course in their induction by PMA , maximal mRNA levels being reached after 60 min of induction . 
These results suggest that expression of c-jun , jun B and jun D genes might be involved in terminal granulocyte differentiation or in regulating granulocyte functionality . 
Platelet-activating factor induces phospholipid turnover , calcium flux , arachidonic acid liberation , eicosanoid generation , and oncogene expression in a human B cell line . 
Platelet-activating factor is a potent mediator of the inflammatory response . 
Studies of the actions of platelet-activating factor have centered mainly around neutrophils , monocytes , and platelets . 
In this report we begin to uncover the influence of platelet-activating factor on B lymphocytes . 
Employing the EBV-transformed human B cell line SKW6.4 , we demonstrate that platelet-activating factor significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine , phosphatidylinositol , and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10-LRB--9-RRB- to 10-LRB--6-RRB- M . 
The inactive precursor , lyso-platelet-activating factor , at a concentration as high as 10-LRB--7-RRB- M had no effect on any of the membrane phospholipids . 
We also show that platelet-activating factor from 10-LRB--12-RRB- to 10-LRB--6-RRB- M induced rapid and significant elevation in intracellular calcium levels , whereas lyso-platelet-activating factor was again ineffective . 
We further demonstrate the impact of platelet-activating factor binding to B cells by measuring platelet-activating factor induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production . 
Moreover , platelet-activating factor was capable of inducing transcription of the nuclear proto-oncogenes c-fos and c-jun . 
Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates platelet-activating factor induced arachidonic acid release perhaps acting at the level of phospholipase A2 . 
In summary , platelet-activating factor is shown here to have a direct and profound effect on a pure B cell line . 
A novel HIV-1 isolate containing alterations affecting the NF-kappa B element . 
Three molecular clones of HIV-1 , derived from a single isolate ( AL1 ) , exhibited distinct replicative and cytopathic properties during propagation in a human T cell line . 
The phenotypic differences observed were attributable , in large part , to changes affecting the viral LTR . 
Nucleotide sequence and PCR analyses demonstrated the presence of novel duplications or deletions involving the NF-kappa B motif . 
These changes in the enhancer element were identified in the original AL1 virus stock . 
Subcloning of the variant NF-kappa B segments into LTR-driven CAT expression vectors confirmed a correlation between promoter activity and replicative\/cytopathic capacity . 
Kappa B binding proteins are constitutively expressed in an IL-2 autocrine human T cell line . 
The IL-2 and the IL-2-R alpha genes are both expressed transiently in normal T lymphocytes after Ag or mitogen activation . 
In contrast , the human T cell line , IARC 301 , expresses these two genes constitutively and we have previously demonstrated that its growth depends on the autocrine production of this T cell growth factor and high affinity IL-2R . 
To dissect the molecular basis for the unusual persistent expression of the IL-2 and IL-2-R alpha genes in these IARC 301 T cells , we have analyzed the interactions of constitutively expressed nuclear proteins with the 5' flanking regions of the IL-2 and IL-2-R alpha genes using both DNase I footprinting and gel retardation techniques . 
We have found that a region in both genes ( -276 to -250 for IL-2-R alpha and -203 to -183 for IL-2 ) , which corresponds to a kappa B enhancer element , is specifically protected by nuclear proteins from IARC 301 . 
In agreement with this finding , both the IL-2 and IL-2-R alpha promoters are active in transient transfection assays in IARC 301 cells . 
In contrast , mutation of the kappa B enhancer results in markedly attenuated activities of both promoters . 
Two proteins binding the kappa B sequence , NF-kappa B and KBF1 , are constitutively expressed in IARC 301 nuclei and induced by PMA and PHA in Jurkat . 
They bind to the kappa B motifs with different relative affinities that may reflect their different contribution in the expression of various promoters . 
The functional domains of the murine Thy-1 gene promoter . 
The Thy-1 gene promoter resembles a " housekeeping " promoter in that it is located within a methylation-free island , lacks a canonical TATA box , and displays heterogeneity in the 5'-end termini of the mRNA . 
Using transgenic mice , we show that this promoter does not confer any tissue specificity and is active only in a position-dependent manner . 
It can only be activated in a tissue-specific manner by elements that lie downstream of the initiation site . 
We have analyzed the functional domains of the minimal Thy-1 promoter and show that the dominant promoter elements consist of multiple binding sites for the transcription factor Sp1 , an inverted CCAAT box , and sequences proximal to the transcription start site . 
DNase I and gel mobility shift assays show the binding of a number of nuclear factors to these elements , including Sp1 and CP1 . 
Our results show that the structure of this promoter only permits productive interactions of the two transcription factors Sp1 and CP1 with the basal transcription machinery in the presence of enhancer sequences . 
Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B { published erratum appears in Science 1991 Oct 4 ; 254 ( 5028 ) : 11 } 
A DNA probe that spanned a domain conserved among the proto-oncogene c-rel , the Drosophila morphogen dorsal , and the p50 DNA binding subunit of NF-kappa B was generated from Jurkat T cell complementary DNA with the polymerase chain reaction ( PCR ) and degenerate oligonucleotides . 
This probe was used to identify a rel-related complementary DNA that hybridized to a 2.6-kilobase messenger RNA present in human T and B lymphocytes . 
In vitro transcription and translation of the complementary DNA resulted in the synthesis of a protein with an apparent molecular size of 65 kilodaltons ( kD ) . 
The translated protein showed weak DNA binding with a specificity for the kappa B binding motif . 
This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-kappa B subunit and binding was inhibited by I kappa B-alpha and -beta proteins . 
In addition , the 65-kD protein associated with the p50 subunit of NF-kappa B and the kappa B probe to form a complex with the same electrophoretic mobility as the NF-kappa B-DNA complex . 
Therefore the rel-related 65-kD protein may represent the p65 subunit of the active NF-kappa B transcription factor complex . 
Lymphocyte glucocorticoid receptor number in posttraumatic stress disorder . 
OBJECTIVE : The authors ' objective was to investigate the possibility that glucocorticoid receptor changes may be involved in the dysregulation of the hypothalamic-pituitary-adrenal ( HPA ) axis in posttraumatic stress disorder ( PTSD ) . 
METHOD : They measured the number of lymphocyte cytosolic glucocorticoid receptors and plasma cortisol concentrations in 15 consecutively admitted male combat Vietnam veterans with PTSD and in a normal comparison group of 11 subjects . 
RESULTS : Both the patients and the normal comparison subjects showed a morning-to-afternoon decline in glucocorticoid receptor concentrations , paralleling the normal diurnal decline in cortisol levels . 
The number of glucocorticoid receptors was 63 % greater in the morning and 26 % greater in the afternoon in the patients with PTSD than in the normal subjects . 
No group differences in cortisol levels were observed , nor were glucocorticoid receptor number and cortisol levels correlated . 
The number of morning glucocorticoid receptors was positively correlated with symptoms of PTSD and anxiety . 
CONCLUSIONS : These results provide further evidence for a dysregulation of the HPA axis in PTSD . 
The finding that patients with PTSD had a substantially greater number of lymphocyte glucocorticoid receptors than normal comparison subjects is consistent with the authors ' previous observations of low 24-hour urinary cortisol excretion in subjects with PTSD . 
Furthermore , the receptor changes observed are opposite of those reported in major depressive disorder . 
The present data , along with other findings of HPA abnormalities in PTSD , support the possibility of a greater negative feedback sensitivity at one or more levels of the HPA axis . 
A thymus-specific member of the HMG protein family regulates the human T cell receptor C alpha enhancer . 
The human T cell-specific transcription factor TCF-1 alpha plays a key role in the tissue-specific activation of the T cell receptor ( TCR ) C alpha enhancer and binds to pyrimidine-rich elements ( 5'-PyCTTTG-3' ) present in a variety of other T cell-specific control regions . 
Using amino acid sequence information derived from the DNA affinity-purified protein , we have now isolated cDNA clones encoding TCF-1 alpha . 
The TCF-1 alpha cDNA contains a single 68-amino-acid domain that is homologous to a region conserved among high-mobility group ( HMG ) and nonhistone chromosomal proteins . 
Expression of full-length and mutant cDNA clones in bacteria reveal that the single HMG motif , which is predicted to contain two extended alpha-helical segments , is sufficient to direct the sequence-specific binding of TCF-1 alpha to DNA . 
Northern blot experiments demonstrate further that TCF-1 alpha mRNA is highly tissue specific , found primarily in the thymus or T cell lines . 
The immature CEM T cell line expresses relatively low levels of TCF-1 alpha mRNA , which are increased upon activation of these cells by phorbol esters . 
Interestingly , the cloned TCF-1 alpha protein is a potent transcriptional activator of the human TCR alpha enhancer in nonlymphoid cell lines , whereas the activity of the endogenous protein in T cell lines is strongly dependent on an additional T cell-specific protein that interacts with the core enhancer . 
TCF-1 alpha is currently unique among the newly emerging family of DNA-binding regulatory proteins that share the HMG motif in that it is a highly tissue-specific RNA polymerase II transcription factor . 
A study on the circadian rhythm of glucocorticoid receptor . 
Circadian rhythm in glucocorticoid receptor ( GR ) was studied in the rat liver and human peripheral leukocytes . 
For rats exposed to a natural environmental photic cycle or a 12L : 12D artificial light regime , peak values of hepatic GR were detected between 23:00 and 02:00 h . 
Except for a 4-hour advancement of the peak , a similar circadian rhythm of hepatic GR was detected in rats reared under a reversed lighting regimen ( 12D : 12L ; lights on between 18:30 and 06:30 h ) . 
In human leukocytes , the peak value of GR was found to parallel that of plasma cortisol with high and low values detected at 04:00-08:00 h and 23:00-24:00 h , respectively . 
In patients suffering from Cushing 's syndrome , the circadian rhythm of plasma cortisol either disappeared or was inverted while that of GR did not significantly deviate from the normal subjects . 
For apoplexic patients with lesions localized to the base of the brain as indicated by computerized tomography , the diurnal variation of GR was abolished . 
Conversely , diurnal rhythmicity persisted in apoplexy patients whose lesions were in the cerebral cortex . 
Thus , we postulated that the circadian modification of GR was independent of the diurnal fluctuations in plasma cortisol level or the circadian variations in environmental lighting and that the rhythmicity might be regulated by the ' circadian pacemaker ' located in the human basal brain . 
These diurnal variations in GR might serve to coordinate the reactivity of the target cells to cortisol because the diurnal rhythms of a GR-mediated response , the fractional inhibition of chemotactic migration rate of polymorphonuclear leukocytes by cortisol , were found to be synchronous with those of GR . 
Multiple Oct2 isoforms are generated by alternative splicing . 
The interaction of the Oct2 transcription factor with the cognate octamer motif ATGCAAAT is a critical determinant of the lymphoid-specific expression of immunoglobulin genes . 
Ectopic expression of cloned Oct2 cDNA was shown to be sufficient to reconstitute at least some aspects of this regulation in non-lymphoid cells . 
We describe the isolation and characterization of multiple cDNAs encoding mouse Oct2 from a mature B-cell line and we show that a variety of isoforms of this transcription factor is generated from a single gene by an alternative splicing mechanism . 
All the isoforms retain the previously characterized POU-domain and are therefore able to bind to the octamer motif . 
Different amounts of the various isoforms are present within the same B-cell regardless of the developmental stage of B-cell differentiation and at least some of the isoforms are conserved between mouse and humans . 
In cotransfection experiments we show that all the isoforms are able to activate an octamer containing promoter element in fibroblasts revealing an unexpected functional redundancy . 
Finally , we show that one of the isoforms encodes the previously described lymphoid-specific Oct2B protein which has been suggested to be involved in the function of the octamer motif in the context of the immunoglobulin heavy-chain ( IgH ) enhancer . 
Processing of the precursor of NF-kappa B by the HIV-1 protease during acute infection . 
Transcription of the human immunodeficiency virus type-1 ( HIV-1 ) genome is regulated in part by cellular factors and is stimulated by activation of latently infected T cells . 
T-cell activation also correlates with the induction of the factor NF-kappa B which binds to two adjacent sites in the HIV-1 long terminal repeat . 
This factor consists of two DNA-binding subunits of relative molecular mass 50,000 ( 50K ) associated with two 65K subunits . 
It is located in the nucleus in mature B cells , but is present in other cell types as an inactive cytoplasmic complex . 
External stimuli , including those that activate T cells , result in nuclear translocation of active NF-kappa B . 
The cloning of the complementary DNA for the 50K subunit helped to identify an exclusively cytoplasmic 105K precursor ( p105 ) ( V.B. , P.K. and A.I. , manuscript submitted ) . 
The expression of active NF-kappa B might therefore also be regulated by the extent of processing of p105 . 
Because HIV-1 requires active NF-kappa B for efficient transcription , we tested the effect of HIV-1 infection on the processing of the human 105K precursor . 
We show here that the HIV-1 protease can process p105 and increases levels of active nuclear NF-kappa B complex . 
Vitamin D receptor expression in human lymphocytes . 
Signal requirements and characterization by western blots and DNA sequencing . 
The signals controlling the expression of the receptor protein for 1 alpha,25-dihydroxyvitamin D3 in normal human lymphocytes and the relationship of this protein to the classical vitamin D receptor were examined . 
Lymphocytes activated with the OKT3 antibody to the T-cell antigen receptor expressed fewer binding sites as compared to lymphocytes that were activated by the polyclonal activator phytohemagglutinin ( PHA ) . 
However , combination of OKT3 and phorbol myristate acetate produced a concentration of binding sites similar to the PHA-activated cells . 
The receptor from OKT3 and OKT3 + phorbol myristate acetate-activated lymphocytes exhibited decreased binding to DNA-cellulose compared to PHA-activated lymphocytes . 
In lymphocytes activated either by PHA or OKT3 ( but not in resting cells ) , a 50-kDa species cross-reacting with a monoclonal antibody against the intestinal vitamin D receptor was detected . 
Finally , RNA from activated lymphocytes was amplified by polymerase chain reaction using oligonucleotide primers flanking the 196 base pair long region encoding the DNA-binding domain of the human intestinal receptor . 
The amplified product showed an identical nucleotide sequence to the DNA-binding domain of the human intestinal receptor . 
These findings suggest that expression of the 1,25--LRB-OH-RRB-2D3 receptor in lymphocytes is triggered by distinct and contingent signals , and that the protein and the mRNA encoding it are identical to the classical vitamin D receptor . 
Cloning of murine TCF-1 , a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers . 
CD3-epsilon gene expression is confined to the T cell lineage . 
We have recently identified and cloned a human transcription factor , TCF-1 , that binds to a functional element in the T lymphocyte-specific enhancer of CD3-epsilon . 
In a panel of human cell lines , TCF-1 expression was restricted to T lineage cells . 
TCF-1 belonged to a novel family of genes that contain the so-called high mobility group 1 ( HMG ) box . 
Here we report the cloning of murine TCF-1 . 
Two splice alternatives were identified that were not previously observed in human TCF-1 . 
Murine and human TCF-1 displayed a 95.5 % overall amino acid homology . 
Recombinant murine and human TCF-1 recognized the same sequence motif in the CD3-epsilon enhancer as judged by gel retardation and methylation interference assays . 
With the murine cDNA clones several aspects of TCF-1 were analyzed . 
First , deletion analysis revealed that a region of TCF-1 containing the HMG box was sufficient for sequence-specific binding . 
Second , by high stringency Northern blotting and in situ hybridization , TCF-1 expression was shown to be confined to the thymus and to the T cell areas of the spleen . 
Third , TCF-1 bound specifically to a functional T cell-specific element in the T cell receptor alpha ( TCR-alpha ) enhancer . 
The T lineage-specific expression and the affinity for functional motifs in the TCR-alpha and CD3-epsilon enhancers imply an important role for TCF-1 in the establishment of the mature T cell phenotype . 
Positive and negative regulation of immunoglobulin gene expression by a novel B-cell-specific enhancer element . 
A new B-cell-specific enhancer element has been identified 3' of E4 and the octamerlike motifs in the human immunoglobulin heavy-chain gene enhancer . 
Tandem copies of this 67-bp MnlI-AluI fragment , when fused to the chloramphenicol acetyltransferase gene driven by the conalbumin promoter , stimulated transcription in B cells but not in Jurkat T cells or HeLa cells . 
Footprinting analysis revealed that the identical sequence CCGAAACTGAAAAGG , designated E6 , was protected by nuclear extracts from B cells , T cells , or HeLa cells . 
Gel mobility shift assays using a synthetic E6 motif detected a B-cell-specific complex in addition to a ubiquitous band found also in T cells and HeLa cells . 
In agreement with the results of gel retardation assays , tandem copies of the E6 motif stimulated transcription in ARH77 and Raji cells but not in Jurkat or HeLa cells . 
Furthermore , a mutant E6 motif lost both in vitro binding activity and in vivo enhancer activity . 
In striking contrast to the mouse Ig heavy-chain enhancer , in which the octamer motif acts as a B-cell-specific enhancer element , the human enhancer contains an octamerlike sequence with one base substitution which bound octamer-binding proteins with only very low affinity and showed no enhancer activity of its own . 
Interestingly , the MnlI-AluI fragment could suppress the basal-level activity of the conalbumin promoter in both Jurkat and HeLa cells . 
Moreover , simian virus 40 enhancer activity was blocked by the MnlI-AluI fragment in HeLa cells but not in B cells . 
Thus , the novel enhancer element identified in this study is probably a target site for both positive and negative factors . 
Glucocorticoid receptor characteristics in monocytes of patients with corticosteroid-resistant bronchial asthma . 
The mechanism of corticosteroid resistance in bronchial asthma has been studied by determining the rank order of potency for different corticosteroids in inhibiting the generation of a 3 kD molecule from peripheral blood monocytes isolated from corticosteroid-sensitive ( CS ) and corticosteroid-resistant ( CR ) asthmatic subjects , which augments leukotriene B4 ( LTB4 ) generation by human neutrophils ( PMN ) stimulated by calcium ionophore . 
In addition , binding studies with ( 3H ) dexamethasone have been performed to determine the dissociation constant ( Kd ) and receptor numbers ( Ro ) in the monocytes of these two groups of subjects . 
The concentration of corticosteroid producing 50 % inhibition ( IC50 ) was 600 nM , 70 nM , and 0.5 nM for hydrocortisone , methylprednisolone , and dexamethasone , respectively , in monocytes from CS individuals . 
There was only weak inhibition of the generation of enhancing activity by the corticosteroids in the CR asthmatic individuals . 
The dexamethasone Kd was 2.45 +\/- 0.58 nM ( mean +\/- SEM , n = 6 ) in the CS group and 1.6 +\/- 0.35 nM ( mean +\/- SEM , n = 6 ) in the CR group of patients ( p = 0.14 ) . 
The Ro in the CS group was 3,605 +\/- 984 binding sites per nucleus ( mean +\/- SEM , n = 6 ) and 4,757 +\/- 692 binding sites per nucleus ( mean +\/- SEM , n = 6 ) in the CR group ( p = 0.23 ) . 
These findings indicate that corticosteroid resistance in bronchial asthma can not be explained by abnormalities in corticosteroid receptor characteristics . 
Tissue-specific expression of the platelet GPIIb gene . 
One of the major objectives in the study of thrombogenesis is to determine the mechanisms by which a hematopoietic progenitor is activated and committed to the megakaryocytic lineage . 
Recent development of primary cultures of human megakaryocytes and the molecular cloning of genes that are specific to this lineage offer the possibility of getting some insights into the genetic mechanisms that control megakaryocytopoiesis . 
One gene of interest is the glycoprotein IIb ( GPIIb ) gene ; GPIIb , the alpha subunit of the platelet cytoadhesin GPIIb-IIIa , is produced in megakaryocytes at an early stage of the differentiation , whereas the other subunit of this complex , GPIIIa , is expressed in other cells . 
For these reasons , the 5'-flanking region of the GPIIb gene was used to identify the regions that interact with DNA-binding nuclear factors . 
A fragment extending from -643 to +33 is capable of controlling the tissue-specific expression of the CAT gene in transfection experiments . 
Within this region , we have identified several sequences that are implicated in DNA protein interactions as shown in DNAse I footprints and gel mobility shift assays . 
One region , centered at -54 , is similar to a nuclear factor E1-binding site , and a region located at position -233 contains a CCAAT motif . 
Two domains centered at positions -345 and -540 , respectively , bind proteins that are present in megakaryocytic cells and nonrelated cells as well . 
Finally , two other domains , located at positions -460 and -510 , interact with proteins that are only present in megakaryocytic cells . 
In addition , deletion of the region containing these two domains results in a significant decrease of the promoter activity . 
It is very likely that these domains bind megakaryocyte-specific nuclear proteins acting as positive transcription factors . 
Demonstration of estrogen and progesterone receptors as well as Ki-67 and p-145 antigens in single tumor cells from blood and pleural effusions using a slide assay . 
We describe a slide assay that allows the demonstration of antigens localized in the nucleus from isolated white blood cells as well as from single tumor cells derived from malignant effusions . 
With the antibodies Ki-67 and anti-p-145 an increased rate of nuclear and nucleolar staining resulted in cells from highly malignant lymphomas . 
An almost identical reaction was obtained when tumor cells from malignant effusions were tested . 
Cells isolated from the blood of patients with leukemic spread of lymphomas of low malignancy yielded a weak staining comparable to that of normal mesothelial cells from non-tumorous cavity fluids . 
The detection of estrogen and progesterone receptors ( ER and PR ) localized in the cell nucleus can be achieved by the same assay . 
The reaction is enhanced by incubation of the tumor cells for 30 min at 37 degrees C prior to fixation . 
Pleural effusions from 20 patients with breast cancer were tested . 
ER was positive in 13 and PR was positive in 12 of the 20 samples . 
In 5 cases there was a divergent reaction with ER and PR antibody . 
The hormone receptors of the primary tumor were known in 15 ( ER ) and 14 ( PR ) patients , respectively . 
In each cohort there was only one case with a negative reaction of the primary tumor and a positive reaction with the isolated tumor cells from the pleural effusions . 
These results indicate that the demonstration of hormone receptor proteins in cells from malignant effusions is possible and that there is a correlation with the status of the primary site of cancer . 
Transforming growth factor-beta suppresses human B lymphocyte Ig production by inhibiting synthesis and the switch from the membrane form to the secreted form of Ig mRNA . 
Transforming growth factor-beta ( TGF-beta ) inhibits B cell Ig secretion and reduces B cell membrane Ig expression . 
The addition of TGF-beta to human B lymphocyte cultures stimulated with Staphylococcus aureus Cowan strain I and IL-2 completely inhibited B cell Ig secretion ( greater than 90 % ) and decreased B cell surface IgM , IgD , kappa L chain , and lambda L chain expression . 
In contrast , TGF-beta had only minimal effects on two other B cell membrane proteins , HLA-DR and CD20 . 
Internal labeling with -LCB-35S-RCB-methionine and immunoprecipitation with anti-IgM , anti-kappa , and anti-lambda antibodies revealed a striking reduction in kappa L chain in the presence of TGF-beta . 
A less pronounced reduction in lambda L chain and microH chain was also noted . 
Northern blot analysis of RNA purified from B cells treated with TGF-beta for varying time intervals revealed a significant decrease in steady state kappa and lambda L chain mRNA levels . 
Furthermore , a significant decrease in the switch from the membrane forms of mu and gamma to their respective secreted forms was noted in the presence of TGF-beta . 
Nuclear run-on experiments demonstrated decreased transcription of kappa L chain . 
The effects of TGF-beta on two transcriptional regulatory factors , Oct-2 and nuclear factor ( NF ) kappa B , known to be important in Ig gene transcription were examined . 
Oct-2 mRNA levels and both Oct-2 and NF-kappa B proteins in nuclear extracts were not altered by treatment with TGF-beta . 
In contrast , levels of the transcriptional factor AP-1 , which is not known to be important in B cell Ig production , were reduced by TGF-beta . 
These findings demonstrate that TGF-beta decreases B lymphocyte Ig secretion by inhibiting the synthesis of Ig mRNA and inhibiting the switch from the membrane form to the secreted forms of mu and gamma mRNA . 
The mechanism by which TGF-beta inhibits Ig chain synthesis is unclear although it does not involve inhibition of the binding of NF-kappa B or Oct-2 to their respective target sequences . 
Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia . 
The BZLF1 protein of Epstein-Barr virus ( EBV ) is a key immediate-early protein which has been shown to disrupt virus latency in EBV-infected B cells . 
We have generated a monoclonal antibody , BZ1 , to BZLF1 which reacts in immunohistology , immunoblotting , and immunoprecipitation and which recognizes both the active , dimeric form and the inactive , monomeric form of the protein . 
Biopsies of oral hairy leukoplakia , an AIDS-associated lesion characterized by high-level EBV replication , were examined by immunohistochemistry using the BZ1 monoclonal antibody . 
A differentiation-associated pattern of BZLF1 expression was observed , BZ1 reacting with nuclei of the upper spinous layer of the lesion . 
This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation . 
A comparison of in situ hybridization to EBV DNA and viral capsid antigen staining with BZ1 reactivity suggested that BZLF1 expression precedes rampant virus replication . 
The inability to detect EBV in the lower epithelial layers of oral hairy leukoplakia raises questions concerning the nature of EBV latency and persistence in stratified squamous epithelium . 
The rhombotin family of cysteine-rich LIM-domain oncogenes : distinct members are involved in T-cell translocations to human chromosomes 11p15 and 11p13 . 
A chromosomal translocation in a T-cell leukemia involving the short arm of human chromosome 11 at band 11p15 disrupts the rhombotin gene . 
This gene encodes a protein with duplicated cysteine-rich regions called LIM domains , which show homology to zinc-binding proteins and to iron-sulfur centers of ferredoxins . 
Two homologues of the rhombotin gene have now been isolated . 
One of these , designated Rhom-2 , is located on human chromosome 11 at band 11p13 , where a cluster of T-cell leukemia-specific translocations occur ; all translocation breakpoints at 11p13 are upstream of the Rhom-2 gene . 
Human and mouse Rhom-2 are highly conserved and , like rhombotin , encode two tandem cysteine-rich LIM domains . 
Rhom-2 mRNA is expressed in early mouse development in central nervous system , lung , kidney , liver , and spleen but only very low levels occur in thymus . 
The other gene , designated Rhom-3 , is not on chromosome 11 but also retains homology to the LIM domain of rhombotin . 
Since the Rhom-2 gene is such a common site of chromosomal damage in T-cell tumors , the consistency of translocations near the rhombotin gene was further examined . 
A second translocation adjacent to rhombotin was found and at the same position as in the previous example . 
Therefore , chromosome bands 11p15 ( rhombotin ) and 11p13 ( Rhom-2 ) are consistent sites of chromosome translocation in T-cell leukemia , with the 11p15 target more rarely involved . 
The results define the rhombotin gene family as a class of T-cell oncogenes with duplicated cysteine-rich LIM domains . 
The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression : marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3 . 
The active metabolite of vitamin D , 1,25-dihydroxyvitamin D3 { 1,25--LRB-OH-RRB-2D3 } , is a potent regulator of human monocyte\/macrophage function in vitro . 
To establish a model for 1,25--LRB-OH-RRB-2D3 regulation of human monocyte monokine synthesis , three human cell lines ( U-937 , THP-1 , and HL-60 ) were examined for : 1 ) the presence of functional 1,25--LRB-OH-RRB-2D3 receptors ; 2 ) the accumulation of interleukin-1 beta ( IL-1 beta ) mRNA and IL-1 beta protein in response to lipopolysaccharide ( LPS ) ; and 3 ) the regulation of this response by 1,25--LRB-OH-RRB-2D3 . 
All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS . 
Preincubation of cells with 1,25--LRB-OH-RRB-2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells . 
From these data , and taking into consideration their state of differentiation and relative ease of culture , U-937 was chosen over HL-60 and THP-1 as the cell line 0 we further characterized . 
In U-937 cells , optimum time and dose of pretreatment with 1,25--LRB-OH-RRB-2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25--LRB-OH-RRB-2D3 ( 10 nM ) . 
Preincubation of cells with 1,25--LRB-OH-RRB-2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS . 
However , exposure of U-937 cells to 1,25--LRB-OH-RRB-2D3 increased by 200 % the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o 
Expression of 1,25-LRB-OH-RRB-2D3 receptors on alveolar lymphocytes from patients with pulmonary granulomatous diseases . 
1,25-LRB-OH-RRB-2D3 is known to be produced at sites of granulomatous reactions . 
In order to characterize the cell types that are targets for this immunoregulatory hormone , we have evaluated the expression of 1,25-LRB-OH-RRB-2D3 receptors on peripheral blood T-lymphocytes and those recovered from the lung by bronchoalveolar lavage from patients with pulmonary granulomatous diseases ( tuberculosis and sarcoidosis ) and from normal control subjects using combined autoradiographic and immunohistochemical techniques . 
Lavage T-lymphocytes from patients with tuberculosis or with sarcoidosis , but not those from normal control subjects , expressed 1,25-LRB-OH-RRB-2D3 receptors as demonstrated by binding of -LCB-3H-RCB-1,25-LRB-OH-RRB-2D3 , which was inhibited by the presence of excess unlabeled 1,25-LRB-OH-RRB-2D3 , but not by the presence of unlabeled 25-LRB-OH-RRB-D3 ( receptor-positive lymphocytes : sarcoidosis , 20 +\/- 12 % ; tuberculosis , 31 +\/- 17 % ) . 
In contrast , blood lymphocytes from patients with granulomatous diseases did not express detectable 1,25-LRB-OH-RRB-2D3 receptors . 
The percentage of lavage T-lymphocytes expressing 1,25-LRB-OH-RRB-2D3 receptors was significantly greater for patients with tuberculosis presenting with isolated hilar adenopathy than for patients with pulmonary infiltrates and\/or cavities . 
1,25-LRB-OH-RRB-2D3 receptors were expressed to a greater extent on CD8+ T-lymphocytes than on CD4+ T-lymphocytes in sarcoidosis , whereas a greater proportion of CD4+ than of CD8+ T-lymphocytes from patients with tuberculosis were receptor-positive . 
These findings support the conclusion that the interaction of 1,25-LRB-OH-RRB-2D3 with its receptor on T-lymphocytes may play an important role in the regulation of granulomatous reactions , but because these receptors are expressed on different lymphocyte populations , the net effect of this potent immunoregulatory molecule is likely different in sarcoidosis and tuberculosis . 
Inhibition of transcription factors belonging to the rel\/NF-kappa B family by a transdominant negative mutant . 
The KBF1 factor , which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb , is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B , which regulates a series of genes involved in immune and inflammatory responses . 
The KBF1\/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family , like the c-rel and v-rel ( proto ) oncogenes . 
The dimerization domain of KBF1\/p50 is contained between amino acids 201 and 367 . 
A mutant of KBF1\/p50 ( delta SP ) , unable to bind to DNA but able to form homo- or heterodimers , has been constructed . 
This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family ( KBF1\/p50 , c- and v-rel ) . 
This mutant also functions in vivo as a trans-acting dominant negative regulator : the transcriptional inducibility of the HIV long terminal repeat ( which contains two potential NF-kappa B binding sites ) by phorbol ester ( PMA ) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant . 
Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines . 
These results constitute the first formal demonstration that these genes are regulated by members of the rel\/NF-kappa B family . 
Human erythroid 5-aminolevulinate synthase : promoter analysis and identification of an iron-responsive element in the mRNA . 
5-Aminolevulinate synthase ( ALAS ) catalyzes the first step of the heme biosynthetic pathway . 
cDNA clones for the human erythroid ALAS isozyme were isolated from a fetal liver library . 
It can be deduced that the erythroid ALAS precursor protein has a molecular weight of 64.6 kd , and is similar in size to the previously isolated human housekeeping ALAS precursor of molecular weight 70.6 kd . 
The mature mitochondrial forms of the erythroid and housekeeping ALAS isozymes are predicted to have molecular weights of 59.5 kd and 64.6 kd , respectively . 
The two isozymes show little amino acid identity in their N-terminal signal sequences but have considerable sequence identity in the C-terminal two-thirds of their proteins . 
An analysis of the immediate promoter of the human erythroid ALAS gene revealed several putative erythroid-specific cis-acting elements including both a GATA-1 and an NF-E2 binding site . 
An iron-responsive element ( IRE ) motif has been identified in the 5'-untranslated region of the human erythroid ALAS mRNA , but is not present in the housekeeping ALAS mRNA . 
Gel retardation experiments established that this IRE motif formed a protein-RNA complex with cytosolic extracts from human K562 cells and this binding was strongly competed with IRE transcripts from ferritin or transferrin receptor mRNAs . 
A transcript of the ALAS IRE , mutated in the conserved loop of the IRE , did not readily form this protein-RNA complex . 
These results suggest that the IRE motif in the ALAS mRNA is functional and imply that translation of the mRNA is controlled by cellular iron availability during erythropoiesis . 
Towards a molecular understanding of T-cell differentiation 
Lymphoid differentiation is one of the best studied examples of mammalian development . 
Here Hans Clevers and Michael Owen describe how the cloning of the genes that encode T-cell-specific membrane proteins allows the identification of transcription factors that control the expression of these T-cell genes . 
Such transcription factors play a key role in the development of the mature T-cell phenotype by functioning as ' master regulators of T-cell differentiation ' . 
Regulation of jun and fos gene expression in human monocytes by the macrophage colony-stimulating factor . 
The macrophage colony-stimulating factor ( M-CSF ) is required for the growth and differentiation of mononuclear phagocytes . 
However , the signaling events responsible for these effects remain unclear . 
The present studies have examined the effects of M-CSF on potential signaling pathways involving expression of the jun and fos early response genes . 
Low levels of c-jun transcripts were detectable in resting human peripheral blood monocytes . 
Treatment of these cells with 10-LRB-3-RRB- units\/ml human recombinant M-CSF was associated with rapid and transient increases in c-jun mRNA levels . 
Nuclear run-on assays and mRNA stability studies demonstrated that M-CSF regulates c-jun expression by both an increase in transcription rate and a prolongation in the half-life of c-jun transcripts . 
M-CSF treatment was also associated with a rapid induction of the jun-B gene , although expression of this gene was prolonged compared to that of c-jun . 
We further demonstrate that M-CSF increases c-fos mRNA levels in human monocytes through control at both the transcriptional and posttranscriptional levels . 
Maximal induction of the c-fos gene was followed by that for the fos-B gene . 
Moreover , M-CSF-induced expression of the fos-related gene , fra-1 , was delayed compared to that for both c-fos and fos-B . 
Taken together , the results indicate that M-CSF treatment is associated with differential activation of multiple members of the jun\/fos family and that expression of these genes could contribute to nuclear signaling mechanisms that regulate a specific program of monocyte differentiation . 
Severe 5-fluorouracil toxicity secondary to dihydropyrimidine dehydrogenase deficiency . 
A potentially more common pharmacogenetic syndrome . 
This study describes the inheritance of a defect in pyrimidine catabolism and its association with drug-induced toxicity in a patient receiving 5-fluorouracil ( FUra ) as adjuvant chemotherapy for breast carcinoma . 
The study population included the affected patient ( proband ) , nine of her blood relatives , and seven healthy volunteers . 
The activity of dihydropyrimidine dehydrogenase ( DPD ) , the initial enzyme of pyrimidine ( and FUra ) catabolism , in peripheral blood mononuclear cells was measured in each subject by a specific radiometric assay using FUra as the substrate . 
The proband had no detectable DPD activity . 
When enzyme levels in the proband and relatives were compared with that in controls , an autosomal recessive pattern of inheritance was demonstrated . 
This is the third patient with severe FUra toxicity secondary to an alteration in pyrimidine catabolism and the second from our clinic population suggesting that the frequency of this genetic defect may be greater than previously thought . 
Monitoring DPD activity may be important in the management of patients experiencing severe toxicity secondary to FUra chemotherapy . 
Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity . 
We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes . 
One of the induced transcripts ( MAD-3 ) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10\/ankyrin motif , which is 60 % similar ( 46 % identical ) to the ankyrin repeat region of the precursor of NF-kappa B\/KBF1 p50 . 
The C-terminus has a putative protein kinase C phosphorylation site . 
In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50\/p65 NF-kappa B complex but not that of the p50\/p50 KBF1 factor or of other DNA-binding proteins . 
The MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B , including adhesion-dependent pathways of monocyte activation . 
Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1 . 
Hydrogen peroxide and oxygen radicals are agents commonly produced during inflammatory processes . 
In this study , we show that micromolar concentrations of H2O2 can induce the expression and replication of HIV-1 in a human T cell line . 
The effect is mediated by the NF-kappa B transcription factor which is potently and rapidly activated by an H2O2 treatment of cells from its inactive cytoplasmic form . 
N-acetyl-L-cysteine ( NP ) , a well characterized antioxidant which counteracts the effects of reactive oxygen intermediates ( ROI ) in living cells , prevented the activation of NF-kappa B by H2O2 . 
NP and other thiol compounds also blocked the activation of NF-kappa B by cycloheximide , double-stranded RNA , calcium ionophore , TNF-alpha , active phorbol ester , interleukin-1 , lipopolysaccharide and lectin . 
This suggests that diverse agents thought to activate NF-kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI . 
ROI appear to serve as messengers mediating directly or indirectly the release of the inhibitory subunit I kappa B from NF-kappa B . 
Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1 : distinct patterns of viral growth are determined by T-cell types . 
Starting with a replication-incompetent molecular clone of human immunodeficiency virus type 1 , lacking all the NF-kappa B and Sp1 binding sites present in the native long terminal repeat ( LTR ) , proviruses containing reconstructed LTRs with individual or combinations of NF-kappa B and Sp1 elements were generated and evaluated for their capacity to produce virus progeny following transfection-cocultivation . 
Virus stocks obtained from these experiments exhibited a continuum of replicative capacities in different human T-cell types depending on which element ( s ) was present in the LTR . 
For example , in experiments involving proviral clones with LTRs containing one or two NF-kappa B elements ( and no Sp1 binding sites ) , a hierarchy of cellular permissivity to virus replication ( peripheral blood lymphocytes = MT4 greater than H9 greater than CEM greater than Jurkat ) was observed . 
Of note was the associated emergence of second-site LTR revertants which involved an alteration of the TATA box . 
These results suggest that the human immunodeficiency virus type 1 LTR possesses functional redundancy which ensures virus replication in different T-cell types and is capable of changing depending on the particular combination of transcriptional factors present . 
Enhancement of human immunodeficiency virus 1 replication in monocytes by 1,25-dihydroxycholecalciferol . 
Human immunodeficiency virus ( HIV ) expression and replication are under tight regulatory control . 
We demonstrate that 1,25-dihydroxycholecalciferol { 1,25--LRB-OH-RRB-2D3 } enhances the replication of monocyte- and lymphocyte-tropic strains of HIV-1 up to 10,000-fold in monocyte cell lines , peripheral blood monocytes , and unfractionated peripheral blood mononuclear cells . 
1,25-LRB-OH-RRB-2D3 is therefore one of the most potent regulators of HIV-1 replication described to date . 
Precursors of 1,25-LRB-OH-RRB-2D3 enhance HIV-1 replication in proportion to their affinity for the 1,25-LRB-OH-RRB-2D3 intracellular receptor , suggesting that 1,25-LRB-OH-RRB-2D3 influences HIV-1 replication by mechanisms involving this receptor . 
These studies may have important implications for the design of effective therapy of HIV-1 infection . 
Glucocorticoid receptors in systemic lupus erythematosus . 
Glucocorticosteroids remain the major treatment modality for systemic lupus erythematosus ( SLE ) , but their mechanism of action is unclear . 
Over the past decade it has become clear that glucocorticosteroid receptors play a significant role in the mechanism of glucocorticosteroid action . 
We studied glucocorticosteroid receptor density and affinity on peripheral blood mononuclear cells by the glucocorticosteroid binding assay in 33 patients with SLE who had taken no glucocorticosteroid for the previous 6 months and in 32 healthy controls . 
Patients ' disease activity was measured by the SLE Disease Activity Index ( SLEDAI ) . 
Glucocorticosteroid receptors on leukocytes of patients with SLE were significantly higher than in healthy controls ( 4419 +\/- 306 vs 3369 +\/- 196 , p less than 0.005 ) . 
The binding affinity was not different between patients and controls . 
There was no correlation between glucocorticosteroid receptor number and SLE disease activity . 
{ Changes in leucocytic estrogen receptor levels in patients with gynecomastia } 
The number of estrogen receptor ( ER ) in human peripheral leucocytes in 13 men with gynecomastia were measured by radioligand binding method . 
The results were compared with those of 13 sex- and age-matched healthy subjects . 
It was found that the number of ER in leucocytes was significantly increased in gynecomastia ( Rs of leucocytes were 1054 +\/- 254 sites\/cell ) . 
It suggested that increase of ER levels play an important role in the pathogenesis of gynecomastia . 
{ Changes in levels of leucocytic estrogen receptor in patients with menopausal type II diabetes and its significance } 
The number of estrogen receptors ( ER ) in human peripheral leucocytes in 12 women with menopausal type II diabetes was measured with radio-ligand binding method . 
The results were compared with those of 12 menopausal women without diabetes and 12 normal women of childbearing age . 
It was found that the number of ER in the patients was significantly decreased . 
Our data indicate that decrease of ER level in leukocytes may be related to the pathogenesis of type II diabetes in menopausal period . 
Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A { see comments } 
Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response . 
In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response . 
The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases . 
Binding of the drug inhibits isomerase activity , but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase , and not from inhibition of isomerase activity . 
A transcription factor , NF-AT , which is essential for early T-cell gene activation , seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs , with little or no effect on other transcription factors such as AP-1 and NF-kappa B . 
Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT . 
FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit . 
Regulation of M-CSF expression by M-CSF : role of protein kinase C and transcription factor NF kappa B . 
Macrophage-colony-stimulating factor ( M-CSF ) , also referred to as CSF-1 , regulates the survival , growth , differentiation and functional activity of monocytes by binding to a single class of high-affinity cell surface receptors , known to be the product of the c-fms protooncogene . 
The detection of both M-CSF and c-fms expression by cells of the monocyte lineage has suggested that M-CSF may act by an autocrine mechanism . 
Interestingly , it has been shown that M-CSF can induce the expression of its own gene . 
Although sensitivity to M-CSF can be modulated by regulation of receptor expression and function , M-CSF responsiveness is largely determined at a postreceptor level . 
To date , little is known about the intracellular pathway of M-CSF signal transduction . 
We have therefore investigated the changes in protein kinase C ( PKC ) activity upon exposure of monocytes to M-CSF . 
We show that M-CSF activates and translocates PKC . 
Inhibition of PKC by the isoquinoline derivative H7 abolishes induction of M-CSF by M-CSF . 
Furthermore , activation of PKC was pertussis-toxin-sensitive and was associated with the detection of an NF kappa B protein in nuclear extracts of M-CSF-induced blood monocytes but not in monocytes exposed to medium treatment only . 
The results suggest that M-CSF induction of M-CSF involves G proteins , PKC and NF kappa B . 
NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I . 
The effect of constitutive Tax expression on the interaction of NF-kappa B with its recognition sequence and on NF-kappa B-dependent gene expression was examined in T lymphoid Jurkat cell lines ( 19D and 9J ) stably transformed with a Tax expression vector . 
Tax expressing T cell lines contained a constitutive level of NF-kappa B binding activity , detectable by mobility shift assay and uv cross-linking using a palindromic NF-kappa B probe homologous to the interferon beta PRDII site . 
In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85 , 75 , and 54 kDa , whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced . 
Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters . 
Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment . 
Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters . 
Regulation of glucocorticoid receptors in human mononuclear cells : effects of glucocorticoid treatment , Cushing 's disease and ketoconazole . 
Glucocorticoid receptors ( GcR ) were determined by a whole cell assay in human mononulear leukocytes ( hMNL ) from control subjects , patients receiving glucocorticoid therapy for systemic diseases and Cushing 's disease patients with or without ketoconazole therapy . 
Prolonged corticosteroid treatment resulted in down-regulation of GcR , while the mean level of GcR in Cushing 's disease was normal . 
In this group , however , receptor levels and morning plasma cortisol values showed a negative correlation , indicating a subtle down-regulatory effect . 
Furthermore , GcR were unaltered after these patients received ketoconazole , in spite of a marked reduction in morning plasma cortisol and urinary free cortisol . 
We also observed that ketoconazole was a weak competitor of GcR in intact cells , although it significantly inhibited -LCB-3H-RCB- dexamethasone binding in cytosolic preparations from rat tissues . 
The results suggested that GcR in hMNL are down-regulated by synthetic steroids given in vivo , but they showed very mild down-regulation in hypercortisolemic patients suffering from Cushing 's disease . 
Finally , we did not observed either up-regulation or antagonism of GcR by ketoconazole treatment , at the time that cortisol levels of patients with Cushing 's disease were reduced . 
This indicates that the beneficial effects of ketoconazole in Cushing 's disease are due to adrenal cortisol suppression and not to interaction with GcR of target cells , and that the process of GcR regulation in hMNL is a complex phenomenon awaiting further elucidation . 
Glucocorticoid receptors in lymphocytes in anorexia nervosa . 
OBJECTIVE : The aim was to explore the down-regulation of the glucocorticoid receptors during hypercortisolaemia in anorexia nervosa . 
DESIGN : Urine and plasma samples were obtained for cortisol determination and blood lymphocytes were isolated for receptor binding studies . 
PATIENTS : Sixteen anorexic patients , aged 16-27 years , with a mean +\/- SEM body mass index of 14.2 +\/- 2.0 ( ranging from 11.1 to 17.4 ) , and 15 normal women were studied . 
Six patients were reinvestigated after a significant weight gain . 
MEASUREMENTS : The binding capacity and affinity of the glucocorticoid receptors were measured with dexamethasone as ligand on lymphocytes . 
RESULTS : In patients , both total and free plasma cortisol concentrations were higher than in the normal women , as was their urinary free cortisol ; the number of glucocorticoid receptors per cell ( Ro ) and the binding affinity ( Kd ) for dexamethasone were , however , not significantly different ( Ro : 7687 +\/- 1750 vs 7347 +\/- 1285 sites\/cell ; Kd : 7.7 +\/- 2.4 vs 7.4 +\/- 1.7 nM at 24 degrees C ) . 
After weight gain ( 14 +\/- 2 to 16 +\/- 2 kg\/m2 ) , receptor numbers were 8421 +\/- 2126 ( pre ) and 9011 +\/- 500 ( post ) sites\/cell , which are not significantly different ( P greater than 0.2 ) ; the Kd was unchanged ( 9.3 +\/- 2.6 vs 9.2 +\/- 2.4 nM ) . 
CONCLUSIONS Hypercortisolaemia does not down-regulate the lymphocyte glucocorticoid receptors in anorexia nervosa and a post-receptor defect might be involved in peripheral tissue resistance to the effects of glucocorticoid hormones in undernutrition . 
USF-related transcription factor , HIV-TF1 , stimulates transcription of human immunodeficiency virus-1 . 
The transcription factor HIV-TF1 , which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 ( HIV-1 ) , was purified from human B cells . 
HIV-TF1 had a molecular weight of 39,000 . 
Binding of HIV-TF1 to the HIV long terminal repeat ( LTR ) activated transcription from the HIV promoter in vitro . 
The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor ( USF ) in the adenovirus major late promoter . 
DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF . 
Interestingly , treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity , suggesting that phosphorylation of HIV-TF1 was essential for DNA binding . 
The disruption of HIV-TF1-binding site induced a 60 % decrease in the level of transcription from the HIV promoter in vivo . 
These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1 . 
The effect of toremifene therapy on serum immunoglobulin levels in breast cancer . 
Estrogens and anti-estrogens enhance the number of immunoglobulin ( Ig ) -secreting cells in pokeweed mitogen ( PWM ) -stimulated lymphocyte cultures . 
Lymphocytes from patients who have received anti-estrogen therapy show similar enhancement of Ig-secreting cells after PWM stimulation . 
In this study the effect of anti-estrogen ( toremifene ) therapy on serum immunoglobulin ( IgA , IgM , IgG ) levels in breast cancer patients was investigated . 
Serum Ig levels were followed up to two years after or during the therapy . 
An unexpected finding was that the Ig levels decreased during the follow-up period . 
This decrease was seen in patients who responded to the therapy as well as in those who did not . 
Induction of NF-kappa B during monocyte differentiation is associated with activation of HIV-gene expression . 
Cells of the monocyte-macrophage lineage are important targets of HIV infection . 
We report here that the phenotypic differentiation of monocyte cell lines induced by phorbol esters or tumour necrosis factor alpha ( TNF alpha ) is associated with expression of nuclear factor kappa B ( NF-kappa B ) . 
In parallel with such differentiation , HIV transcription , monitored using an HIV long terminal repeat reporter gene construct , is activated in such cells under the influence of enhanced NF-kappa B expression . 
Also , in a promonocyte cell line chronically infected with HIV , NF-kappa B expression and HIV transcription were enhanced on stimulation with phorbol ester or TNF alpha . 
Thus , stimulation of monocyte cell lines by phorbol esters or TNF alpha induces cell differentiation and activates HIV transcription . 
Such a process may have fundamental implications in AIDS pathogenesis in vivo and may be important in disease progression induced by opportunistic infections directly or indirectly involving macrophages . 
A nuclear factor NF-GM2 that interacts with a regulatory region of the GM-CSF gene essential for its induction in responses to T-cell activation : purification from human T-cell leukemia line Jurkat cells and similarity to NF-kappa B . 
Activation of T cells by antigen , lectin , or a combination of phorbol-12-myristate acetate ( PMA ) and calcium ionophore ( A23187 ) leads to the induction of genes for a set of lymphokines , including granulocyte-macrophage colony-stimulating factor ( GM-CSF ) . 
We demonstrated in earlier studies that the upstream region of the mouse GM-CSF promoter at positions between -95 and -73 is essential for transcriptional activation in response to PMA\/A23187 . 
This region contains two DNA-binding motifs , GM2 and GC-box . 
The GM2 sequence ( GGTAGTTCCC ) is recognized by an inducible factor NF-GM2 ; the other ( CCGCCC ) by constitutive factors A1 , A2 , and B . 
To elucidate the mechanism of GM-CSF gene activation , we have purified the inducible factor NF-GM2 from the nuclear extract of stimulated Jurkat cells on the basis of specific DNA-binding activity . 
The purified NF-GM2 consists of 50 ( p50 ) and 65 kDa ( p65 ) polypeptides and has a binding activity specific for both the GM-CSF and immunoglobulin kappa ( GGAAAGTCCC ) enhancers . 
Electrophoretically purified p50 alone can form a protein-DNA complex , but in the mixture , p50 associates preferentially with p65 to form the NF-GM2 complex . 
In addition , p65 gave per se , with low affinity , a protein-DNA complex that migrated more slowly than native NF-GM2 complex . 
Furthermore , an antiserum against KBF1 ( identical to 50 kDa NF-kappa B protein ) reacted with the p50 of NF-GM2 , indicating that the NF-GM2 polypeptide can not be immunologically differentiated from the 50 kDa subunit of NF-kappa B . 
The purified NF-GM2 activated in vitro transcription from the kappa B enhancer , while it failed to stimulate transcription from the GM-CSF promoter harboring the GM2 sequence . 
This suggests that the activation mechanism of the GM-CSF gene through the GM2\/GC-box sequence is different from that of genes carrying the kappa B enhancer alone . 
Glucocorticoid receptors in normal leukocytes : effects of age , gender , season , and plasma cortisol concentrations . 
We measured glucocorticoid receptors ( GR ) in mononuclear leukocytes ( MNL ) isolated from peripheral blood of 145 apparently healthy volunteers ( 86 men and 59 women ) . 
An age-related decrease in the number of GR was suggested between subjects younger than 20 years and elderly subjects ; there was no apparent seasonal variation in GR . 
Gender difference in the number of GR was not significant , although women showed slightly fewer GR . 
Eight patients with dermatomyositis\/polymyositis were examined to determine whether the number of GR in MNL could be down-regulated by their cognate ligands . 
The number of GR in MNL from these patients was significantly decreased one month after the initiation of prednisolone therapy . 
However , in normal subjects , the GR in MNL did not demonstrate circadian variation , in contrast to concentrations of plasma cortisol . 
Nuclear transcription factors that bind to elements of the IL-2 promoter . 
Induction requirements in primary human T cells . 
Prior studies have identified several elements that contribute to the activity of the IL-2 promoter in the stimulated T cell line , Jurkat . 
The sites and their corresponding nuclear binding factors include : NF-kappa B , AP-1 , AP-3 , OCT-1 , and NF-AT . 
The latter " nuclear factor for activated T cells " likely contributes to the tissue specificity of IL-2 gene expression . 
Using electrophoretic mobility shift assays , we have studied these transcription factors in primary T cells from human blood to verify their presence in a physiologic setting and to identify the signals that stimulate factor activity . 
All factors are induced in the nuclei of T cells upon activation with mitogens but not with exogenous IL-2 growth factor . 
However , the signaling requirements and sensitivity to protein synthesis inhibitors differ considerably . 
Only the activities for NF-AT and AP-1 sites require two signals for optimal induction , i.e. , PMA plus either lectin or antibody to the CD3 or CD28 surface molecules . 
Other factors are induced by lectin , antibody , and\/or PMA alone . 
After appropriate stimulation , both NF-AT and AP-1 are peculiarly sensitive to the protein synthesis inhibitor anisomycin . 
Our data correlate the activity of NF-AT and AP-1 in gel shift assays with the two signals requirements for IL-2 gene expression . 
An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1 . 
The transcription factor GATA-1 is expressed in a subset of hemopoietic cells , where it mediates the cell-type specific expression of several genes . 
We have cloned the mouse and human GATA-1 genes . 
A region upstream to the first exon , and highly conserved between mouse and man , acts as an erythroid specific enhancer in transient assays , if linked to the GATA-1 or to the SV40 promoter . 
The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site . 
T-helper-cell determinants in protein antigens are preferentially located in cysteine-rich antigen segments resistant to proteolytic cleavage by cathepsin B , L , and D . 
We report on a computer algorithm capable of predicting the location of T-helper-cell epitopes in protein antigen ( Ag ) by analysing the Ag amino acid sequence . 
The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B , L , and D . 
These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC , and resistant segments in Ag may , therefore , be expected to contain more T-cell determinants than susceptible segments . 
From information available in the literature on the substrate specificity of the three enzymes , it is clear that a cysteine is not accepted in any of the S2 , S1 , S1' , and S2' subsites of cathepsin B and L , and not in the S1 and S1' subsites of cathepsin D . 
Moreover , we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine , glycine , lysine , leucine , serine , threonine , and valine . 
By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false ( positive ) predictions . 
Furthermore , we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments . 
Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described . 
Synergism between two distinct elements of the HTLV-I enhancer during activation by the trans-activator of HTLV-I . 
We have conducted functional studies of the enhancer elements of human T-cell leukemia virus type I ( HTLV-I ) using the human T-cell lines Jurkat and MOLT 4 , which are negative for HTLV-I , and MT-2 and TL-Mor , which carry the proviral genome of HTLV-I . 
Two distinct elements have been implicated in function of the HTLV-I enhancer . 
One is the 21-base-pair ( bp ) core element that is responsible for trans-activation by the HTLV-I trans-activator p40tax and that has the ability to bind to cyclic-AMP responsive element binding factor ( CREB ) -like factor ( s ) . 
The other is a region interposed between the 21-bp elements . 
In this study we demonstrate that a subfragment ( C26 ) in the region between the 21-bp elements is involved in trans-activation by p40tax , possibly through binding to an NF-kappa B-like nuclear factor or factors . 
Formation of the protein-DNA complex with the C26 subfragment was positively affected by p40tax . 
The C26 element conferred partial responsiveness to p40tax when linked to one copy of the 21-bp element that , by itself , showed little activation in response to p40tax . 
However , the C26 element alone , even when repeated , could not be activated by p40tax , unlike other NF-kappa B-binding elements . 
In contrast , the C26 element itself was profoundly activated upon stimulation with 12-O-tetradecanoylphorbol-13-acetate . 
These findings therefore suggest that the HTLV-I enhancer contains multiple functional elements , including binding sites for at least CREB- and NF-kappa B-like factors , which synergistically cooperate in activation of the HTLV-I enhancer in response to p40tax . 
Our results also demonstrate that TPA-dependent activation of the HTLV-I enhancer may be mediated through the C26 element . 
Inhibition of phorbol ester-induced monocytic differentiation by dexamethasone is associated with down-regulation of c-fos and c-jun ( AP-1 ) . 
Previous studies have shown that treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate ( TPA ) is associated with induction of monocytic differentiation and expression of the c-jun and c-fos early response genes . 
The present work demonstrates that the glucocorticoid dexamethasone inhibits TPA-induced increases in c-jun and c-fos mRNA levels in U-937 leukemia cells . 
These findings were associated with a block in appearance of the monocytic phenotype , including inhibition of TPA-induced increases in lamin A , lamin C , and vimentin transcripts . 
Other studies have demonstrated that TPA-induced monocytic differentiation and expression of the c-jun and c-fos genes in myeloid leukemia cells are regulated by protein kinase C ( PKC ) . 
The finding that dexamethasone has no effect on TPA-induced activation of PKC suggests that this glucocorticoid inhibits signals downstream or parallel to this enzyme . 
Nuclear run-on assays demonstrate that : ( 1 ) induction of c-jun and c-fos expression by TPA is regulated by transcriptional mechanisms , ( 2 ) TPA-induced expression of c-jun and c-fos does not require protein synthesis , and ( 3 ) TPA-induced expression of both genes is inhibited at the transcriptional level by dexamethasone . 
To further define the effects of dexamethasone at the molecular level , we prepared a series of deleted c-jun promoter fragments linked to the chloramphenicol acetyltransferase ( CAT ) gene . 
Increases in CAT activity during transient expression of these constructs in TPA-treated U-937 cells could be assigned to the region ( -97 to -20 ) of the promoter that contains the AP-1 binding site . 
This induction of CAT activity was sensitive to dexamethasone . 
These findings suggest that dexamethasone down-regulates TPA-induced transcription of the c-jun gene during monocytic differentiation by inhibiting activation of the AP-1 site . 
Charybdotoxin-sensitive , Ca-LRB-2+-RRB--dependent membrane potential changes are not involved in human T or B cell activation and proliferation . 
The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding . 
Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding . 
Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium , the hyperpolarization is presumably through opening of Ca-LRB-2+-RRB--stimulated K+ channels . 
We have used charybdotoxin , a known inhibitor of Ca-LRB-2+-RRB--dependent K+ channels , to study the role of these channels in lymphocyte activation and mitogenesis . 
We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion , without affecting changes in cytosolic Ca2+ . 
However , blockade of the Ca-LRB-2+-RRB--activated K+ channel is not associated with changes in cell-cycle gene activation , IL-2 production , IL-2R expression or B and T cell mitogenesis . 
These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca-LRB-2+-RRB--activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis . 
One base pair change abolishes the T cell-restricted activity of a kB-like proto-enhancer element from the interleukin 2 promoter . 
The inducible , T cell-specific enhancers of murine and human Interleukin 2 ( Il-2 ) genes contain the kB-like sequence GGGATTTCACC as an essential cis-acting enhancer motif . 
When cloned in multiple copies this so-called TCEd ( distal T cell element ) acts as an inducible proto-enhancer element in E14 T lymphoma cells , but not in HeLa cells . 
In extracts of induced , Il-2 secreting El4 cells three individual protein factors bind to TCEd DNA . 
The binding of the most prominent factor , named TCF-1 ( T cell factor 1 ) , is correlated with the proto-enhancer activity of TCEd . 
TCF-1 consists of two polypeptides of about 50 kD and 105 kD ; the former seems to be related to the 50 kD polypeptide of NF-kB . 
Purified NF-kB is also able to bind to the TCEd , but TCF-1 binds stronger than NF-kB to TCEd DNA . 
The conversion of the TCEd to a ' perfect ' NF-kB binding site leads to a tighter binding of NF-kB to TCEd DNA and , as a functional consequence , to the activity of the ' converted ' TCEd motifs in HeLa cells . 
Thus , the substitution of the underlined A residue to a C within the GGGATTTCACC motif abolishes its T cell-restricted activity and leads to its functioning in both El4 cells and HeLa cells . 
These results indicate that lymphocyte-specific factors binding to the TCEd are involved in the control of T cell specific-transcription of the Il-2 gene . 
Negative regulation of human immunodeficiency virus type 1 expression in monocytes : role of the 65-kDa plus 50-kDa NF-kappa B dimer . 
Although monocytic cells can provide a reservoir for viral production in vivo , their regulation of human immunodeficiency virus type 1 ( HIV-1 ) transcription can be either latent , restricted , or productive . 
These differences in gene expression have not been molecularly defined . 
In THP-1 cells with restricted HIV expression , there is an absence of DNA-protein binding complex formation with the HIV-1 promoter-enhancer associated with markedly less viral RNA production . 
This absence of binding was localized to the NF-kappa B region of the HIV-1 enhancer ; the 65-kDa plus 50-kDa NF-kappa B heterodimer was preferentially lost . 
Adding purified NF-kappa B protein to nuclear extracts from cells with restricted expression overcomes this lack of binding . 
In addition , treatment of these nuclear extracts with sodium deoxycholate restored their ability to form the heterodimer , suggesting the presence of an inhibitor of NF-kappa B activity . 
Furthermore , treatment of nuclear extracts from these cells that had restricted expression with lipopolysaccharide increased viral production and NF-kappa B activity . 
Antiserum specific for NF-kappa B binding proteins , but not c-rel-specific antiserum , disrupted heterodimer complex formation . 
Thus , both NF-kappa B-binding complexes are needed for optimal viral transcription . 
Binding of the 65-kDa plus 50-kDa heterodimer to the HIV-1 enhancer can be negatively regulated in monocytes , providing one mechanism restricting HIV-1 gene expression . 
Isolation of a candidate repressor\/activator , NF-E1 ( YY-1 , delta ) , that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site . 
We have determined that the developmental control of immunoglobulin kappa 3' enhancer ( kappa E3' ) activity is the result of the combined influence of positive- and negative-acting elements . 
We show that a central core in the kappa E3' enhancer is active at the pre-B-cell stage but is repressed by flanking negative-acting elements . 
The negative-acting sequences repress enhancer activity in a position- and orientation-independent manner at the pre-B-cell stage . 
We have isolated a human cDNA clone encoding a zinc finger protein ( NF-E1 ) that binds to the negative-acting segment of the kappa E3' enhancer . 
This protein also binds to the immunoglobulin heavy-chain enhancer mu E1 site . 
NF-E1 is encoded by the same gene as the YY-1 protein , which binds to the adeno-associated virus P5 promoter . 
NF-E1 is also the human homologue of the mouse delta protein , which binds to ribosomal protein gene promoters . 
The predicted amino acid sequence of this protein contains features characteristic of transcriptional activators as well as transcriptional repressors . 
Cotransfection studies with this cDNA indicate that it can repress basal promoter activity . 
The apparent dual function of this protein is discussed . 
cAMP-dependent regulation of proenkephalin by JunD and JunB : positive and negative effects of AP-1 proteins . 
We demonstrate that JunD , a component of the AP-1 transcription factor complex , activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase , protein kinase A . 
Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP- , phorbol ester- , and Ca-LRB-2+-RRB--inducible enhancer , and JunD is shown to bind the enhancer as a homodimer . 
Another component of the AP-1 transcription complex , JunB , is shown to inhibit activation mediated by JunD . 
As a homodimer JunB is unable to bind the enhancer ; however in the presence of c-Fos , high-affinity binding is observed . 
Furthermore , JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion , further blurring the distinction between these response elements . 
These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways . 
Glucocorticoid resistance in chronic asthma . 
Glucocorticoid pharmacokinetics , glucocorticoid receptor characteristics , and inhibition of peripheral blood T cell proliferation by glucocorticoids in vitro . 
A total of 37 chronic , severe , nonsmoking asthmatic patients with documented reversible airways obstruction were classified as glucocorticoid-sensitive or -resistant on the basis of changes in FEV1 , FVC , and peak expiratory flow ( PEF ) after oral prednisolone . 
The resistant patients showed no significant improvements in airflow limitation . 
Phytohemagglutinin ( PHA ) -induced proliferation of peripheral blood T lymphocytes from the sensitive but not the resistant asthmatic patients was significantly ( p less than 0.01 ) inhibited by dexamethasone ( 10-LRB--7-RRB- mol\/L ) , reflecting a shift of the dose-response curve . 
When all the asthmatic patients were analyzed together , there was a significant correlation between the degree of sensitivity of T cells to dexamethasone and the clinical responsiveness to prednisolone ( p less than 0.01 ) . 
No differences were observed between six of the sensitive and resistant patients in the clearance of plasma prednisolone derived from orally administered prednisone . 
Peripheral blood mononuclear cell glucocorticoid receptors were also characterized in five sensitive and seven resistant patients . 
The numbers and binding affinities of these receptors could not account for the observed difference in the susceptibility of these cells to functional inhibition by dexamethasone in vitro . 
These results suggest that clinical glucocorticoid resistance in chronic asthma does not reflect abnormal glucocorticoid clearance but may be due at least partly to a relative insensitivity of T lymphocytes to glucocorticoids . 
This lack of sensitivity is unexplained but is not attributable to abnormalities of cellular glucocorticoid receptors . 
Identification of transcriptional suppressor proteins that bind to the negative regulatory element of the human immunodeficiency virus type 1 . 
Two different proteins which independently bound to neighboring sequences within the negative regulatory element ( NRE ) of human immunodeficiency virus type 1 ( HIV-1 ) were detected in the nuclear extract of a virus-infected human T cell line . 
One of the factors bound to a novel dyad symmetrical sequence . 
This sequence is well conserved in various HIV-1 isolates and partial homology was found with the promoter region of the human retinoblastoma gene . 
Similar DNA binding activity was detected in a variety of virus-uninfected human T cell lines and HeLa cells by means of a gel mobility shift assay . 
The other factor bound to a putative AP-1 recognition sequence predicted for the HIV-1 NRE . 
However , this factor did not bind to a typical AP-1 site . 
The insertion of multiple copies of the binding site for the former or latter factor into a heterologous promoter reduced the promoter activity to one-tenth or one-third , respectively . 
Thus , each factor may function as a novel negative regulator of transcription . 
Constitutive activation of NF-kB in human thymocytes . 
NF-kB is a eukaryotic transcription regulatory factor . 
In T cells and T cell lines , NF-kB is bound to a cytoplasmic proteic inhibitor , the IkB . 
Treatment of T cells with mitogens ( phorbol esters ) or cytokines ( TNF alpha ) induces NF-kB nuclear translocation and the subsequent expression of NF-kB dependent T cell genes . 
Here we examined the activation of NF-kB in human T cell thymic progenitors . 
We report differences in -LRB-Ca2+-RRB-i requirement for NF-kB activation in thymocytes as compared to mature T cells . 
Furthermore , our results indicated that thymocytes have a constitutively active form of NF-kB , suggesting that they are activated in vivo . 
The role of jun and fos gene family members in 12-O-tetradecanoylphorbol-13-acetate induced hemopoietic differentiation . 
Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression . 
In this study , we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation , with c-jun expression best paralleling differentiation . 
The generation of AP-1 complexes , as measured by DNA binding activity , closely parallels morphological differentiation . 
Furthermore , the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes . 
Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears . 
This tight correlation between c-jun expression , the generation of AP-1 activity , and differentiation suggests a critical role for this gene and transcriptional complex during this process . 
The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells . 
Regulation of replicative functions in the Epstein-Barr virus ( EBV ) genome is mediated through activation of a virally encoded transcription factor , Z ( BZLF1 ) . 
We have shown that the Z gene product , which binds to AP-1 sites as a homodimer and has sequence similarity to c-Fos , can efficiently activate the EBV early promoter , BMRF1 , in certain cell types ( i.e. , HeLa cells ) but not others ( i.e. , Jurkat cells ) . 
Here we demonstrate that the c-myb proto-oncogene product , which is itself a DNA-binding protein and transcriptional transactivator , can interact synergistically with Z in activating the BMRF1 promoter in Jurkat cells ( a T-cell line ) or Raji cells ( an EBV-positive B-cell ) , whereas the c-myb gene product by itself has little effect . 
The simian virus 40 early promoter is also synergistically activated by the Z\/c-myb combination . 
Synergistic transactivation of the BMRF1 promoter by the Z\/c-myb combination appears to involve direct binding by the Z protein but not the c-myb protein . 
A 30-bp sequence in the BMRF1 promoter which contains a Z binding site ( a consensus AP-1 site ) is sufficient to transfer high-level lymphoid-specific responsiveness to the Z\/c-myb combination to a heterologous promoter . 
That the c-myb oncogene product can interact synergistically with an EBV-encoded member of the leucine zipper protein family suggests 0 c-myb is likely to engage in similar interactions with cellularly encoded transcription factors . 
The 29-kDa proteins phosphorylated in thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein . 
Thrombin plays a critical role in platelet activation , hemostasis , and thrombosis . 
Cellular activation by thrombin leads to the phosphorylation of multiple proteins , most of which are unidentified . 
We have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin . 
A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic , unphosphorylated 27-kDa protein . 
Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species . 
Using this antibody , we isolated and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein ( HSP27 ) , a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen . 
The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies . 
Thus , the " estrogen receptor-related protein " is HSP27 , and the three major 29-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27 . 
These data suggest a role for HSP27 in the signal transduction events of platelet activation . 
High affinity aldosterone binding to plasma membrane rich fractions from mononuclear leukocytes : is there a membrane receptor for mineralocorticoids ? 
In vitro effects of aldosterone on the intracellular concentrations of sodium , potassium and calcium , cell volume and the sodium-proton-antiport have been described in intact human mononuclear leukocytes ( HML ) . 
In the present paper , the binding of a -LCB-125I-RCB--labeled aldosterone derivative to plasma membrane rich fractions of HML was studied . 
High affinity binding of the radioligand with an apparent Kd of approximately 0.1 nM was found . 
Aldosterone displaced the tracer at a similar Kd . 
Both canrenone and cortisol were inactive as ligands up to concentrations of 0.1 microM . 
The findings are the first 0 to demonstrate membrane binding sites with a high affinity for aldosterone , but not for cortisol . 
These data are perfectly compatible with major properties of steroidal effects on the sodium-proton-antiport in HML and thus very likely represent membrane receptors for aldosterone . 
Transcription factor requirements for U2 snRNA-encoding gene activation in B lymphoid cells . 
Transcription of a human U2 small nuclear RNA ( snRNA ) -encoding gene in HeLa cells requires a distal enhancer element , which is composed of one octamer motif ( Oct ) and three Sp 1-binding sites . 
To study the transcription factor requirement in B-cells , different U2 enhancer constructions were transfected into the lymphoid cell line , BJA-B . 
The results showed that the activation of U2 snRNA transcription in B-cells also requires an enhancer comprising both the Oct and at least one Sp 1-binding site . 
Deletion of all the Sp 1-binding sites from the enhancer reduces transcription by 80-90 % in HeLa , as well as in BJA-B cells , whereas the removal of the octamer-binding site reduces transcription to levels below detection in both cell types . 
Enhancers containing a single Oct have , nevertheless , the capacity to partially activate U2 snRNA transcription in both HeLa cells , in which only OTF-1 is expressed , and in BJA-B cells in which OTF-2 is the predominantly expressed octamer-binding factor . 
The most likely interpretation of our results is that both the ubiquitous transcription factor , OTF-1 , and the B-cell-specific transcription factor , OTF-2 , can activate U2 snRNA transcription . 
The results also revealed a similar functional cooperation between the transcription factors which bind to the Oct and the adjacent Sp 1-binding site in BJA-B cells , as has been observed in HeLa cells , since a template which contains a weak binding site for OTFs expresses wild-type levels of U2 snRNA in both cell types when the weak octamer-binding site is combined with a Sp 1-binding site . 
Cortisol receptor resistance : the variability of its clinical presentation and response to treatment . 
Primary ( partial ) cortisol receptor resistance was previously reported in a total of 7 patients and 14 asymptomatic family members . 
Its occurrence is considered to be extremely rare . 
In the present study we report on 6 patients ( 2 males and 4 females ) with the syndrome . 
The first male patient presented with mild hypertension . 
Hydrochlorothiazide therapy resulted in life-threatening hypokalemia . 
The second male patient had slight hypertension without hypokalemia . 
All four female patients presented between the age of 20-30 yr with acne , hirsutism , and irregular menstruations . 
Low dose dexamethasone therapy ( 1-1.5 mg\/day ) was of clinical benefit in these patients . 
All patients showed insufficient suppression of serum cortisol concentrations in the overnight 1-mg dexamethasone test . 
The diurnal rhythm of ACTH and cortisol was intact , albeit at an elevated level . 
There was a normal increase in ACTH , cortisol , and GH ( except in one obese patient ) in response to insulin-induced hypoglycemia , while cortisol production was elevated in three patients . 
Circulating adrenal androgen levels were increased in all patients . 
Glucocorticoid receptors were investigated in a whole cell dexamethasone binding assay in mononuclear leukocytes . 
In the first male patient , the number of receptors was very low , while the affinity was lower than that in controls . 
A lowered affinity to dexamethasone was found in one female patient , while a lowered number of receptors was found in three patients . 
In the second male patient , no abnormalities were found . 
As a bioassay for glucocorticoid action we also measured dexamethasone suppressibility of mitogen-stimulated incorporation of -LCB-3H-RCB-thymidine in mononuclear leukocytes . 
In the male patient with normal receptor status , dexamethasone suppressibility of -LCB-3H-RCB-thymidine incorporation was significantly lower than that in healthy controls with respect to both maximal suppression and IC50 . 
Partial cortisol receptor resistance might be less rare than previously thought . 
In the six patients presented , at least three different forms can be recognized . 
Therapy with dexamethasone was successful in female patients with acne and hirsutism , as the secondary increase in the production of adrenal androgens was effectively controlled . 
A novel mitogen-inducible gene product related to p50\/p105-NF-kappa B participates in transactivation through a kappa B site . 
A Rel-related , mitogen-inducible , kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells . 
The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein . 
This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B . 
Like the 105-kDa precursor , it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats . 
In vitro-translated proteins , truncated downstream of the Rel domain and excluding the repeats , bind kappa B sites . 
We refer to the kappa B-binding , truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97 . 
p50B is able to form heteromeric kappa B-binding complexes with RelB , as well as with p65 and p50 , the two subunits of NF-kappa B . 
Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site . 
The data imply the existence of a complex family of NF-kappa B-like transcription factors . 
{ Changes in leucocytic estrogen receptor levels in patients with climacteric syndrome and therapeutic effect of liuwei dihuang pills } 
The numbers of estrogen receptor ( ER ) in human peripheral leucocytes in 22 women with climacteric syndrome were measured by radioligand method . 
The results were compared with those of 12 normal child-bearing-age women . 
It wat found that the contents of leucocytic ER in climacteric syndrome patients were significantly lower than normal child-bearing-age women . 
The authors used a Chinese prescription -- Liuwei Dihuang Pills ( LDP ) to treat the patients for 2 months . 
The numbers of leucocytic ER were significantly increased after treatment . 
The data indicate that decrease of ER levels in cell may involve in the pathogenesis of climacteric syndrome . 
LDP not only increases plasma estradiol levels , but also increases the leucocytic ER levels . 
This may be the basis of the therapeutic effect on the disease . 
Activity of the kappa B enhancer of the interleukin-2 receptor alpha chain in somatic cell hybrids is accompanied by the nuclear localization of NF-kappa B . 
The two nuclear proteins NF-kappa B ( consisting of subunits p50 and p65 ) and the DNA-binding subunit of NF-kappa B ( p50 ) by itself , also called KBF1 , are constitutively expressed and localized in the nucleus of the human T-cell line IARC 301.5 . 
In order to define the roles of these two factors , which bind to the same kappa B enhancers , in transcription activation we have prepared somatic cell hybrids between IARC 301.5 and a murine myeloma . 
Most hybrids express both KBF1 and NF-kappa B in their nuclei , but one hybrid expresses only KBF1 . 
The kappa B enhancer of the gene encoding the interleukin-2 ( IL-2 ) receptor alpha chain ( IL-2R alpha ) is functional only in the hybrids expressing nuclear NF-kappa B . 
These findings show that nuclear NF-kappa B is necessary to activate the kappa B enhancer , while KBF1 by itself is not sufficient . 
We propose that KBF1 is a competitive inhibitor of NF-kappa B and discuss how these factors may be involved in the transient expression of IL-2 and IL-2 alpha genes during the immune response . 
Influence of estradiol and tamoxifen on susceptibility of human breast cancer cell lines to lysis by lymphokine-activated killer cells . 
The design of combination hormonal and immunotherapeutic protocols for breast cancer patients may be facilitated by analysis of preclinical in vitro model systems . 
Estrogen receptor positive ( ER+ : MCF-7 ) and negative ( ER- : MDA-MB-231 ) human breast cancer cell lines were utilized to evaluate the effects of tamoxifen ( TAM ) and estradiol ( E2 ) on modulation of breast cancer target susceptibility to lysis by lymphokine-activated killer ( LAK ) cells . 
E2-stimulated ER+ cells were more susceptible to lysis by LAK cells than corresponding TAM-treated or control cells , while treatment of ER- cells with either E2 or TAM alone did not alter from control their susceptibility to this immune-mediated lysis . 
All ER+ and ER- cells tested remained sensitive after treatment with TAM to lysis by LAK cells . 
In addition , an adenocarcinoma reactive human-mouse chimeric monoclonal antibody ( ING-1 ) was able to significantly boost in vivo generated LAK cell-mediated lysis of control , E2-treated , and TAM-treated ER+ and ER- cells . 
These in vitro results provide a preclinical rationale for in vivo testing of TAM , interleukin-2 ( IL-2 ) , and breast cancer reactive antibody-dependent cellular cytotoxicity facilitating antibody in patients with refractory or high risk breast cancer . 
Glucocorticoid receptor and inhibition of 3-O-methyl-D-glucose uptake by glucocorticoids in peripheral blood leukocytes from normal humans : correlation between receptor level and hormone effect in vitro . 
We have measured the glucocorticoid receptor concentration in mononuclear and polymorphonuclear leukocytes , both of which were isolated from peripheral blood from ten healthy male volunteers . 
In parallel , the inhibitory effect of dexamethasone on 3-O-methyl-D-glucose uptake was assayed in the corresponding mononuclear leukocytes . 
The glucocorticoid receptor levels in mononuclear leukocytes correlated with those in polymorphonuclear leukocytes , and there was a linear relationship between the cellular glucocorticoid receptor levels and glucocorticoid-mediated inhibition of the uptake of 3-O-methyl-D-glucose in mononuclear leukocytes . 
When mononuclear leukocytes were incubated in the presence of 8-bromo-cAMP , cellular glucocorticoid receptor levels increased and a more pronounced inhibitory effect of dexamethasone was observed on the transport of 3-O-methyl-D-glucose . 
We conclude that the cellular glucocorticoid receptor levels in peripheral blood leukocytes reflect in vitro responsiveness to glucocorticoids in mononuclear leukocytes from healthy males , and that the individual responsiveness may alter upon changes in the cellular levels of glucocorticoid receptor . 
Structure function analysis of vitamin D analogs with C-ring modifications . 
Analogs of 1 alpha,25-dihydroxyvitamin D3 ( 1 alpha,25--LRB-OH-RRB- 2D3 ) with substitutions on C-11 were synthesized . 
Small apolar substitutions ( 11 alpha-methyl , 11 alpha-fluoromethyl ) did not markedly decrease the affinity for the vitamin D receptor , but larger ( 11 alpha-chloromethyl or 11 alpha- or 11 beta-phenyl ) or more polar substitutions ( 11 alpha-hydroxymethyl , 11 alpha--LRB-2-hydroxyethyl } decreased the affinity to less than 5 % of that of 1 alpha,25-OH-RRB-2D3 . 
Their affinity for the vitamin D-binding protein , however , increased up to 4-fold . 
The biological activity of 11 alpha-methyl-1 alpha,25--LRB-OH-RRB-2D3 closely resembled that of the natural hormone on normal and leukemic cell proliferation and bone resorption , whereas its in vivo effect on calcium metabolism of the rachitic chick was about 50 % of that of 1 alpha,25--LRB-OH-RRB-2D3 . 
The 11 beta-methyl analog had a greater than 10-fold lower activity . 
The differentiating effects of the other C-11 analogs on human promyeloid leukemia cells ( HL-60 ) agreed well with their bone-resorbing activity and receptor affinity , but they demonstrated lower calcemic effects in vivo . 
Large or polar substitutions on C-11 of 1 alpha,25--LRB-OH-RRB-2D3 thus impair the binding of the vitamin D receptor but increase the affinity to vitamin D-binding protein . 
The effects of many C-11-substituted 1 alpha,25--LRB-OH-RRB-2D3 analogs on HL-60 cell differentiation exceeded their activity on calcium metabolism . 
Transcriptional regulation during T-cell development : the alpha TCR gene as a molecular model . 
The regulation of gene expression during lymphocyte differentiation is a complex process involving interactions between multiple positive and negative transcriptional regulatory elements . 
In this article , transcriptional regulation of the archetypal T-cell-specific gene , alpha TCR , is discussed . 
Major recent developments , including the identification of novel families of transcription factors that regulate multiple T-cell genes during thymocyte ontogeny and T-cell activation , are described . 
Induction of monocytic differentiation and NF-kappa B-like activities by human immunodeficiency virus 1 infection of myelomonoblastic cells . 
The effects of human immunodeficiency virus 1 ( HIV-1 ) infection on cellular differentiation and NF-kappa B DNA binding activity have been investigated in a new model of myeloid differentiation . 
PLB-985 cells represent a bipotential myelomonoblastic cell population capable of either granulocytic or monocytic differentiation after induction with appropriate inducers . 
By virtue of the presence of CD4 on the cell surface , PLB-985 cells were chronically infected with HIV-1 strain IIIB . 
PLB-IIIB cells clearly possessed a more monocytic phenotype than the parental myeloblasts , as determined by differential staining , increased expression of the myeloid-specific surface markers , and transcription of the c-fms proto-oncogene . 
NF-kappa B binding activity was inducible by tumor necrosis factor and phorbol myristate acetate in PLB-985 . 
However , in PLB-IIIB cells , constitutive expression of a novel NF-kappa B complex was detected , composed of proteins ranging between 70 and 110 kD . 
These proteins interacted specifically with the symmetric NF-kappa B site from the interferon beta ( IFN-beta ) promoter . 
Mutations affecting the 5' guanine residues of the kappa B site were unable to compete for these NF-kappa B-related proteins . 
Inducibility of endogenous IFN-beta and IFN-alpha RNA was also increased in PLB-IIIB cells . 
These studies indicate that HIV-1 infection of myelomonoblastic cells may select for a more mature monocytic phenotype and that unique subunit associations of NF-kappa B DNA binding proteins may contribute to differential NF-kappa B-mediated gene expression . 
The AP-1 site at -150 bp , but not the NF-kappa B site , is likely to represent the major target of protein kinase C in the interleukin 2 promoter . 
Stimulation of T cells with antigen results in activation of several kinases , including protein kinase C ( PKC ) , that may mediate the later induction of activation-related genes . 
We have examined the potential role of PKC in induction of the interleukin 2 ( IL-2 ) gene in T cells stimulated through the T cell receptor\/CD3 complex . 
We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC , and abrogates induction of IL-2 mRNA and protein . 
Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene . 
The IL-2 promoter contains binding sites for nuclear factors including NFAT-1 , Oct , NF-kappa B , and AP-1 , which are all potentially sensitive to activation of PKC . 
We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment , and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter . 
In contrast , mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter , and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion . 
Moreover , cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells . 
Our results indicate that the AP-1 site at -150 bp represents a major , if not the only , site of PKC responsiveness in the IL-2 promoter . 
Interleukin 6-induced differentiation of a human B cell line into IgM-secreting plasma cells is mediated by c-fos . 
The role of the protooncogene c-fos in interleukin ( IL ) 6-induced B cell differentiation was assessed . 
Treatment of SKW 6.4 cells with IL 6 induced a transient and early stimulation of c-fos sense mRNA expression . 
The effect appeared within 30 min and returned to basal levels after 2 h . 
The addition of antisense oligonucleotides to c-fos significantly inhibited IL 6-induced IgM production by SKW 6.4 cells ( p less than 0.001 ) , whereas control oligonucleotides had no inhibitory effect . 
These results indicate that activation of c-fos is involved in IL 6-induced differentiation of SKW 6.4 cells into IgM-secreting cells . 
Gangliosides suppress tumor necrosis factor production in human monocytes . 
Both normal and malignant cells contain gangliosides as important cell membrane constituents that , after being shed , may influence cells of the immune system . 
We have studied the impact of gangliosides on the expression of TNF in blood monocytes and in the monocytic cell line Mono Mac 6 . 
Although under standard culture conditions , bovine brain gangliosides ( 100 micrograms\/ml ) suppressed LPS-stimulated TNF production 5-fold in PBMC and 10-fold in Mono Mac 6 cells , suppression was more efficient under serum-free conditions . 
Looking at highly purified gangliosides , GD3 , GD1a , GM3 , GM2 , and GM1 were all effective in reducing TNF production in PBMC , and in Mono Mac 6 by factor 10 to 50 . 
The suppressive activity was lost in molecules , lacking the sugar moiety or the lipid moiety . 
Gangliosides appear to act at an early step of activation in that TNF transcripts were reduced and the mobilization of the nuclear factor kappa B was blocked . 
Furthermore , in time kinetics , gangliosides were effective for up to 30 min after addition of LPS , but not thereafter . 
However , the expression of the CD14 Ag , a receptor molecule for LPS-LPS binding protein complexes , was unaffected by gangliosides . 
Finally , when using Staphylococcus aureus or platelet activating factor as a stimulus , gangliosides were able to suppress TNF production in Mono Mac 6 cells by factor 5 to 10 , as well . 
On the other hand , phorbol ester-induced production of O2- was similar in cells treated with and without gangliosides . 
Taken together , our data demonstrate that TNF gene expression in monocytes induced by different types of stimuli can be blocked by gangliosides at an early step of signal transduction . 
T cell-specific negative regulation of transcription of the human cytokine IL-4 . 
IL-4 secreted by activated T cells is a pleiotropic cytokine affecting growth and differentiation of diverse cell types such as T cells , B cells , and mast cells . 
We investigated the upstream regulatory elements of the human IL-4 promoter . 
A novel T cell-specific negative regulatory element ( NRE ) composed of two protein-binding sites were mapped in the 5' flanking region of the IL-4 gene : -311CTCCCTTCT-303 ( NRE-I ) and -288CTTTTTGCTT-TGC-300 ( NRE-II ) . 
A T cell-specific protein Neg-1 and a ubiquitous protein Neg-2 binding to NRE-I and NRE-II , respectively , were identified . 
Furthermore , a positive regulatory element was found 45 bp downstream of the NRE . 
The enhancer activity of the PRE was completely suppressed when the NRE was present . 
These data suggest that IL-4 promoter activity is normally down-regulated by an NRE via repression of the enhancer positive regulatory element . 
These data may have implications for the stringent control of IL-4 expression in T cells . 
Human T cell activation through the activation-inducer molecule\/CD69 enhances the activity of transcription factor AP-1 . 
The induction of the AP-1 transcription factor has been ascribed to the early events leading to T cell differentiation and activation . 
We have studied the regulation of AP-1 activity in human peripheral blood T lymphocytes stimulated through the activation inducer molecule ( AIM ) \/CD69 activation pathway . 
Phorbol esters are required to induce AIM\/CD69 cell-surface expression as well as for triggering the proliferation of T cells in conjunction with anti-AIM mAb . 
Mobility shift assays showed that addition of anti-AIM mAb to PMA-treated T lymphocytes markedly enhanced the binding activity of AP-1 to its cognate sequence , the phorbol ester response element . 
In contrast , anti-AIM mAb did not induce any change in the binding activity of NF-kappa B , a transcription factor whose activity is also regulated by protein kinase C . 
The increase in AP-1-binding activity was accompanied by the marked stimulation of the transcription of c-fos but not that of c-jun . 
Blockade of the DNA-binding complexes with an anti-Fos mAb demonstrated a direct participation of c-Fos in the AP-1 complexes induced by anti-AIM mAb . 
Most of the AP-1 activity could be eliminated when the anti-AIM mAb was added to the culture medium in the presence of cycloheximide , suggesting that de novo protein synthesis is crucial for the induction of AP-1-binding activity . 
These data provide the evidence that activation of human peripheral blood T cells through the AIM activation pathway regulate the activity of AP-1 . 
Therefore , this pathway appears as a crucial step in the initiation of early T cell activation events . 
cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein , Elf-1 . 
The recent definition of a consensus DNA binding sequence for the Ets family of transcription factors has allowed the identification of potential Ets binding sites in the promoters and enhancers of many inducible T-cell genes . 
In the studies described in this report , we have identified two potential Ets binding sites , EBS1 and EBS2 , which are conserved in both the human and murine interleukin-2 enhancers . 
Within the human enhancer , these two sites are located within the previously defined DNase I footprints , NFAT-1 and NFIL-2B , respectively . 
Electrophoretic mobility shift and methylation interference analyses demonstrated that EBS1 and EBS2 are essential for the formation of the NFAT-1 and NFIL-2B nuclear protein complexes . 
Furthermore , in vitro mutagenesis experiments demonstrated that inducible interleukin-2 enhancer function requires the presence of either EBS1 or EBS2 . 
Two well-characterized Ets family members , Ets-1 and Ets-2 , are reciprocally expressed during T-cell activation . 
Surprisingly , however , neither of these proteins bound in vitro to EBS1 or EBS2 . 
We therefore screened a T-cell cDNA library under low-stringency conditions with a probe from the DNA binding domain of Ets-1 and isolated a novel Ets family member , Elf-1 . 
Elf-1 contains a DNA binding domain that is nearly identical to that of E74 , the ecdysone-inducible Drosophila transcription factor required for metamorphosis ( hence the name Elf-1 , for E74-like factor 1 ) . 
Elf-1 bound specifically to both EBS1 and EBS2 in electrophoretic mobility shift assays . 
It also bound to the purine-rich CD3R element from the human immunodeficiency virus type 2 long terminal repeat , which is required for inducible virus expression in response to signalling through the T-cell receptor . 
Taken together , these results demonstrate that multiple Ets family members with apparently distinct DNA binding specificities regulate differential gene expression in resting and activated T cells . 
Mineralocorticoids and mineralocorticoid receptors in mononuclear leukocytes in patients with pregnancy-induced hypertension . 
To examine the role of mineralocorticoids in the pathophysiology of pregnancy-induced hypertension ( PIH ) , we studied plasma aldosterone and 18-hydroxycorticosterone levels in 25 women with PIH and 25 normal pregnant women , as controls . 
Furthermore , we evaluated the mineralocorticoid receptor ( MR ) status in mononuclear leukocytes in the 2 groups . 
MR count was significantly ( P less than 0.0005 ) decreased in the PIH group ( 148 +\/- 9 binding sites\/cell ) compared with the control group ( 300 +\/- 17 binding sites\/cell ; mean +\/- SEM ) . 
Plasma aldosterone in women with PIH was 281 +\/- 61 pmol\/L ; in normal pregnant women it was 697 +\/- 172 pmol\/L ( P less than 0.025 ) . 
Plasma 18-hydroxycorticosterone was also significantly ( P less than 0.025 ) lower ( PIH , 1071 +\/- 149 pmol\/L ; controls , 1907 +\/- 318 pmol\/L ) . 
These values were determined at the onset of clinical symptoms of PIH . 
These results can not be explained by receptor down-regulation due to higher levels of mineralocorticoids in PIH ; a hitherto unknown mineralocorticoid may , thus , be responsible for the hypertension and altered MR status . 
A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells . 
The proto-oncoprotein c-Rel is a member of the nuclear factor kappa B transcription factor family , which includes the p50 and p65 subunits of nuclear factor kappa B . 
We show here that c-Rel binds to kappa B sites as homodimers as well as heterodimers with p50 . 
These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various kappa B motifs . 
In particular , the c-Rel homodimer has a high affinity for interleukin-6 ( IL-6 ) and beta interferon kappa B sites . 
In spite of its association with p50 in vitro , however , we found a lymphoid cell-specific nuclear factor in vivo that contains c-Rel but not p50 epitopes ; this factor , termed IL-6 kappa B binding factor II , appears to contain the c-Rel homodimer and preferentially recognizes several IL-6 kappa B-related kappa B motifs . 
Although it has been previously shown that the IL-6 kappa B motif functions as a potent IL-1\/tumor necrosis factor-responsive element in nonlymphoid cells , its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line . 
We also present evidence that IL-6 kappa B binding factor II functions as a repressor specific for IL-6 kappa B-related kappa B motifs in lymphoid cells . 
Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors : a possible target for v-erbA oncogene action . 
The v-erbA oncogene , a mutated version of the thyroid hormone receptor alpha ( c-erbA\/TR-alpha ) , inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes , suggesting a normal function of the proto-oncogene c-erbA in erythropoiesis . 
Here we demonstrate that the endogenous thyroid hormone receptor alpha ( c-erbA\/TR-alpha ) and the closely related retinoic acid receptor alpha ( RAR-alpha ) play a role in the regulation of normal erythroid differentiation . 
Retinoic acid ( RA ) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene . 
When added pulsewise to immature cells , differentiation was accelerated while more mature cells underwent premature cell death . 
Thyroid hormone ( T3 ) alone caused similar but weaker effects . 
Interestingly , T3 strongly enhanced the action of RA , suggesting cooperative action of the two receptors in modulating erythroid differentiation . 
Expression of the human RAR-alpha in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells , thus showing that the RAR-alpha is essential for the RA effect . 
Likewise , enhanced expression of exogenous c-erbA\/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3 , suggesting a similar function of both receptors . 
An 11-base-pair DNA sequence motif apparently unique to the human interleukin 4 gene confers responsiveness to T-cell activation signals . 
We have identified a DNA segment that confers responsiveness to antigen stimulation signals on the human interleukin ( IL ) 4 gene in Jurkat cells . 
The human IL-4 gene , of 10 kilobases , is composed of four exons and three introns . 
A cis-acting element ( P sequence ) resides in the 5' upstream region ; no additional DNA segments with enhancer activity were identified in the human IL-4 gene . 
For further mapping purposes , a fusion promoter was constructed with the granulocyte\/macrophage colony-stimulating factor basic promoter containing 60 base pairs of sequence upstream from the cap site of the mouse granulocyte\/macrophage colony-stimulating factor gene and various lengths of the 5' upstream sequence of the IL-4 gene . 
The P sequence was located between positions -79 and -69 relative to the transcription start site of the human IL-4 gene , and this location was confirmed by base-substitution mutations . 
The plasmids carrying multiple copies of the P sequence showed higher responsiveness to the stimulation . 
The binding protein ( s ) that recognize the P sequence of the IL-4 gene were identified by DNA-mobility-shift assays . 
The binding of NF-LRB-P-RRB- ( a DNA binding protein that specifically recognizes the P sequence ) to the P sequence was abolished when oligonucleotides carrying base substitutions were used , indicating that the NF-LRB-P-RRB- interaction is sequence-specific and that binding specificity of the protein paralleled the sequence requirements for IL-4 expression in vivo . 
The P sequence does not share homology with the 5' upstream sequence of the IL-2 gene , even though surrounding sequences of the IL-4 gene share high homology with the IL-2 gene . 
We conclude that a different set of proteins recognize IL-2 and IL-4 genes . 
Glucocorticoid receptor binding in three different cell types in major depressive disorder : lack of evidence of receptor binding defect . 
1 . In order to further understand the apparent glucocorticoid resistance in major depressive disorder , circadian variation in cortisol concentration , dexamethasone suppression and glucocorticoid receptor binding in mononuclear leukocytes , polymorphonuclear leukocytes and cultured skin fibroblasts were measured in rigidly defined major depressive disorder patients and non-depressed psychiatric controls . 
2 . Mononuclear leukocytes binding to glucocorticoid correlated significantly with polymorphonuclear leukocytes binding to glucocorticoid , but both determinations failed to differentiate major depressive disorder and control subjects . 
3 . Initial and post-dexamethasone in vitro fibroblast binding to glucocorticoid was not different between major depressive disorder and non-depressed control subjects . 
4 . The phenomenon of glucocorticoid resistance in major depressive disorder remains unexplained . 
Kinetics of nuclear translocation and turnover of the vitamin D receptor in human HL60 leukemia cells and peripheral blood lymphocytes -- coincident rise of DNA-relaxing activity in nuclear extracts . 
High affinity receptors ( VDR ) for 1,25-dihydroxycholecalciferol ( calcitriol ) are expressed in HL60 human leukemia cells and in low numbers in peripheral blood lymphocytes ( PBL ) . 
HL60 cells , expressing some characteristics of promyelocytes , can be induced to monocytoid differentiation by calcitriol . 
Specific nuclear translocation of -LCB-3H-RCB-calcitriol\/VDR was examined after exposure of whole cells to 10-LRB--9-RRB- M\/l calcitriol in the presence and absence of a 500-fold excess of unlabeled ligand and subsequent isolation of nuclei . 
Specific nuclear translocation of -LCB-3H-RCB-calcitriol\/VDR was found to be time dependent reaching a maximum of approximately 2100 binding sites\/nucleus after 3 h of incubation in HL60 cells , whereas a maximum of approximately 310 binding sites\/nucleus was found after 3 h in PBL . 
Pulse exposure of HL60 to radiolabeled hormone for 3 h followed by culture in medium without serum and calcitriol lead to nuclear retention of approximately 1600 radiolabeled VDR by 8 h and approximately 1000 VDR by 24 h . 
Radiolabeled VDR disappeared from the nuclear compartment with a halflife of approximately 30 min if cells were cultured with identical concentrations of unlabeled hormone after the pulse ( pulse\/chase-experiments ) . 
No difference of VDR retention in pulse and pulse\/chase-experiments was seen in PBL , where VDR halflife was approximately 30 min . 
No specific translocation into the nuclear compartment was seen when isolated nuclei were incubated in -LCB-3H-RCB-calcitriol . 
Radiolabeled hormone\/receptor complexes of nuclei isolated from cells exposed for 3 h to radiolabeled hormone -- in contrast to identical experiments with intact cells -- did not disappear from the nuclear compartment upon incubation of nuclei with identical concentrations of the unlabeled compound . 
The activity of DNA relaxing enzymes ( e.g. topoisomerases I and II ) in nuclear extracts was measured using a PBR 322-relaxation-assay . 
Enhanced overall enzyme activity was found in nuclear extracts by 1 h after incubation with calcitriol ( final ethanol concentration 0.0001 % v\/v ) in HL60 and PBL . 
The enhanced activity disappeared after 2 h in PBL , whereas it was still enhanced by 4 h in HL60 . 
No effect was seen in ethanol treated controls . 
We conclude that a specific nuclear translocation mechanism exists for calcitriol in both cell types examined , most likely due to translocation of receptor proteins after hormone binding . 
Translocated hormone\/receptor complexes compete for a limited number of specific nuclear binding sites . 
Enhanced activity of topoisomerases in nuclear extracts upon translocation of VDR might reflect interaction of both within the nuclear compartment , thus initiating DNA-unwinding , a prerequisite of transcription initiation . 
{ Regulation of intracellular cholesterol synthesis in hypercholesterolemia by glucocorticoids } 
The rate of endogenous cholesterol synthesis in blood lymphocytes and skin fibroblasts from patients with type IIa hyperlipidemia was found to be increased in comparison with healthy donors . 
The cells of hyperlipidemic patients had lowered levels of glucocorticoid receptors concomitantly with a partial loss of their sensitivity to glucocorticoids . 
In fibroblasts from patients with hereditary hypercholesteremia of homozygous type the number of glucocorticoid receptors did not exceed 10 % of their content in normal cells . 
The decrease of the number of glucocorticoid receptors in patients with type IIa hyperlipidemia seems to be a compensatory response of cells culminating in activation of endogenous cholesterol synthesis . 
Transcription factor activation and functional stimulation of human monocytes . 
Activation of expression of genes encoding transcription factors : c-fos and c-jun and formation of AP1 transcriptional complex in human monocytes was investigated . 
It was found that lipopolysaccharide induced strongly both c-fos and c-jun expression as well as AP1 formation . 
Interferon gamma activated strongly c-fos and weakly c-jun and AP1 . 
Tumor necrosis factor induced slightly c-fos and had almost no effect on c-jun and AP1 . 
The data suggest that differences in functional responses elicited in monocytes by all three factors may be dependent on different routes on nuclear signalling employed by the factors . 
Stable expression of HB24 , a diverged human homeobox gene , in T lymphocytes induces genes involved in T cell activation and growth . 
A diverged homeobox gene , HB24 , which is known to be induced following lymphocyte activation , was introduced into Jurkat T cells under the control of a constitutive promoter . 
Stable transfectants of HB24 were established that expressed high levels of HB24 mRNA and possessed an altered phenotype suggestive of activated T cells . 
A number of genes known to be induced following T cell activation and associated with cell growth were increased in the transfectants , including c-fos , c-myc , c-myb , HLA-DR , lck , NF-kappa B , interleukin-2 and interleukin-2 receptor alpha ( IL-2R alpha ) . 
Analysis of IL-2R alpha expression by transient transfection of IL-2R alpha promoter constructs into the HB24 transfectants revealed constitutive expression ( about 60 % of phytohemagglutinin- and phorbol ester-activated Jurkat cells ) that was dependent on the kappa B site in the IL-2R alpha promoter . 
Furthermore , as a consequence of the increased HB24 mRNA levels , the Jurkat HB24 transfectants proliferated more rapidly than control cell lines . 
Thus , stable expression of HB24 confers an activation phenotype on a human T cell line , implicating this gene as an important transcriptional factor during T cell activation and growth . 
Studies on the biological activity of triiodothyronine sulfate . 
Hepatic microsomes and isolated hepatocytes in short term culture desulfate T3 sulfate ( T3SO4 ) . 
We , therefore , wished to determine whether T3SO4 could mimic the action of thyroid hormone in vitro . 
T3SO4 had no thyromimetic effect on the activity of Ca-LRB-2+-RRB--ATPase in human erythrocyte membranes at doses up to 10,000 times the maximally effective dose of T3 ( 10\/-LRB--10-RRB- mol\/L ) . 
In GH4C1 pituitary cells , T3SO4 failed to displace -LCB-125I-RCB-T3 from nuclear receptors in intact cells or soluble preparations . 
Thus , T3SO4 was not directly thyromimetic in either an isolated human membrane system or a pituitary cell system in which nuclear receptor occupancy correlates with GH synthesis . 
Thyroid hormones inhibit -LCB-3H-RCB-glycosaminoglycan synthesis by cultured human dermal fibroblasts , and T3SO4 displayed about 0.5 % the activity of T3 at 72 h . 
Human fibroblasts contained roughly the same level of microsomal p-nitrophenyl sulfatase activity as that previously observed in hepatic microsomes . 
Propylthiouracil ( 50 mumol\/L ) did not affect the action of T3SO4 , suggesting that deiodination was not important for this activity of T3SO4 . 
Thus , it appears 0 T3SO4 has no intrinsic biological activity , but , under certain circumstances , may be reactivated by desulfation . 
Nuclear factor of activated T cells contains Fos and Jun . 
The nuclear factor NF-AT ( ref. 1 ) is induced in T cells stimulated through the T-cell receptor\/CD3 complex , and is required for interleukin-2 ( IL-2 ) gene induction . 
Although NF-AT has not been cloned or purified , there is evidence that it is a major target for immunosuppression by cyclosporin A ( CsA ) and FK506 ( refs 2-7 ) . 
NF-AT induction may require two activation-dependent events : the CsA-sensitive translocation of a pre-existing component and the CsA-resistant synthesis of a nuclear component . 
Here we report that the newly synthesized nuclear component of NF-AT is the transcription factor AP-1 . 
We show that the inducible nuclear form of NF-AT contains Fos and Jun proteins . 
Furthermore , we identify a pre-existing NF-AT-binding factor that is present in hypotonic extracts of unstimulated T cells . 
On the basis of binding , reconstitution and cotransfection experiments , we propose that activation of NF-AT occurs in at least two stages : a CsA-sensitive stage involving modification and\/or translocation of the pre-existing NF-AT complex , and a CsA-insensitive stage involving the addition of newly synthesized Fos or Fos\/Jun proteins to the pre-existing complex . 
The mechanism of action of cyclosporin A and FK506 . 
CsA and FK506 are powerful suppressors of the immune system , most notably of T cells . 
They act at a point in activation that lies between receptor ligation and the transcription of early genes . 
Here , Stuart Schreiber and Gerald Crabtree review recent findings that indicate 0 CsA and FK506 operate as prodrugs : they bind endogenous intracellular receptors , the immunophilins , and the resulting complex targets the protein phosphatase , calcineurin , to exert the immunosuppressive effect . 
{ Plasma cortisol concentration and blood leukocyte content of glucocorticoid receptors in patients with deficiency-cold vs deficiency-heat syndromes } 
Plasma cortisol concentration and blood leukocyte content of glucocorticoid receptors ( GCR ) were assayed in 20 patients with deficiency syndromes , 10 cold in property ( deficiency-cold ) , the other 10 hot in property ( deficiency-heat ) , and also in 10 healthy individuals as normal control for the purpose of investigating the nature of cold and heat syndromes . 
As a result , the cases of deficiency-cold syndrome ( DCS ) had a normal concentration of plasma cortisol but a lowered content of GCR in leukocytes when compared with the normal control ( P less than 0.05 ) ; the cases of deficiency-heat syndrome ( DHS ) had a higher concentration of plasma cortisol than the normal control ( P less than 0.05 ) and a slightly higher content of GCR in leukocytes . 
It was concluded that the DCS is characterized by diminished biological effects of adrenocortical activity , while the DHS , by augmented biological effects of adrenocortical activity . 
Activation of the human immunodeficiency virus type 1 enhancer is not dependent on NFAT-1 . 
The function of a putative NFAT-1 site in the human immunodeficiency virus type 1 enhancer has been analyzed . 
Activation by the T-cell antigen receptor is minimal in Jurkat cells and is mediated by the kappa B sites . 
The putative NFAT-1 region is not required for the response to anti-CD3 or to mitogens in T-cell , B-cell , or monocyte\/macrophage leukemia lines , nor is it a cis-acting negative regulatory element . 
Protein kinase C activation and protooncogene expression in differentiation\/retrodifferentiation of human U-937 leukemia cells . 
Human U-937 leukemia cells differentiate along the monocytic lineage following 3-day exposures to 12-O-tetradecanoylphorbol-13-acetate ( TPA ) . 
This induction of differentiation is accompanied by adherence and loss of proliferation , as well as expression\/repression of differentiation-associated genes . 
Long term culture of TPA-differentiated U-937 cells in the absence of phorbol ester for 32-36 days resulted in a process of retrodifferentiation . 
The retrodifferentiated cells detached from the substrate and reinitiated proliferation . 
Other cellular parameters , such as glycosidase activities , cytokine release , and filament expression , returned to levels similar to that observed in uninduced cells . 
Treatment of U-937 cells with TPA resulted in a rapid translocation of protein kinase C ( PKC ) from the cytosol to cell membrane fractions within 2-8 min . 
Increased levels of membrane-associated PKC activity persisted until 17-29 days . 
However , longer periods of incubation were associated with a return to the distribution of PKC in control cells . 
Activation of PKC has been implicated in the regulation of certain immediate early response genes , and in the present studies , TPA rapidly induced c-fos and c-jun gene expression . 
Levels of c-fos and c-jun transcripts remained elevated during periods of PKC activation and also returned to levels observed in control cells by 30-36 days , when the cells entered retrodifferentiation . 
Staurosporine , a nonspecific inhibitor of PKC , partially blocked TPA-induced adherence and growth inhibition and concomitantly prevented TPA-induced c-fos and c-jun gene expression . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP . 
The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation . 
Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines . 
The human CD19 gene has been cloned , and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies . 
In particular , a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites . 
Moreover , this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells . 
This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments . 
In addition , BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter . 
Together , this evidence strongly implicates BSAP in the regulation of the CD19 gene . 
Eicosanoids in breast cancer patients before and after mastectomy . 
In 19 patients with a malignant breast tumor , tumor tissue and blood were taken to determine the eicosanoid profile and platelet aggregation . 
Values were compared with those of patients with benign tumors ( n = 4 ) , or undergoing a mammary reduction ( n = 7 ) . 
Postoperatively , blood was taken as well in order to compare pre- and postoperative values . 
Eicosanoids were measured in peripheral blood monocytes and mammary tissue by means of HPLC ; furthermore , TXA2 , 6-keto-PGF1 alpha , and PGE2 were determined by RIA . 
Differences in pre- and postoperative values of cancer patients were seen in plasma RIA values : PGE2 and 6-k-PGF1 alpha were significantly higher preoperatively when compared with postoperatively , however , such differences were seen in the control groups as well . 
Compared to benign tumor or mammary reduction test material the eicosanoid profile of tissue obtained from malignant mammary tumors showed important differences . 
Except for PGF2 alpha , HHT and 15-HETE no detectable quantities of eicosanoids were found in the non-tumor material , whereas in the malignant tumor material substantial quantities of a number of eicosanoid metabolites were present . 
Statistically significant correlations could be established between patient\/histopathology data and the results of the platelet aggregation assays , e.g. between menopausal status and ADP aggregation ; oestrogen receptor ( +\/- ) and collagen and arachidonic acid aggregation , inflammatory cell infiltration score and arachidonic acid aggregation and fibrosis score and ADP aggregation . 
The results show that eicosanoid synthesis in material from mammary cancer patients is different from that in benign mammary tissue . 
The implications , in particular , in relation to future prognosis of the patient , remain obscure . 
c-myc mRNA expression in minor salivary glands of patients with Sjogren 's syndrome . 
c-myc protooncogene is implicated in the pathogenesis of B cell lymphoid malignancies and high levels of c-myc mRNA expression are observed in activated blood mononuclear cells . 
Sjogren 's syndrome ( SS ) is characterized by lymphocytic infiltrates of exocrine glands , remarkable B cell hyperreactivity and a strong predisposition to B cell neoplasia . 
In this study , c-myc protooncogene mRNA expression in 29 labial minor salivary gland biopsies from patients with primary SS and 15 controls was examined using in situ hybridization histochemistry . 
Two 40mer oligonucleotides from the 1st and the 2nd exon of the c-myc gene , labeled with 35S , were used as probes . 
To detect the origin of the cell hybridized with a c-myc probe , a combined immunochemistry in situ hybridization histochemistry technique was used . 
High c-myc mRNA expression was detected on acinar epithelial cells . 
c-myc did not correlate with c-fos and c-jun protein expression . 
Stronger c-myc mRNA expression was detected in labial salivary glands of patients with longer disease duration ( p less than or equal to 0.002 ) and more intense T lymphocyte infiltrates ( p less than 0.05 ) although these patients revealed no hypergammaglobulinemia . 
No correlation was observed between c-myc mRNA and B lymphocyte monoclonicity or lymphoma . 
In conclusion , strong c-myc mRNA expression was observed on epithelial cells of labial salivary glands from patients with primary SS . 
Our findings may indicate the presence of a reactivated virus hosted in these cells . 
Every enhancer works with every promoter for all the combinations tested : could new regulatory pathways evolve by enhancer shuffling ? 
The promoters and enhancers of cell type-specific genes are often conserved in evolution , and hence one might expect that a given enhancer has evolved to work best with its own promoter . 
While this expectation may be realized in some cases , we have not found evidence for it . 
A total of 27 combinations of different promoters and enhancers were tested by transfection into cultured cells . 
We found that the relative efficiency of the enhancers is approximately the same , irrespective of the type of promoter used , i.e. , there was no strong preference for any given enhancer\/promoter combination . 
Notably , we do not see particularly strong transcription when the immunoglobulin kappa enhancer ( or the immunoglobulin heavy chain enhancer ) is used to activate a kappa gene promoter . 
We propose that a generally permissive enhancer\/promoter interaction is of evolutionary benefit for higher eukaryotes : by enhancer shuffling , genes could be easily brought under a new type of inducibility\/cell type specificity . 
Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity . 
Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif . 
Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells , however . 
We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line . 
Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells . 
This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation . 
Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation . 
Moreover , this domain conferred enhancing activity when fused to the Oct1 protein , which by itself was unable to stimulate from a remote position . 
The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position . 
{ Changes in plasma interleukin-1 and their possible relationship with the changes in glucocorticoid receptor in aged long-distance runner } 
For the study of the changes in plasma interleukin-1 ( IL-1 ) and their possible relationship with the changes in glucocorticoid receptor ( GR ) , plasma IL-1 and GR in peripheral blood leukocytes in aged long-distance runner were measured simultaneously . 
The activity of IL-1 was expressed as its ability to stimulate 3H-TdR incorporation in the thymocytes of C57 mice . 
GR was determined by whole cell assay with 3H-Dex . 
The results showed that the activity of plasma IL-1 in aged long-distance runner was 209 % , 223 % and 145 % of the control at 14.7-18.7 , 3.8-7.0 and 1.5-2.6 KD fractions . 
The GR in peripheral blood leukocytes in aged runner was 65 % of the control . 
Possible relationship between the changes in IL-1 and GR in aged long-distance runner and its physiological significance are discussed . 
Transcription factor AP-2 activates gene expression of HTLV-I . 
The HTLV-I LTR contains three conserved regulatory elements known as 21 base pair repeats which are required for stimulation of gene expression by the transactivator protein tax . 
Mutagenesis indicates that the 21 bp repeats can be subdivided into three motifs , A , B and C , each of which influences the level of tax activation . 
The A site in the 21 bp repeat has strong homology with previously described binding sites for the transcription factor AP-2 . 
We demonstrated that AP-2 mRNA was present in T-lymphocytes and that cellular factors from both non-transformed and transformed T-lymphocytes specifically bound to the consensus motif for AP-2 in each 21 bp . 
To determine the role of AP-2 in the regulation of the HTLV-I LTR gene expression , we used an AP-2 cDNA in DNA binding and transient expression assays . 
Gel retardation and methylation interference studies revealed that bacterially produced AP-2 bound specifically and with high affinity to all three 21 bp repeats , and that it required the core sequence AGGC for specific binding . 
Binding of AP-2 prevented the subsequent binding of members of the CREB\/ATF family to an adjacent regulatory motif in the 21 bp repeat . 
Transfection of an AP-2 expression construct into T-lymphocytes activated gene expression from the HTLV-I LTR . 
At least two 21 bp repeats were required for high levels of AP-2 activation and mutagenesis of the AP-2 consensus binding sequences in the 21 bp repeats eliminate this activation . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
{ Age-related changes in glucocorticoid and mineralocorticoid receptors in lymphocytes of healthy persons and patients with hypertension } 
It has been found that the number of glucocorticoid receptors in lymphocytes of the peripheral blood of healthy elderly subjects increases , while the number of mineralocorticoid receptors decreases . 
The mechanisms of hormone-receptor interactions in hypertension are activated : the number of glucocorticoid and mineralocorticoid binding sites grows in hypertensive patients . 
Still a more essential rise in the number of receptors is observed in mid-age hypertensive patients than in elderly ones . 
In vivo footprint analysis of the HLA-DRA gene promoter : cell-specific interaction at the octamer site and up-regulation of X box binding by interferon gamma . 
Analysis of the major histocompatibility complex class II gene promoter DRA has previously identified at least five cis-acting regions required for maximal expression . 
We have examined the DRA promoter for protein-DNA interactions in the intact cell , which may mediate transcriptional activation . 
Using in vivo genomic footprinting we identified interactions in B-cell lines at the octamer site and the Y , X1 , and X2 boxes . 
Class II antigen expressing T-cell lines maintained contacts identical to B-cell lines , while class II-negative T-cell lines exhibited no interactions . 
In lymphoid cell lines , the octamer site is occupied and required for maximal expression . 
This is most likely due to the presence of the lymphoid-specific OTF-2 factor . 
In contrast , the class II-positive nonlymphoid glioblastoma cell line does not exhibit interactions at the octamer site despite the presence of the ubiquitous OTF-1 factor and an open binding site . 
Thus , the DRA promoter discriminates against OTF-1 activation at the level of DNA binding in the glioblastoma line . 
Interferon gamma induces class II expression in this glioblastoma cell line and , in parallel , up-regulates X1 and X2 box protein-DNA interactions , while all other interactions remain unchanged . 
These results suggest that interferon gamma functions on a poised promoter by altering weak , nonproductive interactions at the X boxes to strong interactions . 
These findings provide direct in vivo evidence to strongly suggest that the modulation of X1 and X2 interactions is an important constituent of the interferon gamma induction pathway . 
Simple derivation of TFIID-dependent RNA polymerase II transcription systems from Schizosaccharomyces pombe and other organisms , and factors required for transcriptional activation . 
Resolution of whole cell extract through two chromatographic steps yields a single protein fraction requiring only the addition of TFIID for the initiation of transcription at RNA polymerase II promoters . 
This approach allows the convenient generation of RNA polymerase II transcription systems from Saccharomyces cerevisiae , human lymphocytes , and Schizosaccharomyces pombe . 
TFIIDs from all three organisms are interchangeable among all three systems . 
The S. cerevisiae and Sch. pombe systems support effects of acidic activator proteins , provided 0 a further protein fraction from S. cerevisiae is supplied . 
This further fraction is distinct from the mediator of transcriptional activation described previously and represents a second component in addition to general initiation factors that may facilitate a response to acidic activators . 
The development of functionally responsive T cells . 
The work reviewed in this article separates T cell development into four phases . 
First is an expansion phase prior to TCR rearrangement , which appears to be correlated with programming of at least some response genes for inducibility . 
This phase can occur to some extent outside of the thymus . 
However , the profound T cell deficit of nude mice indicates that the thymus is by far the most potent site for inducing the expansion per se , even if other sites can induce some response acquisition . 
Second is a controlled phase of TCR gene rearrangement . 
The details of the regulatory mechanism that selects particular loci for rearrangement are still not known . 
It seems that the rearrangement of the TCR gamma loci in the gamma delta lineage may not always take place at a developmental stage strictly equivalent to the rearrangement of TCR beta in the alpha beta lineage , and it is not clear just how early the two lineages diverge . 
In the TCR alpha beta lineage , however , the final gene rearrangement events are accompanied by rapid proliferation and an interruption in cellular response gene inducibility . 
The loss of conventional responsiveness is probably caused by alterations at the level of signaling , and may be a manifestation of the physiological state that is a precondition for selection . 
Third is the complex process of selection . 
Whereas peripheral T cells can undergo forms of positive selection ( by antigen-driven clonal expansion ) and negative selection ( by abortive stimulation leading to anergy or death ) , neither is exactly the same phenomenon that occurs in the thymic cortex . 
Negative selection in the cortex appears to be a suicidal inversion of antigen responsiveness : instead of turning on IL-2 expression , the activated cell destroys its own chromatin . 
The genes that need to be induced for this response are not yet identified , but it is unquestionably a form of activation . 
It is interesting that in humans and rats , cortical thymocytes undergoing negative selection can still induce IL-2R alpha expression and even be rescued in vitro , if exogenous IL-2 is provided . 
Perhaps murine thymocytes are denied this form of rescue because they shut off IL-2R beta chain expression at an earlier stage or because they may be uncommonly Bcl-2 deficient ( cf. Sentman et al. , 1991 ; Strasser et al. , 1991 ) . 
Even so , medullary thymocytes remain at least partially susceptible to negative selection even as they continue to mature . 
SRC-related proto-oncogenes and transcription factors in primary human T cells : modulation by cyclosporin A and FK506 . 
Activation of T lymphocytes induces transcription of genes encoding for lymphokines . 
Interleukin-2 ( IL-2 ) gene expression is controlled transcriptionally by the cooperative activity of specific trans-activating factors that bind to the IL-2 enhancer . 
Cyclosporin A ( CsA ) and FK506 inhibit the production of IL-2 in T lymphocytes at the level of gene transcription . 
A member of the src gene family , the lymphocyte-specific protein tyrosine kinase , p56lck , has been implicated in IL-2 production . 
CsA was found not to inhibit lck gene expression , nor the activity of the lck gene product . 
However , CsA and FK506 inhibit the appearance of DNA binding activity of factors that bind to the NF-AT and AP-1 sites in the IL-2 enhancer . 
Since the induction of NF-AT and AP-1 is induced by the same stimuli that stimulate IL-2 production , these results indicate that the immunosuppressant action of CsA and FK506 is exerted at the level of these trans-activating factors . 
Bcl-2 : a repressor of lymphocyte death . 
The genes and mechanisms that control programmed cell death are currently the subject of intense study . 
The bcl-2 gene , a repressor of lymphocyte death , is perhaps the best understood of the programmed cell death associated genes . 
Here , Stanley Korsmeyer provides a brief overview of bcl-2 , concentrating on its roles in B- and T-cell development and in oncogenesis . 
Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels , AP-1 binding and AP-1 transcriptional activity . 
We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 ( AP-1 ) binding and enhancer activity in Jurkat T-cells . 
Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation . 
Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C ( PKC ) , the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested . 
PMA , which directly activates PKC , mimicked the effect of the lectins on c-Fos and c-Jun , but elevation of either intracellular Ca2+ or cAMP levels had little or no effect . 
The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7 , a kinase inhibitor with relatively high specificity for PKC , and less efficiently by H-8 , a structurally related kinase inhibitor less active on PKC , but more active on cyclic nucleotide-dependent kinases . 
Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence , TRE , and AP-1 enhancer activity , in Jurkat cells . 
PMA was also found to increase the AP-1 enhancer activity , whereas elevation of Ca2+ or cAMP had only minor effects . 
We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels , AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC . 
Functional interaction between the two zinc finger domains of the v-erb A oncoprotein . 
The v-erb A oncogene of avian erythroblastosis virus is a mutated and virally transduced copy of a host cell gene encoding a thyroid hormone receptor . 
The protein expressed by the v-erb A oncogene binds to DNA and acts as a dominant negative inhibitor of both the thyroid hormone receptor and the closely related retinoic acid receptor . 
The v-erb A protein has sustained two amino acid alterations within its DNA-binding domain relative to that of c-erb A , one of which , at serine 61 , is known to be important for v-erb A function in the neoplastic cell . 
We report here that the second alteration , at threonine 78 , also plays an important , although more indirect , role : alteration of the sequence at threonine 78 such that it resembles that of c-erb A can act as an intragenic suppressor and can partially restore function to a v-erb A protein rendered defective due to a mutation at position 61 . 
Threonine 78 lies within the D-box of the v-erb A protein , a region thought to mediate receptor-receptor dimerizations , and is not in physical proximity to the serine at position 61 . 
It therefore appears that an indirect interaction occurs between these two sites and that this interaction is crucial for v-erb A function . 
Okadaic acid is a potent inducer of AP-1 , NF-kappa B , and tumor necrosis factor-alpha in human B lymphocytes . 
Treatment of human B lymphocytes with an optimal concentration of okadaic acid , an inhibitor of phosphatases 1 and 2A , resulted in the induction of the transcription factor , AP-1 and a marked increase in NF-kappa B levels . 
In contrast , no effect on the levels of the octamer binding proteins , Oct-1 or Oct-2 , were found . 
Since both AP-1 and NF-kappa B have been reported to be important in the induction of the tumor necrosis factor-alpha ( TNF-alpha ) gene we examined the effects of okadaic acid on TNF-alpha mRNA levels . 
Treatment with okadaic acid resulted in a striking increase in TNF-alpha mRNA transcripts within 1 h of stimulation and large amounts of TNF-alpha were released into the culture media . 
Although okadaic acid provides a potent inductive signal for AP-1 and NF-kappa B it did not induce either B cell proliferation or immunoglobulin secretion . 
A novel Ets-related transcription factor , Elf-1 , binds to human immunodeficiency virus type 2 regulatory elements that are required for inducible trans activation in T cells . 
Human immunodeficiency virus type 1 ( HIV-1 ) and HIV-2 are structurally related retroviruses which both cause AIDS in humans . 
Although both viruses establish latency in quiescent human-peripheral-blood T cells , the asymptomatic phase of HIV-2 infection may be more prolonged than that of HIV-1 . 
The latent phases of both HIV-1 and HIV-2 infection have been shown to be disrupted by T-cell activation , a process that requires host cell transcription factors . 
In the case of HIV-1 , the transcription factor NF-kappa B is sufficient for inducible transcriptional activation . 
In contrast , factors in addition to NF-kappa B are required to activate HIV-2 transcription in infected T cells . 
In this report , we demonstrate that a novel Ets-related transcription factor , Elf-1 , binds specifically to two purine-rich motifs in the HIV-2 enhancer . 
Mutagenesis experiments demonstrated that these Elf-1 binding sites are required for induction of HIV-2 transcription following T-cell-receptor-mediated T-cell activation . 
Moreover , Elf-1 is the only factor present in activated T-cell nuclear extracts that binds to these sites in electrophoretic mobility shift assays . 
Thus , Elf-1 is a novel transcription factor that appears to be required for the T-cell-receptor-mediated trans activation of HIV-2 gene expression . 
These results may explain differences in the clinical spectra of diseases caused by HIV-1 and HIV-2 and may also have implications for the design of therapeutic approaches to HIV-2 infection . 
{ Effect of antihypertensive therapy with captopril on gluco- and mineralocorticoid receptors of peripheral blood lymphocytes in hypertensive patients of various age } 
Binding of 3H-dexamethasone and 3H-aldosterone by peripheral lymphocyte receptors was investigated in healthy persons and hypertensive patients before and after 2-week captopril treatment . 
The number of glucocorticoid and mineralocorticoid binding sites was increased in hypertensives vs normotensives . 
The treatment with the ACE inhibitor captopril led to activation of hormone-receptor interactions . 
There was a more marked rise of the number of receptors in middle-aged ( 44-55 years ) hypertensives vs elderly ( 61-80 years ) subjects after captopril treatment . 
Leukotriene B4 transcriptionally activates interleukin-6 expression involving NK-chi B and NF-IL6 . 
Leukotriene B4 ( LTB4 ) is a notable participant in inflammation and chemotaxis . 
It is , however , still unclear whether LTB4 acts in this regard directly or indirectly by stimulating the release of chemotactic and inflammatory cytokines . 
Here we report that LTB4 induces synthesis of interleukin ( IL ) -6 by human blood monocytes through transcriptional activation of the IL-6 gene . 
We furthermore demonstrate that this process involves activation of the transcription factor NF-chi B and , to a lesser extent , of NF-IL6 , while the activity of the transcription factor AP-1 , shown to otherwise confer IL-6 inducibility , appeared to be unaffected by LTB4 . 
Involvement of NF-chi B and NF-IL6 in induction of IL-6 transcription by monocytes was demonstrated using deleted forms of the IL-6 promoter . 
Activation of the IL-6 promoter by LTB4 was not only associated with accumulation of the respective transcripts but resulted in synthesis of functional IL-6 protein as well . 
In addition , LTB4 mediated transactivation of a heterologous promoter construct containing the NF-chi B or the NF-IL6 enhancer , but not the AP-1 enhancer . 
The signaling events mediating this effect appeared to involve the release of H2O2 , since LTB4 failed to induce NF-chi B or NF-IL6 in the presence of the scavenger of H2O2 , N-acetyl-L-cysteine . 
Estrogen binding sites in peripheral blood monocytes and effects of danazol on their sites in vitro . 
1 . This study was designed to investigate the presence of estrogen type I ( high affinity , low capacity ) and type II ( low affinity , high capacity ) binding sites in human peripheral blood monocytes and the effects of danazol on these sites . 
2 . These two types of estrogen binding sites existed in human peripheral blood monocytes . 
3 . Danazol bound to these sites in high concentration ( 10-LRB--6-RRB- M , clinical serum concentration during danazol therapy ) and decreased the number of both sites . 
4 . It is suggested that danazol has an anti-estrogenic action to the monocytes through the competition and suppression of estrogen binding sites as seen in the estrogen target organ . 
Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication . 
The human immunodeficiency virus ( HIV ) Rev protein is essential for viral structural protein expression ( Gag , Pol , and Env ) and , hence , for viral replication . 
In transient transfection assays , mutant forms of Rev have been identified that inhibit wild-type Rev activity and therefore suppress viral replication . 
To determine whether such transdominant Rev proteins could provide long-term protection against HIV infection without affecting T cell function , T leukemia cell lines were stably transduced with a retroviral vector encoding a transdominant mutant of the Rev protein , M10 . 
While all the M10-expressing cell lines remained infectable by HIV-1 , these same cells failed to support a productive replication cycle when infected with a cloned isolate of HIV-1 . 
In addition , two out of three M10-expressing CEM clones were also resistant to highly productive infection by a heterogeneous HIV-1 pool . 
Expression of M10 did not affect induction of HIV transcription mediated by the kappa B regulatory element or Tat . 
Importantly , constitutive expression of Rev M10 did not alter the secretion of interleukin 2 in response to mitogen stimulation of EL-4 and Jurkat cells . 
The inhibition of HIV infection in cells stably expressing a transdominant Rev protein , in the absence of any deleterious effect on T cell function , suggests that such a strategy could provide a therapeutic effect in the T lymphocytes of acquired immunodeficiency syndrome patients . 
Glucocorticoid receptor in patients with lupus nephritis : relationship between receptor levels in mononuclear leukocytes and effect of glucocorticoid therapy . 
We investigated the clinical significance of glucocorticoid receptor determination in 20 patients with systemic lupus erythematosus ( SLE ) who afterwards developed nephrotic syndrome . 
Glucocorticoid receptor concentrations in mononuclear leukocytes ( MNL ) in these patients were comparable with those in both other patients with SLE and healthy persons . 
Improvement in urinary protein excretion and in disease activity , which was scored according to the SLE Disease Activity Index system of the University of Toronto , closely related to the glucocorticoid receptor concentrations in MNL isolated from the corresponding patients . 
In summary , glucocorticoid receptor determination in patients with lupus nephritis may be a predictive clue for assessing responsiveness to glucocorticoid therapy . 
Characterization of a new tissue-specific transcription factor binding to the simian virus 40 enhancer TC-II ( NF-kappa B ) element . 
We have biochemically and functionally characterized a new transcription factor , NP-TCII , which is present in nuclei from unstimulated T and B lymphocytes but is not found in nonhematopoietic cells . 
This factor has a DNA-binding specificity similar to that of NF-kappa B but is unrelated to this or other Rel proteins by functional and biochemical criteria . 
It can also be distinguished from other previously described lymphocyte-specific DNA-binding proteins . 
The candidate oncoprotein Bcl-3 is an antagonist of p50\/NF-kappa B-mediated inhibition . 
The candidate oncogene bcl-3 was discovered as a translocation into the immunoglobulin alpha-locus in some cases of B-cell chronic lymphocytic leukaemias . 
The protein Bcl-3 contains seven so-called ankyrin repeats . 
Similar repeat motifs are found in a number of diverse regulatory proteins but the motifs of Bcl-3 are most closely related to those found in I kappa B proteins in which the ankyrin repeat domain is thought to be directly involved in inhibition of NF-kappa B activity . 
No biological function has yet been described for Bcl-3 , but it was noted recently that Bcl-3 interferes with DNA-binding of the p50 subunit of NF-kappa B in vitro . 
Here we demonstrate that Bcl-3 can aid kappa B site-dependent transcription in vivo by counteracting the inhibitory effects of p50\/NF-kappa B homodimers . 
Bcl-3 may therefore aid activation of select NF-kappa B-regulated genes , including those of the human immunodeficiency virus . 
A microtitre assay system for glucocorticoid receptors : decreased receptor concentration in myocardial infarction . 
A major difficulty in determination of glucocorticoid receptor sites is the very complicated assay procedure . 
Therefore , we describe a microtitre assay system for glucocorticoid receptors which is a whole-cell competitive binding radioassay using -LCB-3H-RCB--dexamethasone as radioligand . 
This modification of a previously described protocol simplifies and reduces laboratory work and allows assay reproducibility to be controlled more reliably . 
Thus enabled to perform the test on multiple blood samples in parallel , we investigated cardiac infarction patients over a 12-day period to test if glucocorticoid receptor binding is altered in this ' stressful ' disease . 
On the first day of the disease , glucocorticoid receptor capacity was significantly decreased without alteration of the receptor-ligand affinity , whereas on days 4 and 12 the number of receptor sites was normal again . 
This result fits well into the general observation of stress-induced down-regulation of immune responses . 
Activation of NF-kappa B by interleukin 2 in human blood monocytes . 
We report here that interleukin 2 ( IL-2 ) acts on human blood monocytes by enhancing binding activity of the transcription factor NF-kappa B to its consensus sequence in the 5' regulatory enhancer region of the IL-2 receptor alpha chain ( p55 ) . 
Similarly , IL-2 activates NF-kappa B in the human monocytic cell line U 937 , but not in resting human T-cells . 
This effect is detectable within 15 min and peaks 1 h after exposure to IL-2 . 
Enhanced NF-kappa B binding activity is followed by functional activation in that inducibility of the IL-2 receptor alpha chain is mediated by enhanced NF-kappa B binding and that a heterologous promoter containing the NF-kappa B consensus sequence ( -291 to -245 ) of the IL-2 receptor alpha chain gene is activated . 
In addition , IL-2 is capable of increasing transcript levels of the p50 gene coding for the p50 subunit of the NF-kappa B transcription factor , whereas mRNA levels of the p65 NF-kappa B gene remained unchanged . 
Single point estimation of glucocorticoid receptors in lymphocytes of normal subjects and of children under long term glucocorticoid treatment . 
A single point assay of glucocorticoid receptors ( GR ) in human lymphocytes based on the measurement of specific dexamethasone binding has been developed and compared with a common multi-point Scatchard analysis . 
The assay conditions - concentration of the ligand 20 nmol\/l , incubation time 2 h and the cell count 2-6 mil. cells\/tube in the assay volume 0.25 ml were found to be optimal . 
An attempt was also undertaken to use a cell harvester for the separation of cells from unbound ligand . 
Though specifically bound dexamethasone measured by whole-cell assay and that using cell harvester correlated well , almost by one order lower values obtained with the latter method render it non-applicable for receptor quantitation . 
The results from 9 healthy volunteers ( average GR concentration 7131 +\/- 1256 sites\/cell ) correlated excellently with those obtained by the Scatchard analysis . 
The single point assay has been also applied for determination of GH in 10 children treated with large doses of prednisone . 
The average values from healthy volunteers did not differ significantly from those found in these children , though much broader range was found in patients . 
The regulation of the human tumor necrosis factor alpha promoter region in macrophage , T cell , and B cell lines . 
The 1311-base pair human tumor necrosis factor ( TNF ) alpha promoter region was fused to the luciferase ( Luc ) reporter gene and studied in a transient transfection system in three TNF producing cell lines , the U937 macrophage cell line , the MLA 144 T cell line , and the 729-6 B cell line . 
This full length promoter construct can be induced by phorbol 13-myristate acetate ( PMA ) in each of these cell types . 
Analysis of a series of 5'-truncations showed several peaks of basal and PMA induced activity suggesting the presence of several positive and negative regulatory elements . 
A PMA responsive element was localized to a region between -95 and -36 bp relative to the transcription start site . 
Within this region , single AP-2- and AP-1-like consensus sequences were noted . 
These AP-2 and AP-1 sites were each modified with a double point mutation . 
A modest ( 20-50 % ) reduction in TNF promoter activity was observed with the AP-2 site mutation . 
However , mutation of the AP-1 site markedly diminished both the basal and PMA-activated promoter activity . 
Also co-transfections of the wild-type promoter construct with an AP-1\/c-jun expression vector resulted in augmented basal and PMA-induced promoter activity . 
Redox status of cells influences constitutive or induced NF-kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines . 
We have tested the hypothesis that cellular activation events occurring in T lymphocytes and monocytes and mediated through translocation of the transcription factor NF-kappa B are dependent upon the constitutive redox status of these cells . 
We used phenolic , lipid-soluble , chain-breaking antioxidants ( butylated hydroxyanisole ( BHA ) , nordihydroquairetic acid , or alpha-tocopherol ( vitamin E ) to show that peroxyl radical scavenging in unstimulated and PMA- or TNF-stimulated cells blocks the functions depending on NF-kappa B activation . 
BHA was found to suppress not only PMA- or TNF-induced , but also constitutive , HIV-enhancer activity concomitant to an inhibition of NF-kappa B binding activity in both lymphoblastoid T ( J.Jhan ) and monocytic ( U937 ) cell lines . 
This was also true for KBF ( p50 homodimer ) binding activity in U937 cells . 
Secretion of TNF , the product of another NF-kappa B-dependent gene , was abolished by BHA in PMA-stimulated U937 cells . 
The anti-oxidative effect of BHA was accompanied by an increase in thiol , but not glutathione , content in stimulated and unstimulated T cell , whereas TNF stimulation itself barely modified the cellular thiol level . 
Oxidative stress obtained by the addition of H2O2 to the culture medium of J.Jhan or U937 cells could not by itself induce NF-kappa B activation . 
These observations suggest that TNF and PMA do not lead to NF-kappa B activation through induction of changes in the cell redox status . 
Rather , TNF and PMA can exert their effect only if cells are in an appropriate redox status , because prior modification toward reduction with BHA treatment prevents this activation . 
It appears that a basal redox equilibrium tending toward oxidation is a prerequisite for full activation of transduction pathways regulating the activity of NF-kappa B-dependent genes . 
Expression of c-fos , c-jun and jun B in peripheral blood lymphocytes from young and elderly adults . 
The expression of c-fos , c-jun and jun B proto-oncogenes was studied in phytohemagglutinin ( PHA ) activated peripheral blood lymphocytes ( PBL ) from young and aged humans . 
Specific mRNAs for c-fos and c-jun were detectable within 30 min after cell activation and reached maximal levels within 2 h . 
Both c-fos and jun B mRNAs decreased to pre-activation levels within 6 h , while c-jun mRNA remained elevated . 
In PHA-activated PBL , no age-related differences were observed in c-fos or jun B mRNA expression . 
However , c-jun mRNA levels decreased significantly ( 1.73 +\/- 0.08 vs. 1.16 +\/- 0.09 arbitrary units , P &lt; 0.01 , young vs. old ) in PBL from elderly individuals activated with PHA . 
Because previous work has demonstrated that T cells from elderly individuals may display normal proliferative responses when activated via the anti-CD2 pathway , c-jun and jun B mRNA expression was also studied in anti-CD2-activated purified T cells . 
No age-related differences were found in the expression of either of these two proto-oncogenes by anti-CD2 activated T cells . 
These results suggest that the decreased IL-2 production and proliferative response displayed by PHA-activated PBL from elderly adults may be related to age-related changes in c-jun mRNA expression and in the ratio of c-fos to c-jun mRNA . 
A mechanism for the antiinflammatory effects of corticosteroids : the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1 . 
Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined . 
The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes { e.g. , endothelial-leukocyte adhesion molecule 1 ( ELAM-1 ) and intercellular adhesion molecule 1 ( ICAM-1 ) } . 
We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils ( polymorphonuclear leukocytes ) . 
Preincubation of endothelial cells with endotoxin { lipopolysaccharide ( LPS ) , 1 microgram\/ml } led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes ( P &lt; 0.0001 , n = 10 ) to endothelial cells , an increase that was markedly attenuated when endothelial cells were treated with dexamethasone ( IC50 &lt; 1 nM , P &lt; 0.0001 , n = 6 or 7 ) during preincubation with LPS . 
Moreover , the steroid receptor agonist cortisol ( 10 microM ) , but not its inactive metabolite tetrahydrocortisol ( 10 microM ) , diminished LPS-induced endothelial cell adhesiveness . 
Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors { human glucocorticoid receptors ( hGRs ) } was provided by experiments utilizing the steroid antagonist RU-486 . 
RU-486 ( 10 microM ) , which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90 , completely aborted the effect of dexamethasone on adhesiveness of endothelial cells ( P &lt; 0.0005 , n = 3 ) . 
Treatment of endothelial cells with LPS ( 1 microgram\/ml ) stimulated transcription of ELAM-1 , as shown by Northern blot analysis , and expression of membrane-associated ELAM-1 and ICAM-1 , as shown by quantitative immunofluorescence ( both P &lt; 0.001 , n = 9 ) . 
Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 ( IC50 &lt; 10 nM , both P &lt; 0.001 , n = 4-9 ) ; inhibition of expression by dexamethasone was reversed by RU-486 ( both P &lt; 0.005 , n = 4-6 ) . 
As in the adhesion studies , cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 ( both P &lt; 0.005 , n = 3 or 4 ) . 
In contrast , sodium salicylate ( 1 mM ) inhibited neither adhesion nor expression of these adhesion molecules . 
These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription . 
Membrane receptors for aldosterone : a novel pathway for mineralocorticoid action . 
Rapid nongenomic in vitro effects of aldosterone on intracellular electrolytes , cell volume , and Na-LRB-+-RRB--H+ antiport have been found in human mononuclear leukocytes ( HML ) . 
Binding of 125I-labeled aldosterone to plasma membranes of HML shares important features with these functional data . 
This includes a very low apparent dissociation constant ( Kd ) of 0.1 nM for both aldosterone and the effect on the Na-LRB-+-RRB--H-LRB-+-RRB--antiport , a high turnover rate , and the almost exclusive binding selectivity for aldosterone . 
Dexamethasone , RU 26988 , corticosterone , ouabain , amiloride , and 18-hydroxyprogesterone were inactive as ligands . 
Deoxycorticosterone acetate had an intermediate activity with an apparent Kd of 100 nM . 
These findings are the first 0 to demonstrate membrane binding of aldosterone being compatible with major aspects of its nongenomic effects . 
Natural variants of the HIV-1 long terminal repeat : analysis of promoters with duplicated DNA regulatory motifs . 
Sequence variation in the long terminal repeat ( LTR ) region of HIV-1 was analyzed in viral isolates of 17 infected individuals . 
Two classes of LTR size variants were found . 
One HIV-1 variant was detected containing an additional binding site for the transcription factor Sp1 . 
Another LTR size variation was observed in four patients in a region just upstream of the NF-kappa B enhancer . 
This variation was the result of a duplication of a short DNA sequence ( CTG-motif ) . 
Cell culture experiments demonstrated that the natural variant with four Sp1 sites had a slightly higher promoter activity and viral replication rate than the isogenic control LTR with three Sp1 sites . 
No positive effect of the duplicated CTG-motif could be detected . 
In order to measure small differences in virus production more accurately , equal amounts of a size variant and the wild-type plasmid were cotransfected into T-cells . 
The virus with four Sp1 sites did outgrow the three Sp1 virus in 35 days of culture and CTG-monomer virus outcompeted the CTG-dimer virus in 42 days . 
Based on these results we estimate a 5-10 % difference in virus production of the LTR variants when compared to that of wild-type . 
SCL and related hemopoietic helix-loop-helix transcription factors . 
The helix-loop-helix ( HLH ) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals . 
Five members of this family ( MYC , SCL , TAL-2 , LYL-1 and E2A ) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations . 
Although activated in T cell leukemias , expression of SCL and LYL-1 is low or undetectable in normal T cell populations . 
SCL is expressed in erythroid , megakaryocyte and mast cell populations ( the same cell lineages as GATA-1 , a zinc-finger transcription factor ) . 
In addition , both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells , thus implying a role for SCL in erythroid differentiation events . 
However , in contrast to GATA-1 , SCL is expressed in the developing brain . 
Studies of the function of SCL suggest 0 it is also important in proliferation and self-renewal events in erythroid cells . 
Transcription of the hypersensitive site HS2 enhancer in erythroid cells . 
In the human genome , the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream beta-like globin genes 10-50 kilobases away . 
The mechanism of HS2 enhancer function is not known . 
The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase ( CAT ) plasmids . 
In erythroid K562 cells in which the HS2 enhancer is active , the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene . 
In nonerythroid HL-60 cells in which the HS2 enhancer is inactive , long enhancer transcripts are not detectable . 
Splitting the HS2 enhancer between two tandem Ap1 sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene . 
In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism , the HS2 enhancer may ( i ) open up the chromatin structure of a gene domain and ( ii ) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site . 
Ablation of transplanted HTLV-I Tax-transformed tumors in mice by antisense inhibition of NF-kappa B { published erratum appears in Science 1993 Mar 12 ; 259 ( 5101 ) : 1523 } 
Mice transgenic for the human T cell leukemia virus ( HTLV-I ) Tax gene develop fibroblastic tumors that express NF-kappa B-inducible early genes . 
In vitro inhibition of NF-kappa B expression by antisense oligodeoxynucleotides ( ODNs ) inhibited growth of these culture-adapted Tax-transformed fibroblasts as well as an HTLV-I-transformed human lymphocyte line . 
In contrast , antisense inhibition of Tax itself had no apparent effect on cell growth . 
Mice treated with antisense to NF-kappa B ODNs showed rapid regression of transplanted fibrosarcomas . 
This suggests that NF-kappa B expression may be necessary for the maintenance of the malignant phenotype and provides a therapeutic approach for HTLV-I-associated disease . 
Inhibition of anti-CD3 monoclonal antibody-induced T-cell proliferation by dexamethasone , isoproterenol , or prostaglandin E2 either alone or in combination . 
1 . The purpose of these studies was to investigate the modulation of the proliferation of human T cells obtained from peripheral blood by dexamethasone ( DEX ) , isoproterenol ( ISO ) , and prostaglandin E2 ( PGE2 ) . 
The former two substances interact with T cells via the glucocorticoid and beta-adrenergic receptors respectively . 
When occupied by their natural ligands , glucocorticosteroids and catecholamines , these receptors have a role in modulating T-cell function during stress . 
During the inflammatory response increased levels of PGE2 bind to their receptors on T cells and thus alter responsiveness . 
Proliferation of T cells was induced by immobilized anti-CD3 monoclonal antibody ( mAb ) in the presence or absence of an additional costimulatory signal delivered by anti-CD28 mAb . 
2 . Various physiologic concentrations of DEX , ISO , or PGE2 were added at the time of initiation of the cultures and subsequent proliferation of the unstimulated T cells was determined . 
The results demonstrate that physiologic concentrations of all three of these agents inhibit the anti-CD3 mAb-induced proliferation of T cells . 
3 . Although DEX and PGE2 were equipotent in suppressing T-cell proliferation , ISO was much less effective . 
4 . Because concomitant elevations in the peripheral levels of these substances may occur , experiments were performed to determine the T-cell inhibitory effects of DEX together with either PGE2 or ISO . 
Synergistic suppression of T-cell proliferation was observed when various concentrations of DEX and PGE2 , but not DEX and ISO , were added to cultures . 
This synergistic suppression could not be explained by an increase in cAMP accumulation in T cells stimulated with DEX and PGE2 . 
5 . Finally , the addition of anti-CD28 mAb to anti-CD3 mAb-stimulated T cells overcame much of the suppression of proliferation induced by PGE2 or ISO but less so than that induced by DEX . 
Mutations in the Pit-1 gene in children with combined pituitary hormone deficiency . 
Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone and prolactin genes . 
In three unrelated Japanese children with combined pituitary hormone deficiency , we identified three point mutations in the Pit-1 gene , Pro24Leu , Arg143Gln , and Arg271Trp , located on the major transactivation region , POU-specific domain , and POU-homeodomain , respectively . 
Calcitriol : a hematolymphopoietrope ? { editorial } 
A MEDLINE search of the English-language literature was conducted using the indexing terms ' immunology , calcitriol and vitamin D ' to identify studies indicating a role for calcitriol as a primary immunomodulator . 
Sixty-six papers published between January 1956 and June 1991 were identified . 
Forty-five of these reports are cited in this review . 
The data strongly suggest an endocrine , autocrine and\/or paracrine role for calcitriol in immune regulation . 
No unifying hypothesis has yet emerged explaining this collection of data . 
This paper provides a brief review of immune properties currently attributed to calcitriol . 
Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells . 
We have earlier found that in Jurkat cells activation of protein kinase C ( PKC ) enhances the cyclic adenosine monophosphate ( cAMP ) accumulation induced by adenosine receptor stimulation or activation of Gs . 
Here we have therefore examined the effect of the phorbol ester PMA ( phorbol 12-myristate 13-acetate ) which stimulates PKC and a combination of the adenosine receptor agonist NECA ( 5'--LRB-N-ethyl-RRB--carboxamido adenosine ) and forskolin to raise cAMP , on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor , activator protein-1 ( AP-1 ) . 
PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect . 
Both PMA and the combination of NECA and forskolin acted together either to increase ( c-Fos ) or decrease ( Jun ) protein levels as well as increasing AP-1 binding , as judged by gel-shift assay , and AP-1 transcriptional activity . 
Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents . 
The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line , suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription . 
Involvement of Alu sequences in the cell-specific regulation of transcription of the gamma chain of Fc and T cell receptors . 
The Fc epsilon RI-gamma chains are expressed in a variety of hematopoietic cells where they play a critical role in signal transduction . 
They are part of the high affinity IgE receptor in mast cells , basophils , Langerhans cells , and possibly other cells ; a component of the low affinity receptor for IgG ( Fc gamma RIIIA or CD16 ) in natural killer cells and macrophages ; and part of the T cell antigen receptor in subsets of T cells . 
Here we have investigated the transcriptional regulation of the gamma chain gene by analyzing the 2.5-kilobase sequence upstream of the transcription start site . 
This sequence contains a promoter specific to cells of hematopoietic lineage . 
However , the tissue specificity of this promoter is only partial because it is active in all of the hematopoietic cells tested here , regardless of whether they constitutively express Fc epsilon RI-gamma chain transcripts . 
We have identified two adjacent cis-acting regulatory elements , both of which are part of an Alu repeat . 
The first ( -445\/-366 ) is a positive element active in both basophils and T cells . 
The second ( -365\/-264 ) binds to nuclear factors , which appear to be different in basophils and T cells , and acts as a negative element in basophils and as a positive one in T cells . 
Thus , this Alu repeat ( 90 % identical to Alu consensus sequences ) has evolved to become both a positive and negative regulator 
Human immunodeficiency viruses containing heterologous enhancer\/promoters are replication competent and exhibit different lymphocyte tropisms . 
The human immunodeficiency virus ( HIV ) type 1 long terminal repeat ( LTR ) contains binding sites for nuclear factor kappa B ( NF-kappa B ) and the constitutively expressed transcription factor Sp1 , both of which are highly conserved in HIV and simian immunodeficiency virus isolates . 
To delineate the effects of these motifs on the replicative capacity of HIV and to explore the possibility of extending the virus host range , known heterologous enhancer\/promoters were inserted into the HIV-1 LTR in place of the NF-kappa B and Sp1 binding sites . 
The effects of these substitutions on viral replication in transfected HeLa cells and on HIV infection of human peripheral blood lymphocytes or continuous T-leukemia cell lines were evaluated . 
HIVs in which the NF- kappa B\/Sp1 enhancer plus the downstream TATA element were replaced with heterologous enhancer\/promoters were also constructed . 
Viruses containing the human cytomegalovirus immediate-early enhancer exhibited infectious kinetics similar to that of wild-type HIV in activated human peripheral blood lymphocytes and AA2 cells but replicated less efficiently in H9 and CEM cells . 
These studies indicate that heterologous enhancer elements are capable of restoring Tat responsiveness to the HIV LTR in the context of directing reporter gene expression as well as in the production of infectious progeny virions . 
Photoaffinity labeling of plasma membrane receptors for aldosterone from human mononuclear leukocytes . 
Non-genomic effects of aldosterone on the sodium-proton-antiport have been shown in human mononuclear leukocytes which could be related to a new aldosterone membrane receptor . 
In the present paper plasma membranes from human mononuclear leukocytes were covalently photolabeled with a -LCB-125I-RCB--aldosterone derivative . 
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed significant aldosterone binding at a molecular weight of approximately 50000 Dalton which was absent with 1 microM cold aldosterone , but not cortisol in the binding media . 
The presence of the sulfhydryl agent dithiothreitol did not affect results suggesting the absence of disulfide bridges in the steroid binding domain of the receptor . 
These data are the first 0 to define the molecular weight of the membrane receptor for aldosterone . 
Alpha-lipoic acid is a potent inhibitor of NF-kappa B activation in human T cells . 
Acquired immunodeficiency syndrome ( AIDS ) results from infection with a human immunodeficiency virus ( HIV ) . 
The long terminal repeat ( LTR ) region of HIV proviral DNA contains binding sites for nuclear factor kappa B ( NF-kappa B ) , and this transcriptional activator appears to regulate HIV activation . 
Recent findings suggest an involvement of reactive oxygen species ( ROS ) in signal transduction pathways leading to NF-kappa B activation . 
The present study was based on reports that antioxidants which eliminate ROS should block the activation of NF-kappa B and subsequently HIV transcription , and thus antioxidants can be used as therapeutic agents for AIDS . 
Incubation of Jurkat T cells ( 1 x 10-LRB-6-RRB- cells\/ml ) with a natural thiol antioxidant , alpha-lipoic acid , prior to the stimulation of cells was found to inhibit NF-kappa B activation induced by tumor necrosis factor-alpha ( 25 ng\/ml ) or by phorbol 12-myristate 13-acetate ( 50 ng\/ml ) . 
The inhibitory action of alpha-lipoic acid was found to be very potent as only 4 mM was needed for a complete inhibition , whereas 20 mM was required for N-acetylcysteine . 
These results indicate that alpha-lipoic acid may be effective in AIDS therapeutics . 
A human putative lymphocyte G0\/G1 switch gene homologous to a rodent gene encoding a zinc-binding potential transcription factor . 
G0S24 is a member of a set of genes ( putative G0\/G1 switch regulatory genes ) that are expressed transiently within 1-2 hr of the addition of lectin or cycloheximide to human blood mononuclear cells . 
Comparison of a full-length cDNA sequence with the corresponding genomic sequence reveals an open reading frame of 326 amino acids , distributed across two exons . 
Potential phosphorylation sites include the sequence PSPTSPT , which resembles an RNA polymerase II repeat reported to be a target of the cell cycle control kinase cdc2 . 
Comparison of the derived protein sequence with those of rodent homologs allows classification into three groups . 
Group 1 contains G0S24 and the rat and mouse TIS11 genes ( also known as TTP , Nup475 , and Zfp36 ) . 
Members of this group have three tetraproline repeats . 
Groups 1 and 2 have a serine-rich region and an " arginine element " ( RRLPIF ) at the carboxyl terminus . 
All groups contain cysteine- and histidine-rich putative zinc finger domains and a serine-phenylalanine " SFS " domain similar to part of the large subunit of eukaryotic RNA polymerase II . 
Comparison of group 1 human and mouse genomic sequences shows high conservation in the 5' flank and exons . 
A CpG island suggests expression in the germ line . 
G0S24 has potential sites for transcription factors in the 5' flank and intron ; these include a serum response element . 
Protein and genomic sequences show similarities with those of a variety of proteins involved in transcription , suggesting that the G0S24 product has a similar role . 
Ras oncogene transformation of human B lymphoblasts is associated with lymphocyte activation and with a block of differentiation . 
The p21ras small GTP binding proteins participate in signal transduction from cell surface receptors and affect neoplastic transformation and development in many different cell types . 
In the present study , we examined the relationship between ras transformation and differentiation of human B lymphocytes . 
We show that the constitutive expression of the T24 Ha-ras oncogene in EBV-immortalized B lymphoblasts was associated with the induction of the interleukin 2 receptor alpha subunit , with an impaired immunoglobulin gene expression , altered adhesion properties and increased survival in serum-free medium . 
Since induction of the IL-2 receptor alpha subunit is a hallmark of lymphocyte activation , we suggest that p21ras naturally triggers B cell activation . 
The ras-transformed lymphocytes displayed a fully functional IL-2r , as assessed by c-fos induction following treatment with IL-2 ; nevertheless , they were not growth stimulated by this lymphokine . 
The decreased expression of immunoglobulin genes indicates that the ras oncogene blocks terminal differentiation to plasma cells , possibly by inhibiting the activity of lymphocyte-specific transcription factors . 
Somewhat unexpectedly , the constitutive p21ras activity did not cause an increased DNA binding of transcription factors PEA1 ( AP1 ) , PEA3 , Oct-2 or NF-kB . 
Transcription factor GATA-1 and erythroid development . 
In summary , we derived an experimental system that allows us to dissect the function of GATA-1 in red cell development at a genetic level . 
We have established the essential nature of GATA-1 during both primitive and definitive erythropoiesis . 
By ablating the expression of the endogenous GATA-1 gene , we are in a position 0 to introduce a variety of constructs that harbor subtle modifications in flanking or protein-coding sequences . 
We can now study regulatory regions and functional domains of the protein in the context of a true erythroid environment , experiments that have not been possible heretofore . 
Although the assay involves the dramatic loss of red cell production , it should be possible to define important regulatory domains that can then be assayed using less stringent systems , such as cell-free extracts for in vitro transcription . 
The ideal situation would be analyses conducted in GATA-1- erythroid cells . 
However , these cells have been impossible 0 to generate given the requirement of GATA-1 for Epo receptor expression and red cell viability ( C. Simon and S. Orkin , unpublished observations ) . 
It may be possible to produce such cells by first expressing the Epo receptor under the influence of a constitutive promoter and then targeting the GATA-1 gene . 
If GATA-1- -red cells were available , the analyses would involve the actual transcription of or chromatin structure surrounding the globin genes . 
Structure-function studies of the GATA-1 protein could be greatly simplified and a larger number of mutants studied . 
However , the ES cell system can be used as an alternative until targeted erythroleukemia cells become available . 
Other applications involve the introduction of other GATA-binding protein family members 0 to determine whether they rescue the mutation . 
If they can not , chimeric proteins can be tested to identify which amino acids distinguish the different family members . 
We feel that these experiments are vital to understanding the function of GATA-1 during erythroid ontogeny . 
How does GATA-1 regulate red cell genes like globin or the Epo receptor ? 
Once we identify the functional domains of the GATA-binding proteins , we hope to learn what proteins GATA-1 binds to in the basic transcription machinery or in chromatin . 
Is GATA-1 necessary for globin gene switching ? 
GATA-1 may be modified differently during development so that the locus control region can interact with different globin promoters . 
We may find that one region of the protein is required for embryonic expression and another for adult globin gene expression . 
Activation of lymphokine genes in T cells : role of cis-acting DNA elements that respond to T cell activation signals . 
Activation of T cells is initiated by the recognition of antigen on antigen presenting cells 0 to exert the effector functions in immune and inflammatory responses . 
Two types of helper T cell ( Th ) clones ( Th1 and Th2 ) are defined on the basis of different patterns of cytokine ( lymphokine ) secretion . 
They determine the outcome of an antigenic response toward humoral or cell-mediated immunity . 
Although lymphokine genes are coordinately regulated upon antigen stimulation , they are regulated by the mechanisms common to all as well as those which are unique to each gene . 
For most lymphokine genes , a combination of phorbol esters ( phorbol 12-myristate 13 acetate , PMA ) and calcium ionophores ( A23187 ) is required for their maximal induction . 
Yet phorbol ester alone or calcium ionophore alone produce several lymphokines . 
The production of the granulocyte-macrophage colony stimulating factor ( GM-CSF ) is completely dependent on the two signals . 
We have previously found a cis-acting region spanning the GM-CSF promoter region ( positions -95 to +27 ) that confers inducibility to reporter genes in transient transfection assays . 
Further analysis identified three elements required for efficient induction , referred to as GM2 , GC-box and conserved lymphokine element ( CLE0 ) . 
GM2 defines a binding site for protein ( s ) whose binding is inducible by PMA . 
One protein , NF-GM2 is similar to the transcription factor NF-kB . 
GC-box is a binding site for constitutively bound proteins . 
CLEO defines a binding site for protein ( s ) whose optimum binding is stimulated by PMA and A23187 . 
Viral trans-activators such as Tax ( human T cell leukemia virus-1 , HTLV-1 ) and E2 ( bovine papilloma virus , BPV ) proteins are other agents which activate lymphokine gene expression by bypassing T cell receptor ( TCR ) mediated signaling . 
The trans-activation domain of E2 and Tax is interchangeable although they have no obvious sequence homology between them . 
The viral trans-activators appear to target specific DNA binding protein such as NF-kB and Sp1 to cis-acting DNA site and promote lymphokine gene expression without TCR-mediated stimulation . 
Expression of tal-1 and GATA-binding proteins during human hematopoiesis . 
Tal-1 rearrangements are associated with nearly 30 % of human T acute lymphoblastic leukemia . 
Tal-1 gene encodes a putative transcription factor with a basic helix-loop-helix domain and is known to be predominantly expressed in hematopoietic cells . 
We investigated the pattern of tal-1 expression in purified human hematopoietic cells by in situ hybridization and reverse transcriptase polymerase chain reaction analysis . 
Both methods demonstrated that the tal-1 gene is expressed in megakaryocytes and erythroblasts as well as in basophilic granulocytes . 
In addition , our results indicate that the tal-1 1A promoter , which contains two consensus GATA-binding sites , is active mainly in these lineages . 
Because the GATA-1 gene is known to transactivate several genes specific for the erythroid , megakaryocytic , and mastocytic\/basophilic lineages , we studied GATA-1 expression in these purified hematopoietic cells . 
We found that GATA-1 and tal-1 genes are coexpressed in these three lineages . 
Remarkably , the expression of both genes is downmodulated during erythroid and megakaryocytic terminal maturation . 
In immature hematopoietic cells , tal-1 and GATA-1 genes are coexpressed in committed progenitors cells ( CD34+\/CD38-LRB-2+-RRB- ) , whereas they are not detectable in the most primitive cells ( CD34-LRB-2+-RRB-\/CD38- ) . 
In contrast , GATA-2 is strongly expressed in both most primitive and committed progenitors cells , whereas GATA-3 is mostly detected in most primitive ones . 
Altogether our results strongly suggest that GATA-1 modulates the transcription of tal-1 during the differentiation of the erythroid , megakaryocytic , and basosophilic lineages . 
Cell-type-specific transactivation of the parathyroid hormone-related protein gene promoter by the human T-cell leukemia virus type I ( HTLV-I ) tax and HTLV-II tax proteins . 
The human T-cell leukemia virus type I ( HTLV-I ) and HTLV-II Tax proteins are potent transactivators of viral and cellular gene expression . 
Using deletion mutants , the downstream parathyroid hormone-related protein ( PTHrP ) promoter is shown to be responsive to both HTLV-I and HTLV-II Tax as well as the AP1\/c-jun proto-oncogene . 
Transactivation of PTHrP by Tax was seen in T cells but not in B-cell lines or fibroblasts . 
A carboxy terminal Tax deletion mutant was deficient in transactivation of both the PTHrP and IL2R alpha promoters but not the HTLV-I long terminal repeat ( LTR ) . 
Exogenous provision of NFkB rescued IL2R alpha expression but not the PTHrP promoter . 
Thus , HTLV-I Tax , HTLV-II Tax , and c-jun transactivate PTHrP and may contribute to the pathogenesis of hypercalcemia in adult T-cell leukemia . 
I kappa B\/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding . 
The active nuclear form of the NF-kappa B transcription factor complex is composed of two DNA binding subunits , NF-kappa B p65 and NF-kappa B p50 , both of which share extensive N-terminal sequence homology with the v-rel oncogene product . 
The NF-kappa B p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappa B , termed I kappa B . 
In contrast , NF-kappa B p50 alone fails to stimulate kappa B-directed transcription , and based on prior in vitro studies , is not directly regulated by I kappa B . 
To investigate the molecular basis for the critical regulatory interaction between NF-kappa B and I kappa B\/MAD-3 , a series of human NF-kappa B p65 mutants was identified that functionally segregated DNA binding , I kappa B-mediated inhibition , and I kappa B-induced nuclear exclusion of this transcription factor . 
Results from in vivo expression studies performed with these NF-kappa B p65 mutants revealed the following : 1 ) I kappa B\/MAD-3 completely inhibits NF-kappa B p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappa B enhancer in human T lymphocytes , 2 ) the binding of I kappa B\/MAD-3 to NF-kappa B p65 is sufficient to retarget NF-kappa B p65 from the nucleus to the cytoplasm , 3 ) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappa B p65 disrupts its ability to engage I kappa B\/MAD-3 , and 4 ) the unique C-terminus of NF-kappa B p65 attenuates its own nuclear localization and contains sequences that are required for I kappa B-mediated inhibition of NF-kappa B p65 DNA binding activity . 
Together , these findings suggest that the nuclear localization signal and transactivation domain of NF-kappa B p65 constitute a bipartite system that is critically involved in the inhibitory function of I kappa B\/MAD-3 . 
Unexpectedly , our in vivo studies also demonstrate that I kappa B\/MAD-3 binds directly to NF-kappa B p50 . 
This interaction is functional as it leads to retargeting of NF-kappa B p50 from the nucleus to the cytoplasm . 
However , no loss of DNA binding activity is observed , presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappa B p65 . 
Surrogate thyroglobulin receptors and T cell proliferation in Hashimoto 's thyroiditis . 
Immunoglobulin molecules on the surface of a B lymphocyte are the endogenous " receptors " to which specific antigens bind . 
Studies in mice have shown that a monoclonal antibody , conjugated with palmitate to provide a lipid tail , can be inserted into the cell membrane to provide a " surrogate " antigen receptor . 
We have investigated whether a palmitate conjugate of a human monoclonal antibody specific for thyroglobulin ( TG ) could function as a surrogate TG receptor on blood mononuclear cells separated into fractions enriched for T cells or depleted of T cells ( non-T cells ) . 
Using flow cytometry , we detected surrogate TG receptors on non-T ( but not on T ) cells from 11 of 11 individuals studied ( 5 Hashimoto patients and 6 control donors ) . 
In contrast , endogenous TG receptors could only be detected on non-T cells from 1 of 3 Hashimoto patients and from 0 of 4 control donors . 
Because of the efficient binding of TG by surrogate receptors on non-T cells , we assessed the ability of such cells to present TG to T cells . 
Proliferation in response to TG was observed in T cells from only 1 of 5 Hashimoto patients . 
This low frequency of response was no different from that previously detected using cultures of T cells and autologous dendritic cells . 
Therefore , the successful generation of surrogate receptors on non-T cells is not associated with more efficient TG presentation of T cells . 
Furthermore , the significance of the present study is that the T cells , not the antigen-presenting cells , are likely to be the limiting element in the T cell proliferative response to TG and other thyroid autoantigens . 
ras protein activity is essential for T-cell antigen receptor signal transduction . 
In a Jurkat cell model of T-cell activation an interleukin-2 promoter\/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1 . 
Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C ( PKC ) with phorbol esters in combination with calcium mobilization by an ionophore . 
In cotransfection experiments , oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1 . 
Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT . 
A dominant inhibitory ras mutant specifically blocked antigen receptor agonism , indicating that ras activity is required for antigen receptor signaling . 
In addition , an inhibitor of PKC blocked both activated ras and phorbol ester stimulation , suggesting a role for ras upstream of PKC . 
The c-rel protooncogene product represses NF-kappa B p65-mediated transcriptional activation of the long terminal repeat of type 1 human immunodeficiency virus . 
The long terminal repeat ( LTR ) of the type 1 human immunodeficiency virus ( HIV-1 ) and the 5' regulatory region of the gene encoding the interleukin 2 receptor alpha subunit ( IL-2R alpha ) share functional kappa B enhancer elements involved in the regulation of these inducible transcription units during T-cell activation . 
These kappa B enhancer elements are recognized by a structurally related family of interactive proteins that includes p50 , p65 , and the product of the c-rel protooncogene ( c-Rel ) . 
Recent biochemical studies have shown that p65 and p50 form the prototypical NF-kappa B complex , which is rapidly translocated from the cytoplasm to the nucleus during T-cell activation . 
This intracellular signaling complex potently stimulates kappa B-directed transcription from either the HIV-1 LTR or the IL-2R alpha promoter via the strong transactivation domain present in p65 . 
We now demonstrate that nuclear expression of human c-Rel , which is induced by either phorbol ester or tumor necrosis factor alpha with delayed kinetics relative to p65 , markedly represses p65-mediated activation of these transcription units . 
These inhibitory effects of c-Rel correlate with its DNA-binding activity but not with its ability to heterodimerize with p50 , suggesting that c-Rel inhibition involves competition with p50\/p65 for occupancy of the kappa B enhancer element . 
Together , these findings suggest that one function of c-Rel is as a physiologic repressor of the HIV-1 LTR and IL-2R alpha promoters , serving to efficiently counter the strong transcriptional activating effects of p65 . 
Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the -201 to -130 region of the long terminal repeat . 
In previous transfection analyses using the chloramphenicol acetyltransferase reporter gene system , we determined that linker substitution ( LS ) mutations between -201 and -130 ( relative to the transcription start site ) of the human immunodeficiency virus type 1 long terminal repeat ( LTR ) caused moderate decreases in LTR transcriptional activity in a T-cell line ( S.L.Zeichner , J.Y.H. Kim , and J.C.Alwine , J.Virol.65 : 2436-2444 , 1991 ) . 
In order to confirm the significance of this region in the context of viral replication , we constructed several of these LS mutations ( -201 to -184 , -183 to -166 , -165 to -148 , and -148 to -130 ) in proviruses and prepared viral stocks by cocultivation of transfected RD cells with CEMx174 cells . 
In addition , two mutations between -93 and -76 and between -75 and -58 were utilized , since they affect the nuclear factor kappa B ( NF-kappa B ) - and Sp1-binding sites and were expected to diminish viral replication . 
Our results suggest that while transfection analyses offer an adequate approximation of the effects of the LS mutations , the analysis of viral replication using a mutant viral stock presents a more accurate picture , which is sometimes at variance with the transfection results . 
Three mutants ( -201\/-184 NXS , -165\/-148 NXS , and -147\/-130 NXS ) had effects on viral replication that were much more severe than the effects predicted from their performance in transfection analyses , and the effects of two LS mutations ( -201\/-184 NXS and -183\/-166 NXS ) were not predicted by their effects in transfection . 
In addition , we observed cell type-specific permissiveness to replication of some mutant viruses . 
In the cell types tested , the LS mutations indicated an apparent requirement not only for the intact NF-kappa B and SP1-binding sites but also for several regions between -201 and -130 not previously associated with viral infectivity . 
Expression of PILOT , a putative transcription factor , requires two signals and is cyclosporin A sensitive in T cells . 
Few known genes ( IL-2 , members of the IL-8 family , interferon-gamma ) are induced in T cells only through the combined effect of phorbol myristic acetate ( PMA ) and a Ca-LRB-2+-RRB--ionophore , and expression of only these genes can be fully suppressed by Cyclosporin A ( CyA ) . 
We have identified a putative transcription factor , designated PILOT , with an identical dual signal requirement for expression . 
Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA . 
The PILOT protein has a calculated M-LRB-r-RRB- of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1 , EGR2 , and pAT 133 . 
In contrast to T cells , in fibroblasts PILOT gene expression requires only one signal ( PMA ) and is not affected by CyA . 
This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts . 
This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it . 
Human CD4 lymphocytes specifically recognize a peptide representing the fusion region of the hybrid protein pml\/RAR alpha present in acute promyelocytic leukemia cells . 
Fusion proteins present in leukemic cells frequently contain a new amino acid at the fusion point . 
We tested whether a peptide ( BCR1\/25 ) encompassing the fusion region of the hybrid molecule pml\/RAR alpha , which is selectively expressed by acute promyelocytic leukemia ( APL ) cells , can be recognized by human T lymphocytes in vitro . 
CD4+ lymphocytes , at both polyclonal and clonal level , recognized peptide BCR1\/25 in an HLA-DR--restricted fashion on presentation by autologous antigen-presenting cell ( APC ) or by APC expressing the HLA-DR11 restricting molecule . 
Control peptides corresponding to the normal pml and RAR alpha proteins were not recognized . 
One clone ( DEG5 ) also exerted a high and specific cytotoxicity against autologous cells pulsed with BCR1\/25 . 
The autologous DE LCL containing a transduced pml\/RAR alpha fusion gene and expressing a bcr1 type of the pml\/RAR alpha hybrid protein induced the proliferation of DE anti-BCR1\/25 T cell clones . 
It is concluded that the bcr1 type-pml\/RAR alpha fusion protein of APL contains an antigenic site , absent from the normal parent molecules and recognized by human CD4+ lymphocytes . 
A serum response element and a binding site for NF-Y mediate the serum response of the human thrombospondin 1 gene . 
The expression of thrombospondin 1 ( TSP 1 ) , a member of the TSP gene family , is rapidly induced by growth factors . 
We tested the ability of human TSP 1-chloramphenicol acetyltransferase constructs to respond to serum in stably transfected NIH-3T3 cells . 
Two transcriptional elements in the TSP 1 promoter , a distal element at -1280 and a proximal element at -65 , were required for the response of the human TSP 1 gene to serum . 
The distal element contains the 5'-CC-LRB-A-SP-+-SP-T-RRB-6GG-3' consensus sequence characteristic of a serum-response element ( SRE ) . 
Deletions or mutations in this element reduced the serum response of the TSP 1 gene by 80-90 % . 
In gel-shift assays , the -1280 element and the c-fos SRE cross-competed , whereas their functional and binding mutants did not . 
The proximal element contains the sequence 5'-GGCCAATGGG-3' , which closely resembles the consensus binding motif for the CCAAT-binding factor NF-Y ( CBF , CP1 , alpha CP1 ) . 
Deletions or mutations in this element also reduced the serum response by 80-90 % . 
Methylation interference analysis of the -65 region identified a pattern of contacts with nuclear factors resembling that for NF-Y , and an NF-Y-binding site and the proximal TSP 1 element cross-competed in gel-shift assays , whereas their binding mutants did not . 
Finally , an abbreviated TSP 1 promoter\/5'-flank , containing the SRE- and NF-Y-binding sites , mediated a serum response that was close in magnitude to that of the parent promoter . 
We conclude that the serum response of the human TSP 1 gene requires the coordinated function of an SRE- and NF-Y-binding site . 
The interleukin 2 CD28-responsive complex contains at least three members of the NF kappa B family : c-Rel , p50 , and p65 . 
Optimal activation of T cells requires at least two signals . 
One signal can be delivered by the antigen-specific T-cell receptor , and the second signal is provided by the costimulatory molecule ( s ) delivered by the antigen-presenting cell . 
CD28 is a T-cell surface molecule and stimulation through this protein plays an important role in delivering the second activation signal . 
In this report , we show that in human peripheral blood T cells , CD28-mediated signal transduction involves the rel family proteins -- c-Rel , p50 , and p65 . 
Treatment of peripheral blood T cells with phorbol 12-myristate 13-acetate ( PMA ) and anti-CD28 monoclonal antibody ( mAb ) results in augmentation of nuclear c-Rel , p50 , and p65 , and this augmentation can occur in the presence of the immunosuppressant cyclosporin A . 
It is also shown in this report that , in response to PMA\/anti-CD28 mAb or anti-CD3\/anti-CD28 mAb , c-Rel , p50 , and p65 are associated with CD28-responsive element present in the promoter of the human interleukin 2 gene . 
The functional significance of c-Rel involvement in the CD28-responsive complex is demonstrated by transient transfection analysis , where cotransfection of c-Rel augments the level of expression of a chloramphenicol acetyltransferase reporter gene linked to the CD28-responsive element . 
Expression of the Tat protein of HIV1 in human promonocytic U937 cells . 
Numerous studies have shown that , upon HIV1 infection , human promonocytic U937 cells were induced to differentiate , as indicated , for example , by increased expression of adhesion molecules . 
One of the viral proteins involved in this process might be the Tat protein . 
Indeed , this viral protein , which is essential for productive infection , has also been shown to display growth-stimulating properties and immunomodulatory activities . 
In order to apprehend the role of the HIV1 tat gene in inducing the differentiation of HIV1-infected U937 cells , we have successfully introduced this gene into U937 cells by infecting them with retroviral particles transducing tat . 
The effect of the Tat protein constitutively expressed by these cells upon their differentiation was then evaluated by looking for the expression of the c-fos and of the c-fms proto-oncogenes which are linked to the differentiation of myelomonoblastic cells . 
Northern blot analysis revealed in these cells , an increase in the transcription of these two proto-oncogenes , and this increase was amplified after treatment with phorbol myristate acetate . 
No such increase was observed in control U937 cells . 
These results indicate that , among HIV1 gene products , the Tat protein appears to trigger monocytic differentiation , and suggests that this viral protein directs progenitors of the monocyte\/macrophage lineage towards a differentiation stage in which production of viral antigens and virions might be more efficient . 
Stimulation of interleukin-1 alpha and interleukin-1 beta production in human monocytes by protein phosphatase 1 and 2A inhibitors . 
Protein phosphatases 1 and 2A are important in regulating cellular functions by controlling the phosphorylation state of their substrates . 
In human monocytes , the inhibitors of these phosphatases , okadaic acid and calyculin A , were found to increase the mRNA accumulation and cytokine production of interleukin-1 beta and interleukin-1 alpha . 
The increased mRNA accumulation was found to be primarily because of the increase in the transcription rate of the interleukin-1 genes . 
Stimulation of interleukin-1 gene transcription may be caused by the stimulation of transcription factor activities , including those of AP-1 , by these protein phosphatase inhibitors . 
Okadaic acid increased the synthesis of the interleukin-1 beta precursor and mature forms and their secretion . 
This increased processing and secretion correlated with the stimulation of IL-1 beta convertase mRNA accumulation . 
The stimulation of interleukin-1 alpha production by okadaic acid was more modest than that of interleukin-1 beta . 
However , the phosphorylation of the precursor interleukin-1 alpha cytokine was increased . 
These results show that protein phosphatase 1 and 2A inhibitors exert multiple effects on cytokine production in human monocytes and suggest that these two phosphatases play important roles in regulating interleukin-1 production . 
Cell type- and stage-specific expression of the CD20\/B1 antigen correlates with the activity of a diverged octamer DNA motif present in its promoter . 
The CD20 ( B1 ) gene encodes a B cell-specific protein involved in the regulation of human B cell proliferation and differentiation . 
Studies with 5' deletion CD20 promoter-CAT constructs have previously revealed two regions of the promoter between bases -186 and -280 and between bases -280 and -454 which contained positive regulatory elements . 
In this study we identified a sequence element present in the most proximal region located between bases -214 and -201 , TTCTTCTAATTAA , which is important in the high constitutive expression of CD20 in mature B cells and the induction of CD20 in pre-B cells . 
This sequence element was referred to as the BAT box and its deletion significantly reduced the activity of a CD20 promoter-CAT construct in B cells . 
Mobility shift assays with various mutant probes and B cell nuclear extracts demonstrated that the core sequence TAAT was essential for binding to this site . 
Cross competition experiments with an octamer sequence from the Ig heavy chain promoter , the BAT box , and a TA-rich sequence present in the CD21 promoter revealed that all three sequences bound the same nuclear proteins suggesting that the BAT box binding proteins were Oct-1 and Oct-2 . 
Southwestern blotting and UV cross-linking studies confirmed that the BAT box binding proteins were Oct-1 and Oct-2 . 
The affinity of the BAT box binding proteins for the BAT box was approximately 25-fold less than for the octamer sequence and the BAT box binding proteins dissociated from the BAT box 10-fold more rapidly than from the octamer sequence . 
Despite this lower affinity , a trimer of the BAT box sequence was as efficiently transactivated by an Oct-2 expression vector as was a trimer of the octamer sequence in HeLa cells . 
The BAT box and Oct-2 were also implicated in the induction of CD20 in the pre-B cell line , PB-697 , via phorbol esters . 
The induction of CD20 mRNA was temporally associated with induction of Oct-2 mRNA and a BAT box-deleted CD20-CAT construct , in contrast to the wild type , was poorly induced by phorbol esters . 
Together these results suggest that the BAT box binding proteins are important in the B cell specific expression of CD20 and perhaps CD21 . 
Transcription factor jun-B is target of autoreactive T-cells in IDDM . 
Target antigens defined by autoantibodies in IDDM include insulin , a putative glycolipid that reacts with islet cell antibodies , and a 64,000-M-LRB-r-RRB- protein recently identified as glutamic acid decarboxylase . 
In addition , some IDDM sera that contain antibodies to glutamic acid decarboxylase also coprecipitate a 38,000-M-LRB-r-RRB- protein from islets . 
This study used a high titer anti-38,000-M-LRB-r-RRB- serum to screen bacteriophage lambda cDNA expression libraries and identified human islet and placental clones encoding jun-B , the nuclear transcription protein , of predicted 38,000 M-LRB-r-RRB- . 
Peripheral blood T-cells exhibited significant proliferation in response to a recombinant fragment of jun-B ( amino acids 1-180 ) in 12 of 17 ( 71 % ) recent-onset IDDM subjects , 8 of 16 ( 50 % ) ICA-positive first-degree relatives of IDDM subjects who were at risk , 3 of 12 ( 25 % ) other autoimmune disease subjects , and 0 of 10 healthy control subjects . 
Proliferation to tetanus toxoid did not differ significantly between the groups . 
Responses to jun-B were not related to age , sex , or human leukocyte antigen status . 
Thus , autoreactive T-cells identify a novel antigen , p38 jun-B , in IDDM and appear to indicate subjects at risk for the development of clinical disease . 
Transcriptional regulation of interleukin 3 ( IL3 ) in primary human T lymphocytes . 
Role of AP-1- and octamer-binding proteins in control of IL3 gene expression . 
We have investigated the molecular and biochemical basis for activation of interleukin 3 ( IL3 ) gene expression in primary human T lymphocytes following CD3 and CD2 receptor stimulation or activation by phytohemagglutinin plus phorbol 12-myristate 13-acetate . 
Using transfection and reporter gene assays specifically designed for primary T lymphocytes in conjunction with gel retardation assays , Western blot analyses and UV cross-linking studies , we found that c-Jun , c-Fos , and octamer-binding proteins play a major role in transcriptional activation of the IL3 gene via their interaction with two specific regions contained within the IL3 5'-flanking sequence . 
Additionally , the region between bases -107 and -59 of the IL3 promoter containing putative AP-2 and Sp1 binding motifs appears necessary for basal level expression of the IL3 gene . 
The data also indicate that CD2 receptor activation and phytohemagglutinin plus phorbol 12-myristate 13-acetate stimulation augment T cell IL3 gene expression through the same cis- and trans-activating signals . 
These results should contribute to a better understanding of the regulation of IL3 gene expression in human T lymphocytes . 
Interaction between NF-kappa B- and serum response factor-binding elements activates an interleukin-2 receptor alpha-chain enhancer specifically in T lymphocytes . 
We find that a short enhancer element containing the NF-kappa B binding site from the interleukin-2 receptor alpha-chain gene ( IL-2R alpha ) is preferentially activated in T cells . 
The IL-2R alpha enhancer binds NF-kappa B poorly and is only weakly activated by the NF-kappa B site alone . 
Serum response factor ( SRF ) binds to a site adjacent to the NF-kappa B site in the IL-2R enhancer , and both sites together have strong transcriptional activity specifically in T cells . 
Surprisingly , the levels of SRF constitutively expressed in T cells are consistently higher than in other cell types . 
Overexpression of SRF in B cells causes the IL-2R enhancer to function as well as it does in T cells , suggesting that the high level of SRF binding in T cells is functionally important . 
The transcriptionally active factors mediating the effect of the HTLV-I Tax transactivator on the IL-2R alpha kappa B enhancer include the product of the c-rel proto-oncogene . 
The transactivator HTLV-I Tax activates the promoter of the gene coding for the interleukin 2 alpha-chain receptor ( IL-2R alpha ) via a kappa B site that can bind several protein species of the rel family . 
Tax1 strongly activates the enhancer activity of this motif , in both epithelial HeLa and lymphoid Jurkat cells . 
This activation was not observed in undifferentiated embryocarcinoma F9 cells . 
Overexpression of the p50 , p65 and Rel proteins in these cells showed that significant activation of the IL-2R alpha kappa B site was observed only with Rel and Rel plus p65 . 
Moreover , whereas both Tax and phorbol 12-myristate 13-acetate ( PMA ) are able to efficiently induce the binding of NF-kappa B to the IL-2R alpha kappa B site , PMA is functionally inactive . 
Using the DNA affinity precipitation assay , we observed that Tax1 is able to efficiently induce the binding of Rel , whereas PMA is not . 
This established a clear difference between both stimuli , indicating that Rel is the functionally active factor . 
We conclude from these results that the functional activity of members of the rel family is regulated by their interaction with DNA and that Rel can be a potent transcriptional activator on specific kappa B sites . 
p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction . 
The Rel\/NF-kappa B family of transcription factors is composed of two distinct subgroups , proteins that undergo proteolytic processing and contain SWI6\/ankyrin repeats in their carboxyl termini ( p105 , p98 ) , and those without such repeats that do not require processing ( p65 , c-Rel , RelB , and Dorsal ) . 
We demonstrate that the p105 and p98 precursors share functional properties with the I kappa B proteins , which also contain SWI6\/ankyrin repeats . 
Both p105 and p98 were found to form stable complexes with other Rel\/NF-kappa B family members , including p65 and c-Rel . 
Association with the precursors is sufficient for cytoplasmic retention of either p65 or c-Rel , both of which are otherwise nuclear . 
These complexes undergo stimulus-responsive processing to produce active p50\/c-Rel and p55\/c-Rel complexes . 
These observations suggest a second pathway leading to NF-kappa B induction , in which processing of the precursors rather than phosphorylation of I kappa B plays a major role . 
Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor , I kappa B-alpha . 
The NK-kappa B transcription factor complex is sequestered in the cytoplasm by the inhibitory protein I kappa B-alpha ( MAD-3 ) . 
Various cellular stimuli relieve this inhibition by mechanisms largely unknown , leading to NF-kappa B nuclear localization and transactivation of its target genes . 
It is demonstrated here with human T lymphocytes and monocytes that different stimuli , including tumor necrosis factor alpha and phorbol 12-myristate 13-acetate , cause rapid degradation of I kappa B-alpha , with concomitant activation of NF-kappa B , followed by a dramatic increase in I kappa B-alpha mRNA and protein synthesis . 
Transfection studies reveal that the I kappa B-alpha mRNA and the encoded protein are potently induced by NF-kappa B and by homodimers of p65 and of c-Rel . 
We propose a model in which NF-kappa B and I kappa B-alpha mutually regulate each other in a cycle : saturating amounts of the inhibitory I kappa B-alpha protein are destroyed upon stimulation , allowing rapid activation of NF-kappa B . 
Subsequently , I kappa B-alpha mRNA and protein levels are quickly induced by the activated NF-kappa B . 
This resurgence of I kappa B-alpha protein acts to restore an equilibrium in which NF-kappa B is again inhibited . 
Induced myeloid differentiation of K562 cells with downregulation of erythroid and megakaryocytic transcription factors : a novel experimental model for hemopoietic lineage restriction . 
The human erythroleukemia cell line K562 can be induced to differentiate along the erythroid and megakaryocytic lineages . 
Here we demonstrate that hexamethylene bisacetamide ( HMBA ) induced K562 cells to differentiate along a third pathway . 
This was accompanied by downregulation of two transcription factors normally expressed in erythroid , mast and megakaryocyte lineages . 
Northern analysis demonstrated coordinate downregulation of alpha globin and gamma globin in addition to the two lineage-restricted transcription factors , SCL and GATA-1 . 
Proliferation of the K562 cells was also suppressed . 
Clonal assay showed that the suppression was irreversible and appeared analogous to the commitment of murine erythroleukemia ( MEL ) cells to terminal differentiation . 
In contrast to MEL cells , however , K562 cells acquired a macrophage-like morphology and exhibited a complete failure to generate benzidine-positive cells . 
Electron microscopy revealed a marked increase in granules resembling those specific for eosinophils . 
Surface marker analysis showed that HMBA-induced cells expressed reduced levels of glycophorin A , CD5 , CD7 and CD11b . 
No upregulation of megakaryocyte or lymphoid markers occurred . 
Thus the response of K562 cells to HMBA may provide a useful experimental system for studying the molecular mechanisms responsible for downmodulation of lineage-restricted transcription factors during hemopoietic lineage commitment . 
Costimulation of peripheral blood T cell activation by human endothelial cells . 
Enhanced IL-2 transcription correlates with increased c-fos synthesis and increased Fos content of AP-1 . 
Endothelial cells ( EC ) act as APC for resting PBL in vitro , and may have important roles in vivo in the pathogenesis of allograft rejection and delayed hypersensitivity . 
We previously reported that human umbilical vein EC provide costimulatory signals to PHA-stimulated PBL via CD2:lymphocyte function-associated Ag-3 and an unidentified ligand pair , resulting in a three- to eight-fold enhancement of IL-2 production . 
The physiologic relevance of this increase was demonstrated by the proliferative advantage provided by EC to PBL suboptimally stimulated with mAb OKT3 . 
We now report that EC costimulation causes increased levels of IL-2 mRNA as a result of increased IL-2 transcription in PBL . 
We therefore examined the effects of EC on T cell nuclear factors known to regulate IL-2 transcription , including c-jun and c-fos-two components of the transcription factor AP-1 , NFAT , and others . 
PBL constitutively express c-jun transcripts , and the level of c-jun mRNA is not altered by PHA activation in the absence or presence of EC . 
In contrast , c-fos mRNA is absent from resting T cells and is induced on PHA activation . 
EC alone do not induce c-fos mRNA but augment the level of c-fos mRNA in PHA-activated T cells by 3- to 10-fold . 
This effect is largely independent of the CD2:lymphocyte function-associated Ag-3 pathway . 
Gel-shift analysis reveals the constitutive presence of nuclear factors in resting PBL that bind to the proximal AP-1 site of the IL-2 promoter and that contain immunoreactive c-Jun but not c-Fos protein . 
In contrast , AP-1 from PHA-activated cells contains c-Jun and low levels of c-Fos . 
Strikingly , costimulation with EC results in a dramatic increase ( up to 15-fold ) in the c-Fos content of AP-1 . 
Levels of other nuclear factors involved in IL-2 regulation were not altered by EC , although NFAT-DNA complexes migrated at a slightly different mobility . 
In summary , our data suggest that changes in the composition of transcription factor AP-1 is a key molecular mechanism for increasing IL-2 transcription and may underlie the phenomenon of costimulation by EC . 
A protein of the AP-1 family is a component of nuclear factor of activated T cells . 
Nuclear factor of activated T cells ( NF-AT ) is a transcriptional activator involved in the induction of IL-2 gene expression . 
The response element for NF-AT is a sequence localized between -285\/-254 in the IL-2 regulatory region . 
The composition of NF-AT protein is still not fully elucidated . 
We demonstrate that , in normal human T cells , an AP-1 protein is a component of the NF-AT protein complex . 
This was evidenced by the ability of the AP-1 site to compete with the NF-AT site for binding to NF-AT and by the capacity of immobilized anti-Jun and anti-Fos antibodies to deplete NF-AT-binding activity from nuclear extracts of activated T cells . 
There was no detectable binding of in vitro translated Jun\/Fos heterodimer ( AP-1 ) to the NF-AT sequence , and the NF-AT sequence was unable to inhibit the binding of Jun\/Fos to the AP-1 sequence . 
The presence of an AP-1 protein in the NF-AT protein complex may regulate NF-AT-binding activity through protein-protein interaction . 
The lytic transition of Epstein-Barr virus is imitated by recombinant B-cells . 
Lytic transition of Epstein-Barr virus ( EBV ) is initiated by distinct immediate early regulators of the viral cycle , in synchronization to temporary , permissive conditions during host cell differentiation . 
We developed eukaryotic vectors suitable to imitate the processes involved in lytic transition in cell culture systems . 
Two stable B cell lines were established : R59Z activator cells were used to induce lytic EBV expression in a constitutive manner by the production of the BZLF 1 trans-activator ( Zta ) . 
R7-57 reporter cells , on the other hand , signaled induced activity of the lytic origin of EBV replication ( ori Lyt ) . 
Different modes , like chemical induction , lytic superinfection with EBV and single gene trans-activation converted the recombinant ori Lyt element in R7-57 reporter cells . 
BZLF 1 , transiently expressed in R7-57 reporter cells , was the only EBV trans-activator found , sufficient in inducing the viral lytic cycle . 
Basing on these experiments , trans-cellular activation of EBV was tested by cocultivation of BZLF 1-expressing R59Z activator cells with the R7-57 reporter line . 
No lytic effect on the reporter cells could be measured , neither by cocultivation of activator cells nor by coincubation of BZLF 1-containing cell lysates . 
Latency breaking activity , however , was transferred from activator to reporter cells when active , exogenous virus was added . 
The cell system described in these experiments provides a tool for the detection of EBV reactivation and demonstrates the potential of the lytic regulatory gene BZLF 1 . 
Immobilization and recovery of fusion proteins and B-lymphocyte cells using magnetic separation . 
A new approach 0 to facilitate immobilization and affinity purification of recombinant proteins and selected human B lymphocytes has been developed . 
Using magnetic beads with attached DNA containing the Escherichia coli lac operator , fusion proteins comprising the DNA-binding lac repressor could be affinity-purified and recovered by gentle elution conditions , such as with a lactose analogue or by enzymatic means using either deoxyribonuclease ( DNase ) or restriction endonucleases . 
The results show for the first time that a DNA-binding protein can be used for affinity purification of fusion proteins as exemplified by the specific and gentle recovery of beta-galactosidase and alkaline phosphatase from bacterial lysates using immunomagnetic separation . 
The approach was further extended to cell separation by the efficient recovery and elution of human CD37 B lymphocytes from peripheral blood . 
Negative transcriptional regulation of human interleukin 2 ( IL-2 ) gene by glucocorticoids through interference with nuclear transcription factors AP-1 and NF-AT . 
IL-2 gene transcription is affected by several nuclear proteins . 
We asked whether dexamethasone ( Dex ) and cyclosporin A ( CsA ) inhibit IL-2 gene transcription by interfering with the activity of nuclear proteins that bind to the IL-2 promoter . 
Nuclear extracts from primary human T lymphocytes were analyzed by electrophoretic DNA mobility shift assays . 
Both Dex and CsA inhibited the binding of transcription factors AP-1 and NF-AT , but not of NF-kB and OCT-1\/OAF , to their corresponding sites on the IL-2 gene promoter . 
To correlate changes in nuclear factor binding in vitro with transcriptional activity in vivo and define the structural requirements for IL-2 promoter repression , we used transient DNA transfections . 
Jurkat cells were transfected with plasmids containing either the intact IL-2 promoter or its AP-1 , NF-AT , and NF-kB motifs . 
Dex inhibited the IL-2 promoter and the AP-1 , but not the NF-AT and NF-kB plasmids . 
In contrast , CsA inhibited the IL-2 promoter and the NF-AT , but not the AP-1 and NF-kB plasmids . 
These results suggest that in human T lymphocytes both Dex and CsA inhibited IL-2 gene transcription through interference with transcription factors AP-1 and NF-AT . 
We propose that , while maximum inhibition may involve interaction with both transcription factors , AP-1 is the primary target of Dex 
Human CD3-CD16+ natural killer cells express the hGATA-3 T cell transcription factor and an unrearranged 2.3-kb TcR delta transcript . 
In this study we analyzed the T cell receptor ( TcR ) delta transcripts expressed by CD3-CD16+ cells and we investigated whether these cells expressed the hGATA-3 T cell transcription factor and the recombination-activating gene ( RAG ) -1 . 
Multiple TcR delta transcripts deriving from an unrearranged TcR delta gene were detected in both polyclonal and clonal CD3-CD16+ natural killer ( NK ) cell lines . 
Two unrearranged TcR delta transcripts had a size similar to that of the functional TcR delta mRNA ( 2.3 and 1.3 kb ) found in TcR gamma\/delta+ T lymphocytes . 
Sequence analysis of nine different 2.3-kb cDNA clones obtained from NK-derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR delta gene . 
These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the J delta 1 segment . 
The sequence located upstream of the J delta 1 segment corresponded to the previously reported germ-line sequence . 
The J delta 1 segment was correctly spliced to C delta ; in addition the four C delta exons were found to be already assembled . 
Two polyadenylation sites were present in the fourth C delta exon . 
However , only that located at the 3' end appeared to be utilized in the 2.3-kb cDNA . 
The expression of hGATA-3 , a T cell-specific factor known to be involved in the regulation of the transcription of TcR delta locus , was analyzed by Northern blot , in cultured NK cell population and clones ( but not in freshly derived cell populations ) . 
All NK clones and cell lines studied were found to express hGATA-3-specific mRNA , suggesting that hGATA-3 may be involved in the regulation of the unrearranged TcR delta gene expression in NK cells . 
Finally , no transcription of the RAG-1 gene could be detected in all NK cell lines or clones analyzed . 
Cell-specific expression of helix-loop-helix transcription factors encoded by the E2A gene . 
The E2A gene encodes transcription factors of the helix-loop-helix family that are implicated in cell-specific gene expression as part of dimeric complexes that interact with E box enhancer elements . 
It has previously been shown that transcripts of the E2A gene can be detected in a wide range of cell types . 
We have now examined expression of the mouse E2A gene at the protein level using polyclonal antisera directed against distinct portions of the E2A protein to probe blots of cellular extracts . 
A 73 kDa protein was identified by this analysis : this protein is highly enriched in cell lines of B lymphoid origin as compared to pancreatic beta-cells and fibroblast cells . 
The detection of this protein selectively in extracts of lymphoid cells correlates with the presence of the E box-binding activity LEF1\/BCF1 in these cells ; this binding activity was previously shown to be efficiently recognized by antiserum directed against E2A gene products . 
Transfection of cells with full length E2A cDNA leads to appearance of protein co-migrating with the 73 kDa protein on SDS gel electrophoresis and co-migrating with LEF1\/BCF1 on mobility shift analysis . 
Our results are consistent with the view that the DNA-binding activity LEF1\/BCF1 is a homodimer of E2A proteins ; the selective appearance of this putative cell-specific transcription factor in B lymphoid cells seems to be attributable , at least in part , to the elevated E2A protein concentrations in these cells . 
Hypertension in pregnancy . 
Pregnancy-induced hypertension ( PIH ) is a frequent cause of maternal and neonatal morbidity and mortality . 
In the present study we focused on the pathophysiology of PIH , mainly on the role of mineralocorticoids , reversed blood pressure patterns , and the resulting necessity of continuous monitoring of the preeclamptic mother . 
Problems of antihypertensive therapy are discussed and the first results of a pilot study with Urapidil are presented . 
To examine the role of mineralocorticoids in the pathophysiology of PIH , we studied plasma aldosterone and 18-hydroxy-corticosterone ( 18-OH-B ) levels in 25 women with PIH and in 25 healthy pregnant women . 
Furthermore , we evaluated the mineralocorticoid receptor ( MR ) count in mononuclear leukocytes in the 2 groups . 
The MR-count was significantly decreased in the PIH-group . 
The values of plasma aldosterone and 18-OH-B were also low . 
These results can not be explained by receptor down-regulation due to higher level of mineralocorticoids of the zona glomerulosa . 
Perhaps deoxycorticosterone or a hitherto unknown mineralocorticoid is responsible for the hypertension and altered MR-status . 
The first results of continuous blood pressure measurements with a noninvasive , real-time blood pressure monitor ( Finapres ) are presented . 
The comparison of the obtained values with intraarterial measurements demonstrates a good correlation between the two methods . 
We also report on the first experiences with Urapidil in the treatment of hypertension in severe preeclampsia . 
The data show that hypertension in preeclamptic women can be treated by Urapidil without side effects or reflex-tachycardia . 
Further studies will have to prove if Urapidil is suited for prepartal treatment of PIH as well . 
Aldosterone-specific membrane receptors and rapid non-genomic actions of mineralocorticoids . 
Functional studies in extrarenal , non-epithelial cells such as smooth muscle cells and more recently circulating human lymphocytes have provided increasing evidence that aldosterone produces not only classical genomic effects , but also rapid , non-genomic effects on transmembrane electrolyte movements . 
These involve activation of the sodium\/proton exchanger of the cell membrane at very low , physiological concentrations of aldosterone with an acute onset within 1-2 min . 
A second messenger cascade involved is the inositol 1,4,5-trisphosphate\/calcium pathway which responds over the same rapid time course . 
Such changes clearly can not be explained by genomic mechanisms , which are responsible for later effects than the membrane related rapid responses . 
The mechanisms underlying these rapid effects of aldosterone on electrolytes have been extensively studied in human lymphocytes , which thus may represent valuable tools in the delineation of the receptor-effector mechanisms involved . 
The unique characteristics of this new pathway for steroid action include its rapid time course , 10,000-fold selectivity for aldosterone over cortisol and the ineffectiveness of spironolactones , classical mineralocorticoid antagonists , as antagonists of the response . 
Adenovirus E1A inhibits IFN-induced resistance to cytolysis by natural killer cells . 
Infection of target cells with cytopathic viruses inhibits IFN induction of cytolytic resistance to NK cell-mediated cytolysis { IFN-mediated cytoprotection ( IFN-MCP ) } . 
It has been thought that the virally induced inhibition of IFN-MCP is secondary to the shutdown of cellular macromolecular synthesis that accompanies cytopathic virus infections . 
Group C , adenovirus serotype 5 ( Ad5 ) infection inhibits both IFN-MCP and cellular protein synthesis . 
This study determined if the Ad5-induced inhibition of IFN-MCP was independent of adenovirus ( Ad ) infection and secondary only to the expression of the Ad early region 1A gene ( E1A ) . 
To test this hypothesis , 4-h NK cytolysis assays were performed on IFN-gamma-treated human cells infected with an Ad5 E1A deletion mutant , dl343 , or transfected with the Ad5 E1A gene . 
IFN-MCP was not inhibited by infection with dl343 , despite the production of large amounts of both early ( E1B , p55 ) and late ( hexon ) Ad proteins . 
In contrast to E1A-negative , parental cell lines , IFN-MCP was blocked in Ad5 E1A-transfected epithelial and fibroblastic cell lines . 
Genetic mapping studies within the E1A gene demonstrated that expression of only the first exon of E1A was sufficient to inhibit IFN-MCP . 
DNA sequence homology of E1A genes between different Ad groups ( group A , Ad12 ; group C , Ad5 ) is limited almost entirely to three conserved regions located within the first exon of E1A . 
Because IFN-MCP was also blocked in Ad12 E1A-transfected cell lines , expression of one or more of the E1A-conserved regions may be necessary to inhibit IFN-MCP . 
In summary , the expression of E1A gene products inhibited IFN-MCP independently of virus infection . 
E1A 's inhibition of IFN-MCP has the net effect of promoting the selective NK cell-mediated clearance of Ad-infected or Ad-transformed human cells 
A mutation of the glucocorticoid receptor in primary cortisol resistance . 
The precise molecular abnormalities that cause primary cortisol resistance have not been completely described . 
In a subject with primary cortisol resistance we have observed glucocorticoid receptors ( hGR ) with a decreased affinity for dexamethasone . 
We hypothesize that a mutation of the hGR glucocorticoid-binding domain is the cause of cortisol resistance . 
Total RNA isolated from the index subject 's mononuclear leukocytes was used to produce first strand hGR cDNAs , and the entire hGR cDNA was amplified in segments and sequenced . 
At nucleotide 2,317 we identified a homozygous A for G point mutation that predicts an isoleucine ( ATT ) for valine ( GTT ) substitution at amino acid 729 . 
When the wild-type hGR and hGR-Ile 729 were expressed in COS-1 cells and assayed for -LCB-3H-RCB--Dexamethasone binding , the dissociation constants were 0.799 +\/- 0.068 and 1.54 +\/- 0.06 nM ( mean +\/- SEM ) ( P &lt; 0.01 ) , respectively . 
When the wild-type hGR and hGR-Ile 729 were expressed in CV-1 cells that were cotransfected with the mouse mammary tumor virus long terminal repeat fused to the chloramphenicol acetyl transferase ( CAT ) gene , the hGR-Ile 729 conferred a fourfold decrease in apparent potency on dexamethasone stimulation of CAT activity . 
The isoleucine for valine substitution at amino acid 729 impairs the function of the hGR and is the likely cause of primary cortisol resistance in this subject . 
Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock , yet succumb to L. monocytogenes infection . 
The multiple biological activities of tumor necrosis factor ( TNF ) are mediated by two distinct cell surface receptors of 55 kd ( TNFRp55 ) and 75 kd ( TNFRp75 ) . 
Using gene targeting , we generated a TNFRp55-deficient mouse strain . 
Cells from TNFRp55-\/-mutant mice lack expression of TNFRp55 but display normal numbers of high affinity TNFRp75 molecules . 
Thymocyte development and lymphocyte populations are unaltered , and clonal deletion of potentially self-reactive T cells is not impaired . 
However , TNF signaling is largely abolished , as judged by the failure of TNF to induce NF-kappa B in T lymphocytes from TNFRp55-deficient mice . 
The loss of TNFRp55 function renders mice resistant to lethal dosages of either lipopolysaccharides or S. aureus enterotoxin B . 
In contrast , TNFRp55-deficient mice are severely impaired to clear L. monocytogenes and readily succumb to infection . 
Thus , the 55 kd TNFR plays a decisive role in the host 's defense against microorganisms and their pathogenic factors 
{ The trend of molecular biology study on eosinophils } 
Recently , many investigators have been interested in the study on eosinophil biology since genes association with eosinophils such as interleukin-5 or eosinophil granule proteins ( EPO , ECP , EDN , MBP , and CLC ) , were isolated . 
However , the molecular basis for the commitment of progenitors to the eosinophil lineage has not been determined . 
The mechanism by which eosinophil-specific genes encoding primary and secondary granule proteins ( e.g. ECP , EDN , EPO , MBP , and CLC ) are expressed and regulated during eosinophilopoiesis is also unknown . 
In this paper , I described the characterization of genes encoding eosinophil granule proteins and the mRNA expression of GATA-1 binding transcription factor during eosinophil differentiation . 
Regulation of the Ets-related transcription factor Elf-1 by binding to the retinoblastoma protein . 
The retinoblastoma gene product ( Rb ) is a nuclear phosphoprotein that regulates cell cycle progression . 
Elf-1 is a lymphoid-specific Ets transcription factor that regulates inducible gene expression during T cell activation . 
In this report , it is demonstrated that Elf-1 contains a sequence motif that is highly related to the Rb binding sites of several viral oncoproteins and binds to the pocket region of Rb both in vitro and in vivo . 
Elf-1 binds exclusively to the underphosphorylated form of Rb and fails to bind to Rb mutants derived from patients with retinoblastoma . 
Co-immunoprecipitation experiments demonstrated an association between Elf-1 and Rb in resting normal human T cells . 
After T cell activation , the phosphorylation of Rb results in the release of Elf-1 , which is correlated temporally with the activation of Elf-1-mediated transcription . 
Overexpression of a phosphorylation-defective form of Rb inhibited Elf-1-dependent transcription during T cell activation . 
These results demonstrate that Rb interacts specifically with a lineage-restricted Ets transcription factor . 
This regulated interaction may be important for the coordination of lineage-specific effector functions such as lymphokine production with cell cycle progression in activated T cells . 
Activation of primary human T-lymphocytes through CD2 plus CD28 adhesion molecules induces long-term nuclear expression of NF-kappa B . 
Stimulation of highly purified human T-cells via CD2 and CD28 adhesion molecules induces and maintains proliferation for more than 3 weeks . 
This potent interleukin 2 ( IL-2 ) -dependent activation does not require monocytes or accessory cells . 
Long-lasting IL-2 receptivity is associated with high-level expression of the inducible IL-2 receptor alpha chain ( IL-2R alpha ) gene that is regulated at both transcriptional and posttranscriptional levels . 
Increase of IL-2R alpha gene transcription involves the enhanced binding of the transcription factor NF-kappa B to its consensus sequence in the 5'-regulatory region of the IL-2R alpha gene . 
To dissect the molecular basis for the unusually persistent transcription of the IL-2R alpha gene , we analyzed nuclear NF-kappa B binding to a radiolabeled IL-2R alpha kappa B-specific oligonucleotide probe during the time course of CD2 + CD28 activation . 
Resting T-cell nuclear extracts contained KBF1\/p50 homodimer . 
After stimulation , two new kappa B-specific complexes were identified as NF-kappa B p50-p65 heterodimer and putative c-Rel homodimer or c-Rel-p65 heterodimer . 
Both inducible complexes persisted for at least 3 weeks . 
Their relative levels were very similar for the duration of proliferation . 
In parallel , CD2 + CD28 activation triggered a significant intracellular thiol decrease , suggesting that oxygen radicals are involved in the signaling pathway of adhesion molecules . 
Finally , micromolar amounts of pyrrolidine dithiocarbamate , an oxygen radical scavenger that efficiently blocked the nuclear appearance of NF-kappa B in T-lymphocytes , also inhibited IL-2 secretion , IL-2R alpha cell surface expression , and T-cell proliferation . 
Together , these results suggest that NF-kappa B plays an important role in long-term activation of human primary T-lymphocytes via CD2 + CD28 . 
Expression levels of the thyrotropin receptor gene in autoimmune thyroid disease : coregulation with parameters of thyroid function and inverse relation to major histocompatibility complex classes I and II . 
Using a human TSH receptor ( TSH-R ) cDNA probe , we investigated TSH-R transcript levels in 13 human thyroid fragments by Northern blot analysis ; 7 Graves ' disease , 2 Hashimoto 's disease , 3 endemic goiter , and 1 healthy thyroid gland were studied . 
TSH-R expression levels were variable , but displayed a close correlation to the expression of thyroid peroxidase ( r = 0.703 ; P &lt; 0.05 ) , thyroglobulin ( r = 0.817 ; P &lt; 0.01 ) , and the nuclear oncogene c-fos ( r = 0.935 ; P &lt; 0.001 ) , but not c-myc . 
Overall , TSH-R transcript levels were low or absent in those thyroids in which expression of the major histocompatibility complex class I or II ( MHC I or II ) was high , thus establishing an inverse relation ( MHC I , r = -0.791 ; P &lt; 0.01 ; MHC II , r = -0.784 ; P &lt; 0.01 ) . 
In situ hybridization showed that apart from lymphocytes , thyroid cells themselves were the source of MHC II transcripts . 
gamma-Interferon expression was only detectable in 1 Hashimoto 's goiter . 
Our findings suggest that next to lymphocyte infiltration , active regulatory events in the thyrocyte are responsible for the inverse relation between functional parameters ( TSH-R , thyroid peroxidase , thyroglobulin , and c-fos ) and immunological markers ( MHC I and II ) . 
Calcium dependent activation of the NF-AT transcription factor by p59fyn . 
A reporter gene under the control of a T-cell antigen receptor element was activated in Jurkat cells by antigen receptor triggering or by a combination of phorbol myristate acetate , which activates protein kinase C , and a calcium ionophore . 
Both these signals were necessary for expression of the reporter gene . 
When co-transfected with a construct capable of overexpressing the tyrosine kinase p59fyn , the reporter gene was activated by PMA alone . 
Thus p59fyn could replace the calcium ionophore but not activation of protein kinase C . 
The activation by p59fyn plus PMA was blocked by EGTA and by the immunosuppressant drug cyclosporin A . 
Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription . 
Interaction between protein kinase C ( PKC ) - and glucocorticoid receptor ( GR ) -mediated signaling is suggested by the ability of the PKC activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate ( TPA ) to inhibit GR-dependent transcription of the mouse mammary tumor virus ( MMTV ) long terminal repeat ( LTR ) . 
Here we report that this interference is cell specific , as TPA augmented dexamethasone-induced transcriptional activation of the MMTV LTR in several T cell lines but was inhibitory in NIH-3T3 fibroblasts . 
TPA-GR synergism was determined to have occurred at the GR-responsive element ( GRE ) level by functional analysis of deletion mutants or synthetic GRE oligonucleotides driving chloramphenicol acetyl-transferase expression . 
Synergism required an intact GR DNA-binding domain , whereas amino- or carboxyl-terminal domains were dispensable . 
The effect was abrogated by the PKC inhibitor staurosporine , suggesting a role for PKC . 
Increased c-jun , jun-B , and jun-D expression above basal levels and increased transcriptional activity of AP-1\/TPA responsive elements fused to chloramphenicol acetyl-transferase vectors were observed in T cells treated with TPA alone or in combination with dexamethasone . 
The ability of Jun proteins to cooperate with GR in T cells has been investigated after transfection of c-jun , jun-B , or jun-D expression vectors , which augmented GR-dependent transcription from either MMTV LTR or GRE . 
Conversely , c-jun and jun-B transfection blunted GR-dependent transcription in HeLa cells . 
The presence of c-fos had a negative influence on GR function and correlated with the cell-specific synergistic or antagonistic activity of Jun with respect to GR ; high basal expression of c-fos as well as AP-1 DNA binding and transcriptional activity were observed in HeLa cells , but not in T cells . 
Furthermore overexpression of exogenous c-fos has an inhibitory effect on GR-dependent transcription from GRE in T cells . 
We propose that Jun plays a bifunctional role on GR-dependent transcriptional activation of GRE , selecting either synergistic or antagonistic activity depending on the cell-specific microenvironment . 
In this regard , intracellular levels of c-fos appear to be influential . 
A concatenated form of Epstein-Barr viral DNA in lymphoblastoid cell lines induced by transfection with BZLF1 . 
The replicative form of Epstein-Barr virus ( EBV ) DNA was studied using two lymphoblastoid cell lines , X50-7 and 6F11 , which are latently infected by Epstein-Barr virus . 
The lytic cycle of EBV infection was induced by transfection of the cells with the BRLF1\/BZLF1 coding region of the P3HR-1 defective genome . 
We combined two techniques to identify the productive replicative form of Epstein-Barr viral DNA in the lytic cycle-induced cells . 
Restriction enzyme analysis followed by Southern blot hybridization identified a significant increase in the fused fragment encompassing both ends of EBV DNA . 
This indicates an increase in either episomal DNA or concatameric linear DNA . 
Southern blot analysis of in situ lysing gels revealed that the cellular content of linear EBV DNA was also increased significantly after the initiation of the viral lytic cycle , while the amount of circular DNA remained approximately constant . 
We propose from these results that the source of the fused fragment encompassing both ends of EBV DNA is a concatenated linear EBV DNA molecule , and that such a concatenated molecule most likely represents a replicative form of EBV DNA in productively infected cells . 
Ectopic expression of a conditional GATA-2\/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner . 
The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains . 
One enigmatic aspect of GATA factor expression is that several GATA proteins , which ostensibly share the same DNA-binding site specificity , are coexpressed in erythroid cells . 
To elucidate the roles of individual GATA factors in erythropoiesis , conditional alleles of GATA-1 , GATA-2 , and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor ( ER ) . 
These GATA\/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays . 
When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells , exogenous GATA-2\/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner . 
These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2\/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal\/ER or GATA-3\/ER do not display a hormone-responsive block in differentiation . 
Thus , the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells . 
Lipopolysaccharide induces phosphorylation of MAD3 and activation of c-Rel and related NF-kappa B proteins in human monocytic THP-1 cells . 
Many effects of lipopolysaccharide ( LPS ) on gene expression , including that of human immunodeficiency virus ( HIV ) , in monocytic cells are mediated by activation of kappa B DNA-binding proteins . 
However , the specific members of the NF-kappa B\/Rel transcription factor family involved in the LPS response , and the mechanisms through which LPS-generated signals are transduced remain unclear . 
Here we show that LPS induces nuclear expression of c-Rel\/p50 heterodimers as well as p50\/p65 ( NF-kappa B ) kappa B DNA-binding complexes in human monocytic THP-1 cells . 
Nuclear localization of these proteins occurred concomitantly with a rapid decrease in their cytosolic levels and was independent of phorbol ester-sensitive protein kinase C . 
Within 24 h following LPS stimulation there was a striking increase in the levels of c-Rel , p105 , and p50 in the cytosol . 
The increased levels of these proteins correlated with increases in the amounts of their mRNAs during LPS activation of THP-1 cells . 
LPS activation of THP-1 cells resulted in phosphorylation of MAD3 ( an I kappa B-like protein ) , a rapid increase in MAD3 mRNA , and an increase in MAD3 protein by 2 h . 
Thus , LPS activation of human monocytic cells results in nuclear expression of c-Rel\/p50 and p50\/p65 ( NF-kappa B ) and induces phosphorylation of MAD3 . 
Molecular basis of a multiple lymphokine deficiency in a patient with severe combined immunodeficiency . 
We have previously reported that the T lymphocytes of a child with severe combined immunodeficiency are defective in the transcription of several lymphokine genes that include IL2 , IL3 , IL4 , and IL5 , which encode interleukins 2 , 3 , 4 , and 5 ( IL-2 , -3 , -4 , and -5 ) . 
To determine whether the defect in the patient 's T lymphocytes involved a trans-acting factor common to the affected lymphokine genes , we examined the ability of nuclear factors from the patient 's T lymphocytes to bind response elements present in the regulatory region of IL2 . 
Nuclear factor NF-kB , activation protein 1 ( AP-1 ) , OCT-1 , and NF-IL-2B binding activity were normal . 
In contrast , the binding of the nuclear factor of activated T cells ( NF-AT ) to its response element in the IL2 enhancer and to an NF-AT-like response element present in the IL4 enhancer was abnormal . 
To ascertain whether the abnormal NF-AT binding activity was related to an impaired function , we transfected patient and control T lymphocytes with constructs containing the reporter gene encoding chloramphenicol acetyl transferase ( CAT ) under the control of the entire IL2 regulatory region or of multimers of individual enhancer sequences . 
CAT expression directed by the IL2 regulatory region or by a multimer of the NF-AT-binding site was markedly lower in the patient relative to controls . 
In contrast , CAT gene expression directed by a multimer of the OCT-1 proximal ( OCT-1p ) -binding site was equivalent in patient and controls . 
These results indicate that an abnormality of \/ or influencing NF-AT may underlie the multiple lymphokine deficiency in this patient 
Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils : potential role in gene transcription . 
The expression of the hematopoietic transcription factors GATA-1 , GATA-2 , and GATA-3 was studied in eosinophils and basophils . 
Eosinophils express mRNA for GATA-1 , GATA-2 , and GATA-3 . 
Basophils express GATA-2 and GATA-3 . 
Treatment of HL-60 eosinophilic sublines with either interleukin-5 or butyric acid increased the expression of GATA-1 mRNA concomitant with the expression of eosinophil-specific genes , whereas levels of GATA-2 mRNA remained relatively constant . 
The presence of mRNA for these proteins in eosinophils and basophils suggests that gene transcription in these lineages may be regulated by GATA-binding proteins . 
Proliferation index as a prognostic marker in breast cancer . 
BACKGROUND . 
The proliferative activity of tumors has been extensively investigated with different approaches , among which the use of the monoclonal antibody Ki-67 represents an easy and reliable means of assessing cell proliferation . 
In this study , the proliferative activity of 129 primary breast cancers was investigated , and the results were related to prognosis . 
METHODS . 
Tumor samples , obtained from 129 patients who underwent surgery between January 1987 and December 1988 , were processed for staining by an immunohistochemical procedure ( avidin-biotin complex ) . 
The median time of observation was 42 months ( range , 31-55 months ) . 
Life-table analysis ( Mantel-Cox ) was used to assess the probability of disease-free survival ( DFS ) and overall survival ( OS ) . 
RESULTS . 
Tumors with high Ki-67 proliferation indices ( &gt; 20 % ) were associated with a higher 4-year probability of relapse of disease ( 55.3 % versus 79.1 % ; P = 0.003 ) and death ( 71 % versus 95.6 % ; P = 0.00005 ) when compared with tumors with low Ki-67 values . 
In addition , this proliferative parameter maintained its prognostic significance when the patients were stratified according to lymph node involvement , menopausal status , and nuclear estrogen receptor content . 
CONCLUSIONS . 
Tumor proliferative activity as evaluated by the monoclonal antibody Ki-67 seems to be an effective indicator of prognosis in breast cancer for DFS and OS . 
Differential contribution of herpes simplex virus type 1 gene products and cellular factors to the activation of human immunodeficiency virus type 1 provirus . 
We have previously reported that infection with herpes simplex virus type 1 ( HSV-1 ) activates expression of the human immunodeficiency virus type 1 ( HIV-1 ) provirus in T cells . 
Activation of the HIV-1 provirus correlated with the activation of binding of 55- and 85-kDa proteins to the kappa B enhancer and binding of the 50-kDa HLP-1 protein to the LBP-1 sequences of the HIV-1 long terminal repeat . 
Further examination of this system has shown that the inhibition of HSV-1 replication by the antiviral drug acyclovir does not inhibit HSV-1-mediated induction of HIV-1 provirus . 
Surprisingly , the NF-kappa B and HLP-1 binding activities were substantially inhibited in acyclovir-treated cells . 
In the transient-transfection assay , ICP0 , but not ICP4 , activated the HIV-1 long terminal repeat promoter region and the effect of ICP0 was greatly enhanced in the presence of the NF-kappa B binding proteins , suggesting that induction of the HIV-1 provirus involves cooperation between the HSV-1-activated cellular factor , NF-kappa B , and the virus-encoded transactivator , ICP0 . 
Glucocorticoid receptors in mononuclear cells of patients with sepsis . 
Glucocorticoid receptor ( GR ) hormone-binding activity was studied by a whole-cell method in mononuclear cells ( MNC ) from peripheral blood of 7 patients during the hemodynamic compensatory phase of sepsis . 
4 patients were receiving dopamine , which did not affect the GR count . 
The patients ' plasma cortisol concentrations were normal or slightly elevated . 
Despite a wide range , the mean GR count and affinity in MNC from septic patients did not differ from those in normal controls , suggesting that glucocorticoids could still be effective in the hemodynamic compensatory phase of sepsis . 
Transcriptional activation of the macrophage colony-stimulating factor gene by IL-2 is associated with secretion of bioactive macrophage colony-stimulating factor protein by monocytes and involves activation of the transcription factor NF-kappa B . 
Human peripheral blood monocytes ( Mo ) constitutively display the beta-chain of the receptor for IL-2 , whereas expression of the IL-2R alpha-chain is not constitutive but inducible with IL-2 . 
Here we report that binding of human IL-2 to its binding site leads to transcriptional activation of the macrophage CSF ( M-CSF ) gene in Mo resulting in accumulation of M-CSF mRNA and subsequent release of bioactive M-CSF protein as demonstrated by ELISA and inhibition of IL-2 induced release of an activity-stimulating growth of monocyte-type colonies by a neutralizing anti-M-CSF antibody . 
Transcriptional activation of the M-CSF gene by IL-2 is preceded by enhanced binding activity of the transcription factor NF-kappa B to its recognition sequence in the 5' regulatory enhancer region of the M-CSF gene . 
Moreover , using a heterologous promoter ( herpes thymidine kinase ) construct containing the NF-kappa B consensus sequence , it is shown that NF-kappa B binding by an IL-2-induced monocyte-derived nuclear protein confers reporter gene ( human growth hormone ) activity . 
Taken together , our findings indicate that IL-2 induces gene expression of M-CSF in human blood-derived Mo and provide evidence for involvement of NF-kappa B in transcriptional regulation of this gene . 
Comparative analysis of NFAT ( nuclear factor of activated T cells ) complex in human T and B lymphocytes . 
Nuclear factor of activated T cells ( NFAT ) is a transcriptional activator that binds to sequences in the interleukin-2 ( IL-2 ) promoter and is thought to be largely responsible for the T cell-specific inducibility of IL-2 expression . 
Electrophoretic mobility shift assays ( EMSA ) showed that specific NFAT binding activity could also be induced in human B cells . 
The B cell NFAT complex , however , was not functional , since it failed to activate transcription from an NFAT-driven chloramphenicol acetyltransferase ( CAT ) construct . 
Competition with an AP-1 motif or with anti-Jun and anti-Fos antibodies abolished binding to the NFAT motif in both T and B cells , indicating that Jun and Fos are critical for NFAT complex formation in both cell types . 
Purified recombinant Jun and Fos proteins failed to bind directly to the NFAT motif . 
However , when combined with unstimulated B or T cell extracts , full-length , but not truncated , Jun\/Fos heterodimers were able to form an NFAT complex , indicating the presence of a constitutively expressed nuclear factor ( s ) in B and T cells necessary for the formation of the NFAT complex in both cell types . 
An NFAT oligonucleotide carrying mutations in the 5' purine-rich part of the NFAT sequence failed to form a complex and to compete with the wild type motif for NFAT complex formation in both T and B cells . 
We therefore propose a model whereby a core NFAT complex consisting of Jun , Fos , and a constitutive nuclear factor is formed in both T and B cells , but an additional factor and\/or post-translational modification of a factor , missing in B cells , might be required for transactivation by NFAT . 
1,25-Dihydroxy vitamin D3 and 12-O-tetradecanoyl phorbol-13-acetate synergistically induce monocytic cell differentiation : FOS and RB expression . 
1,25-dihydroxy vitamin D3 and 12-O-tetradecanoyl phorbol-13-acetate ( TPA ) interact synergistically to induce monocytic differentiation of U937 histiocytic lymphoma cells . 
Addition of TPA causes an otherwise ineffective dose of 1,25-dihydroxy vitamin D3 to induce differentiation . 
The induced differentiation depends on the simultaneous ( vs. sequential ) presence of both agents . 
The kinetics of induced differentiation are consistent with a G1 specific cellular response 0 to initiate the metabolic cascade culminating in cell differentiation . 
The induced differentiation occurs with down-regulation of c-fos protein and an accompanying up-regulation of RB protein expression , consistent with a possible need for up-regulated RB expression to maintain a given differentiated phenotype and suppress transcriptional activators that might typically be associated with proliferation . 
Differential autoregulation of glucocorticoid receptor expression in human T- and B-cell lines . 
Regulation of glucocorticoid receptor ( GR ) expression by its cognate ligand was examined in the glucocorticoid-sensitive human leukemic T-cell line 6TG1.1 and in the human B-cell line IM-9 . 
In contrast to the decrease in GR mRNA seen in IM-9 cells after treatment with 1 microM dexamethasone for 16-18 h , treatment of 6TG1.1 cells resulted in an 8-fold increase in GR mRNA , as determined by Northern blot and RNase protection analysis , with a corresponding 3- to 4-fold increase in GR protein . 
Half-maximal induction of GR mRNA and protein in 6TG1.1 cells was observed between 10-100 nM dexamethasone , and inclusion of 1 microM RU 38486 completely blocked the effects of 100 nM dexamethasone , demonstrating that positive autoregulation of GR expression in 6TG1.1 cells is a receptor-mediated response . 
Positive autoregulation of GR expression was also observed in glucocorticoid-resistant CEM-C1 cells , which contain functional GR , but whose growth is unaffected by glucocorticoids . 
Thus , positive autoregulation is neither a consequence nor the sole cause of growth arrest . 
The degree of negative autoregulation in IM-9 cells and positive autoregulation in 6TG1.1 cells was unaffected by inhibition of protein synthesis with cycloheximide . 
Measurement of GR mRNA turnover in 6TG1.1 cells treated with actinomycin-D revealed a half-life of 2.5 h , which was unaffected by dexamethasone treatment . 
A similar half-life was determined in IM-9 cells and was also unaffected by steroid treatment . 
These results are consistent with the interpretation that glucocorticoid-mediated autoregulation of GR expression is a tissue-specific primary transcriptional response . 
The p65 subunit of NF-kappa B regulates I kappa B by two distinct mechanisms . 
Transcription factor NF-kappa B ( p50\/p65 ) is generally localized to the cytoplasm by its inhibitor I kappa B . 
Overproduced I kappa B , free from NF-kappa B , is rapidly degraded . 
Overexpression of p65 increases endogenous I kappa B protein in both carcinoma and lymphoid cells by two mechanisms : protein stabilization and increased transcription of I kappa B mRNA . 
In contrast , p65 delta , a naturally occurring splice variant , fails to markedly augment I kappa B protein levels . 
Both overexpressed p65 and coexpressed p50 are cytoplasmic , whereas p65 delta is partly nuclear , indicating that the I kappa B induced by p65 can maintain NF-kappa B in the cytoplasm . 
Thus , p65 and I kappa B are linked in an autoregulatory loop , ensuring that NF-kappa B is held in the cytoplasm until cells are specifically induced to translocate it to the nucleus . 
Single strand conformation polymorphism analysis of androgen receptor gene mutations in patients with androgen insensitivity syndromes : application for diagnosis , genetic counseling , and therapy . 
Recent studies indicate that mutations in the androgen receptor gene are associated with androgen insensitivity syndromes , a heterogeneous group of related disorders involving defective sexual differentiation in karyotypic males . 
In this report , we address the possibility of rapid mutational analysis of the androgen receptor gene for initial diagnosis , genetic counseling , and molecular subclassification of affected patients and their families . 
DNA from peripheral blood leukocytes of six patients from five families with various degrees of androgen insensitivity was studied . 
Exons 2 to 8 of the androgen receptor gene were analyzed using a combination of single strand conformation polymorphism analysis and direct DNA sequencing . 
Female family members were also studied to identify heterozygote carriers . 
Point mutations in the AR gene were identified in all six patients , and all mutations caused amino acid substitutions . 
One patient with incomplete androgen insensitivity was a mosaic for the mutation . 
Four of the five mothers , as well as a young sister of one patient , were carriers of the mutation present in the affected child . 
Our data show that new mutations may occur in the androgen receptor gene leading to sporadic androgen insensitivity syndrome . 
Molecular genetic characterization of the variant allele can serve as a primary tool for diagnosis and subsequent therapy , and can provide a basis for distinguishing heterozygous carriers in familial androgen resistance . 
The identification of carriers is of substantial clinical importance for genetic counseling . 
Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate . 
We have previously shown that minimally oxidized LDL ( MM-LDL ) activated endothelial cells to increase their interaction with monocytes but not neutrophils , inducing monocyte but not neutrophil binding and synthesis of monocyte chemotactic protein-1 and monocyte colony-stimulating factor ( M-CSF ) . 
In the present studies we have examined the signaling pathways by which this monocyte-specific response is induced . 
Both induction of monocyte binding and mRNA levels for M-CSF by MM-LDL were not inhibited in protein kinase C-depleted endothelial cells . 
A number of our studies indicate that cAMP is the second messenger for the effects of MM-LDL cited above . 
Incubation of endothelial cells with MM-LDL caused a 173 % increase in intracellular cAMP levels . 
Agents which increased cAMP levels , including cholera toxin , pertussis toxin , dibutyryl cAMP , and isoproterenol mimicked the actions of MM-LDL . 
Agents which elevated cAMP were also shown to activate NF kappa B , suggesting a role for this transcription factor in activation of monocyte-endothelial interactions . 
Although endothelial leukocyte adhesion molecule ( ELAM ) mRNA synthesis can be regulated by NF kappa B , ELAM was not expressed and ELAM mRNA was only slightly elevated in response to MM-LDL . 
We present evidence that induction of neutrophil binding by LPS is actually suppressed by agents that elevated cAMP levels . 
Costimulation of cAMP and protein kinase C pathways inhibits the CD3-dependent T cell activation and leads to a persistent expression of the AP-1 transcription factor . 
The effects mediated by a combined stimulation of cAMP- and protein kinase C ( PKC ) -dependent pathways have been investigated in different cellular systems , and it has been shown that they may complement each other in activating cell proliferation and differentiation . 
In this report , we show that upon the stimulation of both pathways T lymphocytes became refractory to activation via the CD3\/T cell receptor ( TcR ) complex . 
T cells preincubated with phorbol 12-myristate 13-acetate ( PMA ) and dibutyryl cAMP ( Bt2cAMP ) displayed a deficient proliferative ability in response to anti-CD3 mAb stimulation , whereas lymphocytes treated individually with either Bt2cAMP or PMA responded comparably to untreated samples . 
We detected an association between the reduced mitogenic response and low expression of both interleukin-2 ( IL-2 ) and the alpha chain ( CD25 ) of the IL-2 receptor ( IL-2R ) . 
Analysis of intracellular Ca2+ mobilization suggested that the CD3\/TcR-dependent signal transduction was impaired in PMA\/Bt2cAMP-treated cells . 
Remarkably , we observed that these samples displayed a persistent expression of the c-fos protooncogene , associated to an increased AP-1 DNA-binding activity , whereas no variations of CREB or NF-kB were detected . 
Neither Bt2cAMP nor PMA individually mediated these sustained effects , which therefore appear as a consequence of the interplay between both metabolic stimuli . 
Altogether , the data provide the evidence that both pathways complement each other in regulating gene expression and , conversely , downregulate the TcR transduction mechanisms 
Nuclear factor kappa B , a mediator of lipopolysaccharide effects . 
Exposure of certain cell types to bacterial lipopolysaccharide ( LPS ) leads to activation of nuclear factor kappa B ( NF-kappa B ) , an inducible transcription factor . 
One of NF-kappa B 's unique properties is its posttranslational activation via release of an inhibitory subunit , called inhibitor of NF-kappa B ( I kappa B ) , from a sequestered cytoplasmic form . 
This event is also triggered under various other conditions of biomedical importance . 
Other bacterial toxins , tumor necrosis factor-alpha ( TNF ) , interleukin-1 ( IL-1 ) , T cell mitogens , UV light , gamma rays and oxidative stress were reported to induce NF-kappa B . 
The activated form of NF-kappa B , which is rapidly taken up into nuclei , initiates transcription from immediate early genes in a wide variety of cell types . 
Most of the target genes for NF-kappa B are of relevance for the immune response and can be grouped into those encoding cytokines , cell surface receptors , acute phase proteins and viral genomes , such as that of human immunodeficiency virus type 1 ( HIV-1 ) . 
We will discuss recent experimental evidences suggesting that LPS might share a pathway of NF-kappa B activation with other inducers of the factor . 
This common pathway may involve reactive oxygen intermediates ( ROI ) as messenger molecules . 
Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells . 
The nuclear factor of activated T cells ( NF-AT ) 3 is an inducible DNA-binding protein that is essential for transcriptional induction of the IL-2 gene during T cell activation . 
NF-AT is thought to consist of two components : a ubiquitous , inducible nuclear component that we have identified as Fos and Jun proteins , and a preexisting , T cell-specific component ( NF-ATp ) which is the target for the immunosuppressive agents cyclosporin A ( CsA ) and FK506 . 
We have previously shown that nuclear extracts from activated T cells form two inducible NF-AT complexes with an oligonucleotide corresponding to the distal NF-AT site of the murine IL-2 promoter , although hypotonic extracts of unstimulated T cells form a single complex containing NF-ATp . 
We show that the ability to detect NF-ATp in a gel shift assay , which is essential for purification and biochemical studies of this protein , is strikingly dependent on the precise sequence of the NF-AT oligonucleotide used as the labeled probe . 
Moreover we present evidence that the component that forms the faster-migrating ( " lower " ) nuclear NF-AT complex is derived by a calcium-dependent , cyclosporin-sensitive , posttranslational modification of NF-ATp , and that Fos and Jun proteins stabilize its interaction with DNA . 
The results are discussed in the context of a model relating the two nuclear NF-AT complexes to NF-ATp . 
Enhancing effect of 17 beta-estradiol on human NK cell activity . 
The in vitro effect of 17 beta-estradiol on NK activity was studied . 
The proliferation and NK activity of YT-N17 ( a human NK-like cell line ) were enhanced by 17 beta-estradiol ( E2 ) , and the enhancement was blocked by tamoxifen ( Tx ) , an antagonist of E2 . 
On the contrary , other steroid hormones such as Tx , progesterone , and testosterone had no effect . 
YT-N17 contained 11.8 fmol\/mg protein of estrogen receptor ( mean of two independent assays ) , a value which was 5-10-fold higher than that of other hematopoietic cell lines . 
An enhancement of NK activity by E2 was also seen in large granular lymphocytes obtained from normal subjects , and it was again suppressed by Tx . 
These data suggest that E2 is one of the activating factors for NK\/LGL cells . 
Cytokine modulation of HIV expression . 
Cytokines , the peptide hormones which control the homeostasis of the immune system and also play a fundamental role in inflammatory and immune mediated reactions , have been involved at multiple levels in the pathogenesis of the acquired immune deficiency syndrome ( AIDS ) . 
Infection with the human immunodeficiency virus ( HIV ) has been shown to induce production of several cytokines both in vitro and in vivo . 
Conversely , several cytokines modulate the levels of HIV expression in infected cells of both T lymphocytic and mononuclear phagocytic lineage . 
Activated mononuclear cells , particularly B cells which are in a state of chronic activation in HIV infected individuals , release HIV-inductive cytokines and thus play a potentially important role in the pathogenesis of HIV infection . 
FK506 and ciclosporin : molecular probes for studying intracellular signal transduction . 
The immunosuppressants ciclosporin and FK506 block the Ca-LRB-2+-RRB--dependent signal-transduction pathway emanating from the T-cell receptor , thereby inhibiting the activation of helper T cells . 
Using these drugs as probes , chemists and biologists have uncovered several intracellular signalling molecules bridging the generation of second-messenger Ca2+ ions and the transcriptional activation of IL-2 , among which are calmodulin , calcineurin and the nuclear factor of activated T cells ( NF-AT ) . 
Hence , Ca2+ binds to calmodulin , leading to the binding of calmodulin to calcineurin ; the activated calcineurin , in turn , may dephosphorylate the cytoplasmic subunit of NF-AT , resulting in its translocation from the cytoplasm into the nucleus 0 to form a competent transcriptional activator . 
As described by Jun Liu , these drugs manifest their effects in an unprecedented fashion . 
They do not directly intercept intracellular signalling molecules . 
Instead , they form tight complexes with two different classes of abundant cytosolic receptors called immunophilins upon entering the cell , and consequently inhibit their peptidyl prolyl cis-trans isomerase activities . 
The two structurally distinct immunophilin-drug complexes bind to , and inhibit , the phosphatase activity of calcineurin . 
The impaired transcription factor AP-1 DNA binding activity in lymphocytes derived from subjects with some symptoms of premature aging . 
The study of human disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence . 
The main feature of cellular senescence in vitro is cessation of cell proliferation . 
Down syndrome ( DS ) and neuronal ceroid-lypofuscinosis ( NCL ) are clinically characterized by the premature onset of numerous features normally associated with human aging . 
Phytohemagglutinin stimulated lymphocytes derived from DS subjects showed a statistically significant diminished proliferation capacity in comparison with lymphocytes derived from NCL and healthy individuals . 
We demonstrated , by applying the electrophoretic mobility shift assay , slightly impaired AP-1 DNA binding activity in NCL lymphocytes and strong in DS ones . 
Our results showed that the same molecular mechanisms of proliferation cessation could exist in fibroblasts characterized by replicative senescence and in lymphocytes derived from individuals with premature aging syndromes ( Down ) . 
Cellular immune and cytokine pathways resulting in tissue factor expression and relevance to septic shock . 
Cells of monocyte lineage serve as effector cells in the cellular immune response . 
In addition , they respond to LPS and cytokines with activation and expression of inflammatory effector gene products similar to those elicited by the antigen driven response . 
The response to antigen proceeds at the T helper cell level through two independent forms of cellular collaboration , contact and lymphokine . 
We review the control of expression of the Tissue Factor ( TF ) gene and the function of the TF protein . 
The enhanced initiation of transcription of the TF gene appears to require engagement of a 56 bp LPS Response Element , an enhancer that is engaged by both AP-1 type heterodimeric complexes as well as NF kappa B like heterodimeric complexes . 
Dissociation of NF kappa B from Ig kappa B by cytokine and LPS stimulation , and possibly activated T cells , may represent a common pathway to induction of the TF and other inflammatory genes . 
Enhancement of expression of TF is observed upon adhesion of Mo to endothelial cells and extracellular matrix proteins , as well as upon engagement of leukocyte integrins . 
The biological effects that follow from expression of TF by vascular cells have been resolved by analysis of function aided by the use of recombinant full length TF and truncated surface domain of TF . 
The rules of assembly of the cognate ligands of TF , namely the zymogen plasma factors VII and the serine protease factor VIIa , with the soluble surface domain of TF in free solution , in the presence of phospholipid surfaces and cell surface and of the anchored TF molecule have been described . 
It is evident that assembly of the surface domain of TF with VIIa 0 to form the binary TF.VIIa complex induces a significant increase in the Kcat of the catalytic domain of VIIa for small peptidyl substrates and more profoundly for protein substrate . 
This provides substantial evidence for an allosteric effect on the catalytic cleft of VIIa that is imparted by binding to TF , its cognate catalytic cofactor . 
It is also evident that the TF.VIIa complex is proteolytically active and can activate the zymogen plasma factor X to the serine protease Xa in free solution , inferring that extended substrate recognition by induced structural loci of the TF.VIIa complex are created from either or both proteins to constitute a new recognition structure . 
It is also evident that association of X with charged phospholipid surfaces enhances the proteolytic activation of this zymogen by increasing recognition and susceptibility of the sessile peptide bond deduced from the markedly decreased Km and increased Kcat . 
Visualization of the endogenous NF-kappa B p50 subunit in the nucleus of follicular dendritic cells in germinal centers . 
NF-kappa B , a 50 kDa\/65 kDa ( p50\/p65 ) heterodimer , is a ubiquitous transcription factor involved in the positive regulation of various immune genes . 
The aim of this study was to determine whether NF-kappa B is related to a particular cell type and\/or differentiation step during immunopoiesis . 
Using in situ hybridization on sections from non HIV hyperplastic lymph nodes , we found that the gene of the 105 kDa precursor of p50 was overexpressed in the light zone of germinal centers , with a network aspect , which suggested the involvement of follicular dendritic cells ( FDC ) . 
By immunohistochemistry , p50 protein was detected in the cytoplasm and nucleus of FDC , confirming the involvement of FDC . 
Furthermore , p50 protein was detected in the cytoplasm of all lymphocytes . 
Thus , we focused our study on isolated FDC clusters from normal tonsils . 
As showed on tissue sections , we detected the p50 in both cytoplasm and nucleus of FDC . 
Nuclei of lymphocytes from FDC clusters were negative . 
We next studied p65 and c-Rel protein expression in FDC clusters . 
p65 was detected in the cytoplasm of FDC , whereas nuclei were negative . 
Furthermore , p65 was detected in the nuclei of some lymphocytes . 
c-Rel protein was detected only in the cytoplasm of lymphocytes and not in the nucleus and cytoplasm of FDC . 
Our results indicated that , in the context of T cell-dependent B cell immunopoiesis occurring in FDC clusters , p50 is mainly related to FDC with a massive overexpression in the nuclei , whereas p65 is expressed in a scattered manner in the nuclei of lymphocytes and c-Rel protein exclusively in the cytoplasm of lymphocytes from FDC clusters . 
These results suggested that the two subunits of NF-kappa B and the c-Rel protein have different roles in different cell types during B cell immunopoiesis . 
Involvement of transcription factor YB-1 in human T-cell lymphotropic virus type I basal gene expression . 
Sequences which control basal human T-cell lymphotropic virus type I ( HTLV-I ) transcription likely play an important role in initiation and maintenance of virus replication . 
We previously identified and analyzed a 45-nucleotide sequence ( downstream regulatory element 1 { DRE 1 } ) , +195 to +240 , at the boundary of the R\/U5 region of the long terminal repeat which is required for HTLV-I basal transcription . 
We identified a protein , p37 , which specifically bound to DRE 1 . 
An affinity column fraction , containing p37 , stimulated HTLV-I transcription approximately 12-fold in vitro . 
We now report the identification of a cDNA clone ( 15B-7 ) , from a Jurkat expression library , that binds specifically to the DRE 1 regulatory sequence . 
Binding of the cDNA fusion protein , similarly to the results obtained with purified Jurkat protein , was decreased by introduction of site-specific mutations in the DRE 1 regulatory sequence . 
In vitro transcription and translation of 15B-7 cDNA produced a fusion protein which bound specifically to the HTLV-I +195 to +240 oligonucleotide . 
The partial cDNA encodes a protein which is homologous to the C-terminal 196 amino acids of the 36-kDa transcription factor , YB-1 . 
Cotransfection of a YB-1 expression plasmid increases HTLV-I basal transcription approximately 14-fold in Jurkat T lymphocytes . 
On the basis of the molecular weight , DNA-binding characteristics , and in vivo transactivation activity , we suggest that the previously identified DRE 1-binding protein , p37 , is YB-1 . 
The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines . 
The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin ( LT ; TNF-beta ) gene induction . 
Tax-expressing cell lines produce LT biologic activity . 
An LT promoter ( LT-293 ) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line , which contains defective HTLV-I but expresses tax . 
The observation that a mutated LT-kappa B construct ( M1-CAT ) was inactive in C81-66-45 , confirmed the importance of NF-kappa B in LT gene expression . 
Tax was transfected into HTLV-I-negative human T-cell lines . 
Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site . 
Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent . 
In JM T cells , tax induced LT-293 activity by two- to four-fold , though there was no induction of M1-CAT . 
The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions . 
These studies , the first 0 to demonstrate induction of LT promoter activity over basal levels , indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter , at least in part through activation of NF-kappa B . 
Transient pseudohypoaldosteronism in obstructive renal disease with transient reduction of lymphocytic aldosterone receptors . 
Results in two affected infants . 
We report two patients with transient pseudohypoaldosteronism due to obstructive renal disease . 
Both patients presented with a salt-losing episode simulating adrenal insufficiency . 
In one patient , transient reduction of aldosterone receptors could be documented , while in the second patient the clinical and biochemical parameters were consistent with transient pseudohypoaldosteronism . 
Aldosterone receptors were normal in both patients when studied after the surgical correction of the obstruction . 
Increased natural killer cell activity correlates with low or negative expression of the HER-2\/neu oncogene in patients with breast cancer . 
BACKGROUND . 
Increased expression of the HER-2\/neu oncogene in breast cancer correlates with decreased estrogen receptor concentration and seems to be an important prognostic factor . 
The authors investigated whether there is a correlation between HER-2\/neu expression and immunologic parameters representing tumor defense in patients with breast cancer . 
METHOD . 
A Western blot analysis was used to investigate HER-2\/neu expression , whereas a chromium-release assay using the K562 cell line as target was used to measure natural killer ( NK ) cell activity . 
RESULTS . 
In patients with breast cancer , NK cell activity was significantly higher compared with patients with benign tumors ( P = 0.006 ) or healthy control subjects ( P = 0.002 ) . 
Moreover , 23.3 % of patients with breast cancer showed an overexpression of HER-2\/neu protein . 
Within this group of patients , NK cell activity was significantly lower ( 45.6 +\/- 16.1 % ) compared with the group with no HER-2\/neu overexpression ( 57.3 +\/- 11.0 % ) . 
NK cell activity did not increase in patients with HER-2\/neu overexpression . 
Thus , there was a statistically significant correlation of cytolytic effector cell function with HER-2\/neu expression of the tumor ( P = 0.003 ) , and HER-2\/neu overexpression correlated with a negative estrogen receptor status ( P = 0.005 ) . 
CONCLUSION . 
These data add further evidence to previous observations from the authors ' laboratory that certain tumor characteristics may be associated with reactions of the host with breast cancer 
Induction of tyrosine phosphorylation and T-cell activation by vanadate peroxide , an inhibitor of protein tyrosine phosphatases . 
Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in the transduction of activation signals to T-lymphocytes . 
The regulatory role of protein tyrosine phosphatases ( PTPases ) in this process was explored by studying the effects of a powerful PTPase inhibitor , vanadate peroxide ( pervanadate ) , on the activation cascade of Jurkat human leukaemic T-cells . 
Pervanadate induced activation of the tyrosine kinases lck and fyn ( 4- and 3-fold respectively ) and a dramatic increase in tyrosine phosphorylation of cellular proteins , notably phospholipase C gamma 1 . 
After this event , we observed a rise in intracellular Ca2+ concentration , corresponding to an influx . 
This effect required surface expression of the CD45 PTPase and was not observed in CD45-deficient variants of Jurkat cells . 
In the CD45-negative variant , the effect of pervanadate on tyrosine phosphorylation was globally decreased and some phosphorylated substrates were specifically missing . 
Pervanadate also stimulated transcription of the c-fos gene and accumulation of its mRNA as well as several other hallmarks of T-lymphocyte activation such as surface expression of the CD69 antigen and the interleukin 2 receptor alpha-chain ( CD25 ) . 
Pervanadate synergized with signals delivered by T-cell antigen receptor engagement or by a phorbol ester to induce interleukin 2 production . 
Pervanadate activated NF-kappa B , as shown by an increase in DNA-binding activity of this transcription factor . 
We thus conclude that PTPases play a crucial role in the negative regulation of signal transduction culminating in T-lymphocyte activation . 
Moreover , induction of tyrosine phosphorylation appears sufficient per se to initiate a complete activation programme . 
Steroid-resistant asthma . 
Cellular mechanisms contributing to inadequate response to glucocorticoid therapy . 
The current study examined whether alterations in glucocorticoid receptor ( GR ) binding contribute to poor response to glucocorticoid therapy in asthma . 
29 asthma patients with forced expiratory volume in 1 s ( FEV1 ) &lt; 70 % predicted were studied . 
Patients were classified as steroid sensitive ( SS ) if their morning FEV1 increased &gt; 30 % after a 1-wk course of oral prednisone 20 mg twice daily and steroid resistant ( SR ) if they failed to increase &gt; 15 % . 
PBMC obtained from these two groups , 17 SR and 12 SS , as well as 12 normal controls were analyzed . 
SR patients had two distinguishable GR binding abnormalities : 15 of the 17 SR patients demonstrated a significantly reduced GR binding affinity , as compared with SS patients ( P = 0.0001 ) and normal controls ( P = 0.0001 ) . 
This defect was localized to T cells and reverted to normal after 48 h in culture media . 
However , incubation with a combination of IL-2 and IL-4 sustained this abnormality . 
The other two SR patients had an abnormally low GR number with normal binding affinity that was not limited to T cells . 
Furthermore , GR number failed to normalize after incubation in media alone or IL-2 and IL-4 . 
Therefore , SR asthma may be due to more than one abnormality , the majority related to a reversible cytokine-induced reduction in GR binding affinity and the second related to an irreversible reduction in GR number . 
These findings may have important implications for the design of alternative treatment approaches for recalcitrant asthma . 
Increased proliferation , cytotoxicity , and gene expression after stimulation of human peripheral blood T lymphocytes through a surface ganglioside ( GD3 ) { published erratum appears in J Immunol 1994 Jul 15 ; 153 ( 2 ) : 910 } 
Previous studies have suggested that gangliosides have an important role in cell signaling and recognition . 
However , their specific function in these processes has not been clearly defined . 
A mAb , R24 , that reacts specifically with a cell surface ganglioside ( GD3 ) has been demonstrated to stimulate proliferation of T cells derived from human peripheral blood . 
In this study , we have investigated the mechanisms by which the R24 mAb affects T cell functions . 
We have observed that the R24 mAb stimulates GD3+ T cell proliferation , cytotoxicity , and surface marker expression of IL-2R alpha-chain , IL-2R beta-chain , HLA-DR , CD11a , and CD11c . 
Additionally , IFN-gamma activity but not IL-1 , IL-2 , or IL-4 activity was present in culture supernatants 72 h after R24 stimulation . 
In some donors , increased IL-6 and TNF-alpha activity also was detected after R24 treatment . 
Furthermore , R24 treatment resulted in translocation of c-rel , but little or no NF kappa B p50 or p65 , from the cytoplasm to the nucleus and an increase of NF kappa B binding complexes containing c-rel and p50 . 
This treatment also caused increased tyrosine phosphorylation of specific protein substrates . 
R24-stimulated increases in proliferation , cytotoxicity , and cell surface protein expression could be blocked by cyclosporin and staurosporin , indicating that cyclophilin\/calcineurin and protein kinase C may be involved in the R24 signaling pathway . 
Additionally , herbimycin A , a tyrosine kinase inhibitor , blocked the R24-stimulated increase in proliferation but not cytotoxicity at concentrations consistent with specificity for tyrosine kinases . 
These results suggest that multiple biochemical pathways are involved in the activation of human T cells by R24 . 
The SCL protein displays cell-specific heterogeneity in size . 
SCL protein production was examined in a variety of hemopoietic cell lines by immunoblotting using specific polyclonal antisera . 
SCL protein was detected in erythroid , megakaryocyte , mast and early myeloid cell lines , as well as in several lymphoid leukemia cell lines which are known to harbor SCL gene rearrangements . 
In most cell lines , proteins of molecular weight 49 and 44 kDa were found , however two myeloid cell lines expressed only lower molecular weight species of 24 and 22 kDa . 
This size discrepancy appeared to be due to cell-specific translational regulation , since overexpression of a retrovirally transfected SCL gene yielded the higher molecular weight forms in most cell lines ( GP+E-86 , AT2.5 , M1 ) but only the 22 kDa form in the myeloid cell line , WEHI-3B\/D+ . 
Overexpression of full-length SCL protein in the lymphoid cell lines , SupT1 and Raji , did not alter cell phenotype and there was no evidence for autoregulation of SCL transcription . 
The restricted pattern of SCL protein synthesis is consistent with the restricted expression of SCL mRNA documented previously . 
In addition , the present results indicate that SCL protein size was determined by regulation of translation in a cell-specific manner . 
Human T-cell leukemia virus type I Tax associates with and is negatively regulated by the NF-kappa B2 p100 gene product : implications for viral latency . 
Human T-cell leukemia virus type I ( HTLV-I ) is the etiologic agent of the adult T-cell leukemia , an aggressive and often fatal malignancy of activated human CD4 T cells . 
HTLV-I encodes an essential 40-kDa protein termed Tax that not only transactivates the long terminal repeat of this retrovirus but also induces an array of cellular genes . 
Tax-mediated transformation of T cells likely involves the deregulated expression of various cellular genes that normally regulate lymphocyte growth produced by altered activity of various endogenous host transcription factors . 
In particular , Tax is capable of modulating the expression or activity of various host transcription factors , including members of the NF-kappa B\/Rel and CREB\/ATF families , as well as the cellular factors HEB-1 and p67SRF . 
An additional distinguishing characteristic of HTLV-I infection is the profound state of viral latency that is present in circulating primary leukemic T cells . 
In this study , we demonstrate that HTLV-I Tax can physically associate with p100 , the product of the Rel-related NF-kappa B2 gene , both in transfected cells and in HTLV-I-infected leukemic T-cell lines . 
Furthermore , the physical interaction of Tax with p100 leads to the inhibition of Tax-induced activation of the HTLV-I and human immunodeficiency virus type 1 long terminal repeats , reflecting p100-mediated cytoplasmic sequestration of the normally nuclearly expressed Tax protein . 
In contrast , a mutant of Tax that selectively fails to activate nuclear NF-kappa B expression does not associate with p100 . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
Regulation of the BZLF1 promoter of Epstein-Barr virus by second messengers in anti-immunoglobulin-treated B cells . 
Initiation of the Epstein-Barr virus ( EBV ) lytic cycle is dependent on the transcription of the BZLF1 gene . 
The BZLF1 gene promoter ( Zp ) was activated by crosslinking of cell surface immunoglobulin ( Ig ) with anti-Ig antibody in B cells , even in the absence of other viral genes . 
We identified several anti-Ig response elements within Zp , which were originally defined as 12-O-tetradecanoylphorbol-13-acetate ( TPA ) response elements ( ZI repeats and ZII , an AP-1-like domain ) . 
Since anti-Ig crosslinking leads to activation of protein kinase C ( PKC ) and an increase in intracellular calcium level , Zp was tested for the response to these cellular factors . 
Treatment with calcium ionophore A23187 increased Zp activity . 
When the calcium ionophore was used in conjunction with TPA , a PKC activator , the Zp induction was synergistically enhanced . 
Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture . 
All-trans retinoic acid ( RA ) is an important morphogen in vertebrate development , a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia . 
We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells ( HPC ) stringently purified from adult peripheral blood . 
In clonogenetic fetal calf serum-supplemented ( FCS+ ) or -nonsupplemented ( FCS- ) culture treated with saturating levels of interleukin-3 ( IL-3 ) granulocyte-macrophage colony-stimulating factor ( GM-CSF ) and erythropoietin ( Ep ) ( combined with c-kit ligand in FCS-LRB---RRB--culture conditions ) , RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation , the latter colonies being essentially represented by granulocytic clones . 
This shift is apparently not caused by a recruitment phenomenon , because in FCS+ culture , the total number of colonies is not significantly modified by RA addition . 
In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3\/GM-CSF , adult HPC undergo unilineage erythropoietic differentiation : Here again , treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway . 
Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective , thus indicating that early but not late HPC are sensitive to its action . 
We then analyzed the expression of the master GATA1 gene , which encodes a finger transcription factor required for normal erythroid development ; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction . 
These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC , and may lead to a shift from the erythroid to granulocytic differentiation pathway . 
This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation . 
Induction of phosphatidylinositol turnover and EGR-1 mRNA expression by crosslinking of surface IgM and IgD in the human B cell line B104 . 
We have previously shown that a human B lymphoma cell line , B104 , expressed surface IgM ( sIgM ) and surface IgD ( sIgD ) , and that crosslinking of sIgM and sIgD by anti-IgM antibody ( Ab ) and anti-IgD Ab , respectively , induced Ca2+ influx to almost the same degree , whereas only sIgM-crosslinking caused B104 cell death . 
Here , we investigated the accumulation of cyclic AMP ( cAMP ) , the hydrolysis of inositol phosphates , protein kinase C ( PKC ) activity and the induction of Egr-1 and c-fos mRNA expression by sIgM- and sIgD-crosslinking to examine differences in the signals mediated through sIgM and sIgD in B104 cells . 
Both sIgM- and sIgD-crosslinking with antibodies induced elevation of cAMP levels , phosphatidylinositol turnover , PKC activation and expression of Egr-1 and c-fos mRNA , although sIgM-crosslinking was more effective than sIgD-crosslinking , presumably due to the higher expression of sIgM than of sIgD . 
Egr-1 mRNA expression induced by sIgM- and sIgD-crosslinking was inhibited by H7 , erbstatin and genistein , but not by HA1004 . 
Erbstatin and genistein inhibited the sIg-crosslinking-induced Egr-1 mRNA expression in a dose-dependent manner parallel to that observed in the inhibition of sIg-crosslinking-induced protein tyrosine phosphorylation . 
Phorbol myristate acetate induced Egr-1 mRNA expression but forskolin and dibutyryl cyclic AMP did not . 
These findings suggest that the Egr-1 mRNA activating signals through sIgM and sIgD are protein tyrosine kinase- and PKC-dependent , but protein kinase A-independent . 
Cyclosporin A ( CsA ) and FK506 rescued B104 cells from death induced by anti-IgM Ab , but did not affect the expression of Egr-1 and c-fos mRNA , showing that CsA and FK506 affect signal transducers differently from or downstream to these molecules . 
The difference in signals transduced through sIgM and sIgD in B104 cells is discussed . 
G-LRB-Anh-RRB-MTetra , a natural bacterial cell wall breakdown product , induces interleukin-1 beta and interleukin-6 expression in human monocytes . 
A study of the molecular mechanisms involved in inflammatory cytokine expression { published erratum appears in J Biol Chem 1994 Jun 17 ; 269 ( 24 ) : 16983 } 
It is believed that induction of cytokine expression by bacterial cell wall components plays a role in the development and course of sepsis . 
However , most attention has been focused on lipopolysaccharide ( LPS ) . 
We studied the ability of N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-isoglutamyl-m-diaminopimelyl-D-alanine ( G-LRB-Anh-RRB-MTetra ) , a naturally occurring breakdown product of peptidoglycan that is produced by soluble lytic transglycosylase of Escherichia coli , to induce cytokine expression in human monocytes . 
G-LRB-Anh-RRB-MTetra was found to strongly induce interleukin ( IL ) -1 beta and IL-6 mRNA expression after 2 h and IL-1 beta and IL-6 protein secretion after 48 h of activation . 
The increase in mRNA accumulation was at least partly due to an increase in the transcription rates of the respective genes and was accompanied by a strong induction of nuclear factor-kappa B and activator protein-1 transcription factor expression . 
Experiments using inhibitors of protein kinase C , protein kinase A , and tyrosine kinase-dependent pathways revealed that G-LRB-Anh-RRB-MTetra-induced IL-1 beta and IL-6 mRNA expression involves activation of an H7-inhibitable pathway . 
By using the protein synthesis inhibitor cycloheximide , it was shown that G-LRB-Anh-RRB-MTetra-induced IL-6 mRNA expression depends on the synthesis of new protein , whereas G-LRB-Anh-RRB-MTetra-induced IL-1 beta mRNA accumulation does not . 
When responses to G-LRB-Anh-RRB-MTetra were compared with those to LPS and muramyldipeptide ( MDP ) , it was found that the optimal response to G-LRB-Anh-RRB-MTetra induction was similar to that of LPS but significantly higher than the response to MDP . 
Furthermore , maximal G-LRB-Anh-RRB-MTetra-induced IL-1 beta and IL-6 mRNA expression could be enhanced by co-stimulation with LPS or MDP , suggesting that different receptors and\/or transduction pathways were involved . 
These results indicate that G-LRB-Anh-RRB-MTetra induces IL-1 beta and IL-6 expression in human monocytes suggesting a possible role for G-LRB-Anh-RRB-MTetra in the release of cytokines during sepsis . 
rel Is rapidly tyrosine-phosphorylated following granulocyte-colony stimulating factor treatment of human neutrophils . 
Stimulation of neutrophils with granulocyte-colony stimulating factor ( G-CSF ) results in an enhanced respiratory burst , prolonged survival , and increased tumor cell killing . 
The effects of G-CSF are mediated by binding to specific , high affinity receptors . 
G-CSF receptors lack intrinsic tyrosine kinase activity , but activation of the receptor results in the rapid induction of tyrosine kinase activity . 
Antiphosphotyrosine immunoblots of whole cell lysates prepared from neutrophils show that the G-CSF rapidly induces prominent tyrosine phosphorylation of a protein of a relative molecular mass of 80 kDa . 
Using monospecific antibodies , the 80-kDa tyrosine-phosphorylated protein has been shown to be p80c-rel , a proto-oncogene belonging to a family of transcriptional regulators which include NF-kB . 
The induction of tyrosine phosphorylation of p80c-rel was unique to G-CSF in that granulocyte-macrophage colony stimulating factor which also stimulates neutrophils and induces tyrosine phosphorylation does not result in tyrosine phosphorylation of p80c-rel . 
The consequences of p80c-rel tyrosine phosphorylation are not yet known ; however , tyrosine-phosphorylated p80c-rel is capable of binding to DNA , and G-CSF stimulation results in an increase in the amount of p80c-rel which binds to DNA . 
These results demonstrate that one of the first biochemical events which occurs in neutrophils following G-CSF stimulation , activation of a tyrosine kinase , leads directly to the tyrosine phosphorylation of p80c-rel . 
Thus , the tyrosine kinase activated by G-CSF appears to directly transduce a signal to a protein which functions as a transcriptional regulator . 
Autoregulation of the NF-kappa B transactivator RelA ( p65 ) by multiple cytoplasmic inhibitors containing ankyrin motifs . 
RelA ( p65 ) functions as the critical transactivating component of the heterodimeric p50-p65 NF-kappa B complex and contains a high-affinity binding site for its cytoplasmic inhibitor , I kappa B alpha . 
After cellular activation , I kappa B alpha is rapidly degraded in concert with the induced nuclear translocation of NF-kappa B . 
The present study demonstrates that tumor necrosis factor alpha-induced degradation of I kappa B alpha in human T cells is preceded by its rapid phosphorylation in vivo . 
However , these effects on I kappa B alpha result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA . 
Subsequent studies have revealed that ( i ) cytoplasmic RelA is stably associated not only with I kappa B alpha but also with other ankyrin motif-rich proteins including the products of the NF-kappa B2 ( p100 ) and NF-kappa B1 ( p105 ) genes ; ( ii ) in contrast to RelA-I kappa B alpha , RelA-p100 cytoplasmic complexes are not dissociated following tumor necrosis factor alpha activation ; ( iii ) p100 functions as a potent inhibitor of RelA-mediated transcription in vivo ; ( iv ) the interaction of RelA and p100 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal of RelA , which is required for I kappa B alpha binding ; ( v ) p100 inhibition of RelA function requires the C-terminal ankyrin motif domain , which mediates cytoplasmic retention of RelA ; and ( vi ) as observed with I kappa B alpha , nuclear RelA stimulates p100 mRNA and protein expression . 
These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-kappa B . 
Comparative mapping of SRY in the great apes . 
Cytogenetic studies of the primate Y chromosomes have suggested that extensive rearrangements have occurred during evolution of the great apes . 
We have used in situ hybridization to define these rearrangements at the molecular level . 
pHU-14 , a probe including sequences from the sex determining gene SRY , hybridizes close to the early replicating pseudoautosomal segment in a telomeric or subtelomeric position of the Y chromosomes of all great apes . 
The low copy repeat detected by the probe Fr35-II is obviously included in Y chromosomal rearrangements during hominid evolution . 
These results , combined with previous studies , suggest that the Y chromosome in great apes has a conserved region including the pseudoautosomal region and the testis-determining region . 
The rest of the Y chromosome has undergone several rearrangements in the different great apes . 
Detection of minimal residual disease in a patient with acute promyelocytic leukemia by RT-PCR : necessity of chemotherapy following ATRA therapy . 
The PML\/RAR alpha fusion gene resulting from the t -LRB-15;17-RRB- translocation is a specific marker for acute promyelocytic leukemia ( APL ) . 
We examined bone marrow cells by reverse transcriptase-polymerase chain reaction ( RT-PCR ) to detect residual PML\/RAR alpha mRNA-containing cells following treatment with all-trans retinoic acid ( ATRA ) and cytotoxic chemotherapy in a patient with APL . 
This RT-PCR assay can detect one leukemic cell in 10-LRB-2-RRB- normal cells in vitro . 
We show that PML\/RAR alpha mRNA was still detectable despite clinical remission following ATRA treatment , but undetectable following consolidation with chemotherapy . 
These data show that this technique is useful for the identification of minimal residual disease in patients with APL and that cytotoxic chemotherapy following ATRA therapy is required for the elimination of APL cells . 
Patterns of Pan expression and role of Pan proteins in endocrine cell type-specific complex formation . 
The Pan gene encodes at least two distinct transcripts , Pan-1 and Pan-2 ( also known as E47 and E12 , respectively ) , by the mechanism of alternative RNA splicing . 
Northern blot analyses performed on rat and mouse tissues have detected ubiquitously expressed Pan transcripts , but the abundance , distribution , and form of Pan proteins have not been clearly defined . 
Studies of cell lines representing endocrine , fibroblast , and lymphoid lineages using polyclonal antisera to detect E2A proteins have suggested that significant E2A protein expression is restricted to B-lymphocytes . 
We have developed a monoclonal antibody , Yae , which is specific for Pan\/E2A proteins , and have used the Yae antibody to examine a variety of endocrine and nonendocrine cell lineages for differences in Pan\/E2A protein expression , subcellular localization , and heteromeric complex formation . 
In contrast to previous results obtained using polyclonal antiseras to detect Pan\/E2A proteins , we report comparable levels of Pan proteins in GH\/PRL- and insulin-producing , B- and T-lymphocyte cells . 
IEF-1 , a pancreatic beta-cell type-specific complex believed to regulate insulin expression , is demonstrated to consist of at least two distinct species , one of which does not contain Pan molecules . 
Although it has been postulated that pituitary endocrine cells and pancreatic endocrine beta-cells share identical Pan\/E2A complexes , native-Western analyses of pituitary and endocrine beta-cells detect Pan proteins in distinct cell type-specific complexes . 
Interferon alpha selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage . 
The human myeloid cell nuclear differentiation antigen ( MNDA ) is expressed constitutively in cells of the myeloid lineage , appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes . 
The human myeloid leukemia cell lines , HL-60 , U937 , and THP-1 , express similar levels of immunochemically detectable MNDA . 
Although , the level of MNDA mRNA in primary monocytes is very low it was up-regulated at 6 h following the addition of interferon alpha . 
The effect of interferon alpha on the MNDA mRNA is also observed in the cell lines HL-60 , U937 , and THP-1 . 
The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon alpha and other agents including interferon gamma , endotoxin , poly-LRB-I-RRB-.poly-LRB-C-RRB- , and FMLP . 
The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents . 
Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level . 
This reduced level of mRNA could then be elevated with subsequent interferon alpha treatment . 
The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed . 
The ability of interferon alpha to up-regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells : involvement of the conserved lymphokine element 0 . 
Granulocyte-macrophage colony-stimulating factor ( GM-CSF ) and interleukin-2 ( IL-2 ) are produced by stimulation with phorbol-12-myristate acetate ( PMA ) and calcium ionophore ( A23187 ) in human T cell leukemia Jurkat cells . 
The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A ( CsA ) and FK506 . 
Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin ( CN ) , a Ca2+\/calmodulin-dependent protein phosphatase , can stimulate transcription from the IL-2 promoter through the NF-AT-binding site . 
In this study , we obtained evidence that transfection of the cDNAs for CN A ( catalytic ) and CN B ( regulatory ) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA . 
The constitutively active type of the CN A subunit , which lacks the auto-inhibitory and calmodulin-binding domains , acts in synergy with PMA to activate transcription from the GM-CSF promoter . 
We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2 . 
By multimerizing the regulatory elements of the GM-CSF promoter , we found that one of the target sites for the CN action is the conserved lymphokine element 0 ( CLE0 ) , located at positions between -54 and -40 . 
Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor , termed NF-CLE0 gamma . 
NF-CLE0 gamma binding is induced by PMA\/A23187 and is inhibited by treatment with CsA . 
These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter , which mediates signals downstream of T cell activation . 
Glucocorticoid-mediated inhibition of interleukin-2 receptor alpha and -beta subunit expression by human T cells . 
To determine the mechanism of glucocorticoid ( GC ) -mediated inhibition of T cell functions , the effect of dexamethasone ( DM ) on T cell proliferation and interleukin-2 receptor ( IL-2R ) generation were studied . 
Dexamethasone inhibited IL-2-induced T cell proliferation by 30 % - 88 % , relative to its concentration within the cultures . 
The effect of DM on expression of IL-2R alpha ( Tac , p55 , CD25 ) and beta ( p75 ) genes in activated T cells was examined next . 
In T cells stimulated with purified phytohemagglutinin ( PHA-p ) and 4 beta-phorbol 12-myristate 13-acetate ( PMA ) addition of DM to the cultures resulted in a 60 % reduction in IL-2R alpha and a 30 % reduction in IL-2R beta membrane expression compared to T cells cultured in the absence of DM ( p &lt; 0.01 ) . 
Inhibition of membrane IL-2R alpha and IL-2R beta expression by 10-LRB--6-RRB- M DM was partially reversible by recombinant human IL-2 ( rhIL-2 ) . 
By Northern blot analysis , DM caused a comparable decrease in IL-2R alpha and in IL-2R beta mRNA levels to membrane receptor expression in mitogen-stimulated T cells . 
By in vitro transcription assays , DM regulated IL-2R alpha gene expression at a transcriptional level while transcription of IL-2R beta gene was unaffected by DM . 
The mechanism of action of DM on IL-2R alpha transcription was examined by determining the mRNA levels of the p50 subunit of nuclear factor kappa B ( NF-kappa B ) , a transcription factor that stimulates IL-2R alpha gene expression . 
The data indicate that 10-LRB--6-RRB- M DM increased T cell p50 NF-kappa B mRNA levels by four-fold compared to the levels obtained in the absence of DM . 
Further , the level of nuclear proteins capable of binding to the NF-kappa B sites in activated T cells increased in response to DM . 
In sum , DM regulates T cell membrane expression of IL-2R by more than one molecular mechanism . 
Increased interleukin 2 transcription in murine lymphocytes by ciprofloxacin . 
The fluoroquinolone antibiotic , ciprofloxacin ( cipro ) , induces hyperproduction of interleukin 2 ( IL-2 ) and interferon-gamma ( IFN-gamma ) in stimulated human peripheral blood lymphocytes . 
In this investigation an enhanced and prolonged IL-2 and IL-2 mRNA response was also detected in both stimulated ( T cell mitogens or alloantigens ) murine splenocytes and in the stimulated murine T cell line EL-4 in the presence of ciprofloxacin ( 5-80 micrograms\/ml ) as compared to control cells without antibiotics . 
However , in contrast to human lymphocytes , IFN-gamma production was inhibited and IFN-gamma mRNA levels were unaffected at 24 h and only slightly upregulated at 48 and 72 h of culture in murine splenocytes incubated with cipro ( 20 micrograms\/ml ) . 
EL-4 cells were transfected with a plasmid containing the IL-2 promoter and enhancer region linked to the chloramphenicol acetyltransferase ( CAT ) reporter gene . 
Analysis of CAT activity revealed that cipro enhanced IL-2 gene induction . 
In addition , EL-4 cells incubated with ciprofloxacin showed an early peak and more activated nuclear factor of activated T cells ( NFAT-1 ) as compared to control cells without antibiotics . 
Cipro did not affect the nuclear transcription factors AP-1 or NFIL-2A . 
Taken together , cipro inhibited IFN-gamma synthesis , but enhanced IL-2 production in murine lymphocytes by means of influencing NFAT-1 and causing an increased IL-2 transcription . 
Multiple prolactin-responsive elements mediate G1 and S phase expression of the interferon regulatory factor-1 gene . 
The interferon regulatory factor-1 ( IRF-1 ) gene is both an immediate-early G1 phase gene and an S phase gene inducible by PRL in rat Nb2 T lymphocytes . 
To understand the mechanism by which PRL regulates the biphasic expression of IRF-1 , we cloned the rat IRF-1 gene and functionally characterized the IRF-1 promoter . 
Upon transfection into Nb2 T cells , 1.7 kilobases ( kb ) of IRF-1 5'-flanking DNA linked to a chloramphenicol acetyl transferase ( CAT ) reporter gene mediated a 30-fold induction of CAT enzyme activity in response to 24 h of PRL stimulation . 
Deletion mutants containing 1.3 , 0.6 , and 0.2 kb 5'-flanking DNA were incrementally less transcriptionally active , although 0.2 kb still mediated a 12-fold induction by PRL . 
The sequence between -1.7 and -0.2 kb linked to a heterologous thymidine kinase promoter failed to respond to PRL stimulation , suggesting that the activity of upstream PRL response elements may require an interaction with promoter-proximal elements . 
By assaying CAT enzyme activity across a 24-h PRL induction time course , we were able to assign G1 vs. S phase PRL responses of the IRF-1 gene to different regions of the IRF-1 5'-flanking and promoter DNA . 
The 0.2-kb IRF-CAT construct was induced by PRL stimulation during the G1 phase of the cell cycle . 
In contrast , the 1.7-kb IRF-CAT construct was inducible by PRL during both G1 and S phase of the cell cycle . 
Hence , the PRL-induced biphasic expression of the IRF-1 gene appears to be controlled by separate PRL-responsive elements : elements in the first 0.2 kb of the IRF-1 promoter region act during early activation , and elements between 0.2 and 1.7 kb act in concert with the proximal 0.2-kb region during S phase progression . 
Description and functional implications of a novel mutation in the sex-determining gene SRY . 
The sex-determining gene SRY was screened for molecular alteration in an XY sex-reversed female by single-strand conformation polymorphism ( SSCP ) technique . 
An A-to-G transition was detected which leads to an exchange of a tyrosine by a cysteine in the SRY protein . 
The affected tyrosine residue located at the C terminus of the DNA binding protein is evolutionarily strongly conserved among the members of the HMG box containing proteins . 
Using gel shift assay and peptide synthesis such a mutation is shown to abolish the SRY protein DNA binding ability . 
The involvement of this particular amino acid in the binding specificity is also discussed . 
Prevalence of aneuploidy , overexpressed ER , and overexpressed EGFR in random breast aspirates of women at high and low risk for breast cancer . 
Breast tissue biomarkers which accurately predict breast cancer development within a 10 year period in high risk women are needed but currently not available . 
We initiated this study to determine 1 ) the prevalence of one or more breast tissue abnormalities in a group of women at high risk for breast cancer , and 2 ) if the prevalence of biomarker abnormalities is greater in high risk than in low risk women . 
Eligible high risk women were those with a first degree relative with breast cancer , prior breast cancer , or precancerous mastopathy . 
Low risk women were those without these or other major identifiable risk factors . 
Ductal cells were obtained via random fine needle aspirations and cytologically classified . 
Biomarkers included DNA ploidy , estrogen receptor ( ER ) , and epidermal growth factor receptor ( EGFR ) . 
The prevalence of DNA aneuploidy was 30 % , overexpression of ER 10 % , and overexpression of EGFR 35 % , in the 206 high risk women whose median 10 year Gail risk ( projected probability ) of developing breast cancer was 4.5 % . 
The prevalence of aneuploidy and overexpressed EGFR was significantly higher in the high risk women than in the 25 low risk controls ( p &lt; 0.002 ) , whose median 10 year Gail risk was 0.7 % . 
The difference in the prevalence of ER overexpression between high and low risk groups was not statistically significant ( p = 0.095 ) . 
This may be due to the low prevalence of overexpressed ER and the small number of controls . 
A significant difference was noted in the prevalence of one or more abnormal biomarkers between the high risk and low risk women ( p &lt; 0.001 ) . 
A large prospective trial is needed to determine if one or more of these biomarkers , is predictive of breast cancer development . 
{ The changes in glucocorticoid receptors in peripheral leukocytes in asthmatic subjects } 
The number of glucocorticoid receptors ( GCR ) in peripheral leukocytes was determined by radioligand-binding assay in extrinsic and intrinsic asthmatics . 
Their corresponding plasma cortisol levels were assessed . 
The results showed that the average number of GCR in asthmatics was significantly lower than that in healthy subjects ( P &lt; 0.01 ) , and there was a linear correlation between the number of GCR and the course of asthma . 
Besides , there was also a linear correlation between the number of GCR and the age of the initial attack of asthma . 
No difference in plasma cortisol level was found between asthmatics and healthy subjects . 
These findings suggest that there is no primary and general impairment of glucocorticoid metabolism in the asthmatics , but the number of GCR in the asthmatics is lower than that in healthy controls . 
The decrease of the number of GCR in asthmatics , we think , is related to heredity and repeated attacks of asthma . 
Alteration of structural order of human erythrocyte ghost membrane by glucocorticoids and the influence of the glucocorticoid receptor antagonist RU 486 . 
High-dose pulse glucocorticoid therapy has been used successfully in the clinic in severe pathological conditions for about 20 years . 
The mode of glucocorticoid action after administration of such megadoses is inexplicable up to now . 
It is supposed that some effects may be due to membrane alterations . 
In the present in-vitro experiments the effect of dexamethasone , of further glucocorticoids , and of the glucocorticoid receptor antagonist RU 486 , on structural order of human erythrocyte ghost membranes was investigated by determining the steady-state fluorescence anisotropy of diphenylhexatriene ( DPH ) . 
Dexamethasone was found to induce a significant decrease in membrane structural order at concentrations of about 10-LRB--6-RRB- M in a concentration-dependent manner . 
We found a correlation between the uptake of dexamethasone by the ghost membranes and the decrease in the structural order . 
The other glucocorticoids tested , methylprednisolone and corticosterone , were also effective at concentrations of 10-LRB--5-RRB- M or greater . 
We observed no change in membrane structural order with RU 486 up to a concentration of 10-LRB--4-RRB- M . 
However , simultaneous incubation of RU 486 with dexamethasone caused a distinct interference of RU 486 with dexamethasone . 
Thus , the glucocorticoid-induced membrane perturbation , the possibility to inhibit it by RU 486 , and the inactivity of the structurally related progesterone , refer to relatively specific binding sites for the glucocorticoids in the membrane of erythrocyte ghosts . 
Cloning and characterization of NF-ATc and NF-ATp : the cytoplasmic components of NF-AT . 
Present evidence indicates a pathway of signal transmission in T cells that is outlined in figure 1 . 
The elevation in intracellular calcium that is induced by interactions at the antigen receptor leads to the activation of the calcium-dependent phosphatase calcineurin . 
This in turn leads to the nuclear association of the cytosolic component of NF-ATc . 
The activation of calcineurin and the nuclear import of NF-ATc can both be blocked by cyclosporin A or FK506 in complex with their respective immunophilins . 
Once in the nucleus , NF-ATc interacts with NF-ATn to form an active transcriptional complex . 
NF-ATn is a ubiquitous protein , can be synthesized in response to PMA , and has many similarities to AP-1 . 
The mechanism by which NF-ATc enters the nucleus is unknown , and although it appears to require calcineurin , NF-ATc has not yet been shown to be an in vivo substrate of calcineurin . 
Alternative mechanisms include the possibility that NF-ATc operates on some cytoplasmic anchor or that other proteins that are controlled by calcineurin carry out the nuclear import of NF-ATc . 
Although NF-ATp copurifies with NF-ATc , there is as yet no understanding of how NF-ATp is functioning in vivo . 
Now that these proteins are purified and cloned , the major goals will be to understand their role and the roles of other family members in thymic development . 
Changes in triiodothyronine ( T3 ) mononuclear leukocyte receptor kinetics after T3 administration and multiple cold-air exposures . 
Repeated cold-air exposures increase human triiodothyronine ( T3 ) plasma clearance rates . 
To study the response of the nuclear T3 receptor ( NT3R ) in this condition , binding characteristics were analyzed in human mononuclear leukocytes ( MNL ) . 
In addition , we supplemented one group of individuals with a daily oral replacement dose of T3 to isolate the influence of serum thyroxine ( T4 ) and thyrotropin ( TSH ) levels on receptor kinetics . 
The subjects were exposed to cold air ( 4 degrees C ) twice\/d , 30 min\/exposure , for a total of 80 exposures . 
The T3- subjects received placebo { n = 8 } and the T3+ subjects received T3 ( 30 micrograms\/d ) { n = 8 } in a double-blind fashion . 
Mononuclear leukocytes were isolated from peripheral blood before the cold exposure and drug regimen began , and then after every 20 exposures . 
The dissociation constant ( Kd ) and maximum binding capacity ( MBC ) of the NT3R values were log transformed to minimize between-subject variability . 
In the T3+ group , serum total thyroxine ( TT4 ) , free T4 ( FT4 ) , and TSH were approx 50 % lower than both basal and T3-values . 
The log10Kd increased 0.304 +\/- 0.139 ( p &lt; 0.04 ) and the log10MBC increased 0.49 +\/- 0.10 ( p &lt; 0.001 ) in the T3+ subjects compared to baseline . 
This change in MBC represents a 311 % increase in the MBC over baseline and a fivefold increase over placebo-treated subjects . 
The T3- group showed no change in MBC over the study . 
These results describe for the first time the rapid modulation of the NT3R in response to the combined influence of cold exposure and reduced circulating T4 and TSH . 
Selective effects of DNA damaging agents on HIV long terminal repeat activation and virus replication in vitro . 
Much attention has recently focused on the observation that UV light can activate the long terminal repeat ( LTR ) of the human immunodeficiency virus ( HIV ) . 
Although the mechanism of LTR activation remains obscure , several lines of investigation have suggested that it is a result of activation of the NF-kappa B transcription factor ( s ) following signaling events related to generalized DNA damage . 
In this report , we present data demonstrating that HIV LTR activation is not a general consequence of cellular DNA damage , but rather a process unique to specific genotoxic stimuli , and that it does not necessarily depend on activation of NF-kappa B . 
Furthermore , we demonstrate that several of these agents can significantly increase HIV replication and accelerate CD4-positive lymphocyte cytotoxicity in vitro . 
These findings , therefore , could have clinical significance to AIDS patients with malignancies who are undergoing radiotherapy and chemotherapy . 
Characterization and purification of a protein kinase C substrate in human B cells . 
Identification as lymphocyte-specific protein 1 ( LSP1 ) . 
Incubation of B-chronic lymphocytic leukemia ( B-CLL ) cells with phorbol esters resulted in the phosphorylation of two major PKC substrates , MARCKS ( myristoylated , alanine-rich C kinase substrate ) and MRP ( MARCKS-related protein ) , and of a third protein , with an apparent m.w. of 60,000 that was the most prominent protein kinase C substrate in these cells . 
p60 phosphorylation was time and PMA dose dependent , and was induced by cell-permeable diacylglycerol , but not by inactive phorbol esters . 
Two-dimensional electrophoretic analysis of the protein phosphorylation pattern from the B cell line CESS demonstrated the identity between the p60 protein expressed in this cell line and that expressed in B-CLL cells . 
p60 was purified from CESS cells and peptide microsequencing of this protein revealed that it was lymphocyte-specific protein 1 ( LSP1 ) , that is here characterized as the most prominent protein kinase C substrate in B cells . 
IL-13 induces phosphorylation and activation of JAK2 Janus kinase in human colon carcinoma cell lines : similarities between IL-4 and IL-13 signaling . 
We have recently reported that IL-13R may share a component with IL-4R . 
Here we report that both IL-4 and IL-13 share signaling events in human colon carcinoma cell lines ( HT-29 and WiDr ) . 
IL-13 caused rapid phosphorylation of the three out of four members of the known Janus family of kinases ( JAKs ) . 
We show that JAK2 kinase is rapidly phosphorylated and activated in response to IL-13 . 
Within 1 min of activation , JAK2 was phosphorylated , and peaked in 10 min . 
In addition , IL-13 phosphorylated insulin response substrate-1 , IL-4R p140 , JAK1 , and Tyk2 , but not JAK3 kinase . 
IL-4 also stimulated all three kinases and substrates , but unlike in immune cells , IL-4 did not involve JAK3 activation for its signaling in colon cancer cell lines . 
Furthermore , JAK2 associated with the IL-4R p140 before and after stimulation with IL-13 . 
Both IL-13 and IL-4 induced phosphorylation of IL-4 STAT ( STAT6 ) but not STAT1 , STAT3 , or STAT5 . 
125I-IL-13 did not bind to colon cancer cell lines , but unlabeled IL-13 competed for the binding of 125I-IL-4 . 
Our data suggest that IL-13 utilizes IL-4R and its signaling pathway , and JAK2 may play an important role in the function of IL-4R and IL-13R in colon cancer cells . 
Effects of interleukin-10 on human peripheral blood mononuclear cell responses to Cryptococcus neoformans , Candida albicans , and lipopolysaccharide . 
Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection . 
We compared the effects of the deactivating cytokine interleukin 10 ( IL-10 ) on human peripheral blood mononuclear cell ( PBMC ) responses to lipopolysaccharide ( LPS ) , Cryptococcus neoformans , and Candida albicans . 
IL-10 effected dose-dependent inhibition of tumor necrosis factor alpha ( TNF-alpha ) release in PBMC stimulated by LPS and C. neoformans , with significant inhibition seen with 0.1 U\/ml and greater than 90 % inhibition noted with 10 U\/ml . 
In contrast , even at doses as high as 100 U\/ml , IL-10 inhibited TNF-alpha release in response to C. albicans by only 50 % . 
IL-10 profoundly inhibited release of IL-1beta from PBMC stimulated by all three stimuli . 
TNF-alpha mRNA and release was inhibited even if IL-10 was added up to 8 h after cryptococcal stimulation . 
In contrast , inhibition of IL-1 beta mRNA was of lesser magnitude and occurred only when IL-10 was added within 2 h of cryptococcal stimulation . 
IL-10 inhibited translocation of NF-kappaB in response to LPS but not the fungal stimuli . 
All three stimuli induced IL-10 production in PBMC , although over 10-fold less IL-10 was released in response to C. neoformans compared with LPS and C. albicans . 
Thus , while IL-10 has deactivating effects on PBMC responses to all three stimuli , disparate stimulus- and response-specific patterns of deactivation are seen . 
Inhibition by IL-10 of proinflammatory cytokine release appears to occur at the level of gene transcription for TNF-alpha and both transcriptionally and posttranscriptionally for IL-1beta . 
Activation of nuclear factor of activated T cells in a cyclosporin A-resistant pathway . 
The mechanism of action of the immunosuppressive drug cyclosporin A ( CsA ) is the inactivation of the Ca2+\/calmodulin-dependent serine-threonine phosphatase calcineurin by the drug-immunophilin complex . 
Inactive calcineurin is unable to activate the nuclear factor of activated T cells ( NFAT ) , a transcription factor required for expression of the interleukin 2 ( IL-2 ) gene . 
IL-2 production by CsA-treated cells is therefore dramatically reduced . 
We demonstrate here , however , that NFAT can be activated , and significant levels of IL-2 can be produced by the CsA-resistant CD28-signaling pathway . 
In transient transfection assays , both multicopy NFAT- and IL-2 promoter-beta-galactosidase reporter gene constructs could be activated by phorbol 12-myristate 13-acetate ( PMA ) \/alpha-CD28 stimulation , and this activation was resistant to CsA . 
Electrophoretic mobility shift assay showed the induction of a CsA-resistant NFAT complex in the nuclear extracts of peripheral blood T cells stimulated with PMA plus alphaCD28 . 
Peripheral blood T cells stimulated with PMA\/alphaCD28 produced IL-2 in the presence of CsA . 
Collectively , these data suggest that NFAT can be activated and IL-2 can be produced in a calcineurin independent manner . 
Transcription factors of T and B lymphocytes -- basic research and clinical perspectives for gastroenterology . 
Tissue specific regulation of gene expression by transcription factors is a fascinating new field in molecular immunology . 
This review summarizes data on specific regulation of promoters and enhancers by nuclear trans-acting factors in lymphocytes . 
The structural classes of transcription factors are described and basic methods for detection and analysis of transcription factors are detailed . 
Furthermore , the most important trans-acting factors of T and B lymphocytes ( e.g. NF-kB , NF-AT and STAT families ) and their functional importance are described . 
Several methods for specific down-regulation of transcription factors are shown that may be relevant to treatment of human disease . 
The data are discussed with regard to their potential clinical relevance for gastroenterology . 
Modulation of the expression of the IFN-gamma receptor beta-chain controls responsiveness to IFN-gamma in human peripheral blood T cells . 
IFN-gamma has potent antiproliferative and apoptotic effects in T cells that are important in determining T cell development and polarized differentiation . 
Therefore , any event that enables T cells to become less responsive to gamma may potentially alter immune responsiveness to Ag . 
In this work , we show that human peripheral blood T cells that are stimulated through the TCR and expanded with IL-2 are unresponsive to IFN-gamma , as determined by a lack of activation of jak kinases and the transcription factor , STAT1-LRB-alpha-RRB- , a signal transducer and activator of transcription . 
This nonresponsiveness occurs because of a lack of expression of the beta-chain ( accessory factor ) of the IFN-gamma receptor , while at the same time maintaining IFN-gamma receptor alpha-chain expression . 
Expression of the beta-chain can be restored by secondary TCR ligation or PMA treatment . 
T cell blasts treated with PMA are now responsive to IFN-gamma . 
When freshly isolated , highly enriched ( &gt; 98 % ) T cells are examined for IFN-gamma responsiveness ; these cells can respond to IFN-gamma and express beta-chain . 
Therefore , as T cells progress from primary TCR activation through IL-2-dependent proliferation , followed by secondary TCR stimulation , their responsiveness to IFN-gamma varies , and this may affect their ability to participate in an ongoing immune response . 
Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2 , IL-12 , and IFN-alpha . 
IL-2- , IL-12- , and IFN-alpha-mediated signaling pathways were analyzed in primary NK cells and in the NK3.3 cell line . 
Gel mobility shift and immunoprecipitation analyses revealed that in addition to activating STAT3 ( signal transducer and activator of transcription-3 ) and STAT5 , IL-2 induced tyrosine and serine phosphorylation of STAT1 alpha , which formed IFN-gamma-activated sequence-binding complexes by itself and with STAT3 . 
Although IL-2 and IFN-alpha activated STAT1 alpha and STAT5 , IL-2 predominantly activated STAT5 , while IFN-alpha predominantly activated STAT1 alpha . 
IL-2 induced less STAT1 alpha activation and IFN-alpha induced greater STAT5 activation in NK3.3 cells compared with preactivated primary NK cells . 
In NK3.3 cells , IL-2 induced comparable formation of c-fos promoter sis-inducible element IFN-gamma-activated sequence-binding complexes containing STAT3 alone with complexes containing STAT3 and STAT1 alpha , while in preactivated primary NK cells , it preferentially induced complexes containing STAT3 and STAT1 alpha . 
Thus , signaling in NK3.3 cells is not always identical with that in primary NK cells . 
In contrast to IL-2 and IFN-alpha , IL-12 induced strong tyrosine phosphorylation of STAT4 and variable weak phosphorylation of STAT3 . 
However , supershift analyses using the c-fos promoter sis-inducible element probe showed that IL-12 activated STAT4 , STAT1 alpha , and STAT3 , and induced complexes containing STAT4 only , STAT4 with STAT1 alpha , STAT3 with STAT1 alpha , or STAT1 alpha only in preactivated primary NK cells . 
STAT1 alpha activation by IL-12 correlated with increased phosphorylation of serine , but not tyrosine . 
Finally , IL-2 induced tyrosine phosphorylation of JAK1 and JAK3 , while IL-12 induced phosphorylation of JAK2 and TYK2 in both preactivated primary NK and NK3.3 cells . 
Differential phosphorylation and consequent differential activation of both separate and overlapping STAT proteins by IL-2 , IL-12 , and IFN-alpha may provide a molecular basis for the similarities and differences in the actions of these cytokines on NK cells . 
Sublethal levels of oxidative stress stimulate transcriptional activation of c-jun and suppress IL-2 promoter activation in Jurkat T cells . 
Sublethal levels of oxidative stress are well known to alter T cell functional responses , but the underlying mechanisms are unknown . 
The current study examined the effects of oxidative stress on transcriptional activities mediated by c-Fos\/c-Jun AP-1 and the nuclear factor of activated T cells ( NF-AT ) . 
The present results show that Jurkat T cells acutely exposed to micromolar concentrations of H2O2 exhibit substantial increases in AP-1 binding activity and the expression of c-jun but not c-fos mRNA . 
The preferential induction of c-jun by H2O2 did not represent redox stabilization of mRNA transcripts , and oxidative signals closely resembled PHA\/PMA stimulation by effectively transactivating the full length c-jun promoter via the proximal jun1 tumor promoter-responsive element ( TRE ) -like promoter element . 
Similarly , the complexes binding the consensus AP-1 TRE and jun TRE-like motifs in cells exposed to oxidative signals or PHA\/PMA were indistinguishable , being composed of c-Fos , c-Jun , and JunD . 
However , PHA\/PMA but not oxidative signals induced the coordinate activation of reporter constructs containing the AP-1-TRE , NF-AT , and IL-2 promoter regions along with IL-2 mRNA expression . 
Furthermore , sublethal levels of H2O2 actively suppressed the transcriptional activation of NF-AT and IL-2 reporters as well as the expression of IL-2 mRNA in cells stimulated with PHA\/PMA . 
Gel shift analysis revealed that oxidative suppression of NF-AT represented inhibition in the early generation of NFAT complexes rather than the binding of preformed NF-AT complexes . 
These results suggest that oxidative signals can positively and negatively regulate T cell transcriptional events and that changes in cellular redox can uncouple AP-1 regulation of c-jun from transcriptional up-regulation of IL-2 via NF-AT . 
Cooperation between core binding factor and adjacent promoter elements contributes to the tissue-specific expression of interleukin-3 . 
Tissue-specific expression of interleukin-3 ( IL-3 ) is mediated via cis-acting elements located within 315 base pairs of the transcription start . 
This is achieved in part through the positive activities of the AP-1 and Elf-1 sites in the IL-3 promoter . 
The contribution to T cell-specific expression by other promoter sites was assessed in a transient expression assay with IL-3 promoter constructs linked to a luciferase gene , focusing initially on the core binding factor ( CBF ) site , which is footprinted in vivo upon T cell activation . 
Activity of the CBF site is shown to be critically dependent on the adjacent activator site Act-1 . 
Together the Act-1 and CBF sites form a functional unit ( AC unit ) with dual activity . 
The AC unit is demonstrated to enhance basal activity of promoters both in fibroblasts and T cells . 
This activity is further inducible in activated T cells , but not in fibroblasts . 
In addition to the already identified NIP repressor site , evidence is presented for a second repressor region that restricts promoter activity in fibroblasts . 
Finally , a novel positive regulatory element has been mapped in the IL-3 promoter between nucleotide -180 and -210 that leads to increased expression in T cells . 
Together these results demonstrate that T cell expression of IL-3 is not specified by the activity of a single tissue-specific element , but instead involves multiple interacting elements that provide both specific positive regulation in T cells and specific negative regulation in fibroblasts . 
A 3' --&gt; 5' XPB helicase defect in repair\/transcription factor TFIIH of xeroderma pigmentosum group B affects both DNA repair and transcription . 
XPB is a subunit of the basal transcription factor TFIIH , which is also involved in nucleotide excision repair ( NER ) and potentially in cell cycle regulation . 
A frameshift mutation in the 3'-end of the XPB gene is responsible for a concurrence of two disorders : xeroderma pigmentosum ( XP ) and Cockayne 's syndrome ( CS ) . 
We have isolated TFIIH from cells derived from a patient ( XP11BE ) who carries this frameshift mutation ( TFIIHmut ) and from the mother of this patient ( TFIIHwt ) to determine the biochemical consequences of the mutation . 
Although identical in composition and stoichiometry to TFIIHwt , TFIIHmut shows a reduced 3' --&gt; 5' XPB helicase activity . 
A decrease in helicase and DNA-dependent ATPase activities was also observed with the mutated recombinant XPB protein . 
The XPB mutation causes a severe NER defect . 
In addition , we provide evidence for a decrease in basal transcription activity in vitro . 
The latter defect may provide an explanation for many of the XP and CS symptoms that are difficult 0 to rationalize based solely on an NER defect . 
Thus , this work presents the first detailed analysis of a naturally occurring mutation in a basal transcription factor and supports the concept that the combined XP\/CS clinical entity is actually the result of a combined transcription\/repair deficiency . 
Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking . 
Glutathione-S-transferase ( GST ) -Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor , Fc gamma RIIA . 
Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets . 
Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1 . 
p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase . 
The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD , which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells . 
The 75-kD protein is recognized by antibodies to SLP-76 , which has recently been isolated from T cells and sequenced . 
Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin , although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets . 
p38 and p63 may provide a docking site for Grb2 , thereby linking Grb2 SH3-binding proteins SOS1 , SLP-76 , and p120 to downstream signalling events . 
Retinoic acid activates interferon regulatory factor-1 gene expression in myeloid cells . 
All-trans-retinoic acid ( ATRA ) is the drug of choice in the treatment of acute promyelocytic leukemia ( APL ) . 
ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes . 
However , the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood . 
The NB4 cell line , which is derived from the bone marrow of a patient with APL during relapse , can be used as a model system 0 to study the growth and differentiation of APL cells . 
Because interferon ( IFN ) regulatory factors ( IRF-1 and IRF-2 ) and other IFN-inducible gene products regulate cell growth , we analyzed the effects of ATRA on the expression of these genes . 
We show that ATRA directly activates IRF-1 gene expression , followed by activation of IRF-2 and 2'-5' oligoadenylate synthetase ( OAS ) gene expression with slower kinetics . 
In addition to NB4 cells , ATRA also activated IRF-1 gene expression in HL-60 , U937 , and THP-1 cells , which all respond to ATRA by growth inhibition . 
A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells . 
ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription ( STAT ) activation pathways , suggesting that an alternate mechanism is involved in IRF-1 gene activation . 
The ATRA-induced expression of IRF-1 , an activator of transcription and repressor of transformation , may be one of the molecular mechanisms of ATRA-induced growth inhibition , and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells . 
The c-Jun delta-domain inhibits neuroendocrine promoter activity in a DNA sequence- and pituitary-specific manner . 
The transcription and transformation activity of c-Jun is governed by a 27-amino acid regulatory motif , labeled the delta-domain , which is deleted in v-Jun . 
We have previously shown that c-Jun is a potent inhibitor of the rat prolactin ( rPRL ) promoter activity induced by either oncogenic Ras or phorbol esters . 
Here , we have characterized the structural and cell-specific requirements for this c-Jun inhibitory response , and we show that this c-Jun inhibitory response mapped to the rPRL footprint II repressor site , was pituitary-specific and required the c-Jun delta-domain . 
Moreover , alteration of any one of these features ( e.g. , cis-element , trans-factor , or cell-specific background ) switched c-Jun to a transcriptional activator of the rPRL promoter . 
In HeLa nonpituitary cells , c-Jun alone activated the rPRL promoter via the most proximal GHF-1\/Pit-1 binding site , footprint I , and synergized with GHF-1 . 
Finally , recombinant GHF-1 interacted directly with c-Jun but not c-Fos proteins . 
These data provide important fundamental insights into the molecular mechanisms by which the c-Jun delta-domain functions as a modulatory switch and further imply that the functional role of c-Jun is dictated by cell-specific influences and the delta-domain motif . 
Constitutive expression of specific interferon isotypes in peripheral blood leukocytes from normal individuals and in promonocytic U937 cells . 
Constitutive expression of IFN-alpha5 and IFN-beta was detected in different lymphoid cells including peripheral blood mononuclear cells from normal individuals following amplification of IFN mRNA by reverse transcriptase-polymerase chain reaction and direct sequencing of the amplified product . 
The activated form of the interferon-induced transcription factor complex ISGF3 was also detected in nuclear extracts from uninduced cells . 
Culture supernatants from uninduced U937 cells were also found to activate an ISRE cloned upstream of the luciferase reporter gene , indicating the presence of endogenous IFN activity equivalent to approximately 0.3 to 0.5 IU\/mL . 
This endogenous IFN was also shown to play a role in maintaining the basal level of expression of the major histocompatibility class I genes in lymphoid cells . 
These results suggest that IFN-alpha5 and IFN-beta are produced at low levels in normal tissues and play an important role in the regulation of cell function and in the maintenance of homeostasis . 
Mechanisms of transactivation by nuclear factor of activated T cells-1 . 
Nuclear factor of activated T cells-family proteins ( NFAT1\/NFATp , NFATc , NFAT3 , and NFAT4\/NFATx/NFATc3 ) play a key role in the transcription of cytokine genes and other genes during the immune response . 
We have defined the mechanisms of transactivation by NFAT1 . 
NFAT1 possesses two transactivation domains whose sequences are not conserved in the other NFAT-family proteins , and a conserved DNA-binding domain that mediates the recruitment of cooperating nuclear transcription factors even when it is expressed in the absence of other regions of the protein . 
The activity of the NH2-terminal transactivation domain is modulated by an adjacent regulatory region that contains several conserved sequence motifs represented only in the NFAT family . 
Our results emphasize the multiple levels at which NFAT-dependent transactivation is regulated , and predict significant differences in the architecture of cooperative transcription complexes containing different NFAT-family proteins . 
Precise alignment of sites required for mu enhancer activation in B cells . 
The lymphocyte-specific immunoglobulin mu heavy-chain gene intronic enhancer is regulated by multiple nuclear factors . 
The previously defined minimal enhancer containing the muA , muE3 , and muB sites is transactivated by a combination of the ETS-domain proteins PU.1 and Ets-1 in nonlymphoid cells . 
The core GGAAs of the muA and muB sites are separated by 30 nucleotides , suggesting that ETS proteins bind to these sites from these same side of the DNA helix . 
We tested the necessity for appropriate spatial alignment of these elements by using mutated enhancers with altered spacings . 
A 4- or 10-bp insertion between muE3 and muB inactivated the mu enhancer in S194 plasma cells but did not affect in vitro binding of Ets-1 , PU.1 , or the muE3-binding protein TFE3 , alone or in pairwise combinations . 
Circular permutation and phasing analyses demonstrated that PU.1 binding but not TFE3 or Ets-1 bends mu enhancer DNA toward the major groove . 
We propose that the requirement for precise spacing of the muA and muB elements is due in part to a directed DNA bend induced by PU.1 . 
C\/EBP activators are required for HIV-1 replication and proviral induction in monocytic cell lines . 
Previous work has shown that C\/EBP sites and C\/EBP transcriptional activators are necessary for HIV-1 LTR activity in monocytes\/macrophages . 
We have investigated the role that C\/EBP proteins play in induction and replication of HIV-1 . 
Ectopic expression of the dominant negative C\/EBP protein LIP inhibited HIV-1 mRNA and virus production in activated U1 cells , demonstrating that C\/EBP proteins are required for provirus induction . 
U1 lines overexpressing C\/EBP activator NF-IL-6 produced more viral mRNA and virus particles following cellular activation than control lines , demonstrating that C\/EBP proteins are limiting for virus transcription . 
HIV-1 harboring mutations within two C\/EBP sites were crippled in their ability to replicate in U937 promonocytic cells , indicating that these sites are required for replication . 
These data identify C\/EBP proteins as regulators of HIV-1 expression in monocytes\/macrophages . 
Reversal of apoptosis by the leukaemia-associated E2A-HLF chimaeric transcription factor . 
The E2A-HLF ( for hepatic leukaemia factor ) fusion gene , formed by action of the t-LRB-17;19-RRB- -LRB-q22;p13-RRB- chromosomal translocation , drives the leukaemic transformation of early B-cell precursors , but the mechanism of this activity remains unknown . 
Here we report that human leukaemia cells carrying the translocation t-LRB-17;19-RRB- rapidly died by apoptosis when programmed to express a dominant-negative suppressor of the fusion protein E2A-HLF , indicating that the chimaeric oncoprotein probably affects cell survival rather than cell growth . 
Moreover , when introduced into murine pro-B lymphocytes , the oncogenic E2A-HLF fusion protein reversed both interleukin-3-dependent and p53-mediated apoptosis . 
The close homology of the basic region\/leucine zipper ( bZIP ) DNA-binding and dimerization domain of HLF to that of the CES-2 cell-death specification protein of Caenorhabditis elegans suggests a model of leukaemogenesis in which E2A-HLF blocks an early step within an evolutionarily conserved cell-death pathway . 
Inhibition of transcription factor Stat1 activity in mononuclear cell cultures and T cells by the cyclic AMP signaling pathway . 
Activation of T cells results in a cascade of gene activation and subsequent proliferation and differentiation into effector phenotypes . 
The regulation of transcription factors belonging to the signal transducer and activator of transcription ( STAT ) family was analyzed in PHA-activated mononuclear cells and in purified T cells activated by cross-linking cell surface CD3 . 
Cell activation resulted in a delayed induction of STAT DNA-binding activity , which was sustained for several days , was composed predominantly of Stat1 and Stat3 , and was blocked by cycloheximide and actinomycin D . 
Increased Stat1 and Stat3 mRNA and protein levels were detected , respectively 4 and 24 h after activation . 
Stimulation of the cAMP signal transduction pathway , which skews cytokine production toward a Th2 pattern , resulted in the preferential suppression of Stat1 activity . 
cAMP inhibited the induction of expression of IL-2 receptor components , but did not inhibit IL-4 receptor alpha-chain and CD69 expression or the induction of activator protein 1 transcription factors . 
cAMP signaling inhibited Stat1 at several different levels , including suppression of DNA binding and down-regulation of Stat1 protein and mRNA levels . 
Our results demonstrate the regulation of STAT activity by a signaling pathway that regulates the T cell functional phenotype and is distinct from the cytokine-activated Janus kinase-STAT signaling pathway . 
oriP is essential for EBNA gene promoter activity in Epstein-Barr virus-immortalized lymphoblastoid cell lines . 
During Epstein-Barr virus latent infection of B lymphocytes in vitro , six viral nuclear antigens ( EBNAs ) are expressed from one of two promoters , Cp or Wp , whose activities are mutually exclusive . 
Upon infection , Wp is initially active , followed by a switch to Cp for the duration of latency . 
In this study , the region upstream of Cp was analyzed for the presence of cis elements involved in regulating the activities of the EBNA gene promoters in established in vitro immortalized lymphoblastoid cell lines ( LCLs ) . 
It was determined that oriP , the origin for episomal maintenance during latency , is essential for efficient transcription initiation from either Cp or Wp in LCLs , as well as in some Burkitt 's lymphoma cell lines . 
Deletion of the EBNA2-dependent enhancer located upstream of Cp resulted in a ca. two- to fivefold reduction in Cp activity in the LCLs assayed . 
More extensive deletion of sequences upstream of Cp , including the EBNA2-dependent enhancer , resulted in nearly complete loss of Cp activity . 
This loss of activity was shown to correlate with deletion of two CCAAT boxes , a proximal CCAAT box located at bp -61 to -65 and a distal CCAAT box located at bp -253 to -257 , upstream of Cp . 
Site-directed mutagenesis of these cis elements demonstrated that Cp activity is highly dependent on the presence of a properly positioned CCAAT box , with the dependence on the distal CCAAT box apparent only when the proximal CCAAT box was deleted or mutated . 
Deletion of the glucocorticoid response elements located at ca. bp -850 upstream of Cp did not result in a significant loss in activity . 
In general , deletions which diminished Cp activity resulted in induction of Wp activity , consistent with suppression of Wp activity by transcriptional interference from Cp . 
The identification of oriP and the EBNA2-dependent enhancer as the major positive cis elements involved in regulating Cp activity in LCL suggests that EBNA gene transcription is largely autoregulated by EBNA 1 and EBNA 2 . 
The human T-cell leukemia virus type 1 posttranscriptional trans-activator Rex contains a nuclear export signal . 
The Rex protein of human T-cell leukemia virus type 1 is required for the nuclear export of unspliced viral mRNA and , therefore , for virus replication . 
In this manuscript , we demonstrate that Rex shuttles between the nucleus and the cytoplasm and that its activation domain constitutes a nuclear export signal that specifies efficient transport to the cytoplasm . 
These findings are consistent with a model for Rex-mediated trans-activation in which Rex-viral mRNA complexes are targeted for nuclear export by the direct action of the activation domain . 
Multiple p21ras effector pathways regulate nuclear factor of activated T cells . 
The transcription factor , Nuclear Factor of Activated T cells ( NFAT ) is a major target for p21ras and calcium signalling pathways in the IL-2 gene and is induced by p21ras signals acting in synergy with calcium\/calcineurin signals . 
One p21ras effector pathway involves the MAP kinase ERK-2 , and we have examined its role in NFAT regulation . 
Expression of dominant negative MAPKK-1 prevents NFAT induction . 
Constitutively active MAPKK-1 fully activates ERK-2 and the transcription factor Elk-1 , but does not substitute for activated p21ras and synergize with calcium\/calcineurin signals to induce NFAT . 
Expression of dominant negative N17Rac also prevents TCR and p21ras activation of NFAT , but without interfering with the ERK-2 pathway . 
The transcriptional activity of the NFAT binding site is mediated by a complex comprising a member of the NFAT group and AP-1 family proteins . 
The induction of AP-1 by p21ras also requires Rac-1 function . 
Activated Rac-1 could mimic activated p21ras to induce AP-1 but not to induce NFAT . 
Moreover , the combination of activated MAPKK-1 and Rac-1 could not substitute for activated p21ras and synergize with calcium signals to induce NFAT . 
Thus , p21ras regulation of NFAT in T cells requires the activity of multiple effector pathways including those regulated by MAPKK-1\/ERK-2 and Rac-1 . 
Protein-tyrosine kinase activation is required for lipopolysaccharide induction of interleukin 1beta and NFkappaB activation , but not NFkappaB nuclear translocation . 
In human monocytes , interleukin 1beta protein production and steady state mRNA levels are increased in response to lipopolysaccharide , predominantly as a result of increased transcription of the interleukin 1beta gene . 
Expression of interleukin 1beta and other cytokines , such as interleukin 6 and tumor necrosis factor alpha , has been shown to be dependent on the activation of the transcription factor , NFkappaB . 
Since recent studies have shown that lipopolysaccharide-induced tyrosine kinase activation is not required for NFkappaB nuclear translocation , we sought to determine whether NFkappaB translocated in the absence of tyrosine kinase activity was active in stimulating transcription . 
We have found that , in the human pro-monocytic cell line , THP-1 , the lipopolysaccharide-induced expression of interleukin 1beta is dependent on tyrosine kinase activation . 
Tyrosine kinases are not required for lipopolysaccharide-mediated nuclear translocation of NFkappaB . 
However , in the absence of tyrosine kinase activity , the ability of NFkappaB to stimulate transcription is impaired . 
This inhibition of transcription is specific for NFkappaB ; in the absence of tyrosine kinase activity , AP-1-dependent transcription is enhanced . 
These results suggest that , while lipopolysaccharide-induced expression of inflammatory mediators requires tyrosine kinase activity , tyrosine kinase activity is not obligatory for lipopolysaccharide signal transduction 
LYSP100-associated nuclear domains ( LANDs ) : description of a new class of subnuclear structures and their relationship to PML nuclear bodies . 
The PML gene is fused to the retinoic acid receptor alpha ( RAR alpha ) gene in t-LRB-15;17-RRB- acute promyelocytic leukemia ( APL ) , creating a PML-RAR alpha fusion oncoprotein . 
The PML gene product has been localized to subnuclear dot-like structures variously termed PODs , ND10s , Kr bodies , or PML nuclear bodies ( PML NBs ) . 
The present study describes the cloning of a lymphoid-restricted gene , LYSP100 , that is homologous to another protein that localizes to PML NBs , SP100 . 
In addition to SP100 homology regions , one LYSP100 cDNA isoform contains a bromodomain and a PHD\/TTC domain , which are present in a variety of transcriptional regulatory proteins . 
By immunofluorescence , LYSP100 was localized to nuclear dots that were surprisingly largely nonoverlapping with PML NBs . 
However , a minority of LYSP100 nuclear dots exactly colocalized with PML and SP100 . 
We term the LYSP100 structures " LANDs , " for LYSP100-associated nuclear domains . 
Although LYSP100 is expressed only in lymphoid cells , LANDs could be visualized in HeLa cells by transfection of a LYSP100 cDNA . 
Immunoelectron microscopy revealed LANDs to be globular , electron-dense structures morphologically distinct from the annular structures characteristic of PML NBs . 
LANDs were most often found in the nucleoplasm , but were also found at the nuclear membrane and in the cytoplasm , suggesting that these structures may traffic between the cytoplasm and the nucleus . 
By double-immunogold labeling of PML and LYSP100 , some LANDs were shown to contain both PML and LYSP100 . 
Thus , PML is localized to a second subnuclear domain that is morphologically and biochemically distinct from PML NBs . 
Abnormalities of p16 , p15 and CDK4 genes in recurrent malignant astrocytomas . 
Abnormalities in the p16 , p15 and CDK4 genes that regulate transition through the G1 phase of the cell cycle have been implicated in the malignant progression of astrocytomas . 
The results of the present study demonstrate that dysfunction of these genes also occurs during recurrence of glial tumors that were highly malignant at first presentation . 
Analysis of 10 matched pairs of high grade malignant astrocytomas and their subsequent recurrences identified three distinct groups . 
The primary and recurrent tumors in Group A did not show structural alterations in the p16 , p15 or CDK4 genes , whereas homozygous codeletion of p16 and p15 was observed in both primary and recurrent tumors in Group B . 
The primary tumors in Group C had a normal profile of p16 , p15 and CDK4 at presentation . 
Upon recurrence , however , the tumors sustained either deletion of p16 alone or codeletion of both p16 and p15 or amplification of CDK4 . 
Analysis of the molecular differences between primary anaplastic astrocytomas\/glioblastomas and their subsequent recurrences , which are clinically indistinguishable , may provide better therapeutic options for treatment . 
{ NGFI-B\/nur77 family involved in T-cell apoptosis } 
NGFI-B\/nur77 is a member of the steroid receptor superfamily . 
NGFI-B\/nur77 and its related genes constitute a family and the NGFI-B\/nur77 family consists of three subtypes , named nur77 alpha , nur77 beta , nur77 gamma . 
We cloned human nur77 beta cDNA , called TINUR . 
Although NGFI-B\/nur77 is essential for TCR-mediated apoptosis in T-cell hybridomas , the reports on nur77 knock-out mice and nur77 dominant negative transgenic mice suggest that there is a functional redundancy among NGFI-B\/nur77 family . 
NGFI-B\/nur77 binds to the response element by monomer or heterodimer with retinoid X receptor ( RXR ) . 
Assuming that 9-cis-retinoic acid ( 9-cis-RA ) inhibits TCR-mediated apoptosis , nur77 may cause apoptosis by monomer in the absence of 9-cis-RA and may inhibit apoptosis by heterodimer with RXR in the presence of 9-cis-RA . 
Interaction of the human T-cell lymphotropic virus type 1 tax transactivator with transcription factor IIA . 
The Tax protein of human T-cell lymphotropic virus type 1 ( HTLV-1 ) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation . 
Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein , NF-kappaB , and serum response factor . 
It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery . 
On the basis of several independent assays , we now report a physical and functional interaction between Tax and the transcription factor , TFIIA . 
First , Tax was found to interact with the 35-kDa ( alpha ) subunit of TFIIA in the yeast two-hybrid interaction system . 
Importantly , two previously characterized mutants with point mutations in Tax , M32 ( Y196A , K197S ) and M41 ( H287A , P288S ) , which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay . 
Second , a glutathione-S-transferase ( GST ) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control . 
Third , a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes , Tax and TFIIA were associated . 
Finally , TFIIA facilitates Tax transactivation in vitro and in vivo . 
In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA . 
In addition , transfection of human T lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct . 
Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation . 
Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction . 
Vaccination with synthetic peptides representing cytotoxic T lymphocyte ( CTL ) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses . 
We now report that vaccination with a CTL epitope derived from the human adenovirus type 5 E1A-region ( Ad5E1A234-243 ) , which can serve as a target for tumor-eradicating CTL , enhances rather than inhibits the growth of Ad5E1A-expressing tumors . 
This adverse effect of peptide vaccination was rapidly evoked , required low doses of peptide ( 10 micrograms ) , and was achieved by a mode of peptide delivery that induces protective T-cell-mediated immunity in other models . 
Ad5E1A-specific CTL activity could no longer be isolated from mice after injection of Ad5E1A-peptide , indicating that tolerization of Ad5E1A-specific CTL activity causes the enhanced tumor outgrowth . 
In contrast to peptide vaccination , immunization with adenovirus , expressing Ad5E1A , induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A-expressing tumors . 
These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses . 
These findings are important for the design of safe peptide-based vaccines against tumors , allogeneic organ transplants , and T-cell-mediated autoimmune diseases . 
Defective transcription of the IL-2 gene is associated with impaired expression of c-Fos , FosB , and JunB in anergic T helper 1 cells . 
Anergic CD4+ Th cells do not produce IL-2 when challenged with Ag-pulsed accessory cells because of a transcriptional defect . 
In this work , we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical IL-2 DNA enhancer elements : NF-AT ( nuclear factor of activated T cells ; a sequence that binds a heterotrimeric NFATp , Fos , and Jun protein complex ) and Activator Protein-1 ( AP-1 ) ( that binds Fos and Jun heterodimers ) . 
Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible AP-1 family members c-Fos , FosB , and JunB . 
However , the reduced expression of these proteins was not the result of a global TCR\/CD3-signaling defect because CD3 cross-linking induced an equivalent increase in intracellular-free calcium ions , as well as NFATp dephosphorylation , translocation to the nucleus , and DNA binding in both normal and anergic T cells . 
Thus , defective IL-2 gene transcription appears to be due , at least in part , to a selective block in the expression of the AP-1 Fos and Jun family members in anergic T cells 
Elevated cyclic AMP inhibits NF-kappaB-mediated transcription in human monocytic cells and endothelial cells . 
The NF-kappaB\/Rel family of transcription factors regulates the inducible expression of many genes in activated human monocytes and endothelial cells . 
In this study , we examined the molecular mechanism by which agents that elevate intracellular cAMP inhibit the expression of the tumor necrosis factor alpha ( TNFalpha ) , tissue factor , endothelial leukocyte adhesion molecule-1 , and vascular cell adhesion molecule-1 genes . 
Both forskolin and dibutyryl cAMP , which elevate intracellular cAMP by independent mechanisms , inhibited TNFalpha and tissue factor expression at the level of transcription . 
Induction of NF-kappaB-dependent gene expression in transiently transfected human monocytic THP-1 cells and human umbilical vein endothelial cells was inhibited by elevated cAMP and by overexpression of the catalytic subunit of protein kinase A ( PKA ) . 
Elevated cAMP did not prevent nuclear translocation of p50\/p65 and c-Rel\/p65 heterodimers , decrease nuclear translocation of p65 , or significantly modify TNFalpha-induced phosphorylation of p65 . 
Functional studies demonstrated that transcriptional activation of a plasmid containing multimerized kappaB sites by p65 was inhibited by agents that elevate cAMP and by overexpression of the catalytic subunit of PKA . 
This study indicates that activation of PKA reduces the induction of a distinct set of genes in monocytes and endothelial cells by inhibiting NF-kappaB-mediated transcription . 
Activation of human monocytic cells by Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides proceeds via a pathway distinct from that of lipopolysaccharide but involves the transcriptional activator NF-kappa B . 
There is increasing evidence that lipoproteins of Treponema pallidum and Borrelia burgdorferi are key inflammatory mediators during syphilis and Lyme disease . 
A principal objective of the present study was to identify more precisely similarities and divergences among lipopolysaccharide ( LPS ) - and lipoprotein-lipopeptide-induced immune cell signaling events . 
Like LPS , purified native B. burgdorferi OspA and synthetic analogs of OspA , OspB , and two T. pallidum lipoproteins ( Tpp47 and Tpp17 ) all induced NF-kappa B translocation in THP-1 human monocytoid cells . 
Acylation of OspA and the synthetic peptides was requisite for cell activation . 
Polymyxin B abrogated only the response to LPS . 
By using 70Z\/3-derived pre-B-cell lines either lacking or expressing human CD14 ( the LPS receptor ) , it was observed that expression of human CD14 imparted responsiveness to LPS but not to OspA or spirochetal lipopeptides ( assessed by induction of NF-kappa B and expression of surface immunoglobulin M ) . 
Finally , the biological relevance of the observation that T. pallidum lipoproteins-lipopeptides induce both NF-kappa B and cytokine production in monocytes was supported by the ability of the synthetic analogs to promote human immunodeficiency virus replication in chronically infected U1 monocytoid cells ; these observations also suggest a potential mechanism whereby a syphilitic chancre can serve as a cofactor for human immunodeficiency virus transmission . 
The combined data lend additional support to the proposal that spirochetal lipoproteins and LPS initiate monocyte activation via different cell surface events but that the signaling pathways ultimately converge to produce qualitatively similar cellular responses . 
Chronic human immunodeficiency virus type 1 infection of myeloid cells disrupts the autoregulatory control of the NF-kappaB\/Rel pathway via enhanced IkappaBalpha degradation . 
Productive human immunodeficiency virus type 1 ( HIV-1 ) infection causes sustained NF-kappaB DNA-binding activity in chronically infected monocytic cells . 
A direct temporal correlation exists between HIV infection and the appearance of NF-kappaB DNA-binding activity in myelomonoblastic PLB-985 cells . 
To examine the molecular basis of constitutive NF-kappaB DNA-binding activity in HIV1 -infected cells , we analyzed the phosphorylation and turnover of IkappaBalpha protein , the activity of the double-stranded RNA-dependent protein kinase ( PKR ) and the intracellular levels of NF-kappaB subunits in the PLB-985 and U937 myeloid cell models . 
HIV-1 infection resulted in constitutive , low-level expression of type 1 interferon ( IFN ) at the mRNA level . 
Constitutive PKR activity was also detected in HIV-1-infected cells as a result of low-level IFN production , since the addition of anti-IFN-alpha\/beta antibody to the cells decreased PKR expression . 
Furthermore , the analysis of IkappaBalpha turnover demonstrated an increased degradation of IkappaBalpha in HIV-1-infected cells that may account for the constitutive DNA binding activity . 
A dramatic increase in the intracellular levels of NF-kappaB subunits c-Rel and NF-kappaB2 p100 and a moderate increase in NF-kappaB2 p52 and RelA-LRB-p65-RRB- were detected in HIV-1-infected cells , whereas NF-kappaB1 p105\/p50 levels were not altered relative to the levels in uninfected cells . 
We suggest that HIV-1 infection of myeloid cells induces IFN production and PKR activity , which in turn contribute to enhanced IkappaBalpha phosphorylation and subsequent degradation . 
Nuclear translocation of NF-kappaB subunits may ultimately increase the intracellular pool of NF-kappaB\/IkappaBalpha by an autoregulatory mechanism . 
Enhanced turnover of IkappaBalpha and the accumulation of NF-kappaB\/Rel proteins may contribute to the chronically activated state of HIV-1-infected cells . 
The role of early growth response gene 1 ( egr-1 ) in regulation of the immune response . 
The induction of immediate early genes in cells of the immune system is critical to determining the ultimate outcome of exposure to antigen . 
The importance of many of these genes relates to the role 0 their transcription factor products play in dictating patterns of expression of downstream , function-related genes . 
Evidence from several systems indicates that the immediate early gene , egr-1 may be of particular importance in the immune system . 
Recently , the egr-1 promoter has been shown to be highly responsive to the diverse biochemical signals generated by antigen and cytokines in cells of the immune system . 
Furthermore , an important role for egr-1 in determining the differentiation pathway of myeloid cell precursors has been recently elaborated . 
Finally , potential targets of regulation by the zinc-finger transcription factor encoded by egr-1 include the interleukin-2 , CD44 , ICAM-1 , and tumor necrosis factor genes . 
The role of egr-1 in regulation of the immune response will be discussed in the context of these recent studies . 
Activation of Stat 5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation . 
Self renewal of normal erythroid progenitors is induced by the receptor tyrosine kinase c-ErbB , whereas other receptors ( c-Kit\/Epo-R ) regulate erythroid differentiation . 
To address possible mechanisms that could explain this selective activity of c-ErbB , we analyzed the ability of these receptors to activate the different members of the Stat transcription factor family . 
Ligand activation of c-ErbB induced the tyrosine phosphorylation , DNA-binding , and reporter gene transcription of Stat 5b in erythroblasts . 
In contrast , ligand activation of c-Kit was unable to induce any of these effects in the same cells . 
Activation of the erythropoietin receptor caused specific DNA-binding of Stat 5b , but failed to induce reporter gene transcription . 
These biochemical findings correlate perfectly with the selective ability of c-ErbB to cause sustained self renewal in erythroid progenitors . 
Calcineurin mutants render T lymphocytes resistant to cyclosporin A . 
The immunosuppressants cyclosporin A ( CsA ) and FK506 have been widely used to prevent and treat graft rejection after human organ and tissue transplantations . 
CsA and FK506 associate with intracellular binding proteins ( i.e. , CsA with cyclophilin A and FK506 with FKBP12 ) to form protein\/drug complexes that suppress the immune system by preventing activation of T cells in response to antigen presentation . 
The common target of CsA and FK506 is calcineurin , a Ca2+\/calmodulin-regulated , serine\/threonine-specific protein phosphatase that regulates the nuclear import of a transcription factor , NF-AT , required for expression of T cell activation genes . 
In previous studies , we identified calcineurin mutations that block binding by the cyclophilin A\/CsA or FKBP12\/FK506 complexes and thereby render yeast cells resistant to the antifungal effects of CsA or FK506 . 
In this report , we demonstrate that the corresponding mutations in murine calcineurin render the T cell receptor signal transduction cascade CsA resistant in human Jurkat T cells . 
Our findings support the recently determined calcineurin X-ray crystal structure , provide evidence that calcineurin is the only CsA-sensitive component limiting signaling from the T cell receptor to the nucleus , and suggest a means 0 to render cells and tissues resistant to the toxic side effects of CsA and FK506 . 
Retinoid differentiation therapy in promyelocytic leukemia . 
Acute promyelocytic leukemia ( APL ) is a specific type of acute myeloid leukemia characterized by the morphology of the blast cells , a specific t-LRB-15;17-RRB- translocation , and risks of definite coagulopathy . 
Recently this leukemia was further characterized by an exquisite sensitivity to all-trans retinoic acid 's differentiation effect and the production of a fusion gene altering the gene of RARalpha and a novel gene PML . 
In vivo differentiation therapy with retinoids in APL patients follows strict guidelines related both to the APL cell and the biodisposal of all-trans retinoic acid . 
IL4 and IL13 receptors share the gamma c chain and activate STAT6 , STAT3 and STAT5 proteins in normal human B cells . 
IL13 induces the same biological effects as IL4 in normal human B cells . 
We show that as in the IL4R complex , both IL4R alpha and IL2R gamma c are components of the IL13R and that both cytokines induced STAT6 , STAT3 and STAT5 activation in B cells . 
In spite of this similar downstream signalling , IL4 and IL13 used a different set of Janus kinases : IL13 is unable to activate JAK1 and JAK3 . 
Attenuated function of a variant form of the helix-loop-helix protein , Id-3 , generated by an alternative splicing mechanism . 
The Id family of helix-loop-helix proteins function as negative regulators of DNA binding , basic helix-loop-helix proteins in the regulation of cell growth and differentiation . 
We report here on the identification of a 17 kDa variant of the 14 kDa Id-3 protein termed Id-3L ( long version ) which possesses a unique 60 amino acid carboxy-terminus generated by read through of a ' coding intron ' and alternative splicing . 
Northern analysis revealed expression of a minor 1.1 kb Id-3L transcript together with the predominant 0.95 kb Id-3 transcript in the majority of adult human tissues analysed . 
The variant Id-3L protein is functionally distinguishable from conventional Id-3 since in in vitro DNA mobility shift assays , it was greatly impaired in its ability to abrogate binding of the basic helix-loop-helix protein , E47 , to an E box recognition sequence . 
Characterization of the human myeloid cell nuclear differentiation antigen gene promoter . 
MNDA ( myeloid cell nuclear differentiation antigen ) is an interferon alpha regulated nuclear protein expressed only in cells of the human myelomonocytic lineage . 
To identify mechanisms responsible for this lineage-specific and interferon-regulated expression , the 5' flanking sequence of the gene has been characterized . 
Two interferon-stimulated response elements ( ISRE ) flank a multiple transcription start site region identifying MNDA as a TATA-less interferon-regulated gene . 
Other DNA elements present include a cluster of Myb sites , several Ets , an Ets related PU.1 site and an Sp1 site located within 600 bp of the transcription start sites . 
In addition , DNA methylation was revealed as one of the possible factors in establishing MNDA expression . 
The 5' flanking sequence has promoter activity which is elevated by interferon alpha . 
The findings indicate that MNDA expression is regulated by mechanisms similar to other myelomonocytic cell specific genes and genes up-regulated by interferon alpha . 
Regulation of GM-CSF gene transcription by core-binding factor . 
GM-CSF gene activation in T cells is known to involve the transcription factors nuclear factor-kappa B , AP-1 , NFAT , and Sp1 . 
Here we demonstrate that the human GM-CSF promoter and enhancer also encompass binding sites for core-binding factor ( CBF ) . 
Significantly , the CBF sites are in each case contained within the minimum essential core regions required for inducible activation of transcription . 
Furthermore , these core regions of the enhancer and promoter each encompass closely linked binding sites for CBF , AP-1 , and NFATp . 
The GM-CSF promoter CBF site TGTGGTCA is located 51 bp upstream of the transcription start site and also overlaps a YY-1 binding site . 
A 2-bp mutation within the CBF site resulted in a 2-3-fold decrease in the activities of both a 69-bp proximal promoter fragment and a 627-bp full-length promoter fragment . 
Stepwise deletions into the proximal promoter also revealed that the CBF site , but not the YY-1 site , was required for efficient induction of transcriptional activation . 
The AML1 and CBF beta genes that encode CBF each have the ability to influence cell growth and differentiation and have been implicated as proto-oncogenes in acute myeloid leukemia . 
This study adds GM-CSF to a growing list of cytokines and receptors that are regulated by CBF and which control the growth , differentiation , and activation of hemopoietic cells . 
The GM-CSF locus may represent one of several target genes that are dysregulated in acute myeloid leukemia . 
Tyloxapol inhibits NF-kappa B and cytokine release , scavenges HOCI , and reduces viscosity of cystic fibrosis sputum . 
Cystic fibrosis ( CF ) patients develop progressive cytokine-mediated inflammatory lung disease , with abundant production of thick , tenacious , protease- and oxidant-rich purulent airway secretions that are difficult 0 to clear even with physiotherapy . 
In the search for a potential treatment , we have tested tyloxapol , an alkylaryl polyether alcohol polymer detergent previously used as a mucolytic agent in adult chronic bronchitis . 
Tyloxapol inhibits activation of the transcription factor nuclear factor-kappa B ( NK-kappa B ) , reduces resting secretion of the cytokine interleukin-8 ( IL-8 ) in cultured human monocytes , and inhibits lipopolysaccharide ( LPS ) -stimulated release of tumor necrosis factor-alpha ( TNF-alpha ) , IL-1 beta , IL-6 , IL-8 , granulocyte-macrophage colony-stimulating factor ( GM-CSF ) , and the eiconsanoids thromboxane A2 and leukotriene B4 ( LTB4 ) . 
We have previously shown that tyloxapol is a potent antioxidant for hydroxyl radicals ( OH ) . 
Tyloxapol ( 0.05 to 0.1 % wt\/vol ) effectively scavenges the oxidant hypochlorous acid ( HOCl ; 1 to 7.5 mM ) in vitro , and protects from HOCl-mediated lung injury in rats . 
Tyloxapol also reduces the viscosity of CF sputum ( from 463 +\/- 133 to 128 +\/- 52 centipoise ) . 
We conclude that tyloxapol is potentially useful as a new antiinflammatory therapy for CF lung disease , and could possibly promote clearance of secretions in the CF airway . 
Severe combined immunodeficiency due to defective binding of the nuclear factor of activated T cells in T lymphocytes of two male siblings . 
Peripheral blood lymphocytes ( PBL ) and alloreactive T cell lines of two male infants born to consanguinous parents and presenting with severe combined immunodeficiency ( SCID ) showed a pronounced deficiency in T cell activation . 
Although phenotypically normal , the proliferative response of the childrens ' T cells was strongly reduced but could be improved by the addition of interleukin-2 ( IL-2 ) . 
Furthermore both childrens ' T cells were unable to produce the cytokines IL-2 , interferon-gamma ( IFN-gamma ) , IL-4 and tumor necrosis factor-alpha ( TNF-alpha ) . 
This multiple cytokine production deficiency could not be restored by IL-2 or co-stimulatory signals provided by antigen-presenting cells ( APC ) . 
Moreover , mRNA for IL-2 and IFN-gamma could not be detected . 
In contrast , expression of the activation-dependent cell surface markers CD25 and CD69 was within normal limits . 
To determine whether the functional defect of the patients ' T cells was due to the absence or abnormal binding of transcription factors involved in cytokine gene expression , electrophoretic mobility shift assays were used to examine the DNA binding of AP-1 , Oct , CREB , SP1 , NF-kappa B and the nuclear factor of activated T cells ( NF-AT ) to their respective response elements in the promoter of the IL-2 gene . 
Whereas AP-1 , NF-kappa B , Oct , CREB and SP1 displayed normal binding activities in nuclear extracts , the binding of NF-AT to its IL-2 promoter response element was barely detectable both before and after T cell stimulation . 
Our results strongly suggest that this NF-AT\/DNA binding defect is responsible for the multiple cytokine deficiency and the SCID phenotype observed in the two infant brothers . 
Requirements for induction of vitamin D-mediated gene regulation in normal human B lymphocytes . 
Mature human lymphocytes are unique targets of 1 alpha,25-dihydroxyvitamin D3 ( 1 alpha,25-LRB-OH-RRB-2D3 ) in that vitamin D receptors ( VDR ) are not constitutively expressed , and specific cellular activation signals are required for both the up-regulation of VDR and establishment of reactivity to the lipophilic ligand . 
Treatment of B lymphocytes with the cytokine IL-4 ( IL-4 ) , in the absence of prior activation , induces a weak up-regulation of VDR expression but fails to generate vitamin D-responsive element ( VDRE ) -reactive nuclear protein complexes or to initiate the genomic transcription of 25-hydroxyvitamin D3 24-hydroxylase . 
Stimulation of B lymphocytes by either ligation of CD40 Ag or cross-linking the Ig receptor is also insufficient to render B lymphocytes responsive to 1 alpha,25-LRB-OH-RRB-2D3 . 
However , this apparent lack of response to the secosterol can be overcome by stimulation of B lymphocytes with a combination of these cellular activation signals , which are sufficient to lead to G1 cell cycle progression . 
In the presence of 1 alpha,25-LRB-OH-RRB-2D3 , cellular activation associated with stimulation of such a progression appears to be sufficient for the up-regulation of VDR message and protein and necessary for the establishment of VDRE binding complexes and the induction of 24-hydroxylase message . 
Furthermore , biologic functions are modulated , in that the hormone inhibits proliferation in a subset of the activated B cells . 
These observations suggest that reactivity to 1 alpha,25-LRB-OH-RRB-2D3 is tightly regulated in B lymphocytes , requiring specific signals for its initiation . 
Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis . 
Three subtypes of retinoic acid receptors ( RAR ) , termed RAR alpha , RAR beta , and RAR gamma , have been described . 
They are composed of different structural domains , including distinct domains for DNA and ligand binding . 
RARs specifically bind all-trans-retinoic acid ( RA ) , 9-cis-RA , and retinoid analogs . 
In this study , we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha ( hRAR alpha-LBD , amino acids 154 to 462 ) . 
All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis , and the mutant proteins were characterized by blocking reactions , ligand-binding experiments , transactivation assays , and protease mapping . 
Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA . 
Interestingly , residue C-235 is specifically important in antagonist binding . 
With respect to arginine residues , only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect . 
For all other arginine mutations , no differences in affinity were detectable . 
The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding . 
From these results , we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding . 
In addition , antagonists show distinctly different requirements for efficient binding , which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha . 
Cloning and characterization of the beta subunit of human proximal sequence element-binding transcription factor and its involvement in transcription of small nuclear RNA genes by RNA polymerases II and III . 
The proximal sequence element ( PSE ) -binding transcription factor ( PTF ) , which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA ( snRNA ) genes , is essential for their transcription . 
We previously reported the purification of human PTF , a complex of four subunits , and the molecular cloning and characterization of PTF gamma and delta subunits . 
Here we describe the isolation and expression of a cDNA encoding PTF beta , as well as functional studies using anti-PTF beta antibodies . 
Native PTF beta , in either protein fractions or a PTF-Oct-1-DNA complex , can be recognized by polyclonal antibodies raised against recombinant PTF beta . 
Immunodepletion studies show that PTF beta is required for transcription of both classes of snRNA genes in vitro . 
In addition , immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein ( TBP ) and TFIIIB90 can weakly associate with PTF at low salt conditions , but this association is dramatically reduced at high salt concentrations . 
Along with our previous demonstration of both physical interactions between PTF gamma \/ PTF delta and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes , these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes , and acts through weak interaction with the four-subunit PTF . 
Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis . 
Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation . 
Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin . 
We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region . 
The positive region can be divided into an upstream and a downstream regulatory region . 
The downstream regulatory region contains a cyclic AMP-responsive element ( CRE ) . 
We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro . 
Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays . 
The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells . 
Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site . 
Treatment of the more immature B-cell line , Ramos , with phorbol esters rescues the cells from calcium-dependent apoptosis . 
bcl-2 expression is increased following phorbol ester treatment , and the increased expression is dependent on the CRE site . 
These stimuli result in phosphorylation of CREB at serine 133 . 
The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A . 
Although the CRE site is necessary , optimal induction of bcl-2 expression requires participation of the upstream regulatory element , suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element . 
The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis . 
It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types , particularly those in which protein kinase C is involved . 
JNK ( c-Jun NH2-terminal kinase ) is a target for antioxidants in T lymphocytes . 
AP-1 has been shown to behave as a redox-sensitive transcription factor that can be activated by both oxidant and antioxidant stimuli . 
However , the mechanisms involved in the activation of AP-1 by antioxidants are largely unknown . 
In this study we show that the structurally unrelated antioxidant agents pyrrolidine dithiocarbamate ( PDTC ) , butylated hydroxyanisole , and Nacetylcysteine activated JNK ( c-Jun NH2-terminal kinase ) in Jurkat T cells . 
This activation differed substantially from that mediated by phorbol 12-myristate 13-acetate ( PMA ) and Ca2+ ionophore or produced by costimulation with antibodies against the T cell receptor-CD3 complex and to CD28 . 
The activation of JNK by classical T cell stimuli was transient , whereas that mediated by PDTC and butylated hydroxyanisole ( but not N-acetylcysteine ) was sustained . 
The kinetics of JNK activation correlated with the expression of c-jun which was transient after stimulation with PMA plus ionophore and prolonged in response to PDTC , which also transiently induced c-fos . 
In addition , JNK activation by PMA plus ionophore was sensitive to inhibitors of signaling pathways involving Ca2+ , protein kinase C , and tyrosine phosphorylation , which failed to inhibit the activation mediated by PDTC . 
Transfection of trans-dominant negative expression vectors of ras and raf , together with AP-1-dependent reporter constructs , as well as Western blot analysis using anti-ERK ( extracellular signal-regulated kinase ) antibodies , indicated that the Ras\/Raf/ERK pathway did not appear to mediate the effect of the antioxidant . 
However , the combined treatment with PDTC and PMA , two agents that synergize on AP-1 activation , resulted in the persistent phosphorylation of ERK-2 . 
In conclusion , our results identify JNK as a target of antioxidant agents which can be regulated differentially under oxidant and antioxidant conditions . 
Cytomegalovirus modulates interleukin-6 gene expression . 
Complications after lung transplantation include the development of rejection and an increased incidence of infection , particularly with cytomegalovirus ( CMV ) . 
Several recent studies have suggested that interleukin ( IL ) -6 may be used to detect both infection and rejection after lung transplantation . 
In addition , IL-6 may play a role in the development of bronchiolitis obliterans after transplantation . 
Because CMV is also associated with the development of bronchiolitis obliterans after transplantation , we determined whether CMV induces IL-6 gene expression . 
We demonstrated that CMV infection increased both IL-6 protein and mRNA in peripheral blood mononuclear cells . 
We also demonstrated that the CMV immediate early 1 gene product increased expression of the IL-6 promoter . 
This effect of the CMV immediate early 1 gene product was dependent upon the presence of specific transcription factor binding sites in the IL-6 promoter . 
These studies demonstrate that CMV may be an important cofactor in the development of rejection and infection after transplantation through its effects on IL-6 . 
Interstitial deletion constitutes the major mechanism for loss of heterozygosity on chromosome 20q in polycythemia vera . 
An acquired deletion of the long arm of chromosome 20 is a recurrent abnormality in myeloproliferative disorders , particularly polycythemia vera and myelodysplastic syndromes . 
The association of 20q deletions with myeloid " stem cell " disorders suggests that the deletions mark the site of one or more genes , loss or inactivation of which plays a role in the regulation of normal hematopoietic progenitors . 
We have recently performed a detailed molecular analysis of 20q deletions in peripheral blood ( PB ) granulocytes and defined a commonly deleted region of 16 to 21 centimorgan ( cM ) . 
To further reduce the size of the common deleted region we have searched for small deletions or mitotic recombination events , neither of which would be detected by conventional cytogenetics . 
We have studied 48 patients with polycythemia vera and four patients with idiopathic myelofibrosis . 
In each case , cytogenetic analysis had either failed or had shown no abnormalities of chromosome 20 . 
Seventeen microsatellite markers that span the common deleted region were used to search for loss of heterozygosity in granulocyte DNA . 
No instance of microsatellite instability was observed in a total of 880 comparisons of granulocyte and T-cell DNA . 
Granulocyte DNA from four patients exhibited allele loss on 20q . 
In each case the allele loss was caused by an interstitial deletion because heterozygosity at distal markers was retained and because quantitative Southern blotting demonstrated hemizygosity . 
Loss of heterozygosity in PB granulocytes would be masked by the presence of significant numbers of normal granulocytes not derived from the malignant clone . 
Therefore , the human androgen receptor assay ( HUMARA ) was used to determine granulocyte clonality . 
In 21 of 27 informative female patients the majority of the granulocytes were clonally derived . 
In 5 patients the granulocytes appeared polyclonal and in 1 patient unilateral X inactivation was observed in both granulocytes and T cells . 
These results show that , in the vast majority of patients presented here , the failure to detect loss of heterozygosity can not be attributed to the presence of normal polyclonal granulocytes . 
Our results therefore show that allele loss on chromosome 20q in polycythemia vera does not commonly involve mitotic recombination or chromosome loss and that microsatellite instability is a rare event in this disorder . 
Transcriptional control of steroid-regulated apoptosis in murine thymoma cells . 
Early studies in murine T cell lines indicated that transcriptional transactivation functions encoded in the glucocorticoid receptor ( GR ) N-terminal domain are required for glucocorticoid-mediated apoptosis . 
However , more recent studies in human T cell lines have suggested that the N-terminal domain is not necessary for steroid-regulated apoptosis and that GR-mediated transrepression may be the more critical mechanism . 
To better understand the contribution of the GR N-terminal transactivation domain in mediating murine thymocyte apoptosis , we stably transfected GR , GR variants , and the androgen receptor ( AR ) into receptor-negative S49 murine thymoma cells . 
GR expression levels were shown to be rate-limiting for initiating the apoptotic pathway , and a positive correlation between steroid sensitivity and GR-mediated induction of an integrated mouse mammary tumor virus ( MMTV ) LTR reporter gene was observed . 
Analysis of GR chimeric receptors containing the potent VP16 and E1A viral transactivation domains in place of the GR N terminus revealed that even low level expression of these receptors resulted in both enhanced steroid sensitivity and MMTV induction , thus supporting a role for transactivation in apoptosis . 
In contrast , we found that AR can initiate apoptosis in S49 cells after treatment with 5 alpha-dihydrotestosterone , despite its relative inability to induce high level expression of MMTV . 
To investigate this further , we examined the steroid-regulated expression of an endogenous thymocyte-specific gene called GIG18 . 
We found that GIG18 was rapidly induced to comparable levels by both AR and GR , demonstrating that AR can indeed function as a transcriptional activator in S49 cells and , moreover , that GIG18 induction may be a marker of early apoptotic events in steroid-treated cells . 
Taken together , these results support our conclusion that transcriptional transactivation is a necessary signaling component of S49 cell apoptosis , although an additional role for GR-mediated transrepression can not be excluded 
Suppression of c-jun by antisense oligonucleotides inhibits cell adhesion but not respiratory burst during phorbol ester-induced differentiation of U937 human monoblastic cells . 
We studied the role of the immediate early gene c-jun in cell proliferation and phorbol 12-myristate 13-acetate ( PMA ) -induced differentiation in U937 human monoblastic cells , using c-jun-specific antisense ( AS ) phosphorothioate oligonucleotides . 
In selecting the most specific and potent oligonucleotide sequence , we performed extensive analyses for the binding specificity between all candidates of c-jun AS oligonucleotides and the whole sequences in GenBank database , using a computer program . 
Among the 20 selected oligonucleotides , two potent 15-mer AS oligonucleotides ( C-JUN AS oligonucleotides ) exhibited significant inhibition of cell growth in a dose-dependent manner between 2 and 10 microM . 
Reverse transcription-PCR and Western blot analysis demonstrated that 10 microM of C-JUN AS oligonucleotides reduced c-jun expression at both the mRNA and protein levels . 
More importantly , C-JUN AS oligonucleotides showed distinct effects on two markers of PMA-induced differentiation ; the C-JUN AS oligonucleotides inhibited cell adhesion , whereas they did not affect another marker of differentiation , respiratory burst ( measured by nitro blue tetrazolium reduction assay ) . 
These results suggest a critical role of c-jun in both cell proliferation and PMA-induced cell adhesion but not in PMA-induced respiratory burst in U937 cells . 
Uneven X inactivation in a female monozygotic twin pair with Fabry disease and discordant expression of a novel mutation in the alpha-galactosidase A gene . 
We describe two female monozygotic ( MZ ) twins heterozygous for Fabry disease , an X linked disorder resulting from the deficient activity of alpha-galactosidase A . 
While one of the twins was clinically affected , the other was asymptomatic . 
Enzymatic assay of alpha-galactosidase in blood leucocytes , skin fibroblasts , Epstein-Barr virus transformed lymphoid cell lines , and hair follicles of the twins and their parents confirmed the heterozygous status of the twins and indicated that Fabry disease had occurred as a result of a de novo mutation . 
The son of the unaffected twin sister was shown to be hemizygous . 
Molecular analysis of the alpha-galactosidase A gene permitted the identification of an as yet undescribed point mutation at position 10182 of exon 5 which causes an Asp to Asn substitution at codon 231 . 
Single strand conformation polymorphism ( SSCP ) analysis again showed the heterozygous status of the twins and a normal pattern in their parents . 
The basis for the discordant expression of this d novo mutation in the twins was investigated by studying their X inactivation status . 
Analysis of the inactive X specific methylation at the androgen receptor gene showed unbalanced inactivation in the twins ' fibroblasts and in opposite directions . 
While the maternally derived X chromosome was preferentially active in the asymptomatic twin , the paternal X chromosome was active in the other , affected twin and was found in her hemizygotic nephew . 
These data suggest that the paternal X chromosome carries the de novo alpha-galactosidase A mutation and that uneven X inactivation is the underlying mechanism for disease expression in this novel female MZ twin pair . 
This is the first documented case of female twins discordant for Fabry disease . 
Signaling by IL-2 and related cytokines : JAKs , STATs , and relationship to immunodeficiency . 
Cytokines that bind to the interleukin-2 ( IL-2 ) receptor common gamma chain ( gamma c ) , including IL-2 , IL-4 , IL-7 , IL-9 , and IL-15 , are important for the growth and differentiation of T and B lymphocytes , natural killer cells , macrophages , and monoctyes . 
These cytokines have overlapping biological effects that in part result from the use of the shared receptor subunit gamma c . 
Recently it has become clear that these cytokines activate a number of important intracellular signaling molecules , including the Janus kinases JAK1 and JAK3 and members of the transcription factor family of signal transducers and activators of transcription ( STATs ) . 
The discovery of these signaling pathways has led to important new insights into their role in lymphocyte maturation , as it has emerged that mutations in the genes encoding both gamma c and JAK3 result in similar forms of severe combined immunodeficiency ( SCID ) . 
In this review we examine the structure and function of cytokine receptors and the signaling pathways involved in their regulation of gene expression . 
Furthermore , we discuss recent advances that have led to a better understanding of how cytokines elicit intracellular responses , as well as their role in normal lymphoid development . 
The suppression of T cell function and NF-LRB-kappa-RRB-B expression by serine protease inhibitors is blocked by N-acetylcysteine . 
Direct evidence that N-acetylcysteine ( NAC ) enhances the immune response of peripheral blood T cells at the level of NF-LRB-kappa-RRB-B is presented . 
In addition , NAC blocks the suppression of T cell mitogenesis and cytokine production by protease inhibitors such as N-tosylphenylalanine chloromethyl ketone ( TPCK ) . 
The proliferative responses of purified CD4+ or CD8+ T cells are suppressed more strongly by TPCK when anti-CD28 rather than the phorbol ester PMA is used as the mitogenic coactivator . 
Cytokine ( IL-2 , IL-6 , INF-gamma ) production is inhibited 95-100 % by concentrations of TPCK that totally suppress the mitogenesis of CD4+ or CD8+ cells . 
Using electrophoretic mobility shift assays , we find that TPCK virtually abolishes ( to less than 1 % ) the levels of NF-LRB-kappa-RRB-B ( but not Oct-1 ) found in nuclear and whole cell extracts of activated T cells . 
Strikingly , the immunosuppressive effects of TPCK are blocked when T cells are pretreated for 15 min with 5 mM NAC . 
NAC not only blocks the effect of TPCK but enhances mitogenesis and cytokine production ( &gt; 2.5-fold in some cases ) upon activation of unsuppressed T cells . 
Our data support the notion that NF ( kappa ) B and I ( kappa ) B proteases play obligate roles in T cell activation and mitogenesis , 
IL-12-induced activation of NK and T cells occurs in the absence of immediate-early activation gene expression . 
The responses of lymphocytes to IL-2 and IL-12 , involving proliferation , differentiation , and cytokine production , are only partially overlapping , and may depend on induced differential expression of specific sets of genes . 
Using reverse-transcription PCR differential display , we isolated an mRNA species expressed in IL-2- but not IL-12-stimulated NK cells . 
This was identified as the mRNA encoding the transcription factor egr-1 , which is expressed with fast kinetics in T and NK cells upon IL-2 , but not IL-12 , stimulation . 
Analysis of the accumulation of mRNA-encoding members of the AP-1 transcription factor family demonstrated that c-fos and junB are also expressed upon stimulation of NK and T cells with IL-2 , but not IL-12 , whereas expression of c-jun and junD is not modified by either cytokine . 
Accordingly , increased AP-1 DNA-binding activity and AP-1-dependent transcriptional activity were detected exclusively in IL-2-stimulated cells . 
Analysis of the expression of genes reported to regulate cytokine-induced proliferation demonstrated that both IL-2 and IL-12 induce c-myc mRNA accumulation in NK and T cells , whereas only IL-2 induces bcl-2 expression . 
Our data provide the first demonstration that IL-12-mediated activation of T and NK cells does not involve expression of members of the immediate-early activation genes family ( egr-1 , c-fos , and junB ) , AP-1 transcriptional activity , or bcl-2 expression . 
This indicates that functional differences observed in IL-2- and IL-12-stimulated cells may depend , at least in part , on differential gene regulation 
Soluble factors secreted by activated T-lymphocytes modulate the transcription of the immunosuppressive cytokine TGF-beta 2 in glial cells . 
Coordination of the immune response to injury or disease in the brain is postulated to involve bi-directional discourse between the immune system and the central nervous system . 
This cross communication involves soluble mediators , including various growth factors , cytokines , and neuropeptides . 
In this report , we demonstrate that the supernatant from activated T-lymphocytes is able to induce the transcription of a potent cytokine , TGF-beta 2 in glial cells . 
The activating stimulus invokes signaling mechanisms distinct from known kinase or protease pathways . 
Activation of TGF-beta 2 transcription correlates with the loss of binding activity for an 80 kDA glial labile repressor protein , GLRP , to a responsive region within the TFG-beta 2 promoter . 
Although GLRP shares some characteristics with the inducible transcription factor AP-1 , it appears to be distinct from known AP-1 family members . 
These data along with previous observations demonstrating the potent immunosuppressive activity of TGF-beta 2 , support a model for a feedback mechanism between the activated T-lymphocytes and astrocytes via TGF-beta 2 0 to regulate the immune response . 
Inorganic lead activates NF-kappa B in primary human CD4+ T lymphocytes . 
Inorganic lead ( Pb ) is a ubiquitous environmental contaminant that produces a variety of effects on humoral and cell mediated immune responses . 
The underlying molecular mechanism for Pb 's complex effects on the immune system remain obscure . 
Many of Pb 's effects on the immune system could be explained through activation of the transcription factor , NF-kappa B . 
NF-kappa B is critical for T lymphocyte function and is a strong inducer of HIV-LTR activation . 
We demonstrate that Pb at physiologically relevant concentrations activates NF-kappa B in primary human CD4+ T lymphocytes . 
Pb-induced activation of NF-kappa B is blocked by antibodies for p65 and p50 subunits but not cRel , indicating that the p65:p50 heterodimer ( NF-kappa B ) is involved . 
Functional activation of gene expression by Pb was confirmed using primary CD4+ T cells transfected with an NF-kappa B dependent reporter gene construct . 
Pb did not activate NF-kappa B in 4 different T cell lines , suggesting that lymphoid cell lines may not be reliable surrogates for the study of transcriptional activation in human T cells . 
These data suggest that NF-kappa B may be an important molecular mediator of Pb-induced immunotoxicity . 
Calcium-dependent immediate-early gene induction in lymphocytes is negatively regulated by p21Ha-ras . 
The induction of immediate-early ( IE ) response genes , such as egr-1 , c-fos , and c-jun , occurs rapidly after the activation of T lymphocytes . 
The process of activation involves calcium mobilization , activation of protein kinase C ( PKC ) , and phosphorylation of tyrosine kinases . 
p21-LRB-ras-RRB- , a guanine nucleotide binding factor , mediates T-cell signal transduction through PKC-dependent and PKC-independent pathways . 
The involvement of p21-LRB-ras-RRB- in the regulation of calcium-dependent signals has been suggested through analysis of its role in the activation of NF-AT . 
We have investigated the inductions of the IE genes in response to calcium signals in Jurkat cells ( in the presence of activated p21-LRB-ras-RRB- ) and their correlated consequences . 
The expression of activated p21-LRB-ras-RRB- negatively regulated the induction of IE genes by calcium ionophore . 
This inhibition of calcium-activated IE gene induction was reversed by treatment with cyclosporin A , suggesting the involvement of calcineurin in this regulation . 
A later result of inhibition of this activation pathway by p21-LRB-ras-RRB- was down-regulation of the activity of the transcription factor AP-1 and subsequent coordinate reductions in IL-2 gene expression and protein production . 
These results suggest that p2l-LRB-ras-RRB- is an essential mediator in generating not only positive but also negative modulatory mechanisms controlling the competence of T cells in response to inductive stimulations . 
CD14-mediated signal pathway of Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts . 
Lipopolysaccharide ( LPS ) induces expression of inflammatory cytokines in monocytes\/macrophages via CD14 , one of the LPS receptors , which is expressed predominantly in these cells . 
It has been demonstrated that Porphyromonas gingivalis LPS ( P-LPS ) also is able to induce inflammatory cytokines in human gingival fibroblasts . 
Therefore , it is important to determine whether CD14 is expressed in gingival fibroblasts and to define the P-LPS-mediated signal-transducing mechanism in the cells . 
In this study , we observed unexpectedly by immunohistochemical , Western blotting ( immunoblotting ) , and Northern ( RNA ) blotting assays that CD14 is expressed at high density in human gingival fibroblasts . 
P-LPS-induced expression of the monocyte chemoattractant protein 1 ( MCP-1 ) gene in the cells was inhibited markedly by treatment with anti-human CD14 antibody and was completely inhibited by herbimycin A , a potent inhibitor of tyrosine kinase . 
The inhibitor also dramatically inhibited monocyte chemotactic activity of and MCP-1 production by the cells . 
Furthermore , P-LPS-induced expression of the MCP-1 gene in the cells also was blocked by inhibitors of two transcription factors , i.e. , curcumin , an inhibitor of AP-1 , and pyrolidine dithiocarbamate , an inhibitor of NF-kappaB . 
Both inhibitors inhibited monocyte chemotactic activity in the culture supernatant of P-LPS-treated cells . 
Gel shift mobility assay showed stimulation of the AP-1 and NF-kappaB contents in P-LPS-treated cells . 
This study is the first 0 to demonstrate the expression of CD14 in human gingival fibroblasts and to show that the signal-transducing pathway of P-LPS in the cells is mediated by CD14 . 
The catalytic domain of pp56-LRB-lck-RRB- , but not its regulatory domain , is sufficient for inducing IL-2 production . 
The lymphoid src kinase pp56-LRB-lck-RRB- has been shown to be essential for the induction of different T lymphocyte responses , including CD4-mediated enhancement of Ag-induced T cell activation , early T cell differentiation , induction of IL-2 production , and cytotoxicity . 
It is assumed that pp56-LRB-lck-RRB- acts on these processes by phosphorylating substrates . 
However , it has been recently reported that the NH2 regulatory domain is sufficient to mediate CD4 accessory function . 
In this report we address the contribution of the regulatory and catalytic domains of pp56-LRB-lck-RRB- to another function of this enzyme independent of CD4 : TCR-induced IL-2 production . 
Two pp56-LRB-lck-RRB- mutants lacking either the entire catalytic domain or the entire NH2 regulatory domain were generated , and their abilities to trigger transactivation of the TCR-regulated nuclear factor of activated T cells ( NF-AT ) region of the IL-2 promoter were compared . 
Only the catalytic , but not the NH2 regulatory , domain of pp56-LRB-lck-RRB- was able to induce NF-AT region transactivation on its own and to cooperate with other intracellular signals to trigger this response . 
Moreover , the catalytic domain of pp56-LRB-lck-RRB- was able to induce IL-2 cytokine production to an extent similar to that of wild-type pp56-LRB-lck-RRB- . 
We conclude that different domains of the pp56-LRB-lck-RRB- molecule contribute to regulate distinct biologic functions . 
In fact , while the NH2 regulatory domain is sufficient to mediate CD4 accessory function , we show here that the catalytic domain of pp56-LRB-lck-RRB- is sufficient for induction of IL-2 production , mimicking TCR ligation . 
Isolation and characterization of murine fra-1 : induction mediated by CD40 and surface Ig is protein kinase C dependent . 
The murine fra-1 gene , encoding Fos-related Ag 1 , was isolated from a splenic cDNA library and sequenced . 
Murine fra-1 was highly homologous to rat and human fra-1 . 
Oligonucleotide primers based on the murine sequence were used to construct a quantitative reverse transcription-PCR assay for gene expression . 
B lymphocyte stimulation via both CD40 and surface Ig ( sIg ) receptors substantially induced fra-1 expression , and for both receptors , induction was protein kinase C ( PKC ) dependent . 
This contrasts with induction of c-fos by both CD40 and sIg , which is PKC independent and indicates that CD40 is capable of signaling through PKC or a closely related kinase . 
Induction of fra-1 following engagement of CD40 did not require protein synthesis , suggesting that the PKC-dependent linkage between CD40 and fra-1 is direct . 
CD40-mediated fra-1 induction did require tyrosine kinase activity . 
These results demonstrate that CD40 , like sIg , may employ PKC in producing select outcomes , that individual B cell receptors may signal downstream events via both PKC-dependent and PKC-independent pathways , and that multiple signal transduction pathways may be used to activate the expression of closely related genes . 
Comparative analysis identifies conserved tumor necrosis factor receptor-associated factor 3 binding sites in the human and simian Epstein-Barr virus oncogene LMP1 . 
Nonhuman primates are naturally infected with a B-lymphotropic herpesvirus closely related to Epstein-Barr virus ( EBV ) . 
These simian EBV share considerable genetic , biologic , and epidemiologic features with human EBV , including virus-induced tumorigenesis . 
However , latent , transformation-associated viral genes demonstrate marked sequence divergence among species despite the conserved functions . 
We have cloned the latent membrane protein 1 ( LMP1 ) homologs from the simian EBV naturally infecting baboons ( cercopithicine herpesvirus 12 , herpesvirus papio ) and rhesus monkeys ( cercopithicine herpesvirus 15 ) for a comparative study with the human EBV oncogene . 
The transmembrane domains are well conserved , but there is striking sequence divergence of the carboxy-terminal cytoplasmic domain essential for B-cell immortalization and interaction with the tumor necrosis factor receptor signaling pathway . 
Nevertheless , the simian EBV LMP1s retain most functions in common with EBV LMP1 , including the ability to induce NF--LRB-kappa-RRB-B activity in human cells , to bind the tumor necrosis factor-associated factor 3 ( TRAF3 ) in vitro , and to induce expression of tumor necrosis factor-responsive genes , such as ICAM1 , in human B lymphocytes . 
Multiple TRAF3 binding sites containing a PXQXT\/S core sequence can be identified in the simian EBV LMP1s by an in vitro binding assay . 
A PXQXT\/S-containing sequence is also present in the cytoplasmic domain of the Hodgkin 's disease marker , CD30 , and binds TRAF3 in vitro . 
The last 13 amino acids containing a PXQXT\/S sequence are highly conserved in human and simian EBV LMP1 but do not bind TRAF3 , suggesting a distinct role for this conserved region of LMP1 . 
The conserved TRAF3 binding sites in LMP1 and the CD30 Hodgkin 's disease marker provides further evidence that a TRAF3-mediated signal transduction pathway may be important in malignant transformation . 
Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression . 
Cells need to distinguish between transient Ca2+ signals that induce events such as muscle contraction , secretion , adhesion and synaptic transmission , and sustained Ca2+ signals that are involved in cell proliferation and differentiation . 
The latter class of events is blocked in lymphocytes by the immunosuppressive drugs cyclosporin A and FK506 , which inhibit calcineurin , a Ca2+-activated serine\/threonine phosphatase necessary for the nuclear import of NF-AT transcription factors . 
Here we report that sustained high concentrations of Ca2+ , but not transient pulses , are required to maintain NF-AT transcription factors in the nucleus , where they participate in Ca2+-dependent induction of genes required for lymphocyte activation and proliferation . 
Furthermore , overexpression and constitutive nuclear localization of NF-AT , but not Jun , Fos , NF-kappaB , Oct or Ets family members , renders the interleukin-2 enhancer in Jurkat T lymphocytes resistant to FK506 and cyclosporin A . 
Thus a primary effect of these immunosuppressive reagents is to control the subcellular localization of the NF-AT family of transcription factors . 
Interferons induce normal and aberrant retinoic-acid receptors type alpha in acute promyelocytic leukemia cells : potentiation of the induction of retinoid-dependent differentiation markers . 
Treatment of the acute promyelocytic ( APL ) cell line NB4 with interferon alpha ( IFN-LRB-alpha-RRB- ) , as well as IFN-LRB-beta-RRB- and gamma , results in an increased expression of the transcripts coding for retinoic-acid receptor type alpha ( RAR-LRB-alpha-RRB- ) and the leukemia-specific retinoic acid receptor PML-RAR . 
Transcriptional induction of the RAR-LRB-alpha-RRB- and PML-RAR mRNAs is rapid and it is parallelled by an increase in the corresponding proteins . 
Up-regulation of RAR-LRB-alpha-RRB- and PML-RAR gene expression by IFN-LRB-alpha-RRB- is accompanied by a strong potentiation in the induction of 2 retinoid-dependent granulocytic markers , i.e. , granulocyte-colony-stimulating factor receptor mRNA and leukocyte alkaline phosphatase . 
However , IFN-LRB-alpha-RRB- does not have any effects on the retinoid-dependent regulation of the myeloid surface markers CD11b and CD33 . 
The IFN-dependent increase in RAR-LRB-alpha-RRB- levels and the enhancing effect of the cytokine on retinoid-dependent granulocytic markers expression may be a characteristic of PML-RAR positive cells , since the phenomena are not observed in HL-60 promyelocytes . 
Interferons as well as retinoids inhibit the growth of NB4 cells , although the 2 classes of compounds do not significantly interact in terms of anti-proliferative activity . 
These results suggest the possible use of combinations between IFNs and retinoic acid in the cyto-differentiating treatment of APL patients . 
An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5 , Elf-1 , HMG-I-LRB-Y-RRB- and a GATA family protein . 
Expression of the human interleukin-2 ( IL-2 ) receptor alpha chain gene is potently upregulated by its own ligand , IL-2 . 
In this study , we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs , two putative Ets binding sites ( EBS ) , one of which overlaps the consensus GAS motif , and a GATA motif , which overlaps the non-consensus GAS motif . 
We demonstrate that although the individual components of this element do not respond to IL-2 , together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter . 
Multiple factors including Stat5 , Elf-1 , HMG-I-LRB-Y-RRB- and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element . 
An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter . 
Thus , IL-2-induced IL-2R alpha promoter activity requires a complex upstream element , which appears to contain binding sites for both positive and negative regulatory factors . 
Tyrosine kinase and cAMP-dependent protein kinase activities in CD40-activated human B lymphocytes . 
In vitro , human B lymphocytes undergo long-term proliferation when activated through CD40 , a protein expressed on their cell surface . 
The nature of CD40-dependent signals in proliferating fresh human Epstein-Barr virus-negative B lymphocytes is currently unknown . 
In this study , a CD40-dependent B cell culture system was used to examine the role of different signal transduction elements . 
Protein kinase C ( PKC ) depletion generated by a long-term phorbol 12 myristate 13-acetate treatment had weak effects on proliferation . 
Rather , tyrosine phosphorylation was shown to be directly involved in mediating CD40-dependent signals . 
The use of the protein tyrosine kinase ( PTK ) -specific inhibitor herbimycin A dramatically decreased cellular proliferation without altering the activity of the human immunodeficiency virus-1 long terminal repeat ( HIV-1 LTR ) , a promoter largely dependent on the binding of nuclear factor kappa B ( NF-kappa B ) . 
In contrast , the cAMP-dependent protein kinase specific inhibitor H-89 totally inhibited HIV-1 LTR activity at a concentration as low as 100 nM without affecting cellular proliferation . 
Electrophoretic mobility shift assay ( EMSA ) and supershift assay using an NF-kappa B binding sequence from the kappa light chain as a probe , revealed that both p65 ( RelA ) and c-Rel were present in CD40-stimulated B cells . 
While PKC depletion did not alter the NF-kappa B level , treatment of B lymphocytes with H-89 or herbimycin A provoked a decrease in the NF-kappa B level . 
These observations establish the importance of different signal transducing pathways leading to CD40 activation of B lymphocytes . 
Chromosome 1 aneusomy with 1p36 under-representation is related to histologic grade , DNA aneuploidy , high c-erb B-2 and loss of bcl-2 expression in ductal breast carcinoma . 
Chromosome 1 abnormalities with loss of 1p36 have been investigated in 95 breast-cancer samples by means of a dual-target fluorescence in-situ hybridization ( FISH ) technique using the pUC 1.77 and p1-79 probes , specific for the 1q12 and 1p36 regions , respectively . 
Abnormalities for one or both probes were detected in 83\/95 samples . 
Relative 1p36 under-representation was found in 79\/95 . 
The clinical relevance of these alterations was studied by comparing the FISH results with several parameters currently used in breast-cancer pathology . 
Distinct patterns of chromosome 1 abnormalities were found among the histologic types of breast carcinoma . 
Lobular or mucinous samples showed few or no alterations , whereas most ductal samples had high chromosome 1 polysomy with under-representation of 1p36 . 
In ductal carcinomas , chromosome 1 alterations increased with histologic grade , DNA aneuploidy , loss of bcl-2 and high c-erb B-2 expression . 
These associations were found to be statistically significant . 
No correlation between chromosome 1 alterations and nuclear grade , age , size , lymph-node involvement , hormonal receptor presence , proliferation activity or p53 protein expression was detected . 
These results indicate the utility of this FISH technique for a better definition of the biological characteristics of ductal carcinomas . 
Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF . 
Globin genes are subject to tissue-specific and developmental stage-specific regulation . 
A switch from human fetal ( gamma ) -to adult ( beta ) -globin expression occurs within erythroid precursor cells of the adult lineage . 
Previously we and others showed by targeted gene disruption that the zinc finger gene , erythroid Kruppel-like factor ( EKLF ) , is required for expression of the beta-globin gene in mice , presumably through interaction with a high-affinity binding site in the proximal promoter . 
To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes ( +\/- ) with mice harboring a human beta-globin yeast artificial chromosome transgene . 
We find that in the absence of EKLF , while human beta-globin expression is dramatically reduced , gamma-globin transcripts are elevated approximately 5-fold . 
Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch . 
Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene . 
Modulatory effects of glucocorticoids and catecholamines on human interleukin-12 and interleukin-10 production : clinical implications . 
Interleukin-12 ( IL-12 ) is a key inducer of differentiation of uncommitted T helper ( TH ) cells toward the TH1 phenotype , which regulates cellular immunity , whereas IL-10 inhibits TH1 functions and potentiates TH2-regulated responses ( i.e. , humoral immunity ) . 
To examine the potential effects of stress on TH1\/TH2 balance , we studied the ability of three prototype stress hormones - dexamethasone ( a synthetic glucocorticoid ) and the catecholamines norepinephrine and epinephrine - to alter the production of IL-12 ( p70 ) and IL-10 induced by bacterial lipopolysaccharide ( LPS ) in human whole blood . 
Dexamethasone inhibited LPS-induced bioactive IL-12 production in a dose-dependent fashion and at physiologically relevant concentrations ; 
Lack of IL-12 signaling in human allergen-specific Th2 cells . 
IL-12 is a powerful skewer of CD4+ T cell responses toward the Th1 phenotype by inducing IFN-gamma production in naive Th cells . 
In the present study we addressed the question of whether IL-12 can reverse established Th2 responses into Th1\/Th0 responses by inducing IFN-gamma production in memory Th2 cells . 
To this aim , allergen-specific CD4+ T cell clones ( TCC ) were generated from the peripheral blood of three atopic patients , and their cytokine profiles were analyzed . 
The majority of these TCC exhibited a strongly polarized Th2 cytokine profile , and the production of IFN-gamma could not be induced by exogenous IL-12 . 
Only those TCC with low IFN-gamma levels in the absence of IL-12 responded to IL-12 by additional enhancement of IFN-gamma production . 
The IL-12 nonresponsiveness of the Th2 clones was further evident by the total lack of IL-12-induced phosphorylation of STAT4 ( signal transducer and activator of transcription-4 ) , a transcription factor that is typically involved in IL-12 signaling . 
Consequently , IL-12 also failed to induce the DNA-binding activity of STAT4-containing complexes in the nuclei of these Th2 clones . 
All TCC expressed equal levels of the low-affinity IL-12R beta1 subunit . 
Our results indicate that human allergen-specific Th cells with strongly polarized Th2 cytokine profiles do not respond to IL-12 and , therefore , can not be induced to produce IFN-gamma . 
The apparent high frequency of IL-12-nonresponsive Th cells within the allergen-specific populations in atopic patients predicts a limited skewing potential of IL-12 in the case of established Th2 responses , but only affecting newly recruited naive Th cells . 
Characterization of a CD43\/leukosialin-mediated pathway for inducing apoptosis in human T-lymphoblastoid cells . 
The monoclonal antibody ( mAb ) J393 induces apoptosis in Jurkat T-cells . 
NH2-terminal amino acid sequence analysis identified the 140-kDa surface antigen for mAb J393 as CD43\/leukosialin , the major sialoglycoprotein of leukocytes . 
While Jurkat cells co-expressed two discrete cell-surface isoforms of CD43 , recognized by mAb J393 and mAb G10-2 , respectively , only J393\/CD43 signaled apoptosis . 
J393\/CD43 was found to be hyposialylated , bearing predominantly O-linked monosaccharide glycans , whereas G10-2\/CD43 bore complex sialylated tetra- and hexasaccharide chains . 
Treatment with soluble , bivalent mAb J393 killed 25-50 % of the cell population , while concomitant engagement of either the CD3.TcR complex or the integrins CD18 and CD29 significantly potentiated this effect . 
Treatment of Jurkat cells with mAb J393 induced tyrosine phosphorylation of specific protein substrates that underwent hyperphosphorylation upon antigen receptor costimulation . 
Tyrosine kinase inhibition by herbimycin A diminished J393\/CD43-mediated apoptosis , whereas inhibition of phosphotyrosine phosphatase activity by bis-LRB-maltolato-RRB-oxovanadium-IV enhanced cell death . 
Signal transduction through tyrosine kinase activation may lead to altered gene expression , as J393\/CD43 ligation prompted decreases in the nuclear localization of the transcriptional regulatory protein NF-kappaB and proteins binding the interferon-inducible regulatory element . 
Since peripheral blood T-lymphocytes express cryptic epitopes for mAb J393 , these findings demonstrate the existence of a tightly regulated CD43-mediated pathway for inducing apoptosis in human T-cell lineages . 
Identification and characterization of a leukocyte-specific component of the nuclear body . 
The nuclear body ( NB ) is a cellular organelle that is involved in the pathogenesis of acute promyelocytic leukemia and viral infection . 
The NB is also a target of antibodies in the serum of patients with the autoimmune disease primary biliary cirrhosis . 
In this study , serum from a patient with primary biliary cirrhosis was used to identify a cDNA encoding a novel component of the NB , a 140-kDa protein designated Sp140 . 
The predicted amino acid sequence of the amino-terminal portion of Sp140 was similar to Sp100 , a previously identified NB protein . 
The carboxyl portion of Sp140 contained a zinc-finger domain and a bromodomain , motifs that are present in proteins regulating gene transcription . 
High levels of Sp140 mRNA were detected in human spleen and peripheral blood leukocytes , but not other human tissues . 
The level of SP140 mRNA in myeloid precursor cell lines HL60 and NB4 markedly increased in response to chemically induced cellular differentiation . 
Immunohistochemical techniques were used to demonstrate that SP140 localized to the NB in differentiated HL60 and NB4 cells . 
The location of Sp140 in the NB , and expression of this gene in cells involved in host defense , suggest that Sp140 may be involved in the pathogenesis of acute promyelocytic leukemia and viral infection . 
T-cell-directed TAL-1 expression induces T-cell malignancies in transgenic mice . 
The TAL-1 gene specifies for a basic domain-helix-loop-helix protein , which is involved in the control of normal hematopoiesis . 
In human pathology , the TAL-1 gene product is expressed in a high percentage of T-cell acute lymphoblastic leukemias in the pediatric age range ; however , it has not been established whether the expression has a causal role in oncogenesis . 
In this report , we describe the phenotype of mouse transgenic lines obtained by inducing tal-1 protein expression in lymphoid tissues using the LCK promoter . 
The survival rate of tal-1 transgenic animals was much lower as compared with control mice . 
Histopathological analysis revealed lymphomas of T-cell type , often comprising a minor B-cell component . 
Some mice showed marked splenic lymphocyte depletion . 
Primary lymphocyte cultures showed partial independence from exogenous growth stimuli and increased resistance to low-serum apoptosis . 
To further unravel the tal-1 oncogenic potential , a strain of tal-1 transgenic mice was crossbred with p53-\/- mice ; the survival rate in these animals was reduced by more than one-half when compared with that of tal-1 mice , and histopathological analysis revealed exclusively T-cell lymphomas . 
These data indicate that TAL-1 , expressed in T cells , is per se a potent oncogene , which may exert a key leukemogenetic role in the majority of T-cell acute lymphoblastic leukemias . 
Naive ( CD45RA+ ) T lymphocytes are more sensitive to oxidative stress-induced signals than memory ( CD45RO+ ) cells . 
Formation of reactive oxygen intermediates ( ROI ) after oxidative stress has been shown to be an activation signal for T lymphocytes , e.g. , expression of IL-2 and its receptor are induced . 
These ROI-induced effects can , to a large extent , be attributed to the activation of the transcription factor NF-kappaB . 
Now we have examined whether naive and memory T lymphocytes differ in their sensitivity to ROI-mediated signals . 
When CD45RA+ ( naive ) and CD45RO+ ( memory ) T lymphocytes were directly stimulated with H2O2 , NF-kappaB nuclear translocation was stronger in naive cells than in memory cells and it could be induced with lower doses . 
The composition of the induced nuclear NF-kappaB ( levels of p50 and RelA proteins ) was similar in these cell types . 
The magnitude and kinetics of intracellular ROI were similar , suggesting that there were no differences in ROI-forming mechanisms or antioxidative capacities . 
The probable regulatory point was the cytoplasmic IkappaB inhibitor : in CD45RA+ cells , H2O2 caused a more profound depression in the levels of IkappaB alpha . 
These findings indicate that T cells representing different activation and\/or differentiation stages can be differentially responsive to ROI-mediated signals . 
Interferon augments PML and PML\/RAR alpha expression in normal myeloid and acute promyelocytic cells and cooperates with all-trans retinoic acid to induce maturation of a retinoid-resistant promyelocytic cell line . 
The PML gene is fused to the retinoic acid receptor alpha gene ( RAR alpha ) in the acute promyelocytic leukemia ( APL ) 15 ; 17 translocation . 
PML is expressed in diverse tissues and cell lines and localized in the nucleus with a typical speckled pattern . 
In the bone marrow , it is preferentially expressed in myeloid cells . 
PML appears to be transcriptionally regulated by class I and II interferons , which raises the possibility that interferons modulate the function and growth and differentiation potential of normal myeloid cells and precursors by activating PML-dependent pathways . 
Similarly , interferons could act on APL cells , alone or in combination with all-trans retinoic acid ( RA ) , especially if the PML\/RAR alpha fusion transcript that results from the t-LRB-15;17-RRB- is induced by interferon . 
We report here that PML is expressed at low levels or not expressed in normal circulating human monocytes , lymphocytes , and polymorphonucleate cells , but is markedly induced by interferon ; that PML and PML\/RAR alpha expression is augmented by interferon in the NB4 APL cell line , which carries the t-LRB-15;17-RRB- , and in APL blasts from patients ; that interferon inhibits growth and survival of NB4 APL cells in cooperation with RA ; that interferons alone have minimal maturation effect on NB4 cells ; and , finally , that interferon gamma , but not alpha or beta , induces maturation and growth suppression of NB4 cells with de novo retinoid resistance , and partially restores RA response . 
Generation of CD1+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes . 
We previously showed that granulocyte-macrophage colony-stimulating factor ( GM-CSF ) and macrophage colony-stimulating factor ( M-CSF ) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages . 
However , in vivo , not only CSF but also many other cytokines are produced under various conditions . 
Those cytokines may modulate the differentiation of monocytes by CSFs . 
In the present study , we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells ( DC ) by the combination of GM-CSF plus interleukin-4 ( IL-4 ) and that they differentiate into tartrate-resistant acid phosphatase ( TRAP ) -positive osteoclast-like multinucleated giant cells ( MGC ) by the combination of M-CSF plus IL-4 . 
However , the monocyte-derived DC were not terminally differentiated cells ; they could still convert to macrophages in response to M-CSF . 
Tumor necrosis factor-alpha ( TNF-alpha ) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor , cfms mRNA , and aborting the potential to convert to macrophages . 
In contrast to IL-4 , interferon-gamma ( IFN-gamma ) had no demonstrable effect on the differentiation of monocytes . 
Rather , IFN-gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4 , respectively . 
Taken together , these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC . 
Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation . 
Differential nuclear localization of p50 , p52 , and RelB proteins in human accessory cells of the immune response in situ . 
The Rel\/NF-kappa B proteins , p50 , p52 , p65 , c-Rel , and RelB , constitute a family of transcription factors involved in the positive regulation of a variety of genes during the immune response . 
Recently , it has been shown that RelB knockout mice have no dendritic cells ( DC ) . 
An overexpression of p50 has been described in follicular dendritic cells ( FDC ) . 
A constitutive NF-kappa B activity has been reported in mature macrophages . 
This led to the hypothesis that some of the Rel\/NF-kappa B proteins were key nuclear factors in functions of accessory cells of the immune response . 
Therefore , we investigated in situ the nuclear localization of Rel\/NF-kappa B proteins in accessory cells of the immune system by immunohistochemistry and double labeling by immunofluorescence from five normal human tonsils and five lymph nodes with follicular hyperplasia . 
Nuclear p65 and c-Rel proteins were found in all cell types including lymphocytes . 
In germinal centers GC , p50 , p52 , and RelB were found in the nuclei of FDC only and were not detected in the nuclei of CD68+ cells . 
In T cell areas , p50 , p52 , and RelB were found in the nuclei of HLA-DR+ cells with an antigen-presenting cell ( APC ) morphology . 
p52 and RelB were detected in the nuclei in both CD1a+ and CD68+ cells from the T cell area , whereas p50 was found only in CD68- and CD1a- cells . 
Cells with nuclear p50 were negative for the CD38 , CD20 and CD2 markers . 
These results show that , physiologically , high levels of nuclear of p50 , p52 and RelB are restricted to accessory cells of the immune system , which include FDC in GC , and DC and macrophages in the T cell zone , that specialized scavenger macrophages from GC do not have detectable levels of p52 and RelB , whereas macrophages from the T cell area , known to present the antigen to T cells , do have both nuclear p52 and RelB , and that in the T cell zone , p52 and RelB are located in nuclei of both CD1a+ , CD68+ or both , cells APC , whereas p50 is restricted to CD1a- and CD68- APC . 
The different patterns of p50 , p52 and RelB protein nuclear localization may provide insight into their different roles during the immune response in vivo . 
Signal transduction by DR3 , a death domain-containing receptor related to TNFR-1 and CD95 . 
Tumor necrosis factor receptor-1 ( TNFR-1 ) and CD95 ( also called Fas or APO-1 ) are cytokine receptors that engage the apoptosis pathway through a region of intracellular homology , designated the " death domain . " 
Another death domain-containing member of the TNFR family , death receptor 3 ( DR3 ) , was identified and was shown to induce both apoptosis and activation of nuclear factor kappaB . 
Expression of DR3 appears to be restricted to tissues enriched in lymphocytes . 
DR3 signal transduction is mediated by a complex of intracellular signaling molecules including TRADD , TRAF2 , FADD , and FLICE . 
Thus , DR3 likely plays a role in regulating lymphocyte homeostasis . 
Cloning and expression of the Epstein-Barr virus-encoded dUTPase : patients with acute , reactivated or chronic virus infection develop antibodies against the enzyme . 
The gene encoding the Epstein-Barr virus ( EBV ) -specific dUTPase was amplified from virus DNA by PCR . 
The active enzyme was expressed in Escherichia coli and in insect cells as a non-fusion protein . 
The protein from E. coli specifically converted dUTP to dUMP and did not react with other dNTPs or NTPs . 
Preliminary experiments yielded a Km value of about 0.8 microM for dUTP . 
MAbs against the dUTPase reacted with a protein of approximately 31 kDa in 12-O-tetradecanoyl-phorbol-13-acetate ( TPA ) -stimulated B cells harbouring either type 1 or type 2 EBV . 
The protein was found in untreated cells at low levels , whereas induction of the lytic replication cycle by TPA treatment or by providing the immediate early transactivator BZLF1 in trans resulted in increased expression . 
We demonstrated that the virus dUTPase isolated from EBV-infected cells is a phosphoprotein . 
The protein expressed in insect cells was used to test for the presence of specific antibodies in sera from normal , healthy carriers and from patients with various diseases . 
While the sera of EBV-negative individuals ( 0\/3 ) or healthy carriers ( 0\/33 ) did not contain detectable levels of antibodies , patients with mononucleosis ( 5\/18 ) , chronic EBV infection ( 2\/7 ) , EBV reactivation ( 7\/20 ) and human immunodeficiency virus infection ( 5\/24 ) showed elevated antibody titres against the enzyme . 
This indicated that the dUTPase is expressed during EBV replication and reactivation . 
The enzyme might therefore be a potential target for drug therapy under conditions of active DNA replication . 
Regulation of cytokine and cytokine receptor expression by glucocorticoids . 
Glucocorticoids ( GCS ) profoundly inhibit several aspects of T cell immunity largely through inhibition of cytokine expression at the transcriptional and posttranscriptional levels . 
GCS were also reported to act indirectly by inducing transforming growth factor-beta expression , which in turn blocks T cell immunity . 
In exerting their antiproliferative effects , GCS diffuse into target cells where they bind their cytoplasmic receptor , which in turn translocates to the nucleus where it inhibits transcription of cytokine genes through direct binding to the glucocorticoid response elements ( GRE ) , which are located in the promoter region of cytokine genes or , alternatively , through antagonism of the action of transcription factors required for optimal transcriptional activation . 
In contrast to their inhibitory effects on cytokine expression , GCS up-regulate cytokine receptor expression that correlates with enhanced cytokine effects on target cells . 
In this review , we summarize the current state of knowledge of the mechanism of action of GCS , including the phenomenon of steroid-induced rebound , which ensues upon GCS withdrawal . 
The Oct-2 transcription factor . 
The Oct-2 transcription factor is a member of the POU ( Pit-Oct-Unc ) family of transcription factors and is expressed only in B lymphocytes and in neuronal cells but not in other cell types . 
The primary RNA transcript of the gene is subject to alternative splicing to yield different variants which can either activate or repress gene expression . 
The forms produced in B lymphocytes have a predominantly activating effect on gene expression whereas those produced in neuronal cells have a predominantly inhibitory effect and can repress the expression of both the herpes simplex virus immediate-early genes and the cellular tyrosine hydroxylase gene . 
Thus Oct-2 plays an important role in the regulation of cellular gene expression in both B cells and neuronal cells as well as in the control of viral latency . 
Cell specific expression of human Bruton 's agammaglobulinemia tyrosine kinase gene ( Btk ) is regulated by Sp1- and Spi-1\/PU.1-family members . 
Bruton 's agammaglobulinemia tyrosine kinase ( Btk ) is a cytoplasmic tyrosine kinase involved in the human disease X-linked agammaglobulinemia ( XLA ) . 
The gene is expressed in all hematopoietic cells with the exception of T-cells and plasma cells . 
For this expression pattern the first 280 bp upstream of the major transcriptional start site seems to be sufficient . 
In vitro footprinting analysis within this part of the promoter revealed two Sp1 binding sites as well as a PU-box . 
The transcription factor Spi-1\/PU.1 as well as the closely related factor Spi-B bound to the PU-box in B-cells . 
In the erythroleukemia cell line K562 , due to the absence of Spi-B , only PU.1 bound to the Btk promoter . 
Mutation of either site reduced the expression in transient transfection experiments . 
However , mutation of the PU box had no effect in the T-cell line Jurkat , where none of the Spi-1 family members is expressed . 
In addition Spi-B as well as PU.1 were able to transactivate Btk expression . 
In fetal liver of PU.1-\/- mice , which lack lymphoid and myeloid cells , expression of Btk was reduced two- to threefold but not abolished . 
Collectively this study shows that expression of the Btk gene is regulated by the combined action of Sp1- and PU.1-family members . 
Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene { published erratum appears in Mol Cell Biol 1997 Apr ; 17 ( 4 ) : 2351 } 
The interleukin 2 receptor alpha-chain ( IL-2R alpha ) gene is a key regulator of lymphocyte proliferation . 
IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli . 
Interleukin 2 ( IL-2 ) stimulates IL-2R alpha. transcription , thereby amplifying expression of its own high-affinity receptor . 
IL-2R alpha transcription is at least in part controlled by two positive regulatory regions , PRRI and PRRII . 
PRRI is an inducible proximal enhancer , located between nucleotides -276 and -244 , which contains NF-kappaB and SRE\/CArG motifs . 
PRRII is a T-cell-specific enhancer , located between nucleotides -137 and -64 , which binds the T-cell-specific Ets protein Elf-1 and HMG-I-LRB-Y-RRB- proteins . 
However , none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2 . 
To find new regulatory regions of the IL-2R alpha gene , 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced . 
We identified an 86-nucleotide fragment that is 90 % identical to the recently characterized murine IL-2-responsive element ( mIL-2rE ) . 
This putative human IL-2rE , designated PRRIII , confers IL-2 responsiveness on a heterologous promoter . 
PRRIII contains a Stat protein binding site that overlaps with an EBS motif ( GASd\/EBSd ) . 
These are essential for IL-2 inducibility of PRRIII\/CAT reporter constructs . 
IL-2 induced the binding of Stat5a and b proteins to the human GASd element . 
To confirm the physiological relevance of these findings , we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element . 
Our data demonstrate a major role of the GASd\/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor . 
Association of TRAF1 , TRAF2 , and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation : role in NF-kappaB activation . 
The Epstein-Barr virus ( EBV ) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor ( TNFR ) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus ( CT ) with TNFR-associated factors ( TRAFs ) . 
We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5 % of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3 . 
TRAF1 , TRAF2 , and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids ( aa ) 199 to 214 , within a domain which is important for B-lymphocyte growth transformation ( aa 187 to 231 ) . 
Further deletional and alanine mutagenesis analyses and comparison with TRAF binding sequences in CD40 , in CD30 , and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT\/S is a core TRAF binding motif . 
The negative effects of point mutations in the LMP1-LRB-1-231-RRB- core TRAF binding motif on TRAF binding and NF-kappaB activation genetically link the TRAFs to LMP1-LRB-1-231-RRB--mediated NF-kappaB activation . 
NF-kappaB activation by LMP1-LRB-1-231-RRB- is likely to be mediated by TRAF1\/TRAF2 heteroaggregates since TRAF1 is unique among the TRAFs in coactivating NF-kappaB with LMP1-LRB-1-231-RRB- , a TRAF2 dominant-negative mutant can block LMP1-LRB-1-231-RRB--mediated NF-kappaB activation as well as TRAF1 coactivation , and 30 % of TRAF2 is associated with TRAF1 in EBV-transformed B cells . 
TRAF3 is a negative modulator of LMP1-LRB-1-231-RRB--mediated NF-kappaB activation . 
Surprisingly , TRAF1 , -2 , or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappaB activation . 
The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappaB activation and primary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling . 
Sterol dependent LDL-receptor gene transcription in lymphocytes from normal and CML patients . 
Sterol regulatory element ( SRE ) has been recognized to regulate various key genes coding for especially low density lipoprotein ( LDL ) -receptor , 3-hydroxy-3-methylglutaryl coenzyme A ( HMG-CoA ) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism . 
The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia ( CML ) . 
Consequently , the present study was addressed to understand this phenomenon and revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients . 
However , this factor appeared when the CML patients achieved complete haematological remission ( CHR ) through alpha-interferon therapy . 
Further , an inverse relationship was also observed between sterol modulated LDL-receptor gene transcription and the binding affinity of this 47 kDa factor to the SRE sequence . 
Based upon these results we propose that alpha-interferon through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of 47 kDa sterol regulatory element binding factor as well as LDL-receptor gene transcription in lymphocytes from CML patients . 
Effects of Ara-C on neutral sphingomyelinase and mitogen- and stress-activated protein kinases in T-lymphocyte cell lines . 
Neutral sphingomyelinase ( SMase ) can be activated by extracellular signals to produce ceramide , which may affect mitogen-activated protein kinase ( MAPK ) activities . 
Neutral SMase activity was assessed in membranes from Jurkat , a human T-cell line , and EL4 , a murine T-cell line . 
Ara-C activated SMase with 10 minutes in both Jurkat and EL4 cells , while phorbol ester ( PMA ) had no effect . 
PMA , but not Ara-C or ceramides , activated ERK MAPKS , in Jurkat and EL4 . 
PMA acted synergistically with ionomycin to activate JNK MAPKs in Jurkat and EL4 within 10 minutes . 
Ara-C activated JNKs only after prolonged incubation ( 90-120 minutes ) . 
Thus , ceramide is not a positive signal for ERK activation in T-cell lines . 
The effects of Ara-C on JNK activity may be mediated through secondary response pathways . 
Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines . 
In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines , including both EBV genome negative and EBV carrying cell lines . 
Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines . 
Thus , constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines . 
In contrast , Egr-1 expression is abrogated in group I Burkitt tumor cells , irrespective of the EBV genome carrying status . 
Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression . 
Several forms of EGR-1 protein are found within the different groups of cell lines , and the binding activity to DNA consensus sequences was investigated . 
Finally , time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte . 
The proximal regulatory element of the interferon-gamma promoter mediates selective expression in T cells . 
Interferon-gamma ( IFN-gamma ) is produced by natural killer cells and certain subsets of T cells , but the basis for its selective expression is unknown . 
Within the region between -108 and -40 base pairs of the IFN-gamma promoter are two conserved and essential regulatory elements , which confer activation-specific expression in T cells . 
This report describes studies indicating that the most proximal of these two regulatory elements is an important determinant of its restricted expression . 
The proximal element is a composite site that binds members of the CREB\/ATF , AP-1 , and octamer families of transcription factors . 
Jun is essential for activation-induced transcription and binds preferably as a heterodimer with ATF-2 . 
In contrast , CREB appears to dampen transcription from this element . 
The CpG dinucleotide in this element is selectively methylated in Th2 T cells and other cells that do not express IFN-gamma , and methylation markedly reduces transcription factor binding . 
As a target for DNA methylation and for binding of transcription factors that mediate or impede transcription , this element appears to play a central role in controlling IFN-gamma expression . 
Activation of nuclear factor-kappaB via T cell receptor requires a Raf kinase and Ca2+ influx . 
Functional synergy between Raf and calcineurin . 
Signals transduced via the TCR activate the transcription factor nuclear factor-kappaB ( NF-kappaB ) , which , in turn , is critical to the transcriptional induction of many genes important for the proliferation and expression of a differentiated phenotype . 
Treatment of T cells with the protein kinase C activator PMA in combination with Ca2+ ionophores mimics this process , and the two agents are often substituted for TCR stimulation , bypassing the TCR . 
Here we identify intracellular signaling components involved in activation of NF-kappaB following TCR stimulation . 
TCR signaling was triggered by treating Jurkat T cells with PHA or anti-CD3 Abs , and NF-kappaB activation was monitored by electrophoretic mobility shift assays and\/or by kappaB-dependent reporter assays . 
Contrary to the idea that protein kinase C is involved in TCR-mediated activation of NF-kappaB , high doses of staurosporine did not interfere with activation of NF-kappaB by PHA , while the same dose of staurosporine completely blocked activation by PMA . 
PHA-induced kappaB-dependent reporter activity was , however , effectively blocked by a dominant negative form of Raf-1 , suggesting a critical role for a Raf kinase . 
The TCR-mediated activation of NF-kappaB was also dependent on a Ca2+ influx , because the Ca2+ channel blocker , SK&amp;F 96365 , as well as other agents that prevented the Ca2+ influx , inhibited NF-kappaB activation . 
Cotransfection of a constitutively active form of calcineurin largely substituted for the Ca2+ requirement and reversed the blockade by SK&amp;F 96365 . 
Consistent with these observations , coexpression of constitutively active forms of Raf-1 and calcineurin synergistically induced kappaB-dependent reporter activity , suggesting a physiologically relevant functional interaction between the kinase and the phosphatase . 
HIV-1 LTR activity in human CD40-activated B lymphocytes is dependent on NF-kappaB . 
CD40-stimulated human B lymphocytes are highly permissive to a productive infection by the human immunodeficiency virus type 1 . 
In these cells , nuclear factors involved in activation of the HIV-1 LTR , which contains the transcriptional control elements of the virus , are unknown . 
Transient expression assays with plasmids containing deleted parts of the LTR region linked to a reporter gene showed that the NF-kappaB binding site was essential for HIV-1 LTR activity in CD40-stimulated B lymphocytes . 
In addition , electrophoretic mobility shift and supershift assays revealed that important NF-kappaB binding activity composed of at least p50 , p65 , and c-Rel NF-kappaB subunits was present in nuclei of CD40-stimulated B cells . 
These results confirm at a molecular level the ability of HIV-1 to replicate in B cells and that this activity is strongly associated with NF-kappaB . 
Mutation of tyrosines 492\/493 in the kinase domain of ZAP-70 affects multiple T-cell receptor signaling pathways . 
The protein-tyrosine kinase ZAP-70 is implicated , together with the Src kinase p56-LRB-lck-RRB- , in controlling the early steps of the T-cell antigen receptor ( TCR ) signaling cascade . 
To help elucidate further the mechanism by which ZAP-70 regulates these initial events , we used a dominant-negative mutant approach . 
We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain . 
This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases . 
Moreover , TCR-induced phosphorylation of pp36-38 , thought to play a role upstream of these pathways , was found to be reduced . 
In contrast , overexpression of wild-type ZAP-70 induced constitutive activation of nuclear factor of activated T cells . 
The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR zeta chain and was able to bind p56-LRB-lck-RRB- . 
This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56-LRB-lck-RRB- with ZAP-70 . 
Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56-LRB-lck-RRB- , which in turn phosphorylates Tyr-492 and\/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity . 
ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation . 
Lymphocytes from CML patients lack a 47 kDa factor having affinity for a genomic sterol regulatory sequence . 
Deranged cellular cholesterol homeostasis has been widely recognized in the initiation as well as progression of various types of cancers including chronic myeloid leukaemia ( CML ) . 
Since the human genomic sterol regulatory element ( SRE ) has been shown to regulate various key genes involved in this phenomenon , the present study revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects , as well as its absence in lymphocytes from untreated CML patients . 
However , this factor appeared when these CML patients achieved complete haematological remission ( CHR ) through alpha-interferon therapy . 
Furthermore , an inverse relationship was also observed between the LDL receptor gene expression at the transcriptional level and the binding affinity of this 47 kDa protein factor to the SRE sequence . 
Based upon these results we propose that this factor may have a role in pathophysiology of chronic myeloid leukaemia . 
Synergistic interactions between overlapping binding sites for the serum response factor and ELK-1 proteins mediate both basal enhancement and phorbol ester responsiveness of primate cytomegalovirus major immediate-early promoters in monocyte and T-lymphocyte cell types . 
Cytomegalovirus ( CMV ) infection is nonpermissive or persistent in many lymphoid and myeloid cell types but can be activated in differentiated macrophages . 
We have shown elsewhere that both the major immediate-early gene ( MIE ) and lytic cycle infectious progeny virus expression can be induced in otherwise nonpermissive monocyte-like U-937 cell cultures infected with either human CMV ( HCMV ) or simian CMV ( SCMV ) by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate ( TPA ) . 
Two multicopy basal enhancer motifs within the SCMV MIE enhancer , namely , 11 copies of the 16-bp cyclic AMP response element ( CRE ) and 3 copies of novel 17-bp serum response factor ( SRF ) binding sites referred to as the SNE ( SRF\/NFkappaB-like element ) , as well as four classical NFkappaB sites within the HCMV version , contribute to TPA responsiveness in transient assays in monocyte and T-cell types . 
The SCMV SNE sites contain potential overlapping core recognition binding motifs for SRF , Rel\/NFkappaB , ETS , and YY1 class transcription factors but fail to respond to either serum or tumor necrosis factor alpha . 
Therefore , to evaluate the mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE ( SRF\/ETS element ) motif found in the HCMV and chimpanzee CMV MIE enhancers , we have examined the functional responses and protein binding properties of multimerized wild-type and mutant elements added upstream to the SCMV MIE or simian virus 40 minimal promoter regions in the U-937 , K-562 , HL-60 , THP-1 , and Jurkat cell lines . 
Unlike classical NFkappaB sites , neither the SNE nor the SEE motif responded to phosphatase inhibition by okadaic acid . 
However , the TPA responsiveness of both CMV elements proved to involve synergistic interactions between the core SRF binding site ( CCATATATGG ) and the adjacent inverted ETS binding motifs ( TTCC ) , which correlated directly with formation of a bound tripartite complex containing both the cellular SRF and ELK-1 proteins . 
This protein complex was more abundant in U-937 , K-562 , and HeLa cell extracts than in Raji , HF , BALB\/c 3T3 , or HL-60 cells , but the binding activity was altered only twofold after TPA treatment . 
A 40-fold stimulation of chloramphenicol acetyltransferase activity mediated by four tandem repeats of the SNE could be induced within 2 h ( and up to 250-fold within 6 h ) after addition of TPA in DNA-transfected U-937 cells , indicating that the stimulation appeared likely to be a true protein kinase C-mediated signal transduction event rather than a differentiation response . 
Slight differences in the sequence of the core SRF binding site compared with that of the classical c-Fos promoter serum response element , together with differences in the spacing between the SRF and ETS motifs , appear to account for the inability of the SCMV SNEs to respond to serum induction . 
Acetylsalicylic acid and sodium salicylate inhibit LPS-induced NF-kappa B\/c-Rel nuclear translocation , and synthesis of tissue factor ( TF ) and tumor necrosis factor alfa ( TNF-alpha ) in human monocytes . 
We have investigated the effects of acetylsalicylic acid and sodium salicylate on the LPS-induced synthesis of the pro-coagulant protein tissue factor ( TF ) and the pro-inflammatory protein tumor necrosis factor-alpha ( TNF-alpha ) , as well as the prostaglandin PGE2 in human monocytes . 
Both drugs dose-dependently inhibited LPS-induced TF and TNF-alpha synthesis at the mRNA and the protein level , and reduced PGE2 production . 
As evidenced by electro mobility shift assay ( EMSA ) and the use of a NF-kappa B prototypic probe , these drugs probably exert their inhibitory effects by interference with the nuclear translocation of NF-kappa B\/c-Rel proteins . 
These data may expand the understanding of the anti-thrombotic and anti-inflammatory effects of these drugs when activation of monocytes occurs . 
Characterization of the murine cyclin-dependent kinase inhibitor gene p27Kip1 . 
The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in regulating cell-cycle progression . 
p27Kip1 directly inhibits the catalytic activity of cyclin\/cdks ( cyclin-dependent kinase ) complexes and\/or interferes physically with cyclin\/cdks activation by CAK . 
Interestingly , the expression level of p27Kip1 mRNA was maximal in resting Go T-cells and rapidly declined following anti-CD3 activation . 
We report here the cloning of p27Kip1 gene from murine genomic DNA and the functional analysis of the promoter of the p27Kip1 gene . 
The gene consists of at least three exons and spans more than 5.6 kb of DNA . 
Primer extension and nuclease S1 protection analysis revealed two major transcription initiation sites . 
The promoter region lacked a TATA box but contained potential binding sites for the transcriptional factors including two Sp1 , CRE , Myb and NFkB located at positions -153 , -178 , -286 , -875 , and -1011 , respectively . 
To analyze the regulatory mechanisms controlling p27Kip1 gene expression , we characterized the 5'-flanking region from nt -1609 to +178 . 
The -326 to -615 region contained positive regulatory elements . 
Glucocorticoid-mediated inhibition of RANTES expression in human T lymphocytes . 
The chemokine RANTES has been implicated in the pathogenesis of allergic inflammatory diseases including asthma and rhinitis which are frequently treated with glucocorticoids . 
We observed that dexamethasone dramatically inhibited RANTES mRNA expression dose dependently in anti-CD3 activated Hut-78 T cells and human PBMCs . 
Inhibition of RANTES expression did not appear to be secondary to IL-2 inhibition and required binding to the intracellular glucocorticoid receptor . 
The down-regulation of RANTES expression by glucocorticoids in T cells may directly contribute to the efficacy of these agents in suppressing cellular infiltration and to their anti-inflammatory properties . 
Effects of glucocorticoids on lymphocyte activation in patients with steroid-sensitive and steroid-resistant asthma . 
BACKGROUND : Glucocorticoids are important medications used to control the airway inflammation associated with asthma . 
Synthetic glucocorticoids vary in their binding affinity for the glucocorticoid receptor ( GCR ) . 
METHODS : We compared hydrocortisone , beclomethasone dipropionate , triamcinolone acetonide , flunisolide , and budesonide with regard to their capacity to inhibit phytohemagglutinin-induced peripheral blood mononuclear cell proliferation from six patients with steroid-sensitive asthma and seven patients with steroid-resistant asthma . 
Peripheral blood mononuclear cell GCR binding affinities for dexamethasone and budesonide were also determined for both patient groups by using a radioligand binding assay and Scatchard analysis . 
RESULTS : Dose-dependent inhibition was demonstrated for all glucocorticoids in both patient groups , with the steroid-resistant group requiring approximately 2 log-fold more glucocorticoids for an equivalent degree of inhibition . 
The mean concentrations necessary to cause 50 % inhibition of lymphocyte proliferation ( IC50s ) for the steroid-sensitive group ranged from 2 x 10-LRB--10-RRB- mol\/L for budesonide to 7 x 10-LRB--8-RRB- mol\/L for hydrocortisone , whereas the mean IC50s for the steroid-resistant group ranged from approximately 2 x 10-LRB--8-RRB- mol\/L for budesonide to greater than 10-LRB--6-RRB- mol\/L for hydrocortisone . 
In addition , a significant correlation was noted between the degree of inhibition of lymphocyte proliferation ( IC50 ) and the binding affinity of dexamethasone to the GCR . 
Patients with steroid-resistant asthma have been shown to have a reduced GCR binding affinity . 
The GCR binding affinity for budesonide was significantly higher in both groups ( i.e. , lower dissociation constant ) than that obtained for dexamethasone . 
CONCLUSION : These data suggest that glucocorticoids such as budesonide , by virtue of their high GCR binding affinities and greater ability to suppress lymphocyte proliferation , may therefore be beneficial in the management of difficult-to-control asthma . 
Involvement of nuclear factor-kappa B activation in IgE synthesis in human B cells . 
Nuclear factor-kappa B ( NF-kappa B ) is a transcription factor that binds to the consensus DNA sequence in the cis-acting elements of various genes . 
Although NF-kappa B activates the expression of many genes involved in immune and inflammatory responses , little is known about the role of NF-kappa B activation in the induction of IgE synthesis in human B cells . 
Therefore we first examined the participation of NF-kappa B in germline C epsilon transcription in a human Burkitt lymphoma B cell line , DND39 . 
Stimulation of DND39 cells with IL-4 or anti-CD40 monoclonal antibody ( mAb ) activated phosphatidylinositol 3-kinase and subsequently induced nuclear expression of NF-kappa B , which was identified by electrophoretic mobility shift assays . 
n-Acetyl-L-cysteine ( NAC ) , a potent antioxidant , blocked NF-kappa B activation caused by IL-4 and by anti-CD40 mAb . 
Although inhibition of IL-4-driven germline C epsilon transcription by NAC was not sufficient , the agent remarkably diminished anti-CD40 mAb-mediated up-regulation of germline C epsilon transcription . 
Second , we studied the effect of NAC on IgE synthesis in human normal B cells costimulated with IL-4 and anti-CD40 mAb . 
NAC was effective in inhibiting mature C epsilon transcription and IgE synthesis in the T cell-independent culture system . 
However , NAC did not significantly affect the spontaneous production of IgE by atopic B cells . 
These results indicate that NF-kappa B activity is commonly inducible in DND39 cells by IL-4 and anti-CD40 mAb and suggest that NF-kappa B sensitive to NAC may play a role in regulating IgE synthesis in B cells . 
{ Molecular mechanisms of age-related lymphocyte dysfunction } 
Aging is classically accompanied by a dysregulation of the immunologic machinery . 
As a consequence , the immune response developed in senescent organisms is usually inappropriate , often inefficient , sometimes aberrant , and potentially detrimental . 
The age-associated immune dysfunction may be implicated to some degree in the extreme susceptibility of the elderly to infection and neoplasia and may even participate in various aspects of senescence . 
The current understanding of the molecular mechanisms underlying immunosenescence is still fragmentary . 
The most extensively studied phenomenon is the progressive decline in the proliferative capacities of T lymphocytes with aging . 
The loss of proliferative potential in response to antigenic challenge is a characteristic feature of immune senescence . 
It is directly implicated in the emergence of the age-related immune deficiency . 
The purpose of this review is to show how the accumulation of various biochemical lesions with advancing age leads to the failure of a critical cell function , namely the activation-induced lymphocyte proliferation . 
The biochemical modifications responsible for the defect in transduction and execution of the proliferative signal are analyzed as a function of age . 
The multiple alterations observed on the various biochemical pathways may appear as a consequence of a unique deleterious mechanism more fundamentally related to the process of senescence such as the inability to cope with oxidative stress . 
Sequence analysis and expression in cultured lymphocytes of the human FOSB gene ( G0S3 ) . 
G0S3 is a member of a set of putative G0\/G1 switch regulatory genes ( G0S genes ) selected by screening cDNA libraries prepared from human blood mononuclear cells cultured for 2 hr with lectin and cycloheximide . 
The sequence shows high homology with the murine FOSB gene , which encodes a component of the AP1 transcriptional regulator . 
Comparison of cDNA and genomic sequences reveals a 4-exon structure characteristic of the FOS family of genes . 
Freshly isolated cells show high levels of FOSB\/G0S3 and FOS\/G0S7 mRNAs , which decline rapidly during incubation in culture medium . 
The kinetics of expression suggest that the high initial levels are caused by the isolation procedure , and do not reflect constitutive expression . 
In cells preincubated for a day , levels of FOS mRNA reach a maximum 20 min after the addition of lectin and decline to control levels over the next 3 hr . 
Levels of FOSB mRNA reach a maximum 40 min after the addition of lectin and decline to control levels over the next 6 hr . 
In freshly isolated cells , both FOS and FOSB mRNAs increase dramatically in response to the protein synthesis inhibitor cycloheximide . 
In preincubated cells , the cycloheximide response is decreased , especially in the case of FOSB . 
These differences in expression of FOS and FOSB suggest different roles and regulation . 
Regions of low base order-dependent stem-loop potential in the region of the gene are defined . 
These indicate where base order has been adapted for purposes other than stem-loop stability ( e.g. , encoding proteins or gene regulation ) . 
Regions of low potential in a 68.5-kb genomic segment containing the FOSB gene suggest that the potential may help locate genes in uncharted DNA sequences . 
Pancreatic development and maturation of the islet B cell . 
Studies of pluripotent islet cultures . 
Pancreas organogenesis is a highly regulated process , in which two anlage evaginate from the primitive gut . 
They later fuse , and , under the influence of the surrounding mesenchyme , the mature organ develops , being mainly composed of ductal , exocrine and endocrine compartments . 
Early buds are characterized by a branching morphogenesis of the ductal epithelium from which endocrine and exocrine precursor cells bud to eventually form the two other compartments . 
The three compartments are thought to be of common endodermal origin ; in contrast to earlier hypotheses , which suggested that the endocrine compartment was of neuroectodermal origin . 
It is thus generally believed that the pancreatic endocrine-lineage possesses the ability to mature along a differentiation pathway that shares many characteristics with those of neuronal differentiation . 
During recent years , studies of insulin-gene regulation and , in particular , the tissue-specific transcriptional control of insulin-gene activity have provided information on pancreas development in general . 
The present review summarizes these findings , with a special focus on our own studies on pluripotent endocrine cultures of rat pancreas . 
Induction of vascular cell adhesion molecule-1 by low-density lipoprotein . 
Low-density lipoprotein ( LDL ) is a well-established risk factor for atherosclerosis . 
When endothelial cells are incubated with this lipoprotein in pathophysiologic amounts , the cells are activated . 
Among the documented cellular responses to LDL is increased recruitment of monocytes , which are believed to play a major role in promoting intimal plaque formation . 
The findings presented here link an atheogenic lipoprotein , LDL , with the induction of an adhesion molecule important in atherogenesis 
Human LDL induces the vascular cell adhesion molecule-1 ( VCAM-1 ) transcriptionally with an increase in mRNA levels through activation of the VCAM promoter . 
This effect is blocked by anti-VCAM antibodies . 
After a 2-day incubation in LDL , the binding of NF-kappa B , which is believed to be a key oxidative-stress sensor for VCAM regulation , remains at basal level . 
In contrast , the binding activities of AP-1 and GATA , on the other hand , are increased by LDL . 
Thus , a component of LDL-enhanced endothelial recruitment of monocytes is attributed to VCAM-1 expression , which appears to be mediated through AP-1 and GATA . 
These data identify LDL as a VCAM-inducer possibly distinct from cytokines and endotoxin . 
The NF-kappa B inhibitor , tepoxalin , suppresses surface expression of the cell adhesion molecules CD62E , CD11b\/CD18 and CD106 . 
Tepoxalin , a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation . 
Its immunosuppressive property is distinct from cyclosporin because only tepoxalin , but not cyclosporin , suppresses NF-kappa B activation . 
Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 ( ICAM-1 , CD54 ) \/MAC-1 ( CD11b\/CD18 ) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells . 
The mechanism of inhibition is related to the surface expression of several cell adhesion molecules . 
Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65\/p50 subunit of NF-kappa B , and then stimulated with PMA , revealed a reduced expression of CD11b\/CD18 on monocytic HL60 cells , and endothelial adhesion molecule-1 ( CD62E ) and vascular adhesion molecule-1 ( CD106 ) on human umbilical vein endothelial cells . 
Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 ( CD11a\/CD18 ) and CD54 were unaffected . 
Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine , IL-8 , a known inducer of CD11b\/CD18 expression . 
Thus the suppression of CD11b\/CD18 expression by tepoxalin may involve IL-8 . 
Our results suggest that by inhibiting NF-kappa B activation , surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation . 
Nuclear NF-ATp is a hallmark of unstimulated B cells from B-CLL patients . 
B lymphocytes from the peripheral blood of patients with chronic lymphocytic leukaemia ( CLL ) were analysed for the nuclear presence and DNA binding of a panel of transcription factors which are involved in the gene control of lymphoid cells . 
The following transcription factors were studied : the Octamer factors Oct-1 and Oct-2 , members of the AP-1 factor family , NF-AT factors , in particular NF-ATp , and members of the Rel\/NF-kB family . 
We show that the constitutive nuclear translocation of NF-ATp , a member of the growing family of NF-AT factors , is a hallmark of nonstimulated B cells from CLL patients that distinguishes B-CLL cells from ' normal ' B lymphocytes . 
Constitutive nuclear appearance was also observed for NF-kB2\/p52 . 
Constitutive binding of further factor proteins to DNA , such as JunD , c-Fos and FosB , was detected in several patients whereas the localisation and DNA binding of other factors such as c-Jun , RelA\/p65 and c-Rel was unaltered . 
It is remarkable that in B-CLL cells the nuclear appearance and DNA binding of specific transcription factors is dramatically affected whereas other members of the same factor family remained unaltered in these leukemic cells . 
It remains to be shown which molecular events lead to the specific ' pre-activation ' , i.e. constitutive nuclear translocation and DNA binding , of these members of NF-AT , NF-kB and AP-1 factor families . 
{ The value of the clinical test of glucocorticoid receptors of peripheral blood leukocytes in patients with chronic pulmonary heart disease } 
In order to inquire into the functional state of adrenal cortex in patients with pulmonary heart disease , the number of glucocorticoid receptors ( GCR ) of peripheral blood leukocytes in patients with chronic pulmonary heart disease was determined with radioligand-binding assay and the corresponding plasma cortisol levels were assessed with radioimmune assays . 
The results showed that the number of GCR in the patients was significantly reduced ( P &lt; 0.01 ) and it was increased when their health state was improved . 
However , it was still lower than that in healthy subjects ( P &lt; 0.01 ) . 
The number of GCR in the patients was greatly increased when these patients were treated with oxygen ( P &lt; 0.01 ) . 
No difference in plasma cortisol was found between the patients and the healthy subjects ( P &gt; 0.05 ) . 
These results indicate that the function of adrenal cortex may be improved by the compensation mechanism of the patients , but the lower GCR number was the result of lacking of oxygen in the patients . 
The number of GCR may be improved by inhalation of oxygen . 
Therefore oxygen therapy is helpful in raising the activity of glucocorticoid receptors and controlling the development of the disease . 
Lack of T-cell-mediated recognition of the fusion region of the pml\/RAR-alpha hybrid protein by lymphocytes of acute promyelocytic leukemia patients . 
In previous studies , it was shown that the fusion region of the pml\/RAR-alpha protein , expressed by acute promyelocytic leukemia ( APL ) cells , can be specifically recognized in vitro by donor ( D. E. ) CD4 T cells in a HLA class II DR11-restricted fashion . 
We present here the results on the recognition of several pml\/RAR-alpha peptides by APL patients expressing HLA DR11 . 
The in vitro immunization of peripheral blood lymphocytes from four patients in remission ( S.R. , F.R. , M.M. , P. G. ) with BCR1\/25 , a 25-mer pml\/RAR-alpha , did not elicit either a polyclonal or a clonal immune response specific to the peptide . 
We then generated new donor anti-pml\/RAR-alpha CD4-LRB-+-RRB- T-cell clones . 
These clones were tested for their recognition of BCR1\/25 . 
One clone ( C3\/5 , CD3-LRB-+-RRB- , CD4-LRB-+-RRB- , CD8-LRB---RRB- ) was selected for further analysis . 
Clone C3\/5 showed specific proliferation , cytotoxicity , and cytokine ( tumor necrosis factor alpha , granulocyte-macrophage colony-stimulating factor ) production when challenged with autologous lymphoblastic cell lines pulsed with peptide BCR1\/25 . 
C3\/5 cells developed specific proliferation and cytotoxicity when challenged with peptide-pulsed lymphoblastic cell lines and peripheral blood lymphocytes from the four DR11-LRB-+-RRB- APL patients . 
APL blasts , available only from patients F.R. and P.G. , were not lysed by C3\/5 and were unable to present peptide BCR1\/25 . 
Incubation of APL cells with IFN-gamma failed to induce HLA class II molecules and recognition by the C3\/5 clone . 
Since APL cells do not express HLA class II molecules , we tested in two donors ( D.E. and C.H.R. ) and in patients S.R. and P.G. whether the use of 9-mer peptides ( BCR1\/9 ) would generate a CD8\/HLA class I-restricted response . 
No peptide-specific T-cell line or clone could be generated from both donors and patients . 
These findings are discussed in relation to possible therapeutic approaches to the immunotherapy of APL . 
Induction of NF-KB during monocyte differentiation by HIV type 1 infection . 
The production of human immunodeficiency virus type 1 ( HIV-1 ) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA , and in purified human monocytes and macrophages . 
Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat . 
PMA treatment , and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei . 
In nuclear extracts from monocytes or macrophages , induction of NF-KB occurred only if the cells were previously infected with HIV-1 . 
When U937 cells were infected with HIV-1 , no induction of NF-KB factor was detected , whereas high level of progeny virions was produced , suggesting that this factor was not required for viral replication . 
These results indicate that in monocytic cell lineage , HIV-1 could mimic some differentiation\/activation stimuli allowing nuclear NF-KB expression . 
The NF kappa B independent cis-acting sequences in HIV-1 LTR responsive to T-cell activation . 
The rate of transcription initiation directed by the long terminal repeat ( LTR ) of HIV-1 increases in response to mitogenic stimuli of T cells . 
Here we show that the response of the HIV-1 LTR may be governed by two independent sequences located 5' to the site of transcription initiation sequences that bind either NFAT-1 or NF kappa B . 
The rate of LTR-directed gene expression increased in response to treatment with either a phorbol ester or tumor necrosis factor alpha if either the NFAT-1 or NF kappa B binding sites were deleted , but failed to respond to these mitogenic stimuli if both sequences were absent . 
The HIV-1 mutant virus containing both NF kappa B and NFAT-1 deletion was able to replicate although at a much decreased growth rate , while the deletion of NFAT-1 alone increased the viral growth rate in Jurkat cells . 
Neither deletion of NF kappa B nor deletion of NFAT-1 decreased activation of viral replication by phorbol ester . 
Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes . 
Upon activation , T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells . 
Here we investigated the transcriptional basis for these changes . 
The proenkephalin promoter contains a sequence GGGGACGTCCCC , named B2 , which is similar to the kappa B sequence GGGGACTTTCC , the binding site of the transcription factor nuclear factor ( NF ) -kappa B . 
Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site , concomitant with activation of the proenkephalin promoter . 
Mutations at the B2 site abolish this transcriptional activation . 
The purified homodimer ( two p50s ) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer ( two p65s plus two p50s ) form of the factor . 
Thus , it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B . 
However , as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific , yet another T-cell-specific factor which synergizes with NF-kappa B should be considered . 
1,25-Dihydroxyvitamin D3 receptor RNA : expression in hematopoietic cells . 
1,25-Dihydroxyvitamin D3 { 1,25-LRB-OH-RRB-2D3 } induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients ; these effects are probably mediated through the 1,25-LRB-OH-RRB-2D3 receptor . 
Little is known of expression of 1,25-LRB-OH-RRB-2D3 receptor RNA in hematopoietic cells . 
We examined the expression and modulation of expression of 1,25-LRB-OH-RRB-2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells . 
Constitutive expression of 1,25-LRB-OH-RRB-2D3 receptor RNA was detected in various kinds of hematopoietic cells , including macrophages and activated T lymphocytes , as well as in cell lines KG-1 ( myeloblasts ) , HL-60 ( promyelocytes ) , ML-3 ( myelomonoblasts ) , U937 , THP-1 ( monoblasts ) , K562 ( erythroblasts ) , and S-LB1 ( HTLV-1-transfected T lymphocytes ) . 
Receptor transcripts were 4.6 kilobases ( kb ) , and no variant sizes were observed . 
All cell lines examined in this group also expressed 1,25-LRB-OH-RRB-2D3 receptors . 
Most B lymphocyte lines expressed negligible levels of 1,25-LRB-OH-RRB-2D3 receptor RNA and protein ; however ; analysis of a lymphoid\/myeloid somatic hybrid suggested that suppression of expression of 1,25-LRB-OH-RRB-2D3 receptor RNA in B lymphocytes may be a dominant characteristic . 
HL-60 cells were cultured with 10-LRB--7-RRB- mol\/L 1,25-LRB-OH-RRB-2D3 for 24 to 72 hours , and levels of expression of 1,25-LRB-OH-RRB-2D3 receptor and its RNA were examined . 
Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand . 
Levels of occupied 1,25-LRB-OH-RRB-2D3 receptor protein increased in these HL-60 cells ; but the total number of 1,25-LRB-OH-RRB-2D3 receptors decreased about 50 % at 24 hours and returned toward normal at 72 hours . 
Steady-state levels of 1,25-LRB-OH-RRB-2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages . 
Nondividing macrophages from normal individuals also expressed 1,25-LRB-OH-RRB-2D3 receptor RNA . 
In contrast , nondividing peripheral blood lymphocytes from normal individuals did not express 1,25-LRB-OH-RRB-2D3 receptor RNA ; with stimulation of proliferation of these cells , accumulation of 1,25-LRB-OH-RRB-2D3 receptor RNA increased markedly . 
Half-life ( t1\/2 ) of 1,25-LRB-OH-RRB-2D3 receptor RNA in T lymphocytes was short ( 1 hour ) as determined by measuring decay of the message after addition of actinomycin D . 
Consistent with this short t1\/2 , accumulation of 1,25-LRB-OH-RRB-2D3 receptor RNA increased in cells as their protein synthesis was inhibited . 
Further studies are required to understand the physiologic role of 1,25-LRB-OH-RRB-2D3 receptors in myeloid cells and proliferating T lymphocytes . 
Comparison of constitutive and inducible transcriptional enhancement mediated by kappa B-related sequences : modulation of activity in B cells by human T-cell leukemia virus type I tax gene . 
The kappa B sequence ( GGGACTTTCC ) binds a factor , NF-kappa B , that is constitutively found in its functional , DNA binding form only in B lymphocytes . 
A factor with apparently indistinguishable sequence specificity can be induced in many other cell types , where it is used to regulate inducible gene expression . 
For example , kappa B-related sequences have been shown to be important for the transcription of a few inducible genes , such as the interleukin 2 receptor alpha-chain gene and the beta-interferon gene . 
However , these genes are not constitutively active in B lymphocytes , suggesting that other regulatory mechanisms must play a role in determining the patterns of expression . 
We have investigated the constitutive and inducible transcriptional activity mediated by five kappa B-related sequence elements in two different cell types . 
We show that in S194 plasma cells the activity of each element correlates well with the relative affinity of B-cell-derived NF-kappa B for that element . 
This leads to significantly lower transcription enhancement by sites derived from the interleukin 2 receptor or T-cell receptor genes in S194 cells . 
However , in either EL-4 ( T ) cells or S194 cells , both lower-affinity sites can be significantly induced by the tax gene product of human T-cell leukemia virus type I , showing that NF-kappa B activity can be modulated even in a B-cell line that constitutively expresses this factor . 
Immune response of peripheral blood mononuclear cells to HBx-antigen of hepatitis B virus . 
The hepatitis B virus genome encodes a transcriptional transactivator protein designated HBxAg . 
We have investigated whether this antigen is a target structure for human T-lymphocytes . 
Using recombinant HBxAg protein , we found HBxAg-specific stimulation of peripheral blood mononuclear cells in patients with acute hepatitis B virus infection ( 6 of 6 ) and chronic hepatitis B virus infection ( 6 of 17 ) but not in healthy individuals . 
With HBxAg-specific synthetic polypeptides , several T-cell epitopes were identified . 
Most were located in the carboxyterminal half of the HBxAg protein . 
Five T-cell clones specific for a T-cell epitope located at the carboxyterminal region of HBxAg were established and found to belong to the CD2\/CD4-positive , CD8-negative subtype . 
These data establish for the first time HBxAg as an antigen in the cellular immune response . 
Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer . 
A family of transcriptional activators has recently been identified in chickens ; these transcriptional activators recognize a common consensus motif ( WGATAR ) through a conserved C4 zinc finger DNA-binding domain . 
One of the members of this multigene family , cGATA-3 , is most abundantly expressed in the T-lymphocyte cell lineage . 
Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms . 
The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer . 
We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation . 
HIV enhancer activity perpetuated by NF-kappa B induction on infection of monocytes { see comments } 
Permissiveness to replication of human immunodeficiency virus ( HIV ) differs in T lymphocytes and macrophages . 
In T cells , HIV transcription is poorly detected in vivo . 
Cloned , normal T lymphocytes show very little , if any , basal activity of the HIV enhancer and low nuclear expression of NF-kappa B , a potent transcriptional activator of the HIV enhancer . 
In contrast , fixed tissue macrophages express detectable HIV proteins , indicating permanent virus transcription . 
One explanation for the perpetuation of virus infection in macrophages could be sustained nuclear NF-kappa B expression . 
However , the U937 monocytic cell line , which is fully permissive to HIV replication , is known to express only low levels of nuclear NF-kappa B . 
We show here that chronic HIV infection results in both induction of a nuclear factor with antigenic properties indistinguishable from those of NF-kappa B and permanently increased HIV enhancer activity . 
This phenomenon , which is independent of tumour necrosis factor , is associated with HIV replication , and is thus likely to explain at least in part the perpetuation of HIV infection in monocytes . 
Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection . 
During latent Epstein-Barr virus ( EBV ) infection of human B lymphocytes , six viral nuclear antigen ( EBNAs ) are expressed from long primary transcripts by means of alternative splicing and alternative polyadenylylation sites . 
These transcripts initiate from one of two promoters , Cp or Wp , that function in a mutually exclusive fashion . 
Wp is exclusively utilized during the initial stages of infection of primary B lymphocytes , followed by a switch to Cp usage . 
These studies have been extended to show that ( i ) a mutant EBV strain lacking the gene encoding EBNA 2 fails to switch from Wp to Cp usage in primary B lymphocytes , although the virus contains a functional Cp ; ( ii ) a region from -429 to -245 base pairs upstream of Cp is essential for Cp activity in B lymphocytes , but only in the context of upstream and downstream sequences ; ( iii ) this region contains an EBNA 2-dependent enhancer ; and ( iv ) DNase I protection employing nuclear extracts from B and T lymphocytes revealed a B-cell-specific footprint in the region of the EBNA 2-dependent enhancer . 
These results support a model for viral promoter switching during the initial stages of infection in which Wp activity leads to the expression of EBNA 2 , followed by activation of Cp through the EBNA 2-dependent enhancer . 
Inhibition of protein phosphatases by okadaic acid induces AP1 in human T cells . 
To examine the role of protein phosphatases in T cell activation , Jurkat cells were treated with okadaic acid , an inhibitor of type 1 and 2A phosphatases , and nuclear extracts were examined for the presence of AP1 as a measure of early T cell activation . 
Okadaic acid was found to be a potent inducer of AP1 . 
In contrast to phorbol esters such as phorbol myristate acetate ( PMA ) , the induction of AP1 by okadaic acid occurs predominantly by transcriptional activation of the jun and fos family of proto-oncogenes . 
Surprisingly , while the addition of phytohemagglutinin further enhanced the induction of AP1 , the addition of PMA inhibited it . 
Okadaic acid treatment was found to dramatically increase mRNA transcripts of the jun family of proto-oncogenes including c-jun , junD , and junB and to a lesser extent the fos family including c-fos and fra-1 . 
By comparison , PMA is a very inefficient inducer of the jun gene family in Jurkat cells . 
Similar to its effect on the induction of AP1 by okadaic acid , PMA inhibits the induction of c-jun mRNA by okadaic acid . 
Transfection of c-jun promoter constructs confirmed the marked difference between PMA and okadaic acid in inducing c-jun transcription . 
The induction of AP1 by okadaic acid suggests that protein phosphatases 1 and 2A ( PP1 and PP2A ) may be involved in T cell activation as important negative regulators of the transcription factor AP1 . 
Transactivation of the human immunodeficiency virus promoter by human herpesvirus 6 ( HHV-6 ) strains GS and Z-29 in primary human T lymphocytes and identification of transactivating HHV-6 ( GS ) gene fragments . 
Human herpesvirus 6 ( HHV-6 ) can activate the human immunodeficiency virus ( HIV ) promoter and accelerate cytopathic effects in HIV-infected human T cells . 
This study examines the regions of the HIV promoter required for HHV-6 transactivation in a heterogeneous population of primary human T lymphocytes with or without antigenic stimulation . 
Two different strains of HHV-6 , GS and Z29 , transactivated the HIV promoter . 
The GS strain transactivated the promoter in both stimulated and resting T cells , while the Z29 strain increased HIV promoter activity only in stimulated T cells . 
Three DNA clones containing HHV-6 ( GS ) genomic fragments transactivated the HIV promoter in cotransfected T cells . 
A 21.4-kb DNA clone , pZVB70 , showed the highest transactivating ability , while two other DNA fragments , pZVB10 ( 6.2 kb ) and pZVH14 ( 8.7 kb ) , showed lower activity . 
One of these clones , pZVH14 , activated the HIV promoter construct containing a mutation in the NF kappa B site . 
However , this mutated NF kappa B promoter was not transactivated during HHV-6 ( GS ) infection or after cotransfection with pZVB70 or pZVB10 . 
These data indicate that the NF kappa B sites of the HIV promoter are essential for its transactivation during HHV-6 ( GS ) infection . 
By increasing HIV promoter activity in primary T lymphocytes , HHV-6 may consequently increase HIV replication , leading to an increase in the cytopathic effect on coinfected human T cells . 
Cortivazol mediated induction of glucocorticoid receptor messenger ribonucleic acid in wild-type and dexamethasone-resistant human leukemic ( CEM ) cells . 
Cortivazol is a phenylpyrazolo glucocorticoid of high potency and unusual structure . 
In both wild-type and highly dexamethasone ( dex ) -resistant clones of the human leukemic cell line CEM , exposure to cortivazol leads to cell death . 
It has been shown recently that in wild-type CEM cells but not in a dex-resistant , glucocorticoid receptor ( GR ) -defective clone ICR-27 TK-3 , dex induces GR mRNA . 
To test the hypothesis that cortivazol acts in dex-resistant cells by making use of the residual GR found there , wild-type and dex-resistant clones were treated with various concentrations of cortivazol and induction of GR mRNA was studied . 
Cortivazol significantly induced GR mRNA in the normal CEM-C7 as well as in two classes of dex-resistant clones , although the dex-resistant clones needed at least 10 times more cortivazol than the normal cells for significant GR mRNA induction . 
Increased levels of GR mRNA were noticed as early as 3 h after treatment . 
A general correlation between induction of GR mRNA and lysis of the normal and dex-resistant cells was found . 
Positive induction of GR mRNA might be one of the earliest crucial steps in the lysis of normal and dex-resistant CEM cells , or might serve as a marker for the process . 
However , the lysis pathway in the dex-resistant cells is defective in that dex-resistant clones needed significantly more cortivazol than the normal cells for lysis of the cells . 
Human tumor necrosis factor alpha gene regulation in phorbol ester stimulated T and B cell lines . 
The minimal region of the human tumor necrosis factor alpha ( TNF-alpha ) gene promoter necessary for its transcriptional induction by phorbol esters ( PMA ) in human T and B lymphocyte cell lines has been localized between -52 and +89 nucleotides ( nt ) relative to the gene 's transcriptional start site . 
Comparison of these sequences to those required to mediate virus or lipopolysaccharide ( LPS ) induction of the gene reveal significant differences , and thus , the sequence requirements for PMA induction are distinct from those that mediate induction by virus or LPS . 
Although three sites in the TNF-alpha promoter ( kappa 1 , kappa 2 , and kappa 3 ) specifically bind the transcription factor NF-kappa B in lymphoid nuclear extracts , TNF-alpha mRNA induction by PMA does not correlate with NF-kappa B binding activities displayed by different T and B cell lines . 
Moreover , kappa 1 - kappa 3 can each be deleted from the TNF-alpha promoter with little effect on the gene 's inducibility by PMA . 
Therefore , TNF-alpha mRNA induction by PMA , like its induction by virus and LPS , is not primarily mediated by NF-kappa B , but rather is mediated through other sequences and protein factors . 
Surprisingly , multimers of kappa 1 - kappa 3 can confer PMA inducibility on a heterologous promoter in a B ( Raji ) , but not a T ( HUT78 ) cell line . 
However they are not functional on a truncated TNF-alpha promoter , indicating that promoter context and cell type specificity influence the PMA inducible function of these NF-kappa B binding sites . 
Cloning of a human homeobox gene that resembles a diverged Drosophila homeobox gene and is expressed in activated lymphocytes . 
A new homeobox gene , HB24 , has been isolated from a human B-lymphocyte cDNA library . 
Northern blot analysis of polyadenylated RNA purified from activated human B cells revealed a single mRNA transcript of approximately 2.3 kb . 
Two cDNA clones were sequenced and provided 2,250 nucleotides ( nt ) of DNA sequence information . 
There is a single methionine codon-initiated open reading frame of 1,458 nt in frame with a homeobox and a CAX repeat , and the open reading frame is predicted to encode a protein of 51,659 daltons . 
When the homeodomain from HB24 was compared to known mammalian and Drosophila homeodomains it was found to be only moderately conserved , but when it was compared to a highly diverged Drosophila homeodomain , H2.0 , it was found to be 80 % identical . 
The HB24 mRNA was absent or present at low levels in normal B and T lymphocytes ; however , with the appropriate activation signal HB24 mRNA was induced within several hours even in the presence of cycloheximide . 
Characterization of HB24 expression in lymphoid and select developing tissues was performed by in situ hybridization . 
Positive hybridization was found in thymus , tonsil , bone marrow , developing vessels , and in fetal brain . 
HB24 is likely to have an important role in lymphocytes as well as in certain developing tissues . 
HTLV-1 Tax induces expression of various immediate early serum responsive genes . 
Human T-cell leukemia virus type 1 ( HTLV-1 ) is an etiological agent of adult T-cell leukemia ( ATL ) . 
We showed here by mobility-shift assay that T-cell lines transformed with the virus contained high levels of AP-1 activities . 
Consistent with this result , these cell lines expressed increased levels of mRNAs encoding the AP-1 proteins , c-Fos , Fra-1 , c-Jun , JunB , and JunD . 
Previously , transcription of the c-fos gene has been reported to be transactivated by the viral transcription factor , Tax1 . 
By using the human T-cell line ( JPX-9 ) , in which expression of the Tax1 is inducible , we showed that expression of mRNAs for Fra-1 , c-Jun , and JunD was also transactivated by Tax1 . 
Moreover , Tax1 activated expression of two other transcription factors having zinc finger motifs , Egr-1 and Egr-2 , in the same cells . 
The Tax1-inducible transcription factors identified here are encoded by the members of immediate early genes under the control of growth signals . 
Thus , Tax1 was suggested to replace growth signals , at least in part , by this mechanism . 
Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives . 
HIV-1 proviral DNA contains two binding sites for the transcription factor NF-kappa B . 
HIV-1-infected individuals have , on average , abnormally high levels of tumour necrosis factor alpha ( TNF alpha ) and abnormally low plasma cysteine levels . 
We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-kappa B expression . 
The experiments in this report show that cysteine or N-acetylcysteine ( NP ) raise the intracellular glutathione ( GSH ) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells . 
However , inhibition of HIV-1 replication appears not to be directly correlated with GSH levels . 
Cysteine and NP also inhibit NF-kappa B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase ( CAT ) gene expression under control of NF-kappa B binding sites in uninfected cells . 
This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-kappa B-dependent genes and enhance HIV-1 replication . 
NP may be considered for the treatment of HIV-1-infected individuals . 
Lymphocyte glucocorticoid receptor binding during depression and after clinical recovery . 
Lymphocyte glucocorticoid receptor binding parameters were studied in 15 severely depressed patients during depression and after clinical recovery , and in 15 healthy controls . 
There was no difference in glucocorticoid receptor number or affinity between depressed patients and recovered or control subjects . 
Afternoon ACTH and cortisol concentrations did not differ significantly between the three groups . 
No relationship could be established between glucocorticoid receptor binding and antidepressant medication . 
These data support the view of an impaired ligand-induced plasticity of glucocorticoid receptor regulation rather than the hypothesis of decreased glucocorticoid receptor numbers during depression . 
NF-kappa B activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A , protein kinase C , and Ca-LRB-2+-RRB--regulated kinases . 
NF-kappa B is a DNA-binding regulatory factor able to control transcription of a number of genes , including human immunodeficiency virus ( HIV ) genes . 
In T cells , NF-kappa B is activated upon cellular treatment by phorbol esters and the cytokine tumor necrosis factor alpha ( TNF alpha ) . 
In the present work , we investigated the molecular events leading to NF-kappa B activation by TNF alpha in a human T cell line ( Jurkat ) and its subclone JCT6 , which presents a deficiency in the PKA transduction pathway . 
We found that in both cell lines , both phorbol ester and TNF alpha were able to activate NF-kappa B . 
Phorbol activation was positively modulated by Ca2+ influx while TNF alpha activation was not . 
Furthermore , while PMA activation was inhibited by the PKC inhibitor staurosporin , the TNF alpha effect was unchanged . 
TNF alpha did not activate cAMP production and its signal was not modulated by cAMP activators . 
Moreover , cAMP activators did not activate NF-kappa B in Jurkat cells . 
Thus , TNF alpha-induced NF-kappa B activation was found to be mediated by none of the major signal-mediating kinases such as protein kinase C ( PKC ) , protein kinase A , or Ca-LRB-2+-RRB--regulated kinases . 
Furthermore , we found that cytoplasmic acidification facilitated NF-kappa B activation by both TNF alpha and PKC , by a mechanism that increases NF-kappa B\/I kappa B dissociation without affecting the NF-kappa B translocation step . 
Tumor necrosis factor-alpha mRNA accumulation in human myelomonocytic cell lines . 
Role of transcriptional regulation by DNA sequence motifs and mRNA stabilization . 
The cytokine TNF mediates many of the pathologic signs of cachexia , inflammation , and sepsis . 
The current work describes the regulation of TNF in human myelomonocytic cell lines after PMA stimulation . 
The cell lines exhibit a low level of constitutive TNF mRNA expression . 
Within 2 to 4 h of PMA exposure , steady state levels of TNF mRNA are markedly elevated in all myelomonocytic cell lines studied . 
This rise is due to increased mRNA stability , which increased by almost twofold , and to an overall increase in transcription , which rises by more than sixfold . 
At the level of the genomic TNF gene , a DNase I hypersensitive site is detected within the TNF promoter between -200 to -100 bp relative to the transcription initiation site . 
Although absent in nonexpressing erythroleukemia cell lines , the DNase I site is present in uninduced myelomonocytic cell lines and is not changed after PMA induction . 
The PMA induction of c-fos mRNA correlated well with TNF gene induction ; expression of genes encoding other proteins in the AP-1 complex ( junB and junD ) were also induced by PMA . 
The nuclear extracts from resting and induced ML-1 cells contain proteins binding specifically to the AP-1 , AP-2 , and NF kappa B sequence located within the TNF promoter . 
PMA induction increases the level of a number of specific binding complexes relative to the resting cells . 
The regulatory mechanisms of the human and murine TNF genes are discussed . 
HIV1 infection of human monocytes and macrophages promotes induction or translocation of NF-KB-related factors . 
In 1991 , we demonstrated , using electrophoretic mobility shift assays , that 3 different factors ( termed B1 , B2 and B3 ) with affinity for the KB-enhancer target sequence were specifically detected in nuclear extracts from HIV1-infected monocytes and macrophages . 
The B2 factor was induced in the nuclei of these cells only upon HIV1 infection . 
The B3 factor was only slightly evident in nuclei of uninfected cells but was readily detectable in nuclei of infected monocytes . 
Its expression remained very low in nuclei of HIV1-infected macrophages . 
In this paper , we demonstrate that the B2 factor is expressed in the cytosol of monocytes and macrophages as a DNA-binding protein , indicating that it is not associated with an inhibitor ( IKB ) . 
This factor remained clustered in the cytosol and was translocated to the nuclei only after HIV1 infection . 
The B3 factor is detected in the cytosol only when cells are HIV1-infected . 
The role of HIV1 infection in the expression and the translocation of these factors is discussed . 
Anti-CD2 receptor antibodies activate the HIV long terminal repeat in T lymphocytes . 
The CD2 T lymphocyte glycoprotein surface molecule mediates both cell to cell adhesion and T cell activation , two processes that are involved in the spread of HIV infection . 
Treatment of chronically HIV-infected PBMC with anti-CD2 mAb has been shown to induce the expression of infectious virus from these cultures . 
In this study we investigated the mechanisms whereby anti-CD2 antibodies stimulate viral production . 
We demonstrate that treatment of transiently transfected T lymphocytes with anti-CD2 antibodies results in activation of the HIV long terminal repeat . 
Furthermore , CAT assays using mutated HIV long terminal repeat-CAT constructs and gel shift assays demonstrate that this activation is dependent on the NF-kappa B enhancer . 
These studies suggest that interaction of CD2 with its natural ligand , LFA-3 , may play a role in regulation of HIV expression . 
Demonstration of a 1,25-dihydroxyvitamin D3-responsive protein in human lymphocytes : immunologic crossreactivity and inverse regulation with the vitamin D receptor . 
Using Western blot analysis with a monoclonal antibody recognizing a 17-amino acid epitope of the 1,25-dihydroxyvitamin D3 { 1,25-LRB-OH-RRB-2D3 } receptor , we have detected two crossreacting proteins in activated normal human lymphocytes . 
The smaller of the two proteins ( 50 kDa ) was indistinguishable from the classical 1,25-LRB-OH-RRB-2D3 receptor and , similar to the classical 1,25-LRB-OH-RRB-2D3 receptor , was upregulated in a dose-dependent fashion by 1,25-LRB-OH-RRB-2D3 . 
The larger crossreacting protein exhibited an electrophoretic mobility of 80 kDa , was localized in the cell cytosol , and appeared to be specific for activated lymphocytes since it was not detected in several other human cells including monocytes . 
More strikingly , the 80-kDa protein was downregulated in a dose-dependent fashion by 1,25-LRB-OH-RRB-2D3 ; this effect was independent of the mode of lymphocyte activation and specific for the 1,25-LRB-OH-RRB-2D3 metabolite of vitamin D3 . 
However , two potent immunosuppressive agents , glucocorticoids and cyclosporin A , also inhibited the 80-kDa protein . 
v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII . 
The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone ( T3\/T4 ) receptor c-erbA ( type alpha ) . 
It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes . 
Here , we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II ( CAII ) , a gene whose transcription is efficiently suppressed by v-erbA . 
This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein . 
In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA\/T3 receptor at an approximately equimolar ratio , c-erbA activity is dominant over v-erbA . 
T3 efficiently induced erythroid differentiation in these cells , thus overcoming the v-erbA-mediated differentiation arrest . 
Likewise , T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site . 
The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA . 
Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein . 
Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor . 
We have used a DNA-binding\/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor ( hGR ) , generated in rabbit reticulocyte lysates , to bind DNA . 
In vitro translated hGR was indistinguishable from native hGR , as determined by migration on sodium dodecyl sulfate-polyacrylamide gels , sedimentation on sucrose density gradients , and reactivity with antipeptide antibodies generated against hGR . 
In addition , cell-free synthesized hGR was capable of specific binding to glucocorticoid response element ( GRE ) -containing DNA fragments . 
Using this assay system , we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors . 
In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid . 
Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C ( no activation ) . 
In contrast , addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors . 
Agonist ( dexamethasone ) was slightly more effective in supporting specific DNA binding than antagonist ( RU486 ) . 
DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation . 
Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes . 
The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat . 
Oligonucleotides containing the consensus GRE were the most efficient competitors , and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive , whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding , except when competitor was present at extremely high concentrations . 
Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties , including GRE-specific DNA binding , observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues . 
Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding . 
Clone pAT 133 identifies a gene that encodes another human member of a class of growth factor-induced genes with almost identical zinc-finger domains . 
We report the structure and regulation of a gene represented by clone pAT 133 , which is induced upon transition from a resting state ( G0 ) through the early phase of the cell cycle ( G1 ) . 
The pAT 133 gene is immediately induced , with FOS-like kinetics , in human T cells and in fibroblasts . 
Primary structure analysis showed that the encoded protein contains three tandem zinc-finger sequences of the type Cys2-Xaa12-His2 . 
This zinc-finger region , which is thought to bind DNA in a sequence-specific manner , is similar ( greater than 80 % on the amino acid level ) to two previously described transcription factors pAT 225\/EGR1 and pAT 591\/EGR2 . 
Except for the conserved zinc-finger domains , the amino acid sequences of the three proteins are distinct . 
This structural similarity suggests that the pAT 133 gene encodes a transcription factor with a specific biological function . 
Comparing the regulation of these related zinc-finger-encoding genes showed coordinate induction upon mitogenic stimulation of resting T lymphocytes and of resting fibroblasts . 
However , upon transition from a proliferating ( G1 ) to a resting state of the cell cycle the three genes were differently regulated . 
In human histiocytic U937 cells mRNA of clone pAT 133 was constitutively expressed , whereas mRNA of pAT 225\/EGR1 was induced upon induction of terminal differentiation . 
In contrast mRNA representing pAT 591\/EGR2 was not expressed in these cells . 
This difference in gene regulation suggests distinct biological roles in the control of cell proliferation for the respective proteins . 
Regulation of interleukin-1 beta production by glucocorticoids in human monocytes : the mechanism of action depends on the activation signal . 
Glucocorticoids are known to downregulate interleukin-1 beta production in monocytic cells by two different mechanims : direct inhibition of the gene transcription and destabilization of the preformed interleukin-1 beta mRNA . 
Now we have examined the effect of the nature of the monocyte activating signal on these two inhibitory mechanims . 
When human monocytes were preincubated with dexamethasone for 1 hour and then stimulated either with bacterial lipopolysaccharide or phorbol myristate , it was found that dexamethasone inhibited the lipopolysaccharide-induced interleukin-1 beta protein production , but the phorbol myristate-induced production was increased 3-10 fold . 
This difference was also seen at the mRNA level . 
When dexamethasone was added to the cultures 3 hours after the stimulators , it clearly decreased the interleukin-1 beta mRNA levels regardless of the stimulator used ( although the effect was clearly weaker on the PMA-induced mRNA ) . 
Thus these data suggest that the phorbol myristate-induced signal ( prolonged protein kinase C activation ? ) can not be inhibited by prior incubation with dexamethasone and it also protects the induced mRNA for the degradative action of dexamethasone . 
TCF-1 , a T cell-specific transcription factor of the HMG box family , interacts with sequence motifs in the TCR beta and TCR delta enhancers . 
We have recently identified and cloned TCF-1 , a T cell-specific transcription factor with specificity for the AACAAAG motif in the CD3 epsilon enhancer and for the TTCAAAG motif in the TCR alpha enhancer . 
TCF-1 belongs to the family of transcription-regulating proteins which share a region of homology termed the HMG-box . 
Here , we show by gel retardation analysis that TCF-1 specifically recognizes the T beta 5 element of the TCR beta enhancer and the T delta 7 element of the TCR delta enhancer . 
Comparison of the sequences of all elements recognized by TCF-1 defines a consensus motif A\/T A\/T C A A\/G A G . 
These observations imply that TCF-1 is involved in the control of several T cell-specific genes and might thus play an important role in the establishment and maintenance of the mature T cell phenotype . 
Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein . 
Dimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression . 
Hepatocyte nuclear factor-1 alpha ( HNF-1 alpha ) is a homeodomain-containing protein that functions as a dimer . 
A dimerization cofactor of HNF-1 alpha ( DCoH ) was identified that displayed a restricted tissue distribution and did not bind to DNA , but , rather , selectively stabilized HNF-1 alpha dimers . 
The formation of a stable tetrameric DCoH-HNF-1 alpha complex , which required the dimerization domain of HNF-1 alpha , did not change the DNA binding characteristics of HNF-1 alpha , but enhanced its transcriptional activity . 
However , DCoH did not confer transcriptional activation to the GAL4 DNA binding domain . 
These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex . 
Kappa B-specific DNA binding proteins are differentially inhibited by enhancer mutations and biological oxidation . 
Kappa B ( kappa B ) enhancer binding proteins isolated from the nuclei of activated human T cells produce two distinct nucleoprotein complexes when incubated with the kappa B element from the interleukin-2 receptor-alpha ( IL-2R alpha ) gene . 
These two DNA-protein complexes are composed of at least four host proteins ( p50 , p55 , p75 , p85 ) , each of which shares structural similarity with the v-rel oncogene product . 
Nuclear expression of these proteins is induced with distinctly biphasic kinetics following phorbol ester activation of T cells ( p55\/p75 early and p50\/p85 late ) . 
DNA-protein crosslinking studies have revealed that the more rapidly migrating B2 complex contains both p50 and p55 while the more slowly migrating B1 complex is composed of p50 , p55 , p75 , and p85 . 
Site-directed mutagenesis of the wild-type IL-2R alpha kappa B enhancer ( GGGGAATCTCCC ) has revealed that the binding of p50 and p55 ( B2 complex ) is particularly sensitive to alteration of the 5' triplet of deoxyguanosine residues . 
In contrast , formation of the B1 complex , reflecting the binding of p75 and p85 , critically depends upon the more 3' sequences of this enhancer element . 
DNA binding by all four of these Rel-related factors is blocked by selective chemical modification of lysine and arginine residues , suggesting that both of these basic amino acids are required for binding to the kappa B element . 
Similarly , covalent modification of free sulfhydryl groups with diamide ( reversible ) or N-ethylmaleimide ( irreversible ) results in a complete loss of DNA binding activity . 
In contrast , mild oxidation with glucose oxidase selectively inhibits p75 and p85 binding while not blocking p50 and p55 interactions . 
These findings suggest that reduced cysteine thiols play an important role in the DNA binding activity of this family of Rel-related transcription factors . 
A novel primer extension method 0 to detect the number of CAG repeats in the androgen receptor gene in families with X-linked spinal and bulbar muscular atrophy . 
X-linked spinal and bulbar muscular atrophy ( SBMA ) , an adult-onset form of motor neuron disease , was recently reported to be caused by amplification of the CAG repeats in the androgen receptor gene . 
We report here a simple and rapid strategy 0 to detect the precise number of the CAGs . 
After the DNA fragment containing the CAG repeats is amplified by the polymerase chain reaction , a primer extension is carried out ; the extension of the end-labelled reverse primer adjacent to 3' end of CAG repeats stops at the first T after CAG repeats with the incorporation of dideoxy ATP in the reaction mixture . 
The resultant primer products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography . 
This method could be quite useful to detect not only CAG repeats in SBMA but also other polymorphic dinucleotide and trinucleotide repeats . 
Stimulation of interferon beta gene transcription in vitro by purified NF-kappa B and a novel TH protein . 
The human interferon beta ( IFN-beta ) regulatory element consists of multiple enhanson domains which are targets for transcription factors involved in inducible expression of the promoter . 
To further characterize the protein-DNA interactions mediating IFN-beta induction , positive regulatory domain ( PRD ) II binding proteins were purified from phorbol ester induced Jurkat T-cells and from IFN primed , cycloheximide\/polyinosinic-polycytidylic acid treated HeLa S3 cells . 
From HeLa cells , two major proteins of 52 and 45 kilodaltons ( kD ) copurified with DNA binding activity , whereas from T-cells , four proteins -- a major protein of 52 kD and three minor proteins of 82 , 67 , and 43-47 kD -- were purified . 
Also , an induction specific DNA binding protein was purified from HeLa cells that interacted with the ( AAGTGA ) 4 tetrahexamer sequence and the PRDI domain . 
This protein is immunologically distinct from IRF-1\/ISGF2 . 
Uninduced or Sendai virus induced HeLa extracts were used to examine transcription in vitro using a series of IFN beta promoter deletions . 
Deletions upstream of the PRDII element increased transcription in the uninduced extract , indicating predominantly negative regulation of the promoter . 
A 2-4-fold increase in IFN-beta promoter transcription was observed in Sendai virus induced extracts , and deletion of PRDI and PRDII elements decreased this induced level of transcription . 
When purified PRDII and tetrahexamer binding proteins were added to the induced extract , a 4-fold increase in transcription was observed . 
These experiments demonstrate that it is possible to modulate IFN-beta transcription in vitro but indicate that additional proteins may be required to fully activate IFN-beta transcription . 
Cortisol resistance in acquired immunodeficiency syndrome . 
This study concerns 9 iv drug abusers with acquired immunodeficiency syndrome ( AIDS ) who developed hypercortisolism without the clinical signs or metabolic consequences of hypercortisolism . 
All patients were characterized by an Addisonian picture ( weakness , weight loss , hypotension , hyponatremia , and intense mucocutaneous melanosis ) . 
An acquired form of peripheral resistance to glucocorticoids was suspected . 
We , therefore , examined glucocorticoid receptor characteristics on mononuclear leukocytes by measuring -LCB-3H-RCB-dexamethasone binding and the effect of dexamethasone on -LCB-3H-RCB-thymidine incorporation , which is one of the effects of glucocorticoid receptor activation . 
Glucocorticoid receptor density was increased in AIDS patients with an Addisonian picture ( group 1 ; 16.2 +\/- 9.4 fmol\/million cells ) compared to values in 12 AIDS patients without an Addisonian picture ( group 2 ; 6.05 +\/- 2.6 fmol\/million cells ; P less than 0.01 ) and sex- and age-matched controls ( 3.15 +\/- 2.3 fmol\/million cells ; P less than 0.01 ) . 
The affinity of glucocorticoid receptors ( Kd ) was strikingly decreased ( 9.36 +\/- 3.44 nM in group 1 ; 3.2 +\/- 1.5 nM in group 2 ; 2.0 +\/- 0.8 nM in controls ; P less than 0.01 ) . 
-LCB-3H-RCB-Thymidine incorporation was decreased dose-dependently by dexamethasone in controls and patients ; the effect was significantly blunted ( P less than 0.05 ) in group 1 patients , which suggests that activation of glucocorticoid receptor is impaired as a result of the glucocorticoid receptor abnormality . 
In conclusion , AIDS patients with hypercortisolism and clinical features of peripheral resistance to glucocorticoids are characterized by abnormal glucocorticoid receptors on lymphocytes . 
Resistance to glucocorticoids implies a complex change in immune-endocrine function , which may be important in the course of immunodeficiency syndrome . 
Binding of erythroid and non-erythroid nuclear proteins to the silencer of the human epsilon-globin-encoding gene . 
To clarify the molecular mechanisms involved in the developmental control of hemoglobin-encoding genes we have been studying the expression of these genes in human cells in continuous culture . 
We have previously reported the presence of a transcriptional control element with the properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin-encoding gene { Cao et al. , Proc.Natl.Acad.Sci.USA 86 ( 1989 ) 5306-5309 } . 
We also showed that this silencer has stronger inhibitory activity in HeLa cells , as compared to K562 human erythroleukemia cells . 
Using deletion mutants and cis-cloned synthetic oligodeoxyribonucleotides in transient expression assays , nucleotide sequences responsible for this effect have now been further delimited to 44 bp located from -294 to -251 bp . 
Gel electrophoresis mobility shift assays and DNaseI footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells . 
Two binding proteins are detected in K562 nuclear extracts , while only one is found in extracts from HeLa cells . 
Possible mechanisms by which these proteins may regulate transcription of the epsilon-globin-encoding gene in erythroid and non-erythroid cells are discussed . 
{ Regulatory effect of insulin on glucocorticoid receptor in human peripheral leukocytes } 
The regulatory effect of insulin on the specific binding power of glucocorticoid receptor ( GR ) of human leukocytes was assessed by the unoccupied receptor sites capable of combining with -LCB-3H-RCB- labelled dexamethasone measured at 3 and 24 h after incubation with various concentrations of insulin added to the medium . 
After 3 h incubation the specific binding power with -LCB-3H-RCB- Dex was decreased by 23.3 +\/- 10.0 , 32.2 +\/- 13.2 and 54.3 +\/- 9.2 % ( P greater than 0.05 , P greater than 0.05 and P less than 0.01 as compared with the control value of 100 in the absence of insulin ) respectively in the presence of 20 mU\/L ( physiological testing concentration ) , 200 mU\/L ( physiological upper limit ) and 2,000 mU\/L ( pharmacological concentration ) insulin in the incubation medium . 
After 24 h incubation the decrease of these values increased respectively to 43.5 +\/- 19.0 , 56.1 +\/- 20.7 and 80.2 +\/- 15.5 ( P less than 0.05 , P less than 0.01 and P less than 0.01 compared with control ) . 
Thus the inhibitory effect of insulin on the GR binding power is both dose- and time-dependent , which strongly suggests that GR is tonically controlled by insulin concentration change under physiological conditions . 
Mineralocorticoid effector mechanism in preeclampsia . 
Mineralocorticoid effector mechanisms were evaluated in 29 patients with preeclampsia and in 25 uncomplicated pregnancies by measurement of plasma aldosterone , levels of mineralocorticoid receptor ( MR ) in mononuclear leucocytes , and subtraction potential difference ( SPD ; rectal minus oral values ) . 
Mean values for plasma aldosterone were not different between the two groups , but significant differences were observed for MR ( preeclampsia , 81 +\/- 44 receptors\/cell ; controls , 306 +\/- 168 ) and SPD ( preeclampsia , 65 +\/- 7 mV ; controls , 12 +\/- 5 mV ) . 
In six cases we determined MR , plasma aldosterone , and SPD in patients with preeclampsia before and 3 months after delivery . 
MR were reduced before delivery ( 96 +\/- 27 receptors\/cell ) , and SPD increased ( 64 +\/- 8 mV ) , with both parameters normalizing after delivery ( MR , 242 +\/- 79 ; SPD , 14.0 +\/- 4 mV ) . 
Aldosterone levels returned to normal nonpregnant values after delivery . 
These data suggest an important role for abnormalities in mineralocorticoid effector mechanisms in the etiology of preeclampsia and could be an useful marker for diagnosis . 
Leukotriene B4 stimulates c-fos and c-jun gene transcription and AP-1 binding activity in human monocytes . 
We have examined the effect of leukotriene B4 ( LTB4 ) , a potent lipid proinflammatory mediator , on the expression of the proto-oncogenes c-jun and c-fos . 
In addition , we looked at the modulation of nuclear factors binding specifically to the AP-1 element after LTB4 stimulation . 
LTB4 increased the expression of the c-fos gene in a time- and concentration-dependent manner . 
The c-jun mRNA , which is constitutively expressed in human peripheral-blood monocytes at relatively high levels , was also slightly augmented by LTB4 , although to a much lower extent than c-fos . 
The kinetics of expression of the two genes were also slightly different , with c-fos mRNA reaching a peak at 15 min after stimulation and c-jun at 30 min . 
Both messages rapidly declined thereafter . 
Stability of the c-fos and c-jun mRNA was not affected by LTB4 , as assessed after actinomycin D treatment . 
Nuclear transcription studies in vitro showed that LTB4 increased the transcription of the c-fos gene 7-fold and the c-jun gene 1.4-fold . 
Resting monocytes contained nuclear factors binding to the AP-1 element , but stimulation of monocytes with LTB4 induced greater AP-1-binding activity of nuclear proteins . 
These results indicate that LTB4 may regulate the production of different cytokines by modulating the yield and\/or the function of transcription factors such as AP-1-binding proto-oncogene products . 
The B cell-specific nuclear factor OTF-2 positively regulates transcription of the human class II transplantation gene , DRA . 
The promoter of the major histocompatibility class II gene DRA contains an octamer element ( ATTTGCAT ) that is required for efficient DRA expression in B cells . 
Several DNA-binding proteins are known to bind this sequence . 
The best characterized are the B cell-specific OTF-2 and the ubiquitous OTF-1 . 
This report directly demonstrates that OTF-2 but not OTF-1 regulates the DRA gene . 
In vitro transcription analysis using protein fractions enriched for the octamer-binding protein OTF-2 demonstrate a positive functional role for OTF-2 in DRA gene transcription . 
In contrast , OTF-1-enriched protein fractions did not affect DRA gene transcription although it functionally enhanced the transcription of another gene . 
Recombinant OTF-2 protein produced by in vitro transcription\/translation could also enhance DRA gene transcription in vitro . 
In vivo transient transfection studies utilizing an OTF-2 expression vector resulted in similar findings : that OTF-2 protein enhanced DRA gene transcription , and that this effect requires an intact octamer element . 
Together these results constitute the first direct evidence of a positive role for the lymphoid-specific octamer-binding factor in DRA gene transcription . 
Corticosteroid receptors and lymphocyte subsets in mononuclear leukocytes in aging . 
Plasma cortisol and aldosterone levels and number of related receptors in mononuclear leukocytes were measured in 49 healthy aged subjects ( 62-97 yr ) and in 21 adult controls ( 21-50 yr ) . 
In all subjects , in addition , lymphocyte subsets were determined as an index of corticosteroid action . 
The mean number of type I and type II receptors was significantly lower in aged subjects than in controls ( respectively , 198 +\/- 96 and 272 +\/- 97 receptors\/cell for type I , and 1,794 +\/- 803 and 3,339 +\/- 918 for type II receptors ) . 
Plasma aldosterone and cortisol and lymphocyte subsets were not different in the two groups . 
All of the parameters were also tested for correlation , and a significant inverse correlation was found between age and type I and type II receptors when all subjects were plotted and between aged and CD4 and age and CD4\/CD8 in the aged group . 
These data show that aged subjects have reductions of corticosteroid receptors that are not associated with increase of related steroids and that this situation probably represents a concomitant of the normal aging process . 
Interferon-gamma potentiates the antiviral activity and the expression of interferon-stimulated genes induced by interferon-alpha in U937 cells . 
Binding of type I interferon ( IFN-alpha\/beta ) to specific receptors results in the rapid transcriptional activation , independent of protein synthesis , of IFN-alpha-stimulated genes ( ISGs ) in human fibroblasts and HeLa and Daudi cell lines . 
The binding of ISGF3 ( IFN-stimulated gene factor 3 ) to the conserved IFN-stimulated response element ( ISRE ) results in transcriptional activation . 
This factor is composed of a DNA-binding protein ( ISGF3 gamma ) , which normally is present in the cytoplasm , and other IFN-alpha-activated proteins which preexist as latent cytoplasmic precursors ( ISGF3 alpha ) . 
We have found that ISG expression in the monocytic U937 cell line differs from most cell lines previously examined . 
U937 cells express both type I and type II IFN receptors , but only IFN-alpha is capable of inducing antiviral protection in these cells . 
Pretreatment with IFN-gamma potentiates the IFN-alpha-induced protection , but IFN-gamma alone does not have any antiviral activity . 
ISG15 mRNA accumulation in U937 cells is not detectable before 6 h of IFN-alpha treatment , peaks at 24 h , and requires protein synthesis . 
Although IFN-gamma alone does not induce ISG expression , IFN-gamma pretreatment markedly increases and hastens ISG expression and transcriptional induction . 
Nuclear extracts assayed for the presence of ISRE binding factors by electrophoretic mobility shift assays show that ISGF3 is induced by IFN-alpha within 6 h from undetectable basal levels in untreated U937 cells . 
Activation of ISGF3 alpha , the latent component of ISGF3 , occurs rapidly . 
However , the increase in ISGF3 activity ultimately correlates with the accumulation of ISGF3 gamma induced by IFN-alpha or IFN-gamma . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
Specific NF-kappa B subunits act in concert with Tat to stimulate human immunodeficiency virus type 1 transcription . 
NF-kappa B is a protein complex which functions in concert with the tat-I gene product to stimulate human immunodeficiency virus ( HIV ) transcription . 
To determine whether specific members of the NF-kappa B family contribute to this effect , we have examined the abilities of different NF-kappa B subunits to act with Tat-I to stimulate transcription of HIV in Jurkat T-leukemia cells . 
We have found that the p49 ( 100 ) DNA binding subunit , together with p65 , can act in concert with Tat-I to stimulate the expression of HIV-CAT plasmid . 
Little effect was observed with 50-kDa forms of p105 NF-kappa B or rel , in combination with p65 or full-length c-rel , which do not stimulate the HIV enhancer in these cells . 
These findings suggest that the combination of p49 ( 100 ) and p65 NF-kappa B can act in concert with the tat-I gene product to stimulate the synthesis of HIV RNA . 
Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway . 
We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120 , yet show a markedly reduced susceptibility to infection with HIV-1 . 
Two subclones were found to have an abnormal response to the protein kinase C ( PKC ) activator PMA . 
PMA treatment induced CD3 and CD25 ( IL-2R ) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones , but not on these two mutants . 
Direct assays of PKC activity were conducted . 
Total cellular PKC enzymatic activity was found to be normal in these subclones . 
PMA-induced CD4 down-modulation occurred normally . 
In addition , activation of c-raf kinase was normal . 
Since HIV-1 long terminal repeat contains two functional nuclear factor kB ( NF-kB ) regulatory elements , we studied the ability of PMA to induce NF-kB binding activity by different assays . 
Chloramphenicol acetyl transferase ( CAT ) assays using the HIV-1 ( -139 ) long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones ( unlike the parental line and other subclones ) . 
Okadaic acid , an inhibitor of phosphatases 1 and 2A , did not overcome the defect in these subclones . 
Gel retardation assays , using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells , showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone . 
Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools . 
Thus , reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth , but correlated with a pronounced reduction in their susceptibility to HIV-1 infection . 
Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes . 
B lymphocytes and macrophages express closely related immunoglobulin G ( IgG ) Fc receptors ( Fc gamma RII ) that differ only in the structures of their cytoplasmic domains . 
Because of cell type-specific alternative messenger RNA splicing , B-cell Fc gamma RII contains an insertion of 47 amino acids that participates in determining receptor function in these cells . 
Transfection of an Fc gamma RII-negative B-cell line with complementary DNA 's encoding the two splice products and various receptor mutants indicated that the insertion was responsible for preventing both Fc gamma RII-mediated endocytosis and Fc gamma RII-mediated antigen presentation . 
The insertion was not required for Fc gamma RII to modulate surface immunoglobulin-triggered B-cell activation . 
Instead , regulation of activation involved a region of the cytoplasmic domain common to both the lymphocyte and macrophage receptor isoforms . 
In contrast , the insertion did contribute to the formation of caps in response to receptor cross-linking , consistent with suggestions that the lymphocyte but not macrophage form of the receptor can associate with the detergent-insoluble cytoskeleton . 
Induction of the POU domain transcription factor Oct-2 during T-cell activation by cognate antigen . 
Oct-2 is a transcription factor that binds specifically to octamer DNA motifs in the promoters of immunoglobulin and interleukin-2 genes . 
All tumor cell lines from the B-cell lineage and a few from the T-cell lineage express Oct-2 . 
To address the role of Oct-2 in the T-cell lineage , we studied the expression of Oct-2 mRNA and protein in nontransformed human and mouse T cells . 
Oct-2 was found in CD4+ and CD8+ T cells prepared from human peripheral blood and in mouse lymph node T cells . 
In a T-cell clone specific for pigeon cytochrome c in the context of I-Ek , Oct-2 was induced by antigen stimulation , with the increase in Oct-2 protein seen first at 3 h after activation and continuing for at least 24 h . 
Oct-2 mRNA induction during antigen-driven T-cell activation was blocked by cyclosporin A , as well as by protein synthesis inhibitors . 
These results suggest that Oct-2 participates in transcriptional regulation during T-cell activation . The relatively delayed kinetics of Oct-2 induction suggests that Oct-2 mediates the changes in gene expression which occur many hours or days following antigen stimulation of T lymphocytes . 
NF-kappa B-dependent induction of the NF-kappa B p50 subunit gene promoter underlies self-perpetuation of human immunodeficiency virus transcription in monocytic cells . 
The molecular mechanisms underlying the sustained nuclear translocation of NF-kappa B observed in U937 monocytic cells chronically infected with human immunodeficiency virus ( HIV ) were studied . 
The activity of the promoter regulating the synthesis of the p105 precursor of the NF-kappa B p50 subunit was enhanced in these cells . 
Deletions in this promoter indicated that this upregulation was mediated through the NF-kappa B- but not the AP-1-binding motif , by bona fide p50\/p65 heterodimers . 
Analysis of cytosolic extracts indicated that NF-kappa B levels were increased in HIV-infected cells . 
In contrast to the transient NF-kappa B activation induced by phorbol ester , the permanent NF-kappa B translocation induced by HIV infection was not dependent on PKC isoenzymes alpha and beta as shown by the use of a specific inhibitor ( GF 109203X ) . 
These observations indicate that during chronic HIV infection of U937 cells , continuous NF-kappa B ( p50\/p65 ) translocation results in p105 promoter upregulation with subsequent cytosolic NF-kappa B accumulation , ready for further translocation . 
This HIV-mediated mechanism results in a self-perpetuating loop of NF-kappa B production . 
TAR-independent transactivation by Tat in cells derived from the CNS : a novel mechanism of HIV-1 gene regulation . 
The Tat protein of human immunodeficiency virus type 1 ( HIV-1 ) is essential for productive infection and is a potential target for antiviral therapy . 
Tat , a potent activator of HIV-1 gene expression , serves to greatly increase the rate of transcription directed by the viral promoter . 
This induction , which seems to be an important component in the progression of acquired immune deficiency syndrome ( AIDS ) , may be due to increased transcriptional initiation , increased transcriptional elongation , or a combination of these processes . 
Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR ( transactivation responsive ) which is present in the leader sequence of all HIV-1 mRNAs . 
This interaction is believed to be an important component of the mechanism of transactivation . 
In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway . 
A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR . 
Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat ( LTR ) previously identified as the HIV-1 enhancer , or NF-kappa B domain . 
DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells . 
Further , we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes , the developing CNS , and adult testis . 
BSAP has been identified previously as a transcription factor that is expressed at early , but not late , stages of B-cell differentiation . 
Biochemical purification and cDNA cloning has now revealed that BSAP belongs to the family of paired domain proteins . 
BSAP is encoded by the Pax-5 gene and has been highly conserved between human and mouse . 
An intact paired domain was shown to be both necessary and sufficient for DNA binding of BSAP . 
Binding studies with several BSAP recognition sequences demonstrated that the sequence specificity of BSAP differs from that of the distantly related paired domain protein Pax-1 . 
During embryogenesis , the BSAP gene is transiently expressed in the mesencephalon and spinal cord with a spatial and temporal expression pattern that is distinct from that of other Pax genes in the developing central nervous system ( CNS ) . 
Later , the expression of the BSAP gene shifts to the fetal liver where it correlates with the onset of B lymphopoiesis . 
BSAP expression persists in B lymphocytes and is also seen in the testis of the adult mouse . 
All of this evidence indicates that the transcription factor BSAP may not only play an important role in B-cell differentiation but also in neural development and spermatogenesis . 
Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells . 
Human immunodeficiency virus type 1 ( HIV-1 ) can establish a persistent and latent infection in CD4+ T lymphocytes ( W.C.Greene , N.Engl.J. Med.324 : 308-317 , 1991 ; S.M.Schnittman , M.C.Psallidopoulos , H.C. Lane , L.Thompson , M.Baseler , F.Massari , C.H.Fox , N.P.Salzman , and A.S.Fauci , Science 245 : 305-308 , 1989 ) . 
Production of HIV-1 from latently infected cells requires host cell activation by T-cell mitogens ( T.Folks , D.M.Powell , M.M.Lightfoote , S.Benn , M.A. Martin , and A.S.Fauci , Science 231 : 600-602 , 1986 ; D.Zagury , J. Bernard , R.Leonard , R.Cheynier , M.Feldman , P.S.Sarin , and R.C. Gallo , Science 231 : 850-853 , 1986 ) . 
This activation is mediated by the host transcription factor NF-kappa B { G.Nabel and D.Baltimore , Nature ( London ) 326 : 711-717 , 1987 } . 
We report here that the HIV-1-encoded Nef protein inhibits the induction of NF-kappa B DNA-binding activity by T-cell mitogens . 
However , Nef does not affect the DNA-binding activity of other transcription factors implicated in HIV-1 regulation , including SP-1 , USF , URS , and NF-AT . 
Additionally , Nef inhibits the induction of HIV-1- and interleukin 2-directed gene expression , and the effect on HIV-1 transcription depends on an intact NF-kappa B-binding site . 
These results indicate that defective recruitment of NF-kappa B may underlie Nef 's negative transcriptional effects on the HIV-1 and interleukin 2 promoters . 
Further evidence suggests that Nef inhibits NF-kappa B induction by interfering with a signal derived from the T-cell receptor complex . 
{ Mechanism of action of steroid hormones . I . Estrogens } 
The steroid hormone are very versatile molecules : although they are related among them by their chemical structure , they have very diverse functions and including antagonic . 
Their action mechanism is not completely cleared . 
The estrogens participate in the regulation of practically all the reproductive and sexual events of the female , although the intracellular actions by which they take place are not well known and the proposed models do not adequately satisfy the questions . 
Currently it is accepted the existence of a cytoplasmic and\/or nuclear receptor , without explaining satisfactorily how the hormones come to the nucleus . 
The endocrine events that are rapidly expressed ( seconds ) are due to a possible interaction with cellular membrane . 
The purpose of this review is to analyze and concilliate the reported data on the mechanism of action of estrogens . 
Selection of optimal kappa B\/Rel DNA-binding motifs : interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation . 
Analysis of the p50 and p65 subunits of the NF-kappa B transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo- or heterodimer formation . 
In addition , the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65 . 
However , these studies have used a limited number of known kappa B DNA motifs , and the question of the optimal DNA sequences preferred by each homodimer has not been addressed . 
Using purified recombinant p50 , p65 , and c-Rel proteins , optimal DNA-binding motifs were selected from a pool of random oligonucleotides . 
Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins , and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins . 
Contrary to previous assumptions , we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers . 
Differential binding affinities were also obtained with p50- and c-Rel-selected sequences . 
Using either a p50- or p65-selected kappa B motif , which displayed differential binding with respect to the other protein , little to no binding was observed with the heterodimeric NF-kappa B complex . 
Similarly , in transfection experiments in which the selective kappa B binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct , the p65- and p50-selected motifs were activated only in the presence of p65 and p50\/65 ( a chimeric protein with the p50 DNA binding domain and p65 activation domain ) expression vectors , respectively , and neither demonstrated a significant response to stimuli that induce NF-kappa B activity . 
These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappa B complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically , depending on the specific kappa B motifs present . 
Regulation of c-jun expression during induction of monocytic differentiation by okadaic acid . 
The present work has examined the effects of okadaic acid , an inhibitor of type 1 and 2A protein phosphatases , on the regulation of c-jun expression during monocytic differentiation of U-937 leukemia cells . 
The results demonstrate that okadaic acid treatment is associated with induction of a differentiated monocyte phenotype characterized by : ( a ) growth arrest ; ( b ) increases in Mac-1 cell surface antigen expression ; ( c ) down-regulation of c-myc transcripts ; and ( d ) induction of tumor necrosis factor gene expression . 
This induction of monocytic differentiation was associated with transient increases in c-jun mRNA levels , which were maximal at 6 h . 
Similar effects were obtained for the c-fos gene . 
Run-on analysis demonstrated detectable levels of c-jun transcription in U-937 cells and that this rate is increased approximately 40-fold following okadaic acid exposure . 
c-jun mRNA levels were superinduced in cells treated with both okadaic acid and cycloheximide , whereas inhibition of protein synthesis had little , if any , effect on okadaic acid-induced c-jun transcription . 
The half-life of c-jun mRNA was similar ( 45-50 min ) in both untreated and okadaic acid-induced cells . 
In contrast , treatment with both okadaic acid and cycloheximide was associated with stabilization ( t 1\/2 = 90 min ) of c-jun transcripts . 
Taken together , these findings indicate that the induction of c-jun transcription by okadaic acid is controlled primarily by a transcriptional mechanism . 
Since previous studies have demonstrated that the c-jun gene is autoinduced by Jun\/AP-1 , we also studied transcription of c-jun promoter ( positions -132\/+170 ) -reporter gene constructs with and without a mutated AP-1 element . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors . 
A novel B cell-restricted activity , required for high levels of octamer\/Oct-dependent transcription from an immunoglobulin heavy chain ( IgH ) promoter , was detected in an in vitro system consisting of HeLa cell-derived extracts complemented with fractionated B cell nuclear proteins . 
The factor responsible for this activity was designated Oct coactivator from B cells ( OCA-B ) . 
OCA-B stimulates the transcription from an IgH promoter in conjunction with either Oct-1 or Oct-2 but shows no significant effect on the octamer\/Oct-dependent transcription of the ubiquitously expressed histone H2B promoter and the transcription of USF- and Sp1-regulated promoters . 
Taken together , our results suggest that OCA-B is a tissue- , promoter- , and factor-specific coactivator and that OCA-B may be a major determinant for B cell-specific activation of immunoglobulin promoters . 
In light of the evidence showing physical and functional interactions between Oct factors and OCA-B , we propose a mechanism of action for OCA-B and discuss the implications of OCA-B for the transcriptional regulation of other tissue-specific promoters . 
Characterization of a novel T lymphocyte protein which binds to a site related to steroid\/thyroid hormone receptor response elements in the negative regulatory sequence of the human immunodeficiency virus long terminal repeat . 
We have previously identified a T lymphocyte protein which binds to a site within the LTR of the human immunodeficiency virus type 1 ( HIV-1 ) and exerts an inhibitory effect on virus gene expression . 
The palindromic site ( site B ) recognized by this protein is related to the palindromic binding sites of members of the steroid\/thyroid hormone receptor family . 
Here we characterize the T cell protein binding to this site as a 100 kD protein which is most abundant in T cells and which binds to site B as a 200 kD complex . 
This protein is distinct from other members of the steroid\/thyroid hormone receptor family including the COUP protein which has a closely related DNA binding specificity . 
Reticuloendotheliosis virus long terminal repeat elements are efficient promoters in cells of various species and tissue origin , including human lymphoid cells . 
Promiscuous transcriptional activity of the reticuloendotheliosis virus ( REV ) long terminal repeat ( LTR ) was detected in transient expression assays using LTR-chloramphenicol acetyltransferase-encoding gene chimeras , and cells of diverse species and tissue type ; levels of expression from two different REV LTRs correlate with reports of pathogenicity of the respective viruses in vivo . 
REVs do not encode a transactivator targeted to the viral LTR , and cells infected with Marek 's disease virus , a herpesvirus with an overlapping host range , do not express factors that preferentially enhance expression from REV or avian sarcoma\/leukemia virus LTRs . 
REV LTRs work efficiently in human lymphoid cells , and are viable alternatives to promoters commonly used for expression of cloned genes . 
They may also prove useful in the identification of new , ubiquitous cellular transcription factors . 
Phorbol ester reduces constitutive nuclear NF kappa B and inhibits HIV-1 production in mature human monocytic cells . 
NF kappa B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic leukemias . 
There is little information available on the response of NF kappa B to cytokines in normal human monocytes . 
We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus ( HIV-1 ) long terminal repeat , which contains a tandem repeat of the NF kappa B binding sequence , as a probe in a gel retardation assay to study this transcription factor . 
Using this assay , we have detected NF kappa B in extracts of nuclei from normal human monocytes . 
Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate ( TPA ) for 4-24 h caused the complete disappearance of NF kappa B from nuclear extracts of monocytes . 
A similar result was obtained with the mature monocytic leukemia cell line THP-1 . 
The constitutive transcription factor SP1 was unaffected by addition of TPA . 
The disappearance of NF kappa B from the nucleus was concentration dependent between 10 and 50 ng\/ml of phorbol ester . 
In THP-1 cells , TPA also induced a new , faster-migrating NF kappa B species not induced in monocytes . 
Protein kinase C inhibitor staurosporine , but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004 , also dramatically reduced constitutive levels of nuclear NF kappa B . 
Finally , TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication , as determined by reverse transcriptase assays , in a concentration-dependent manner . 
These results are in striking contrast to the increase in nuclear NF kappa B and HIV-1 replication induced by phorbol esters in promonocytic leukemia cells U937 and HL-60 , and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes . 
Targeted degradation of c-Fos , but not v-Fos , by a phosphorylation-dependent signal on c-Jun . 
The proto-oncogene products c-Fos and c-Jun heterodimerize through their leucine zippers to form the AP-1 transcription factor . 
The transcriptional activity of the heterodimer is regulated by signal-dependent phosphorylation and dephosphorylation events . 
The stability of c-Fos was found to also be controlled by intracellular signal transduction . 
In transient expression and in vitro degradation experiments , the stability of c-Fos was decreased when the protein was dimerized with phosphorylated c-Jun . 
c-Jun protein isolated from phorbol ester-induced cells did not target c-Fos for degradation , which suggests that c-Fos is transiently stabilized after stimulation of cell growth . 
v-Fos protein , the retroviral counterpart of c-Fos , was not susceptible to degradation targeted by c-Jun . 
The use of interferon-gamma-treated U937 cells in chemiluminescence assays 0 to detect red cell , platelet and granulocyte antibodies of potential clinical significance . 
The chemiluminescent ( CL ) response of interferon-gamma-treated U937 ( IFN-U937 ) cells to sensitized target cells has been used to detect red cell , platelet and granulocyte antibodies . 
A clone of U937 cells was selected which expressed Fc receptor I ( Fc gamma RI ) and which , after incubation with IFN-gamma for 72 h , was capable of generating high levels of lucigenin-enhanced CL . 
The CL responses of IFN-U937 cells and peripheral blood human monocytes to sensitized red cells , platelets or granulocytes were then compared . 
Assays using monocytes or IFN-U937 cells were of comparable sensitivity for detection of antibodies against all three types of target cell . 
In addition , the use of IFN-U937 cells reduced interassay variation and simplified assay performance . 
The potential clinical usefulness of these CL assays was suggested by the ability of both monocytes and IFN-U937 cells to respond to red cells , platelets or granulocytes sensitized with sera from pregnant women whose babies had either haemolytic disease of the newborn ( HDN ) , alloimmune thrombocytopenia or alloimmune neutropenia respectively . 
In addition , monocytes and IFN-U937 cells both responded to red cells sensitized with antibodies against a variety of specificities of assumed ( although not documented ) clinical significance for blood transfusion recipients . 
In contrast , monocytes and IFN-U937 cells responded only weakly to red cells sensitized with either anti-D in sera from mothers of babies unaffected by HDN , or with antisera containing high titre antibodies with specificities not normally associated with significantly reduced red cell survival . 
Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B- lymphocyte precursors { published erratum appears in Proc Natl Acad Sci U S A 1993 Apr 15 ; 90 ( 8 ) : 3775 } 
Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway . 
We show that in B-lymphocyte precursors , irradiation with gamma-rays leads to ( i ) stimulation of phosphatidylinositol turnover ; ( ii ) downstream activation by covalent modification of multiple serine-specific protein kinases , including protein kinase C ; and ( iii ) activation of nuclear factor kappa B . 
All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A . 
Thus , tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors . 
Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation . 
Interleukin-4 inhibits the lipopolysaccharide-induced expression of c-jun and c-fos messenger RNA and activator protein-1 binding activity in human monocytes . 
We studied the effect of interleukin-4 ( IL-4 ) on the lipopolysaccharide ( LPS ) induction of two immediate early genes c-fos and c-jun . 
These genes encode proteins that form the dimeric complex activator protein-1 ( AP-1 ) , which is active as a transcriptional factor . 
Maximal accumulation of either c-fos and c-jun messenger RNA ( mRNA ) occurred 30 minutes after LPS addition . 
When cells were treated with IL-4 for 5 hours before LPS activation , both the c-fos and the c-jun mRNA expression was decreased . 
The inhibition of c-fos and c-jun expression by IL-4 in LPS-treated cells was shown to be due to a lower transcription rate of the c-fos and c-jun genes . 
IL-4 did not affect the stability of the c-fos and c-jun transcripts . 
Finally , using electrophoretic mobility shift assays , evidence was obtained that IL-4 inhibits LPS-induced expression of AP-1 protein . 
These data indicate that IL-4 suppresses the induction of transcription factors in human activated monocytes . 
The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage . 
We have isolated cDNA clones of myeloid differentiation primary response ( MyD ) genes , activated in the absence of de novo protein synthesis following induction for differentiation along either the macrophage or granulocyte lineage in human myeloblastic leukemia HL-60 cells . 
One cDNA clone of a primary response gene , expressed upon macrophage differentiation , encoded for Egr-1 , a zinc finger transcription factor . 
The Egr-1 gene was observed to be transcriptionally silent in HL-60 cells , but active in U-937 and M1 cells , the latter two being predetermined for macrophage differentiation . 
Egr-1 antisense oligomers in the culture media blocked macrophage differentiation in both myeloid leukemia cell lines and normal myeloblasts . 
HL-60 cells constitutively expressing an Egr-1 transgene ( HL-60Egr-1 ) could be induced for macrophage , but not granulocyte , differentiation . 
These observations indicate that expression of Egr-1 is essential for and restricts differentiation of myeloblasts along the macrophage lineage . 
Interleukin-3 expression by activated T cells involves an inducible , T-cell-specific factor and an octamer binding protein . 
Interleukin-3 ( IL-3 ) is exclusively expressed by activated T and natural killer cells , a function that is tightly controlled both in a lineage-specific and in a stimulation-dependent manner . 
We have investigated the protein binding characteristics and functional importance of the ACT-1-activating region of the IL-3 promoter . 
This region binds an inducible , T-cell-specific factor over its 5' end , a site that is necessary for the expression of IL-3 in the absence of other upstream elements . 
Over its 3' end , it binds a factor that is ubiquitously and constitutively expressed . 
This factor is Oct-1 or an immunologically related octamer-binding protein , and it plays a role in coordinating the activity of several regulatory elements . 
These characteristics make the ACT-1 site analogous to the activating ARRE-1 site in the IL-2 promoter . 
Furthermore , and despite a lack of sequence homology , the promoters of IL-3 and IL-2 share an organizational pattern of regulatory elements that is likely to be important for the T-cell-specific expression of these genes . 
The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter . 
The Epstein-Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B-cells by EBV . 
We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP , TP1 and TP2 . 
The promoter of the TP1 gene was chosen as a model system 0 to study the molecular mechanism of EBNA2 mediated transactivation . 
To identify an EBNA2 dependent cis-acting element , various TP1 promoter-reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR1 . 
We were able to delineate an 81 bp EBNA2 responsive region between -258 and -177 relative to the TP1 RNA start site . 
The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter . 
Electrophoretic mobility shift assays revealed three specific protein-DNA complexes formed with sequences of the EBNA2 responsive element . 
Two of these were not cell type specific , but the third was detected only in EBNA2 positive cell extracts . 
Gel-shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex . 
Thus , these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis-element of the TP1 promoter . 
Protease treatment of nuclear extracts distinguishes between class II MHC X1 box DNA-binding proteins in wild-type and class II-deficient B cells . 
The X box region is critical for directing the expression of class II major histocompatibility complex genes in B lymphocytes . 
Although several class II promoter-specific DNA binding factors have been described , only the X box region factor , RFX , shows a genetic correlation with class II expression , being deficient in some B cell lines derived from patients with class II-deficient congenital immunodeficiency . 
To further evaluate the role of X box DNA-binding proteins in class II gene expression , the role of the X box region was examined in both class II-positive and -negative lymphoid cells . 
In addition to the wild-type B cell line Raji , two class II transcriptional mutant cell lines , SJO and RJ2.2.5 , and Jurkat , a class II negative T cell line , were examined . 
In contrast to wild-type B cells , neither of the class II mutant cell lines could use the X box region to direct the expression of a transiently transfected reporter gene , indicating that the X box-dependent transcriptional pathway is defective in these cells . 
The binding activity of the X1 box DNA-binding protein RFX was examined and found to be present in wild-type B cells and the mutant RJ2.2.5 but was absent in SJO and Jurkat . 
However , other X1 box-specific activities were detected in all these cell lines . 
To determine whether these different X1 box activities represented distinct DNA binding proteins or multimeric forms of the same factor ( s ) , protease treatment of the crude nuclear extracts followed by DNA-binding assays were carried out and demonstrated that B cell extracts contain at least two X1-specific factors . 
One of these cleaved products ( band 1 pk ) correlates with RFX activity . 
A similar comparison with protease-treated extracts prepared from Jurkat cells demonstrated the presence of the band 1pk activity despite an absence of the native RFX activity . 
In contrast , protease treatment and analysis of SJO extracts showed no detectable levels of the band 1pk activity . 
These results demonstrate that multiple X1 box-specific DNA-binding activities exist in all lymphoid cells , but the presence of an actively binding RFX species correlates with class II transcription . 
The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction . 
In these studies , we have identified DNA sequences and specific protein interactions necessary for transcriptional regulation of the human prointerleukin 1 beta ( proIL-1 beta ) gene . 
A cell-type-independent lipopolysaccharide ( LPS ) -responsive enhancer element located between -3757 and -2729 bp upstream from the transcription start site ( cap site ) consisted of at least six discrete subregions which were essential to the maximal induction by LPS in transfected monocytes . 
The enhancer also appeared to mediate phorbol myristate acetate induction in monocytes and IL-1 responsiveness in fibroblasts . 
Deletion and base substitution mutations along with DNA binding studies demonstrated that the enhancer contained a minimum of three functional protein binding sequences , two of which appeared to be important for gene induction . 
One of the essential proteins which bound to the enhancer was similar or identical to members of the C\/EBP family of transcription factors required for both IL-1- and LPS-specific induction of the IL-6 gene ( i.e. , the NF-IL6 proteins ) . 
When ligated to the proIL-1 beta cap site-proximal region ( located between -131 to +12 ) , both the proIL-1 beta and the simian virus 40 enhancer elements functioned more efficiently in monocytes than in HeLa cells , which are not normally competent for IL-1 beta expression . 
When ligated to the murine c-fos promoter , however , the proIL-1 beta enhancer was inducible in phorbol myristate acetate-stimulated HeLa cells , suggesting the existence of a proIL-1 beta promoter-proximal requirement for tissue specificity . 
Suppression of a cellular differentiation program by phorbol esters coincides with inhibition of binding of a cell-specific transcription factor ( NF-E2 ) to an enhancer element required for expression of an erythroid-specific gene . 
Induction by hemin increases , while induction with 12-O-tetradecanoylphorbol-13-acetate ( TPA ) represses , erythroid-specific gene expression in the human cell line K562 . 
We analyzed the effects of hemin or TPA induction on the binding and activity of transcription factors at a regulatory element found within the transcriptional regulatory sequences of many erythroid-specific genes . 
TPA induction increases the binding of ubiquitous AP-1 factors to this element . 
TPA induction inhibits the binding of the lineage limited transcription factor NF-E2 to this transcriptional control element . 
Hemin induction of K562 cells does not facilitate the binding of NF-E2 to its recognition site . 
Hemin induction appears to nonspecifically increase the expression of transiently transfected genes in K562 cells . 
Beyond this nonspecific increase in gene expression , hemin induction acts to increase the activity of the lineage limited transcription factor NF-E2 . 
The divergent effects of hemin and TPA on gene expression in K562 cells are mediated , in part , by their contrasting effects on the transcription factor NF-E2 . 
Transcriptional regulation of the pyruvate kinase erythroid-specific promoter . 
Mammal pyruvate kinases are encoded by two genes . 
The L gene produces the erythroid ( R-PK ) or the hepatic ( L-PK ) isozymes by the alternative use of two promoters . 
We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter . 
A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene . 
Within this region , we define a minimal promoter ( -62 to +54 ) that displays erythroid-specific activity and contains two DNA binding sites . 
One , located at -50 , binds members of the CCACC\/Sp1 family and the other , located at -20 , binds the erythroid factor GATA-1 . 
Although the -20 GATA binding site ( AGATAA ) is also a potential TFIID binding site , it does not bind TFIID . 
Furthermore , the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity . 
Mutations and deletions of both sites indicate that only the association of CCACC\/Sp1 and GATA binding sites can drive efficient and tissue-specific expression of this R-PK minimal promoter . 
Finally , by co-transfection experiments , we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells . 
Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element ( HS-40 ) : functional role of specific nuclear factor-DNA complexes . 
We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element ( HS-40 ) located 40 kb upstream of the zeta 2 globin gene . 
It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells , an erythroid cell line of embryonic and\/or fetal origin . 
Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids , they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter . 
Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104 , respectively . 
The functional domains of HS-40 were also mapped . 
Bal 31 deletion mapping data suggested that one GATA-1 motif , one GT motif , and two NF-E2\/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter . 
Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2\/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor . 
Finally , we did genomic footprinting of the HS-40 enhancer region in K562 cells , adult nucleated erythroblasts , and different nonerythroid cells . 
All sequence motifs within the functional core of HS-40 , as mapped by transient expression analysis , appeared to bind a nuclear factor ( s ) in living K562 cells but not in nonerythroid cells . 
On the other hand , only one of the apparently nonfunctional sequence motifs was bound with factors in vivo . 
In comparison to K562 , nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region . 
These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal\/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer ( HS-40 ) and the globin promoters . 
NF-kappa B controls expression of inhibitor I kappa B alpha : evidence for an inducible autoregulatory pathway . 
The eukaryotic transcription factor nuclear factor-kappa B ( NF-kappa B ) participates in many parts of the genetic program mediating T lymphocyte activation and growth . 
Nuclear expression of NF-kappa B occurs after its induced dissociation from its cytoplasmic inhibitor I kappa B alpha . 
Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-kappa B is associated with both the degradation of performed I kappa B alpha and the activation of I kappa B alpha gene expression . 
Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B . 
Association of the newly synthesized I kappa B alpha with p65 restores intracellular inhibition of NF-kappa B DNA binding activity and prolongs the survival of this labile inhibitor . 
Together , these results show that NF-kappa B controls the expression of I kappa B alpha by means of an inducible autoregulatory pathway . 
The Sp1 transcription factor binds the CD11b promoter specifically in myeloid cells in vivo and is essential for myeloid-specific promoter activity . 
The myeloid integrin CD11b is expressed selectively on the surface of mature macrophages , monocytes , neutrophils , and natural killer cells . 
Lineage-specific expression is controlled at the level of mRNA transcription . 
Recent isolation of the CD11b promoter shows that 92 base pairs ( bp ) of 5'-flanking DNA are sufficient to direct myeloid-specific expression of a reporter gene . 
To characterize regulatory sequences important for promoter activity , we performed linker scanning analysis of the 92-bp CD11b promoter and demonstrate that a sequence at bp -60 is essential for CD11b promoter activity . 
We show that this sequence binds the transcription factor Sp1 in vitro and in vivo . 
In vivo the Sp1 site is bound only in myeloid ( U937 ) cells , not in cervical carcinoma ( HeLa ) cells . 
In addition , the macrophage transcription factor PU.1 binds the CD11b promoter in vitro and in vivo close to the Sp1 site . 
We propose a model in which binding of a myeloid-specific factor ( PU.1 ) allows a general factor ( Sp1 ) to bind in a tissue-specific fashion thereby contributing to the myeloid-specific expression of CD11b . 
Functional antagonism between vitamin D3 and retinoic acid in the regulation of CD14 and CD23 expression during monocytic differentiation of U-937 cells . 
1,25 alpha-Dihydroxicholecalciferol ( VitD3 ) and retinoic acid ( RA ) are important regulators of the proliferation and differentiation of several cell types . 
This paper describes how the expression of the monocyte-macrophage Ag , CD14 , and the low affinity Fc receptor for IgE , CD23 , were inversely regulated during VitD3- and RA-induced monocytic differentiation of human U-937 monoblasts . 
PMA induced the expression of both CD14 and CD23 mRNA and protein . 
Exposure to VitD3 rapidly induced the de novo expression of CD14 mRNA and protein . 
The addition of cycloheximide completely blocked the VitD3 induction of CD14 mRNA expression , indicating that the induction was dependent on ongoing protein synthesis . 
While inducing CD14 expression , VitD3 concomitantly suppressed the basal , PMA- , and RA-inducible CD23 expression in a dose-dependent manner . 
In contrast , U-937 cells induced by RA strongly increased their expression of CD23 mRNA and protein , whereas they completely lacked detectable CD14 cell surface or mRNA expression . 
Furthermore , the VitD3- and the PMA-induced CD14 expression was inhibited as a temporal consequence of the RA-induced differentiation . 
The results suggest that there exists a functional antagonism between VitD3 and RA that may have important implications for the regulation of certain immune and inflammatory responses through their inverse effects on CD14 and CD23 gene expression . 
Lymphocytes from the site of disease suggest 0 adenovirus is one cause of persistent or recurrent inflammatory arthritis . 
The assessment of synovial lymphocyte reactivity to adenovirus antigen stimulation was undertaken in patients with persistent or recurrent inflammatory arthritis . 
The 3H-thymidine uptake procedure was employed , incorporating multiple microbiological antigens . 
Five patients were found with repeated maximal responses to adenovirus antigen ; in one of these adenovirus nucleotide sequences were present in a synovial biopsy specimen . 
It is concluded that adenovirus may be one cause of persistent or recurrent inflammatory arthritis . 
HIV-1 Nef protein inhibits the recruitment of AP-1 DNA-binding activity in human T-cells . 
The human immunodeficiency virus type 1 long terminal repeat , HIV-1-LTR , contains binding sites for several cellular transcription factors which contribute to HIV-1 gene expression . 
Our previous studies on the function of the HIV-1-encoded Nef protein suggested that Nef may be an inhibitor HIV-1 transcription . 
To determine whether Nef affects the binding of cellular factors implicated in HIV-1 regulation , 32P-labeled oligonucleotides corresponding to the binding sites were incubated with nuclear extracts prepared from Nef-expressing T-cell lines that were not stimulated or were stimulated with T-cell mitogens . 
We found that Nef inhibited the recruitment of AP-1 DNA-binding activity in mitogen-stimulated human T-cells . 
Additionally , Nef expressing cells were transiently transfected with a plasmid in which HIV-1 AP-1 DNA recognition sequences were cloned downstream of the chloramphenicol acetyltransferase ( CAT ) gene . 
Mitogen-mediated transcriptional activation of the CAT gene in this construct was inhibited in Nef-expressing cells but not in control cells . 
These studies suggest that , by inhibiting AP-1 activation , Nef may play a role in regulating HIV-1 gene expression in infected T-cells 
A chimeric type II \/ type I interleukin-1 receptor can mediate interleukin-1 induction of gene expression in T cells . 
The type I interleukin-1 receptor ( IL-1R ) is capable of transducing a signal resulting in promoter activation in T cells . 
This signal transduction is dependent on the cytoplasmic domain , which consists of 213 amino acids . 
In contrast to the type I receptor , the type II IL-1R has a small cytoplasmic tail , and it is not clear whether this receptor is a signal-transducing or a regulatory molecule . 
Here we report that the type II IL-1R does not mediate gene activation in Jurkat cells . 
However , a hybrid receptor composed of the extracellular and transmembrane regions of the human type II interleukin-1 fused to the cytoplasmic domain of the human type I IL-1R was capable of transducing a signal across the membrane resulting in a pattern of gene activation identical to that mediated by the type I IL-1R . 
Our results indicated that the extracellular domain of the type II IL-1R was capable of functionally interacting with interleukin-1 and transmitting the resulting signal to a heterologous cytoplasmic domain . 
Cloning and functional characterization of early B-cell factor , a regulator of lymphocyte-specific gene expression . 
Early B-cell factor ( EBF ) was identified previously as a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific mb-1 gene . 
Partial amino acid sequences obtained from purified EBF were used to isolate cDNA clones , which by multiple criteria encode EBF . 
The recombinant polypeptide formed sequence-specific complexes with the EBF-binding site in the mb-1 promoter . 
The cDNA hybridized to multiple transcripts in pre-B and B-cell lines , but transcripts were not detected at significant levels in plasmacytoma , T-cell , and nonlymphoid cell lines . 
Expression of recombinant EBF in transfected nonlymphoid cells strongly activated transcription from reporter plasmids containing functional EBF-binding sites . 
Analysis of DNA binding by deletion mutants of EBF identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors . 
DNA-binding assays with cotranslated wild-type and truncated forms of EBF indicated that the protein interacts with its site as a homodimer . 
Deletions delineated a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins . 
Together , these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression . 
Oxidoreductive regulation of nuclear factor kappa B . 
Involvement of a cellular reducing catalyst thioredoxin . 
We have investigated an oxidoreductive regulatory pathway for the DNA binding activity of a pleiotropic cellular transcription factor , nuclear factor kappa B ( NF kappa B ) , has been investigated by using NF kappa B prepared from the nucleus and the cytosol of the primary human T lymphocytes . 
We show that a cellular reducing catalyst thioredoxin ( Trx ) plays a major role in activation of the DNA binding of NF kappa B in vitro and stimulation of transcription from the NF kappa B-dependent gene expression . 
We demonstrate evidence suggesting that redox regulation of NF kappa B by Trx might be exerted at a step after dissociation of the inhibitory molecule I kappa B , a cytosolic-anchoring protein for NF kappa B . 
To examine the effect of Trx in intact cells , we performed transient assay with a chloramphenicol acetyltransferase-expressing plasmid under the control of human immunodeficiency virus ( HIV ) long terminal repeat and an effector plasmid expressing human Trx . 
The promoter activity from HIV long terminal repeat was greatly augmented by co-transfecting the Trx-expressing plasmid , whose effect was dependent on the NF kappa B-binding sites . 
These findings have suggested that cysteine residue ( s ) of NF kappa B might be involved in the DNA-recognition by NF kappa B and that the redox control mechanism mediated by Trx might have a regulatory role in the NF kappa B-mediated gene expression . 
These results may also provide a clue to understanding of the molecular process of AIDS pathogenesis and its possible biochemical intervention . 
Synergism between the CD3 antigen- and CD2 antigen-derived signals . 
Exploration at the level of induction of DNA-binding proteins and characterization of the inhibitory activity of cyclosporine . 
We have demonstrated earlier that the crosslinkage of the CD3\/TCR complex with the CD2 antigen results in the proliferation of normal human T cells . 
The effect of this synergism was perceptible at the level of induction of the IL-2 gene , a process critical for T cell growth . 
To further understand the molecular and nuclear basis for this synergism , we have explored the induction of DNA-binding proteins in highly purified normal human T cells signaled via the CD3 and\/or CD2 proteins . 
The effect of transmembrane signaling of T cells with ionomycin , and\/or sn-1,2 dioctanoyl glycerol , was also determined . 
The emergence of nuclear binding proteins was investigated using interleukin-2 sequence specific oligonucleotide probes in the electrophoretic mobility shift assay . 
Our studies demonstrate for the first time that CD3 antigen-derived signals and CD2 antigen-derived signals are synergistic in inducing the emergence of transcription factors that bind to the NF-AT1 , AP-1 , and NF-kB sites located in the promoter\/enhancer region of the IL-2 gene . 
Moreover , cyclosporine , at concentrations readily accomplished in clinical practice , was found to inhibit the emergence of these DNA-binding proteins in normal human T cells signaled via cell surface proteins implicated in antigen-dependent T cell activation and in T cells stimulated by mobilization of cellular calcium and activation of protein kinase C . 
Regulation of the beta-globin locus . 
Transcription of the human beta-globin gene cluster depends upon upstream regulatory sequences , which are collectively termed the locus control region . 
Recent studies have provided new insights into how the individual genes of the cluster are regulated through development . 
The crux of transcriptional activation is how the locus control region communicates with the gene-proximal regulatory elements . 
Induction of CD8 antigen and suppressor activity by glucocorticoids in a CEM human leukemic cell clone . 
The relationship between glucocorticoid effect and regulation of cell surface antigens was investigated in two models of leukemic cell lines , CEM C7 denoted ( r+ , ly+ ) and CEM C1 ( r+ , ly- ) . 
The reactivity of murine monoclonal antibodies , anti-CD4-FITC , anti-CD8-FITC , anti-CD2-FITC and anti-calla-FITC , were analyzed using flow cytometry . 
The suppressor function was determined using -LCB-3H-RCB-thymidine incorporation into phytohemagglutinin-activated peripheral blood lymphocytes . 
Dexamethasone treatment of a human leukemic cell clone CEM C7 caused an increase in a subset of cells expressing the surface antigen CD8 , which is present on suppressor and cytotoxic T-lymphocytes . 
By comparison , there was no modification of the expression of CD4 antigen , which is expressed at high levels in these cells . 
After two days of treatment with 5 x 10-LRB--8-RRB--SP-M dexamethasone , CEM C7 cells showed a two-fold increase in suppressor activity compared to untreated cells . 
In contrast , there was no regulation by glucocorticoids of either the CD8 or CD4 antigens in the leukemic clone CEM C1 . 
Furthermore , no modification of the suppressor function in CEM C1 cells by dexamethasone was observed . 
In the human leukemic cells studied here , the ability to induce CD8 antigen expression in a CD4+ cells correlates with the ability to induce cell lysis in a glucocorticoid receptor positive cell population . 
Defective translocation of protein kinase C in multidrug-resistant HL-60 cells confers a reversible loss of phorbol ester-induced monocytic differentiation . 
Previous studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters . 
This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype . 
The present studies have examined the effects of vincristine-selected , multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate ( TPA ) -induced HL-60 cell differentiation . 
The results demonstrate that multidrug-resistant HL-60 cells , designated HL-60\/vinc , fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation . 
By contrast , treatment of HL-60\/vinc cells with okadaic acid , an inhibitor of serine\/threonine protein phosphatases , induces c-jun transcription , growth arrest , and expression of the c-fms gene . 
Studies were also performed with an HL-60\/vinc revertant ( HL-60\/vinc/R ) line that has regained partial sensitivity to vincristine . 
The finding that HL-60\/vinc/R cells respond to TPA with induction of a monocytic phenotype , but not c-jun expression , suggests that c-jun induction is not obligatory for monocytic differentiation . 
Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60\/vinc and HL-60\/vinc/R cells , whereas c-fos expression is attenuated in the HL-60\/vinc line . 
Since TPA activates protein kinase C ( PKC ) , we examined translocation of PKC from the cytosol to the membrane fraction . 
Although HL-60 and HL-60\/vinc/R cells demonstrated translocation of PKC activity , this subcellular redistribution was undetectable in HL-60\/vinc cells . 
Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60\/vinc cells , but not in response to okadaic acid . 
Taken together , these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun\/fos early response gene expression . 
Regulation of lymphoid-specific immunoglobulin mu heavy chain gene enhancer by ETS-domain proteins . 
The enhancer for the immunoglobulin mu heavy chain gene ( IgH ) activates a heterologous gene at the pre-B cell stage of B lymphocyte differentiation . 
A lymphoid-specific element , microB , is necessary for enhancer function in pre-B cells . 
A microB binding protein is encoded by the PU.1\/Spi-1 proto-oncogene . 
Another sequence element , microA , was identified in the mu enhancer that binds the product of the ets-1 proto-oncogene . 
The microA motif was required for microB-dependent enhancer activity , which suggests that a minimal B cell-specific enhancer is composed of both the PU.1 and Ets-1 binding sites . 
Co-expression of both PU.1 and Ets-1 in nonlymphoid cells trans-activated reporter plasmids that contained the minimal mu enhancer . 
These results implicate two members of the Ets family in the activation of IgH gene expression . 
Cell cycle analysis of E2F in primary human T cells reveals novel E2F complexes and biochemically distinct forms of free E2F . 
The transcription factor E2F activates the expression of multiple genes involved in cell proliferation , such as c-myc and the dihydrofolate reductase gene . 
Regulation of E2F involves its interactions with other cellular proteins , including the retinoblastoma protein ( Rb ) , the Rb-related protein p107 , cyclin A , and cdk2 . 
We undertook a detailed analysis of E2F DNA-binding activities and their cell cycle behavior in primary human T cells . 
Three E2F DNA-binding activities were identified in resting ( G0 ) T cells with mobilities in gel shift assays distinct from those of previously defined E2F complexes . 
One of these activities was found to be a novel , less abundant , Rb-E2F complex . 
The most prominent E2F activity in resting T cells ( termed complex X ) was abundant in both G0 and G1 but disappeared as cells entered S phase , suggesting a possible role in negatively regulating E2F function . 
Complex X could be dissociated by adenovirus E1A with a requirement for an intact E1A conserved region 2 . 
However , X failed to react with a variety of antibodies against Rb or p107 , implicating the involvement of an E1A-binding protein other than Rb or p107 . 
In addition to these novel E2F complexes , three distinct forms of unbound ( free ) E2F were resolved in gel shift experiments . 
These species showed different cell cycle kinetics . 
UV cross-linking experiments suggested that a distinct E2F DNA-binding protein is uniquely associated with the S-phase p107 complex and is not associated with Rb . 
Together , these results suggest that E2F consists of multiple , biochemically distinct DNA-binding proteins which function at different points in the cell cycle . 
Antisense oligonucleotides to the p65 subunit of NF-kappa B block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes . 
NF-kappa B is a pleiotropic regulator of a variety of genes implicated in the cellular response to injury . 
This function has been attributed to the coordinated binding of subunits of NF-kappa B to distinct regions of the promoter elements of numerous genes , including cytokines , growth factor receptors , and adhesion molecules . 
Antisense phosphorothioate oligonucleotides to the p50 and p65 subunits of the NF-kappa B complex were used to define the physiologic role of this transcription factor in resting and stimulated granulocytes . 
A reduction in the expression of p65 was produced by treatment with the phosphorothioate antisense oligodeoxynucleotide . 
This reduction was accompanied by rapid changes in the cellular adhesion of dimethyl sulfoxide-differentiated HL-60 leukemia cells stimulated by 12-O-tetradecanoylphorbol 13-acetate ( TPA ) . 
These effects were characterized by a marked reduction in CD11b integrin expression on the surface of treated cells . 
Furthermore , the p65 antisense oligomer effectively abolished an upregulation of CD11b that was produced by formyl-met-leu-phe and TPA . 
However , the p65 antisense phosphorothioate oligodeoxynucleotide had no significant effect on the production of reactive oxygen intermediates or on phagocytosis by these cells . 
These findings indicate that antisense oligomers to p65 can be used to define the role of NF-kappa B in the activation pathways of neutrophils . 
Human T cell transcription factor GATA-3 stimulates HIV-1 expression . 
A family of transcriptional activating proteins , the GATA factors , has been shown to bind to a consensus motif through a highly conserved C4 zinc finger DNA binding domain . 
One member of this multigene family , GATA-3 , is most abundantly expressed in T lymphocytes , a cellular target for human immunodeficiency virus type 1 ( HIV-1 ) infection and replication . 
In vitro DNase I footprinting analysis revealed six hGATA-3 binding sites in the U3 region ( the transcriptional regulatory domain ) of the HIV-1 LTR . 
Cotransfection of an hGATA-3 expression plasmid with a reporter plasmid whose transcription is directed by the HIV-1 LTR resulted in 6- to 10-fold stimulation of LTR-mediated transcription , whereas site specific mutation of these GATA sites resulted in virtual abrogation of the activation by hGATA-3 . 
Further , deletion of the hGATA-3 transcriptional activation domain abolished GATA-dependent HIV-1 trans-activation , showing that the stimulation of viral transcription observed is a direct effect of cotransfected hGATA-3 . 
Introduction of the HIV-1 plasmids in which the GATA sites have been mutated into human T lymphocytes also caused a significant reduction in LTR-mediated transcription at both the basal level and in ( PHA- plus PMA- ) stimulated T cells . 
These observations suggest that in addition to its normal role in T lymphocyte gene regulation , hGATA-3 may also play a significant role in HIV-1 transcriptional activation . 
Tumor necrosis factor receptor expression and signal transduction in HIV-1-infected cells . 
OBJECTIVE : To examine the inter-relationship between HIV-1 infection and the cell surface receptors for tumor necrosis factor ( TNF ) -alpha , an immunoregulatory cytokine that can enhance HIV-1 replication . 
DESIGN : Infected promyelocytic and promonocytic cells were examined because they normally express both types of TNF receptors . 
METHODS : TNF receptor surface expression was determined by specific monoclonal antibody recognition and flow cytometry , and signal transduction was detected by gel shift analysis . 
HIV-1 activation and expression was quantitated by reverse transcriptase assay . 
RESULTS : In the OM-10.1 promyelocytic model of chronic infection , TNF-alpha-induced HIV-1 expression also resulted in a substantial increase in 75 kd TNF receptor ( TR75 ) expression although 55 kD TNF receptor ( TR55 ) levels were not dramatically altered . 
A series of uninfected parental HL-60 subclones all reduced TR75 surface expression in response to TNF-alpha treatment . 
Enhanced TR75 expression on OM-10.1 cells followed the same TNF-alpha-dose dependency as that observed for HIV-1 production . 
An increase in TR75 expression was also evident during the peak of an acute HIV-1 infection of HL-60 promyelocytes . 
Although TR55 expression was unaltered during TNF-alpha-induced HIV activation , this receptor was still involved in the viral activation process . 
Antibody cross-linking of TR55 , in the absence of exogenous TNF-alpha , induced maximal HIV-1 expression , an up-modulation of surface TR75 , and nuclear NF-kappa B activity in OM-10.1 cultures . 
Surprisingly , this was the case even when an antagonistic anti-TR55 antibody was used . 
Anti-TR55 antibody cross-linking in chronically infected U1 promonocytic cultures could only partially substitute for TNF-alpha-induced HIV-1 expression . 
CONCLUSIONS : Our results demonstrated that HIV-1 infection can selectively influence the surface expression of TNF receptors , potentially influencing its own expression and altering normal immunoregulatory signal transduction . 
Inhibition of HIV-1 latency reactivation by dehydroepiandrosterone ( DHEA ) and an analog of DHEA . 
The initial infection with human immunodeficiency virus type 1 ( HIV-1 ) in most individuals usually results in the establishment of a latent or chronic infection before eventual progression toward acquired immunodeficiency syndrome . 
HIV-1 can also establish a latent or persistent infection in some T cell lines that show minimal constitutive virus expression . 
However , activation of the T cell lines leading to enhanced HIV-1 replication can be induced by antigens , mitogens , and cytokines ( tumor necrosis factor alpha { TNF-alpha } , interleukin 1 , and interleukin-2 ) . 
Various gene products from other viruses ( HTLV-1 , HSV , EBV , CMV , HBV , and HHV-6 ) can also enhance HIV-1 long terminal repeat ( LTR ) -driven reporter gene activity . 
On the basis of these observations , it has been proposed that reactivation of latent HIV-1 harbored in chronically infected T lymphocytes , monocytes , or macrophages plays an important role in the pathogenesis of AIDS . 
So far , there are no drugs or therapy available that can provide protection against HIV-1 latency reactivation . 
ACH-2 , derived from a human T cell line ( CEM ) , is chronically infected with HIV-1 , with low levels of constitutive virus expression . 
ACH-2 can be converted to productive infection by stimulation of the cells with 12-O-tetradecanoylphorbol-13-acetate ( TPA ) , mitogen or cytokines ( TNF-alpha ) , or infection with HSV . 
Therefore the ACH-2 cell line is a good candidate for studying the effects of drugs on HIV-1 activation . 
Previously , we have reported that DHEA and synthetic analogs of DHEA can be modest inhibitors of HIV-1 IIIB replication in phytohemagglutinin-stimulated peripheral blood lymphocyte cultures . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
Chlorinated dibenzo-p-dioxins and dibenzofurans and the human immune system . 
1 . Blood cell receptors in volunteers with moderately increased body burdens . 
Using monoclonal antibodies ( mAbs ) and flow cytometry , we studied a variety of surface receptors on lymphocyte subpopulations of workers with moderately increased body burdens of 2,3,7,8-tetrachlorodibenzo-p-dioxin ( TCDD ) and of other polychlorinated dibenzo-p-dioxins and dibenzofurans ( PCDD\/PCDF ) , expressed here as International-Toxicity Equivalencies ( I-TE ) . 
The hypothesis 0 to be tested was whether or not humans exhibit a similar susceptibility to PCDDs\/PCDFs with respect to the surface receptors found previously to respond to small doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin ( TCDD ) in Callithrix jacchus . 
These are : helper-inducer ( memory ) T cells ( CD4+CD45R0+CD45RA-CD29highCD11a+ ) , CD20+ B cells , and cytotoxic T cells ( CD8+CD56+\/CD57+ ) . 
Furthermore , 68 triple-labellings with mAbs were performed on the cells of each volunteer to possibly generate further hypotheses . 
It was evaluated whether any of the variables might be used as a biomarker of effects for this class of compounds . 
There were two main goals : ( 1 ) to evaluate whether workers with a moderately increased PCDD\/PCDF-body burden { 25-140 ppt TCDD or 104-522 ppt I-TE in blood fat } exhibit changes in the surface receptors of white blood cells , as observed in previous studies in non-human primates , and ( 2 ) to clarify whether persons at the upper range { 10-23 ppt TCDD or 30-90 ppt I-TE in blood fat } of the body burden reference values of a not particularly exposed population show detectable deviations in these immunological variables , when compared with persons at the lower and medium range { 1-3 ppt TCDD or 9-29 ppt I-TE } of these body burden reference values . 
Regression analysis of our data revealed slight trends for some of the biomarkers ( e.g. CD45R0+ ) . 
With one exception , these were all increases . 
None of the alterations observed are of medical relevance . 
The slight increase in the percentage of CD4+CD45R0+ cells remained significant even after covariant analysis taking age-related changes into account . 
Altogether , the data do not provide any evidence 0 to support an assumption that moderately increased body burdens of PCDDs\/PCDFs in adults induce decreases in the cellular components of the human immune system . 
Adult humans certainly are less susceptible to this action of PCDDs\/PCDFs than adolescent Callithrix jacchus . 
The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor . 
The macrophage colony-stimulating factor ( M-CSF ) receptor is expressed in a tissue-specific fashion from two distinct promoters in monocytes\/macrophages and the placenta . 
In order to further understand the transcription factors which play a role in the commitment of multipotential progenitors to the monocyte\/macrophage lineage , we have initiated an investigation of the factors which activate the M-CSF receptor very early during the monocyte differentiation process . 
Here we demonstrate that the human monocytic M-CSF receptor promoter directs reporter gene activity in a tissue-specific fashion . 
Since one of the few transcription factors which have been implicated in the regulation of monocyte genes is the macrophage- and B-cell-specific PU.1 transcription factor , we investigated whether PU.1 binds and activates the M-CSF receptor promoter . 
Here we demonstrate that both in vitro-translated PU.1 and PU.1 from nuclear extracts bind to a specific site in the M-CSF receptor promoter just upstream from the major transcription initiation site . 
Mutations in this site which eliminate PU.1 binding decrease M-CSF receptor promoter activity significantly in macrophage cell lines only . 
Furthermore , PU.1 transactivates the M-CSF receptor promoter in nonmacrophage cells . 
These results suggest that PU.1 plays a major role in macrophage gene regulation and development by directing the expression of a receptor for a key macrophage growth factor . 
Rhabdomyosarcomas do not contain mutations in the DNA binding domains of myogenic transcription factors . 
Skeletal myogenesis is regulated by a group of transcription factors ( MyoD , myogenin , myf5 , and myf6 ) that are " basic helix-loop-helix " proteins that bind to the promoters of muscle-specific genes and promote their expression . 
We have previously shown that after a mutation of Leu122 to Arg the DNA binding basic domain of MyoD confers c-myc-like functional characteristics to the protein . 
In this study we used single-strand conformation polymorphism analysis to determine whether such mutations occur naturally in rhabdomyosarcomas . 
We have found that the basic domains of all the myogenic factors remain unaltered in rhabdomyosarcomas . 
Selection against such mutations may be the result of functional redundancy of these myogenic transcription factors . 
Activation of the granulocyte-macrophage colony-stimulating factor promoter in T cells requires cooperative binding of Elf-1 and AP-1 transcription factors . 
The granulocyte-macrophage colony-stimulating factor ( GM-CSF ) gene has been studied extensively as a model system of transcriptional induction during T-lymphocyte activation . 
The GM-CSF gene is not expressed in resting peripheral blood T cells but is rapidly induced at the transcriptional level following activation through the cell surface T-cell receptor . 
A highly conserved 19-bp element located immediately 5' of the human GM-CSF TATA box ( bp -34 to -52 ) , herein called purine box 1 ( PB1 ) , has been shown to bind a T-cell nuclear protein complex and to be required for transcriptional induction of the GM-CSF gene following T-cell activation . 
The PB1 sequence motif is highly conserved in both human and murine GM-CSF genes . 
In this report , we demonstrate that the PB1 element alone confers inducibility on a heterologous promoter following transfection into human Jurkat T cells . 
In addition , we identify a major PB1 nuclear protein-binding complex that is not present in resting peripheral blood T cells but is rapidly induced following T-cell activation . 
Sequence analysis revealed that PB1 is composed of adjacent binding sites for Ets and AP-1 transcription factors . 
In vitro mutagenesis experiments demonstrated that both the Ets and AP-1 sites are required for binding of the inducible PB1 nuclear protein complex and for the transcriptional activity of this element and the GM-CSF promoter in activated T cells . 
( ABSTRACT TRUNCATED AT 250 WORDS ) 
Effects of CD45 on NF-kappa B . 
Implications for replication of HIV-1 . 
Increased levels of replication of the HIV type 1 are observed after the activation of infected T cells through the TCR . 
However , anti-CD45 antibodies inhibit these effects in cells from infected individuals . 
In this study , we examined interrelationships between CD45 and HIV-1 further . 
We measured effects on the HIV-1 LTR in T cell lines that were stimulated with antibodies against CD45 and in those that lacked the expression of CD45 on their surfaces . 
First , anti-CD45 antibodies did not affect basal but decreased activated levels of expression from the HIV-1 LTR . 
Second , T cells , which lack CD45 and can not signal via the TCR , supported higher levels of viral replication and gene expression . 
This was due to the presence of active NF-kappa B complexes in the nucleus of CD45- T cells . 
Additionally , infected T cells displayed lower levels of CD45 on their surfaces . 
Thus , CD45 plays an active role in the physiology of T cells and in the replication of HIV-1 . 
Human interferon regulatory factor 2 gene . 
Intron-exon organization and functional analysis of 5'-flanking region . 
Interferon regulatory factor 2 ( IRF-2 ) is a transcriptional regulatory protein that terminates interferon beta expression initiated by interferon regulatory factor 1 . 
In this study , we isolated the genomic DNA for human IRF-2 gene , determined the intron-exon structure of the human IRF-2 gene , mapped the major transcription initiation site , identified a number of potential regulatory elements in the 5'-flanking region , and localized the IRF-2 gene on human chromosome 4 . 
The IRF-2 promoter region contains a CpG island , with several GC boxes , a putative NF-kappa B-binding site , and a CAAT box , but no TATA box . 
When the promoter region was linked with a heterologous reporter gene , we found that the promoter region is inducible by both interferons ( interferon-alpha and -gamma ) and interferon regulatory factor 1 . 
The region which induced these inductions was identified as being confined to 40 nucleotides 5' to the major transcriptional initiation site by testing a series of clones with truncated promoter of IRF-2 . 
This region contains elements which are shared with the transcriptional enhancers of other genes including interferon regulatory factor 1 , interferon beta , and interferon-inducible genes . 
These data suggest that interferon regulatory factor 1 not only triggers the activation of the interferon signal transduction pathway , but also may play a role in limiting the duration of this response by activating the transcription of IRF-2 . 
Direct exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin ( TCDD ) increases infectivity of human erythrocytes to a malarial parasite . 
Direct exposure to 10 nM 2,3,7,8-TCDD caused a 75 % increase and a 2-fold increase in the infectivity of isolated human erythrocytes to P. falciparum after 48 hours when the parasites were in an unsynchronized or synchronized state of growth , respectively . 
Treatment of human erythrocytes with 10 microM sodium orthovanadate ( NaOV ) , an inhibitor of plasma membrane Ca-ATPase and phosphotyrosine phosphatase , decreased parasitemia by 30 % . 
Co-treatment of RBCs with TCDD and NaOV completely blocked the TCDD-induced increase in parasitemia . 
Because erythrocytes are anucleated , these results are discussed as evidence for biochemical changes by TCDD without requiring the activation of gene products . 
Novel aldosterone receptors : specificity-conferring mechanism at the level of the cell membrane . 
Functional studies in extra-renal , nonepithelial cells such as smooth muscle cells and more recently circulating human lymphocytes have provided increasing evidence that aldosterone produces not only classical genomic effects , but also rapid non-genomic effects on transmembrane electrolyte movements . 
These involve activation of the sodium\/proton-exchanger of the cell membrane at very low , physiological concentrations of aldosterone with an acute onset within 1-2 minutes . 
A second messenger cascade involved is the inositol-1,4,5-trisphosphate\/calcium pathway which responds over the same rapid time course . 
Such changes clearly can not be explained by genomic mechanisms , which are responsible for later effects than the membrane-related rapid responses . 
In addition to its rapid time course the unique characteristics of this new pathway for steroid action include a 10000-fold selectivity for aldosterone over cortisol and the ineffectiveness of spironolactones , classical mineralocorticoid antagonists , as antagonists of the response . 
Subsequently binding sites have been demonstrated in the plasma membrane of human lymphocytes which show pharmacological ( aldosterone specificity ) and kinetic ( high turnover ) properties identical with those of the rapid aldosterone effects in the same cells . 
SDS-PAGE analysis of the receptor protein has shown a molecular weight of approximately 50 kD . 
The present paper reviews the data supporting a new , two-step model for non-genomic and genomic aldosterone effects . 
It also suggests a novel specificity-conferring mechanism for mineralocorticoid action at the membrane level . 
Pentoxifylline for the treatment of infection with human immunodeficiency virus . 
Cytokine dysregulation in human immunodeficiency virus type 1 ( HIV-1 ) infection has been documented in numerous studies and has been cited as an important component in the pathogenesis of this retroviral infection . 
Pharmacological modification of cytokine dysregulation , therefore , has been suggested as a therapeutic modality for HIV-1 infection . 
Dr. Dezube of Beth Israel Hospital ( Boston ) concisely reviews the state of our knowledge regarding the effects of pentoxifylline on expression of tumor necrosis factor-alpha , a cytokine known to influence HIV-1 replication and to play a possible role in the clinical manifestations of advanced infection with this virus . 
Pentoxifylline , a trisubstituted xanthine derivative , has been used to decrease blood viscosity and is reasonably well tolerated by most recipients of the drug . 
Results of preliminary studies , many of which were conducted by Dr. Dezube , suggest that use of this agent in combination with antiretroviral compounds may prove useful in the treatment of patients with HIV-1 infection 
Evidence for a trans-acting activator function regulating the expression of the human CD5 antigen . 
Interspecies somatic cell hybrids were generated by fusing the mouse T-lymphoma cell line , BW5147 , with normal human T lymphocytes at different stages of differentiation . 
Thymocytes , activated peripheral T lymphocytes , or an activated T-cell clone were used as human partners , respectively , in three independent fusions . 
Irrespective of the human cell partner used for fusion , a certain number of hybrids lost CD5 surface expression over a period of time in culture . 
Analysis at the phenotype and genetic level showed that lack of CD5 expression was due neither to segregation of human autosome 11 , on which the CD5 gene has been mapped , nor to deletion of the CD5 structural gene . 
Furthermore , loss of CD5 surface expression correlated with the absence of specific mRNA . 
Since these hybrids preferentially segregate human chromosomes , these results indicate the existence of a non-syntenic trans-active locus , or loci , positively controlling the expression of the human CD5 gene . 
Alternate immune system targets for TCDD : lymphocyte stem cells and extrathymic T-cell development . 
We here summarize evidence that thymic atrophy induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin ( TCDD ) can be mediated , at least in part , by damage to extrathymic T-cell precursors in bone marrow and fetal liver . 
This atrophy induction does not involve apoptotic mechanisms in thymocytes affected by the bcl-2 proto-oncogene . 
TCDD mediates atrophy induction through its specific receptor ( the AhR ) and not through effects on the estrogen receptor . 
Both TCDD and estradiol induce extrathymic T-cell differentiation in the liver . 
These extrathymic T-cell populations include cells expressing elevated levels of V beta T-cell receptors that are normally deleted in thymic development . 
BCL-6 and the molecular pathogenesis of B-cell lymphoma . 
The results presented identify the first genetic lesion associated with DLCL , the most clinically relevant form of NHL . 
Although no proof yet exists of a role for these lesions in DLCL pathogenesis , the feature of the BCL-6 gene product , its specific pattern of expression in B cells , and the clustering of lesions disrupting its regulatory domain strongly suggest that deregulation of BCL-6 expression may contribute to DLCL development . 
A more precise definition of the role of BCL-6 in normal and neoplastic B-cell development is the goal of ongoing study of transgenic mice engineered either to express BCL-6 under heterologous promoters or lacking BCL-6 function due to targeted deletions . 
In addition to contributing to the understanding of DLCL pathogenesis , the identification of BCL-6 lesions may have relevant clinical implications . 
DLCL represent a heterogeneous group of neoplasms which are treated homogeneously despite the fact that only 50 % of patients experience long-term disease-free survival ( Schneider et al. 1990 ) . 
The fact that BCL-6 rearrangements identify biologically and clinically distinct subsets of DLCL suggests that these lesions may be useful as markers in selection of differential therapeutic strategies based on different risk groups . 
Furthermore , the BCL-6 rearrangements can be used to identify and monitor the malignant clone with sensitive PCR-based techniques . 
Since clinical remission has been observed in a significant fraction of DLCL cases , these markers may serve as critical tools for sensitive monitoring of minimal residual disease and early diagnosis of relapse ( Gribben et al. 1993 ) . 
Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines . 
IL-4 and IL-10 inhibit the cytokine production and mRNA expression by monocytes\/macrophages . 
To investigate the molecular mechanism of the inhibitory effect on transcriptional or post-transcriptional regulation of IL-6 gene expression by IL-4 and IL-10 , we studied IL-6 production , expression level of IL-6 mRNA , IL-6 promoter activity , transcriptional activity of NF-kappaB and NF-IL-6 , and IL-6 mRNA stability in human monocytic cell lines , THP-1 and U937 , stimulated by PMA and LPS in the absence or the presence of IL-4 or IL-10 . 
Both IL-4 and IL-10 were seen to inhibit IL-6 production and the expression of IL-6 mRNA in both monocytic cell lines studied . 
In chloramphenicol acetyltransferase assays , utilizing the transient transfection of a chloramphenicol acetyltransferase reporter plasmid containing the IL-6 gene promoter , IL-4 , but not IL-10 , suppressed the transcriptional activity of the IL-6 gene promoter stimulated by PMA and LPS . 
Electrophoretic mobility shift assays showed that IL-4 , but not IL-10 , inhibited nuclear NF-kappaB activity , and that IL-4 and IL-10 did not affect NF-IL-6 activity . 
On the other hand , IL-10 enhanced the degradation of IL-6 mRNA in a mRNA stability assay . 
These results suggest that IL-4 may inhibit the transcription of the IL-6 gene by affecting NF-kappaB binding activity , while IL-10 may inhibit the IL-6 mRNA levels post-transcriptionally , without suppressing promoter activity . 
Therefore , we conclude that IL-4 and IL-10 inhibit IL-6 production by different mechanisms in human monocytic cell lines . 
Age-related decreases in IL-2 production by human T cells are associated with impaired activation of nuclear transcriptional factors AP-1 and NF-AT . 
Although transcriptional factors AP-1 and nuclear factor of activated T cells ( NF-AT ) are important for the normal induction of IL-2 , it is unknown if the age-related decline in IL-2 production by activated human T cells may be associated with aberrancies in transcriptional regulatory proteins . 
In the current studies , IL-2 production by T cells from elderly ( mean 78 years ) and young ( mean 37 years ) humans was measured in cultures stimulated with PHA , PHA plus PMA , crosslinked anti-CD3 mAB OKT3 plus PMA , or PMA plus ionomycin . 
Substantial decreases of IL-2 production were observed for cell cultures from 7 of 12 elderly individuals in response to the different stimuli , whereas the levels of IL-2 produced by stimulated T cells from other elderly individuals were equivalent to those observed for stimulated T cells of young subjects . 
Analyses of nuclear extracts by electrophoretic DNA mobility shift assays showed that decreased IL-2 production by stimulated T cells of elderly individuals was closely associated with impairments in the activation of both AP-1 and NF-AT . 
By contrast , T cells from elderly subjects with normal levels of IL-2 production exhibited normal activation of AP-1 and NF-AT . 
In addition , the results of competition experiments analyzing the normal components of NF-AT showed that the age-related reductions in stimulus-dependent NF-AT complexes corresponded to the slow migrating complexes that were composed of c-Fos\/c-Jun AP-1 . 
The resting and stimulated levels of NF kappa B were reduced in T cells from certain elderly individuals ; however , alterations of NF kappa B did not correlate with changes in IL-2 expression . 
Thus , these results show that age-related impairments in the activation of AP-1 and NF-AT are closely associated with decreased expression of IL-2 and further suggest that aberrancies in the signaling pathways important for the induction of transcriptionally active c-Fos\/c-Jun AP-1 may contribute to the impaired activation of NF-AT . 
Recombinant NFAT1 ( NFATp ) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes . 
Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response . 
We have identified two new isoforms of the transcription factor NFAT1 ( previously termed NFATp ) that are the predominant isoforms expressed in murine and human T cells . 
When expressed in Jurkat T cells , recombinant NFAT1 is regulated , as expected , by the calmodulin-dependent phosphatase calcineurin , and its function is inhibited by the immunosuppressive agent cyclosporin A ( CsA ) . 
Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol 12-myristate 13-acetate ; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA . 
Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation . 
When expressed in COS cells , however , NFAT1 is capable of transactivation , but it is not regulated correctly : its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol 12-myristate 13-acetate . 
Recombinant NFAT1 can mediate transcription of the interleukin-2 , interleukin-4 , tumor necrosis factor alpha , and granulocyte-macrophage colony-stimulating factor promoters in T cells , suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response . 
Glucocorticoids and interferon-alpha in the acquired immunodeficiency syndrome . 
Some patients with acquired immunodeficiency syndrome ( AIDS ) develop glucocorticoid resistance characterized by low receptor affinity ( Kd ) for glucocorticoids in mononuclear , cells and high values of ACTH and cortisol . 
As glucocorticoids regulate interferon-alpha ( IFN alpha ) production , we hypothesized that IFN alpha , a cytokine produced predominantly by monocytes in AIDS , should be increased in cortisol-resistant AIDS , attributing the lack of cortisol inhibition to IFN alpha production . 
Therefore , we examined glucocorticoid receptor characteristics on monocytes by -LCB-3H-RCB-dexamethasone binding and measured IFN alpha , cortisol , and ACTH in AIDS patients with ( AIDS-GR ) or without glucocorticoid resistance ( AIDS-C ) and controls ( C ) . 
Monocytes of AIDS-GR patients had a receptor Kd of 10.5 +\/- 4.2 nmol\/L that was higher than that in the AIDS-C group ( 2.9 +\/- 0.8 nmol\/L ) and normal subjects ( 2.0 +\/- 0.8 nmol\/L ; P &lt; 0.01 ) . 
IFN alpha levels were increased in the AIDS-GR group ( 17 +\/- 6 vs. 4 +\/- 1 U\/mL in the AIDS-C group and 2 +\/- 0.5 U\/mL in the C group ; P &lt; 0.01 ) . 
Correlations were found between plasma IFN alpha and receptor Kd on monocytes of AIDS-GR ( r = 0.77 ) and between IFN alpha and plasma cortisol in the same group ( r = 0.74 ) . 
The poly-LRB-I-RRB--poly-LRB-C-RRB--induced IFN alpha production by monocytes was inhibited by glucocorticoids in the C and AIDS-C groups ( approximately 80 % inhibition in both groups ) ; the effect was reversed by the receptor antagonist RU-38486 . 
By contrast , glucocorticoids failed to inhibit IFNalpha production from AIDS-GR monocytes ( approximately 20 % inhibition ) . 
In conclusion , elevated IFN alpha levels in AIDS-GR may be due to the lack of inhibitory effect of cortisol on IFN alpha production due to cortisol resistance in monocytes . 
Signals leading to the activation of NF-kappa B transcription factor are stronger in neonatal than adult T lymphocytes . 
The molecular background of the defects in the immune reactivity of human neonates has not been fully elucidated . 
As the NF-kappa B transcription factor has a central role in the control of transcription of several genes involved in immune and inflammatory responses , the authors have analysed the activation of NF-kappa B in human umbilical cord T lymphocytes . 
The activity was tested by quantitating the nuclear proteins binding to an oligonucleotide containing the consensus kappa B binding sequence ( electrophoretic mobility shift assay ) . 
The data obtained demonstrate that phorbol dibutyrate\/calcium ionophore A23187 ( PDBu\/iono ) combination induced a clearly higher nuclear translocation of NF-kappa B in neonatal than adult T cells . 
This higher NF-kappa B activity was restricted to the CD4+ T-cell subset . 
Analysis of the nuclear extracts with antibodies directed against the major components of NF-kappa B the p50 and RelA ( p65 ) proteins , indicated that the composition of NF-kappa B was similar in neonatal and adult cells . 
These results suggest that neonatal T cells are exposed to oxidative stress-inducing signals during delivery and\/or are inherently more sensitive to NF-kappa B activating signals than adult T cells . 
BCL-6 , a POZ\/zinc-finger protein , is a sequence-specific transcriptional repressor . 
Approximately 40 % of diffuse large cell lymphoma are associated with chromosomal translocations that deregulate the expression of the BCL6 gene by juxtaposing heterologous promoters to the BCL-6 coding domain . 
The BCL6 gene encodes a 95-kDa protein containing six C-terminal zinc-finger motifs and an N-terminal POZ domain , suggesting that it may function as a transcription factor . 
By using a DNA sequence selected for its ability to bind recombinant BCL-6 in vitro , we show here that BCL-6 is present in DNA-binding complexes in nuclear extracts from various B-cell lines . 
In transient transfectin experiments , BCL6 can repress transcription from promoters linked to its DNA target sequence and this activity is dependent upon specific DNA-binding and the presence of an intact N-terminal half of the protein . 
We demonstrate that this part of the BCL6 molecule contains an autonomous transrepressor domain and that two noncontiguous regions , including the POZ motif , mediate maximum transrepressive activity . 
These results indicate that the BCL-6 protein can function as a sequence-specific transcriptional repressor and have implications for the role of BCL6 in normal lymphoid development and lymphomagenesis . 
The Ets protein Spi-B is expressed exclusively in B cells and T cells during development . 
Spi-B and PU.1 are hematopoietic-specific transcription factors that constitute a subfamily of the Ets family of DNA-binding proteins . 
Here we show that contrary to previous reports , PU.1 and Spi-B have very different expression patterns . 
PU.1 is expressed at high levels in B cells , mast cells , megakaryocytes , macrophages , neutrophils , and immature erythroid cells and at lower levels in mature erythrocytes . 
PU.1 is completely absent from peripheral T cells and most T cell lines based on sensitive RT-PCR assays . 
In contrast , Spi-B is expressed exclusively in lymphoid cells and can be detected in early fetal thymus and spleen . 
In situ hybridizations of adult murine tissues demonstrate Spi-B mRNA in the medulla of the thymus , the white pulp of the spleen , and the germinal centers of lymph nodes . 
Spi-B expression is very abundant in B cells and both Spi-B mRNA and protein are detected in some T cells . 
In situ hybridization and Northern blot analysis suggest that Spi-B gene expression increases during B cell maturation and decreases during T cell maturation . 
Gel-retardation experiments show that Spi-B can bind to all putative PU.1 binding sites , but do not reveal any preferred Spi-B binding site . 
Finally , both PU.1 and Spi-B function as transcriptional activators of the immunoglobulin light-chain enhancer E lambda 2.4 when coexpressed with Pip ( PU.1-interaction partner ) in NIH-3T3 cells . 
Taken together , these data suggest that differences in patterns of expression between Spi-B and PU.1 distinguish the function of each protein during development of the immune system . 
Effects of IL-10 and IL-4 on LPS-induced transcription factors ( AP-1 , NF-IL6 and NF-kappa B ) which are involved in IL-6 regulation . 
Interleukin-10 ( IL-10 ) , like IL-4 , is known to inhibit cytokine expression in activated human monocytes . 
We showed that both IL-10 and IL-4 inhibit LPS-induced IL-6 mRNA and protein expression by inhibiting the transcription rate of the IL-6 gene . 
The strong inhibition of the IL-6 transcription rate prompted us to study the effect of IL-10 and IL-4 on the expression of transcription factors . 
We questioned whether or not IL-10 and IL-4 affected the expression of transcription factors that are known to be involved in the control of the IL-6 transcription rate , namely activator protein-1 ( AP-1 ) , nuclear factor IL-6 ( NF-IL6 ) , and nuclear factor kappa B ( NF-kappaB ) . 
In electrophoretic mobility shift assays ( EMSAs ) we showed that IL-10 and IL-4 inhibited LPS-induced AP-1 binding activity . 
The inhibiting effect of IL-4 was slightly more pronounced than that of IL-10 . 
Downregulation of LPS-induced AP-1 was accompanied , and thus possibly explained , by a reduced expression at mRNA level of the two major components of the AP-1 complex , namely c-fos and c-jun as determined by Northern experiments . 
Binding activity of NF-IL6 was also strongly inhibited by IL-4 whereas IL-10 showed no effect . 
NF-IL6 mRNA levels were not affected by IL-10 or IL-4 , suggesting that IL-4 affects binding activity of preexisting NF-IL6 . 
Neither IL-10 nor IL-4 inhibited LPS-induced NF-kappa B binding activity . 
In agreement with this finding , Northern experiments where p65 and p105 mRNA levels were determined , demonstrated that expression of these components of the NF-kappa B transcription factor were not affected by IL-10 or IL-4 . 
Furthermore , neither IL-10 nor IL-4 showed any effect on I-kappa B mRNA expression as determined by Northern experiments . 
Thus , IL-10 and IL-4 similarly affect IL-6 expression . 
However , for IL-4 this was accompanied with a reduction of AP-1 and NF-IL6 binding activity whereas IL-10 only inhibited AP-1 binding activity . 
Various modes of basic helix-loop-helix protein-mediated regulation of murine leukemia virus transcription in lymphoid cell lines . 
The transcriptionally regulatory regions of the lymphomagenic Akv and SL3-3 murine leukemia retroviruses ( MLVs ) contain two types of E-box consensus motifs , CAGATG . 
One type , EA\/S , is located in the upstream promoter region , and the other , E-LRB-gre-RRB- , is located in a tandem repeat with enhancer properties . 
We have examined the requirements of the individual E-boxes in MLV transcriptional regulation . 
In lymphoid cell lines only , the E-LRB-gre-RRB--binding protein complexes included ALF1 or HEB and E2A basic helix-loop-helix proteins . 
Ectopic ALF1 and E2A proteins required intact E-LRB-gre-RRB- motifs for mediating transcriptional activation . 
ALF1 transactivated transcription of Akv MLV through the two E-LRB-gre-RRB- motifs equally , whereas E2A protein required the promoter-proximal E-LRB-gre-RRB- motif . 
In T- and B-cell lines , the E-LRB-gre-RRB- motifs were of major importance for Akv MLV transcriptional activity , while the EA\/S motif had some effect . 
In contrast , neither E-LRB-gre-RRB- nor EA\/S motifs contributed pronouncedly to Akv MLV transcription in NIH 3T3 cells lacking DNA-binding ALF1 or HEB and E2A proteins . 
The Id1 protein was found to repress ALF1 activity in vitro and in vivo . 
Moreover , ectopic Id1 repressed E-LRB-gre-RRB--directed but not EA\/S-directed MLV transcription in lymphoid cell lines . 
In conclusion , E-LRB-gre-RRB- motifs and interacting basic helix-loop-helix proteins are important determinants for MLV transcriptional activity in lymphocytic cell lines . 
Granulocyte-macrophage colony-stimulating factor preferentially activates the 94-kD STAT5A and an 80-kD STAT5A isoform in human peripheral blood monocytes . 
Granulocyte-macrophage colony-stimulating factor ( GM-CSF ) induces immediate effects in monocytes by activation of the Janus kinase ( JAK2 ) and STAT transcription factor ( STAT5 ) pathway . 
Recent studies have identified homologues of STAT5 , STAT5A , and STAT5B , as well as lower molecular weight variants of STAT5 . 
To define the activation of the STAT5 homologues and lower molecular weight variant in human monocytes and monocytes differentiated into macrophages by culture in macrophage-CSF ( M-CSF ) , we measured the GM-CSF induced tyrosine phosphorylation of STAT5A , STAT5B , and any lower molecular weight STAT5 isoforms . 
Freshly isolated monocytes expressed 94-kD STAT5A , 92-kD STAT5B , and an 80-kD STAT5A molecule . 
Whereas 94-kD STAT5A was clearly tyrosine phosphorylated and bound to the enhancer element , the gamma response region ( GRR ) , of the Fc gamma RI gene , substantially less tyrosine phosphorylated STAT5B bound to the immobilized GRR element . 
Macrophages lost their ability to express the 80-kD STAT5A protein , but retained their ability to activate STAT5A . 
STAT5A-STAT5A homodimers and STAT5A-STAT5B heterodimers formed in response to GM-CSF . 
Therefore , activation of STAT5A predominates compared to STAT5B when assayed by direct immunoprecipitation and by evaluation of bound STATs to immobilized GRR . 
Selective activation of STAT5 homologues in addition to generation of lower molecular isoforms may provide specificity and control to genes expressed in response to cytokines such as GM-CSF . 
CTL recognition of an altered peptide associated with asparagine bond rearrangement . 
Implications for immunity and vaccine design . 
The extent to which peptides containing chemically and post-translationally modified amino acid side chains are recognized by primed CTL has not been clearly defined . 
We report on the CTL recognition of a MHC class I-restricted peptide containing a cyclized asparagine ( succinimide ) residue . 
This modification of the asparagine side chain is a common intermediate structure during deamidation , isomerization , and bond rearrangements of amide-containing amino acids and also occurs as a side reaction in peptide synthesis . 
The CTL specifically recognized the succinimide-containing peptide showing only weak cross-reactivity at high concentrations of the parent peptide containing unmodified asparagine . 
Similarly , CTL raised against the parent peptide did not recognize the succinimide derivative of this peptide . 
Naturally processed forms of these structures are likely to occur given the importance and frequency of deamidation both in vitro and in vivo . 
Moreover , since succinimide intermediates of deamidated peptides can occasionally be very stable , these peptides have the potential to act as altered self-Ags with significant implications for autoimmunity . 
In addition , unwanted and potentially hazardous specificities may be elicited when using synthetic peptides in subunit vaccines in which succinimide residues may form spontaneously during storage or chemical synthesis . 
Transgenic expression of PML\/RARalpha impairs myelopoiesis . 
The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene ( PML ) on chromosome 15 with the retinoic acid receptor alpha ( RARalpha ) on chromosome 17 . 
This yields a fusion transcript , PML\/RARalpha , a transcription factor with reported dominant negative functions in the absence of hormone . 
Clinical remissions induced with all-trans retinoic acid ( RA ) treatment in acute promyelocytic leukemia are linked to PML\/RARalpha expression in leukemic cells . 
To evaluate the PML\/RARalpha role in myelopoiesis , transgenic mice expressing PML\/RARalpha were engineered . 
A full-length PML\/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice . 
Expression was confirmed in the bone marrow with a reverse transcription PCR assay . 
Basal total white blood cell and granulocyte counts did not appreciably differ between PML\/RARalpha transgenic and control mice . 
Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice . 
However , in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors , especially in those responding to granulocyte\/ macrophage colony-stimulating factor . 
Granulocyte\/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition . 
Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation . 
Following irradiation , PML\/RARalpha transgenic mice , as compared with controls , more rapidly depressed peripheral white blood cell and granulocyte counts . 
As expected , nearly all control mice ( 94.4 % ) survived irradiation , yet this irradiation was lethal to 45.8 % of PML\/RARalpha transgenic mice . 
Lethality was associated with more severe leukopenia in transgenic versus control mice . 
Retinoic acid treatment of irradiated PML\/RARalpha mice enhanced granulocyte recovery . 
These data suggest that abnormal myelopoiesis due to PML\/RARalpha expression is an early event in oncogenic transformation . 
Characterization of a new isoform of the NFAT ( nuclear factor of activated T cells ) gene family member NFATc { published erratum appears in J Biol Chem 1996 Dec 27 ; 271 ( 52 ) : 33705 } 
The cyclosporin A ( CsA ) \/FK506-sensitive nuclear factor of activated T cells ( NFAT ) plays a key role in the inducible expression of cytokine genes in T cells . 
Although NFAT has been recently shown to be inducible in several non-T immune cells , the NFAT gene family members characterized to date have been isolated only from T cells . 
To further characterize NFAT function in human B cells and to demonstrate cytokine gene specificity of NFAT proteins , we report here the isolation and characterization of a cDNA clone from the Raji B cell line . 
The cDNA clone encodes a new isoform , NFATc.beta , of the NFAT gene family member NFATc ( designated here NFATc.alpha ) . 
The amino acid sequence of NFATc.beta differs from that of NFATc.alpha in the first NH2-terminal 29 residues and contains an additional region of 142 residues at the COOH terminus . 
Northern analysis using a probe encompassing a common region of both isoforms showed two mRNA species of 2.7 and 4.5 kilobase pairs , while an NFATc.beta-specific probe detected only the 4.5-kilobase pair mRNA which was preferentially expressed in the spleen . 
Transient expression of NFATc.beta was capable of activating an interleukin-2 NFAT-driven reporter gene in stimulated Jurkat cells in a CsA-sensitive manner . 
However , NFATc.beta neither bound to the kappa3 element ( an NFAT-binding site ) in the tumor necrosis factor-alpha promoter nor activated the tumor necrosis factor-alpha promoter in cotransfection assays . 
These data suggest that different members or isoforms of NFAT gene family may regulate inducible expression of different cytokine genes . 
Sequence-specific DNA binding of the B-cell-specific coactivator OCA-B . 
B-cell-specific transcription of immunoglobulin genes is mediated by the interaction of a POU domain containing transcription factor Oct-1 or Oct-2 , with the B-cell-specific coactivator OCA-B ( Bob-1 , OBF-1 ) and a prototype octamer element . 
We find that OCA-B binds DNA directly in the major groove between the two subdomains of the POU domain , requiring both an A at the fifth position of the octamer element and contact with the POU domain . 
An amino-terminal fragment of OCA-B binds the octamer site in the absence of a POU domain with the same sequence specificity . 
Coactivator OCA-B may undergo a POU-dependent conformational change that exposes its amino terminus , allowing it to recognize specific DNA sequences in the major groove within the binding site for Oct-1 or Oct-2 . 
The recognition of both the POU domain and the octamer sequence by OCA-B provides a mechanism for differential regulation of octamer sites containing genes by the ubiquitous factor Oct-1 . 
Cross talk between cell death and cell cycle progression : BCL-2 regulates NFAT-mediated activation . 
BCL-2-deficient T cells demonstrate accelerated cell cycle progression and increased apoptosis following activation . 
Increasing the levels of BCL-2 retarded the G0 --&gt; S transition , sustained the levels of cyclin-dependent kinase inhibitor p27Kip1 , and repressed postactivation death . 
Proximal signal transduction events and immediate early gene transcription were unaffected . 
However , the transcription and synthesis of interleukin 2 and other delayed early cytokines were markedly attenuated by BCL-2 . 
In contrast , a cysteine protease inhibitor that also blocks apoptosis had no substantial affect upon cytokine production . 
InterleUkin 2 expression requires several transcription factors of which nuclear translocation of NFAT ( nuclear factor of activated T cells ) and NFAT-mediated transactivation were impaired by BCL-2 . 
Thus , select genetic aberrations in the apoptotic pathway reveal a cell autonomous coregulation of activation . 
Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism . 
Epstein-Barr virus ( EBV ) , the causative agent of infectious mononucleosis , is a human herpesvirus associated with epithelial cell malignancies ( nasopharyngeal carcinoma ) as well as B-cell malignancies . 
Understanding how viral latency is disrupted is a central issue in herpesvirus biology . 
Epithelial cells are the major site of lytic EBV replication within the human host , and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas . 
It is known that expression of a single viral immediate-early protein , BZLF1 , is sufficient to initiate the switch from latent to lytic infection in B cells . 
Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency . 
Here we show that , unexpectedly , expression of another viral immediate-early protein , BRLF1 , can disrupt viral latency in an epithelial cell-specific fashion . 
Therefore , the mechanisms leading to disruption of EBV latency appear to be cell-type specific . 
Multifactor cis-dominant negative regulation of IL-2 gene expression in anergized T cells . 
The molecular mechanism underlying IL-2 transcriptional blockade in anergic T cell clones is not fully understood . 
To examine whether an active negative regulatory process occurs , we created a reporter construct containing as an enhancer four copies of the NF-AT site and one copy of the octamer site ( 4X NF-AT-Oct ) . 
This construct was only slightly reduced ( 1.3-fold ) in its expression when stimulated under anergic conditions , while a whole mouse IL-2 enhancer construct showed a reduction of 4.3-fold . 
Addition of the -176 to -96 sequence to the 4X NF-AT-Oct construct did not impart the ability to be affected by anergy , but addition of the -236 to -96 sequence did , demonstrating that anergy is an active inhibitory process and that more than the presence of the -150 AP-1 binding site ( -152 to -147 ) is required to mediate the effect . 
Mutational studies of the -236 to -96 sequence indicated that the presence of both the -130 AP-1-like site ( -187 to -181 ) and the -150 proximal AP-1 site were necessary to observe anergy . 
Because the -180 site is not required for trans-activation , it was possible to confirm by mutation in the normal mouse IL-2 enhancer that this site is absolutely essential for anergy induction . 
The simplest model 0 to explain these results is that anergy is mediated by a complex of multiple transcription factors that exert a cis-acting dominant negative regulatory effect on the trans-activation of the IL-2 gene . 
Abnormality of Oct-1 DNA binding in T cells from Sjogren 's syndrome patients . 
Primary Sjogren 's syndrome ( SS ) is an autoimmune rheumatic disease characterized by T cell hypoactivity . 
To understand the diminished T cell response to activation signals , we measured nucleoprotein DNA-binding activities regulating gene expression during T cell activation using the electrophoretic mobility shift assay . 
Peripheral blood lymphocytes from 9\/19 SS patients were found to be defective in their ability to bind an october sequence ( Oct-1 ) . 
This Oct-1-binding phenotype remained stable in culture for up to 3 days prior to activation . 
This abnormality was not seen in resting T cells nor T cells from patients with systemic lupus erythematosus , rheumatoid arthritis ( RA ) , or SS accompanied by RA . 
The SS Oct-1 DNA-binding abnormality correlated significantly with an inability of cells to exit the Gzero\/G1 cell cycle phase when stimulated in vitro . 
Importantly , nucleoprotein extracts showing decreased DNA-binding activity had normal amounts of Oct-1 proteins as determined by immunoprecipitation , implying a functional defect in the Oct-1 protein . 
Moreover , defective DNA binding was corrected by treatment with acid phosphatase . 
Cell-type-specific regulation of the human tumor necrosis factor alpha gene in B cells and T cells by NFATp and ATF-2\/JUN . 
The human tumor necrosis factor alpha ( TNF-alpha ) gene is one of the earliest genes transcribed after the stimulation of a B cell through its antigen receptor or via the CD-40 pathway . 
In both cases , induction of TNF-alpha gene transcription can be blocked by the immunosuppressants cyclosporin A and FK506 , which suggested a role for the NFAT family of proteins in the regulation of the gene in B cells . 
Furthermore , in T cells , two molecules of NFATp bind to the TNF-alpha promoter element kappa 3 in association with ATF-2 and Jun proteins bound to an immediately adjacent cyclic AMP response element ( CRE ) site . 
Here , using the murine B-cell lymphoma cell line A20 , we show that the TNF-alpha gene is regulated in a cell-type-specific manner . 
In A20 B cells , the TNF-alpha gene is not regulated by NFATp bound to the kappa 3 element . 
Instead , ATF-2 and Jun proteins bind to the composite kappa 3\/CRE site and NFATp binds to a newly identified second NFAT site centered at -76 nucleotides relative to the TNF-alpha transcription start site . 
This new site plays a critical role in the calcium-mediated , cyclosporin A-sensitive induction of TNF-alpha in both A20 B cells and Ar-5 cells . 
Consistent with these results , quantitative DNase footprinting of the TNF-alpha promoter using increasing amounts of recombinant NFATp demonstrated that the -76 site binds to NFATp with a higher affinity than the kappa 3 site . 
Two other previously unrecognized NFATp-binding sites in the proximal TNF-alpha promoter were also identified by this analysis . 
Thus , through the differential use of the same promoter element , the composite kappa 3\/CRE site , the TNF-alpha gene is regulated in a cell-type-specific manner in response to the same extracellular signal . 
Interleukin-6 promotes multiple myeloma cell growth via phosphorylation of retinoblastoma protein . 
Interleukin-6 ( IL-6 ) mediates autocrine and paracrine growth of multiple myeloma ( MM ) cells and inhibits tumor cell apoptosis . 
Abnormalities of retinoblastoma protein ( pRB ) and mutations of RB gene have been reported in up to 70 % of MM patients and 80 % of MM-derived cell lines . 
Because dephosphorylated ( activated ) pRB blocks transition from G1 to S phase of the cell cycle whereas phosphorylated ( inactivated ) pRB releases this growth arrest , we characterized the role of pRB in IL-6-mediated MM cell growth . 
Both phosphorylated and dephosphorylated pRB were expressed in all serum-starved MM patient cells and MM-derived cell lines , but pRB was predominantly in its phosphorylated form . 
In MM cells that proliferated in response to IL-6 , exogenous IL-6 downregulated dephosphorylated pRB and decreased dephosphorylated pRB-E2F complexes . 
Importantly , culture of MM cells with RB antisense , but not RB sense , oligonucleotide ( ODN ) triggered IL-6 secretion and proliferation in MM cells ; however , proliferation was only partially inhibited by neutralizing anti-IL-6 monoclonal antibody ( MoAb ) . 
In contrast to MM cells , normal splenic B cells express dephosphorylated pRB . 
Although CD40 ligand ( CD40L ) triggers a shift from dephosphorylated to phosphorylated pRB and proliferation of B cells , the addition of exogenous IL-6 to CD40L-treated B cells does not alter either pRB or proliferation , as observed in MM cells . 
These results suggest that phosphorylated pRB is constitutively expressed in MM cells and that IL-6 further shifts pRB from its dephosphorylated to its phosphorylated form , thereby promoting MM cell growth via two mechanisms ; by decreasing the amount of E2F bound by dephosphorylated pRB due to reduced dephosphorylated pRB , thereby releasing growth arrest ; and by upregulating IL-6 secretion by MM cells and related IL-6-mediated autocrine tumor cell growth . 
E3 , a hematopoietic-specific transcript directly regulated by the retinoic acid receptor alpha . 
Retinoic acid ( RA ) -induced maturation mediated by the retinoic acid receptor alpha ( RAR alpha ) has been implicated in myeloid development . 
We have used differential hybridization analysis of a cDNA library constructed from the murine RA-inducible MPRO promyelocyte cell line to identify immediate-early genes induced by RA during granulocytic differentiation . 
E3 , one of nine sequences identified , was upregulated in an immediate-early manner , with transcript levels peaking after 60 minutes exposure to RA . 
E3 transcripts were RA-inducible in HL60 cells , but not in an RA-resistant subclone , HL60R , that harbors a mutated RAR alpha gene . 
However , when HL60R cells were transduced with a functional copy of the RAR alpha gene , RA induced a 10-fold increase in E3 mRNA levels . 
E3 transcripts are present in the myeloid , B-lymphoid , and erythroid lineages , absent in nonhematopoietic cells , and encode a highly hydrophobic , potentially phosphorylated polypeptide of unknown function with significant homology to a putative protein expressed in myeloid cells . 
The murine E3 promoter harbors a single bipartite retinoic acid response element which in transient transfection assays conferred RA sensitivity . 
These results indicate that E3 is a hematopoietic-specific gene that is an immediate target for the activated RAR alpha during myelopoiesis . 
Human TAFII 105 is a cell type-specific TFIID subunit related to hTAFII130 . 
We previously characterized Drosophila and human TAF subunits that make up the core TFIID complex found in all cells . 
Here , we report that differentiated B cells contain a novel substoichiometric TAF of 105 kDa not found associated with TFIID isolated from other cell types . 
The cDNA encoding hTAFII105 reveals a highly conserved C-terminal domain shared by hTAFII130 and oTAFII110 , while the N-terminal coactivator domain has diverged significantly . 
All cells tested express TAFII105 mRNA , but only B cells contain significant levels of protein associated with TFIID . 
Transient overexpression of hTAFII105 selectively squelches the transcription of some genes in B cells . 
These properties suggest that TAFII105 is a cell type-specific subunit of TFIID that may be responsible for mediating transcription by a subset of activators in B cells . 
Eosinophil priming by cytokines : from cellular signal to in vivo modulation . 
Eosinophils play an important role in the effector phase of allergic inflammation . 
This review will focus on the conversion of the unprimed eosinophil phenotype in the peripheral blood of normal individuals to the primed phenotype found in the peripheral blood and tissues of allergic patients , a phenomenon called priming . 
Recent data on the signals initiated after cytokine receptor activation on eosinophils will be reviewed . 
A critical role of Sp1- and Ets-related transcription factors in maintaining CTL-specific expression of the mouse perforin gene . 
This study was designed to determine the potential cis-elements involved in transcriptional regulation of the mouse perforin gene . 
DNase I hypersensitive site ( DHS ) mapping revealed that the perforin locus contained six DHS within 7.0 kb of the 5' upstream sequence ( -7.0 kb ) and two DHS in intron 2 . 
The six 5' upstream and one intronic DHS were detected in only perforin-expressing lymphocytes . 
Chloramphenicol acetyltransferase ( CAT ) activities directed by 5' upstream promoter were detected preferentially in perforin-expressing cell lines . 
A construct termed PFP5a containing -795 bp exhibited the highest CAT activity , and PFP9a20 containing only -73 bp also produced significantly high CAT activity in CTLL-R8 cells . 
The proximal region in PFP9a20 contained two potential Sp1 binding sites ( GC box and GT box ) and one Ets binding site ( EBS ) . 
Electrophoretic mobility shift assay showed that each of the cis-elements bound specific protein factors . 
When single-point mutation was introduced to each GC box , EBS , and GT box in PFP9a20 , at least 3-fold less CAT activity was observed in CTLL-R8 cells . 
To confirm the importance of the three cis-acting elements in the perforin gene expression , point mutation was introduced again to each proximal GC box , EBS , and GT box of PFP5a . 
The point mutations resulted in a 2.5- to 3-fold reduction of CAT activity . 
The results suggest that a combination of the three proximal cis-acting elements may constitute a minimal region responsible for CTL-specific expression of perforin . 
Vitamin D3- and retinoic acid-induced monocytic differentiation : interactions between the endogenous vitamin D3 receptor , retinoic acid receptors , and retinoid X receptors in U-937 cells . 
Retinoic acid ( RA ) and 1,25 alpha-dihydroxycholecalciferol ( VitD3 ) are potent regulators of hematopoletic differentiation . 
Yet , little is known as to how the RA and VitD3 receptor network operates in hematopoietic cells , and whether receptor interactions can explain the interplay between the RA- and VitD3-signaling pathways during differentiation . 
Therefore , we analyzed the expression , DNA binding , and transcriptional activity of the endogenous RA and VitD3 receptors { retinoic acid receptors ( RARs ) , retinoid X receptors ( RXRs ) , and VitD3 receptor ( VDR ) } in the U-937 cell line , in which RA and VitD3 induce distinct monocytic differentiation pathways . 
VitD3 induction resulted in the formation of VDR\/RXR DNA-binding complexes on both VitD3 response elements and RA response elements ( RAREs ) . 
However , transcriptional activation was only observed from a VitD3 response element-driven reporter construct . 
Several DNA-binding complexes were detected on RAREs in undifferentiated cells . 
Stimulation by RA resulted in increased RAR beta\/RXR DNA binding , activated RARE-dependent transcription , and increased expression of RAR-beta . 
Concomitant stimulation by VitD3 inhibited the RA-stimulated formation of RAR beta\/RXR heterodimers , favoring VDR\/RXR binding to the RARE . 
Also , VitD3 inhibited the expression of CD23 and CD49f , characteristic markers of retinoid-induced U-937 cell differentiation . 
In contrast , neither the RA-stimulated , RARE-mediated transcription nor the induced RAR-beta expression was suppressed by VitD3 , suggesting that VitD3 selectively inhibited the retinoid-induced differentiation program but not the RARE-mediated signal . 
These results demonstrate a complex role for VitD3 in modifying the retinoid differentiation pathway and may have implications for differentiation-inducing therapy of hematopoietic tumors . 
Tyrosines 113 , 128 , and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity . 
SLP-76 ( SH2 domain leukocyte protein of 76 kDa ) is a recently identified substrate of the TCR-stimulated protein tyrosine kinases that functions in the signal transduction cascade linking the TCR with IL-2 gene expression . 
In this report , we demonstrate that engagement of the TCR results in tyrosine phosphorylation of SLP-76 in its amino-terminal acidic region . 
Two tyrosines ( Y113 and Y128 ) fall within an identical five amino-acid motif and are shown to be phosphorylated upon TCR ligation . 
Although mutation of either Y113 and Y128 has a minimal effect on SLP-76 function , mutation of both residues decreases significantly the ability of SLP-76 to promote T cell activation . 
A third tyrosine within the amino-terminal region ( Y145 ) appears to be the most important for optimal SLP-76 function , as altering it alone to phenylalanine has a potent impact on SLP-76 augmentation of NFAT promoter activity . 
Apoptosis signaling pathways in normal T cells : differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity . 
Fas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells . 
Restimulation of T cell blasts up-regulates Fas and Fas ligand expression , with subsequent interaction leading to cell death . 
Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli , but inhibition of Fas-mediated killing has not been consistently observed . 
To examine the behavior of Bcl-2 in normal cells , T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling . 
Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis , whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited . 
Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing . 
In contrast , interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis . 
These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors . 
Since T cells normally express Bcl-2 and Bcl-xL following activation , their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses . 
Susceptibility to natural killer cells and down regulation of MHC class I expression in adenovirus 12 transformed cells are regulated by different E1A domains . 
All human adenoviruses transform rodent cells in vitro , but only cells transformed by serotypes belonging to subgroups A ( Ad12 ) and B ( Ad3 ) are tumorigenic for immunocompetent animals . 
In these cells , the expression of MHC-class I antigens is repressed and might allow them to escape from recognition by cytotoxic T lymphocytes ( CTL ) and to develop in tumor . 
Furthermore , these cell lines appear resistant to lysis by natural killer ( NK ) cells . 
To determine the E1A domain ( s ) responsible for these properties several cell lines were created by transforming baby rat kidney ( BRK ) cells with a set of plasmids expressing different Ad2\/Ad12 hybrid E1A gene products . 
The MHC class 1 gene expression was inhibited in cells expressing the Ad12 13S mRNA product and in cells transformed with Ad2\/Ad12 hybrid E1A gene product harboring the C-terminal part of the conserved region ( CR ) 3 of Ad12 . 
Susceptibility of these transformed cell lines to NK cells was determined by cytolytic assays . 
The results obtained suggest that two Ad12 E1A domains are required to induce resistance of the cell lines to NK cells . 
Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells . 
In this study the genetic control of major histocompatibility complex ( MHC ) class II gene expression during the transition from B cell to plasma cell has been analyzed . 
Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription . 
We show here that the plasma cell-specific repressor function , designated SIR ( suppressor of immune response genes ) , does not act directly on the transcription of class II genes , but instead on the transcription of the AIR-1 gene , whose product , the class II transactivator ( CIITA ) , is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes . 
This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do not express CIITA-specific transcripts . 
Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids . 
These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers . 
Interestingly , in CIITA-transfected cell hybrids , cell surface expression of the human HLA-DQ heterodimer was not observed . 
This result was not attributable to lack of HLA-DQ alpha or -DQ beta transcription , because both transcripts were present in the CIITA-transfected hybrids , although at reduced levels . 
These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset , which may be thus controlled at the posttranscriptional level by a CIITA-independent mechanism . 
Nasal NK- and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma and Hodgkin 's disease . 
Nasal T\/NK-cell lymphomas can be further separated into those of natural killer ( NK ) cell lineage or of T-cell lineage , with differences in cellular phenotype , T-cell receptor ( TcR ) gene rearrangement and TcR transcript expression . 
Both NK- and T-cell subtypes are closely associated with Epstein-Barr virus ( EBV ) . 
In this study , EBV gene expression was determined in 23 cases of nasal lymphoma ( NL ) by in situ hybridisation ( ISH ) , reverse transcriptase-polymerase chain reaction ( RT-PCR ) and immunohistochemistry ( IH ) . 
Of the 23 cases , 19 were classified as NK-cell and 4 as T-cell tumours . 
ISH for EBV-encoded small non-polyadenylated RNAs showed that all cases , whether NK or T , harboured EBV in virtually all tumour cells . 
RT-PCR demonstrated that NL of both subtypes expressed EBNAI of the QUK splice pattern , the latent membrane proteins , LMP1 and 2 and the BamHI A rightward transcripts in the absence of EBNA2 mRNAs , compatible with the latency type II pattern . 
In addition , analysis of EBV protein expression by IH revealed a heterogeneous pattern of EBV gene expression at the single-cell level consisting of both LMP1+ and LMP1- tumour cells , suggesting a mixture of latency I and II . 
Although 2 early lytic transcripts , BZLF1 and BHRF1 , were also detected in 13 and 10 cases , respectively , the lack of ZEBRA staining in any case indicates that these lytic transcripts are most likely expressed by rare cells in the biopsies entering lytic cycle . 
The viral transcriptional pattern similar to that of nasopharyngeal carcinoma and Hodgkin 's disease suggests that EBV can exploit common regulatory mechanisms for gene transcription in diverse host cell types . 
Down-regulation of immunogenic proteins ( EBNA2-EBNA6 ) in nasal lymphoma may enable tumour cells to evade host cytotoxic T-cell surveillance . 
Stat3 recruitment by two distinct ligand-induced , tyrosine-phosphorylated docking sites in the interleukin-10 receptor intracellular domain . 
Recent work has shown that IL-10 induces activation of the JAK-STAT signaling pathway . 
To define the mechanism underlying signal transducer and activator of transcription ( STAT ) protein recruitment to the interleukin 10 ( IL-10 ) receptor , the STAT proteins activated by IL-10 in different cell populations were first defined using electrophoretic mobility shift assays . 
In all cells tested , IL-10 activated Stat1 and Stat3 and induced the formation of three distinct DNA binding complexes that contained different combinations of these two transcription factors . 
IL-10 also activated Stat5 in Ba\/F3 cells that stably expressed the murine IL-10 receptor . 
Using a structure-function mutagenesis approach , two tyrosine residues ( Tyr427 and Tyr477 ) in the intracellular domain of the murine IL-10 receptor were found to be redundantly required for receptor function and for activation of Stat3 but not for Stat1 or Stat5 . 
Twelve amino acid peptides encompassing either of these two tyrosine residues in phosphorylated form coprecipitated Stat3 but not Stat1 and blocked IL-10-induced Stat3 phosphorylation in a cell-free system . 
In contrast , tyrosine-phosphorylated peptides containing Tyr374 or Tyr396 did not interact with Stat3 or block Stat3 activation . 
These data demonstrate that Stat3 but not Stat1 or Stat5 is directly recruited to the ligand-activated IL-10 receptor by binding to specific but redundant receptor intracellular domain sequences containing phosphotyrosine . 
This study thus supports the concept that utilization of distinct STAT proteins by different cytokine receptors is dependent on the expression of particular ligand-activatable , tyrosine-containing STAT docking sites in receptor intracellular domains . 
Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3 . 
The biological active form of vitamin D3 , 1,25-dihydroxyvitamin D3 ( VD ) , regulates cellular growth and differentiation . 
This provides the hormone with an interesting therapeutic potential . 
However , hypercalcemia is a side effect , which is caused by VD 's classical action , the regulation of calcium homeostasis . 
This made the need for VD analogues with selectively increased cell regulatory properties . 
Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD . 
In this report the biological properties of the compounds ZK161422 and ZK157202 , which are 20-methyl- and 20-methyl-23-eneanalogues , respectively , have been analyzed in comparison with VD . 
Both compounds show about 2-fold lower affinity to the VD receptor ( VDR ) than VD . 
However , compared to VD , their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells . 
Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold , ZK161422 has the same calcium-mobilizing potency as VD . 
Moreover , ZK161422 , but not ZK157202 , showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides ( IP9 ) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides ( DR3 ) . 
This observation supports a model , in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues . 
T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis . 
Rheumatoid arthritis is a multistep disorder associated with autoimmune features of yet unknown etiology . 
Implication of viruses such as Epstein-Barr virus ( EBV ) in rheumatoid arthritis pathogenesis has been suspected on the basis of several indirect observations , but thus far , a direct link between EBV and rheumatoid arthritis has not been provided . 
Here we show that a large fraction of T cells infiltrating affected joints from a patient with chronic rheumatoid arthritis recognizes two EBV transactivators ( BZLF1 and BMLF1 ) in a major histocompatibility complex-restricted fashion . 
Responses to these EBV antigens by synovial lymphocytes from several other chronic rheumatoid arthritis patients were readily detectable . 
Thus these results suggest a direct contribution of EBV to chronic rheumatoid arthritis pathogenesis . 
They also demonstrate for the first time the occurrence of T cell responses against EBV transactivating factors , which might be central in the control of virus reactivation . 
Prostaglandin E2 induction of binding activity to CRE and AP-2 elements in human T lymphocytes . 
Prostaglandins of the E series are immunomodulatory agents which exert inhibitory as well as stimulatory effects on a variety of immune responses . 
Since it is known that PGE2 is able to increase cAMP levels , we investigated whether it can affect gene expression through the activation of the transcription factors which bind enhancer elements in the promoter regions of cAMP-regulated genes . 
Using electrophoretic mobility shift assay , we demonstrated that a short treatment of human T lymphocytes with PGE2 induces specific binding activity to CRE and AP-2 , but not AP-1 , DNA elements . 
Since the okadaic acid , a potent protein phosphatase inhibitor , prolongs the induction of the binding activity , phosphorylation events are likely to occur . 
This activity seems to be due to increased cAMP levels because forskolin and IBMX mimic the effects of PGE2 . 
More interestingly , transfection experiments with CRE-CAT plasmide show that PGE2 activates the transcription of a CRE-containing promoter . 
These data support the positive role for PGE2 on some immune functions . 
Tissue and cell-type specific expression of the tuberous sclerosis gene , TSC2 , in human tissues . 
TSC2 is a gene on chromosome 16p13.3 associated with the autosomal dominant neurocutaneous disorder , tuberous sclerosis complex ( TSC ) . 
By using a partial nucleotide sequence from the cloned TSC2 and polymerase chain reaction methodology , we constructed a digoxigenin-labeled complementary DNA probe to examine TSC2 gene expression in autopsy- or biopsy-derived human tissues by in situ hybridization . 
TSC2 messenger RNA was widely expressed in various cell types throughout the body , including epithelia , lymphocytes , and cells with endocrine functions , e.g. , adrenal cortex and anterior pituitary . 
It was prominently and selectively ( within the central nervous system ) expressed in pyramidal cells of the cerebral cortex and other motor neurons , e.g. , in spinal cord and brainstem nuclei . 
Visceral TSC2 expression was comparable in autopsy tissues from patients with and without TSC ; TSC2 messenger RNA expression was most prominent in cells with a rapid mitotic rate and turnover , e.g. , epithelia and lymphocytes , with central nervous system pyramidal cells and other neurons being an obvious exception , and\/or in cells with important secretory\/transport functions . 
This widespread expression of the TSC2 gene supports the view that it encodes a protein vital to cell growth and metabolism or one that functions as a tumor\/growth suppressor . 
CD40 , but not lipopolysaccharide and anti-IgM stimulation of primary B lymphocytes , leads to a persistent nuclear accumulation of RelB . 
In this study we analyzed the effect of CD40 stimulation on the activity and nuclear appearance of Rel\/nuclear factor kappaB ( NF-kappaB ) factors in primary murine B lymphocytes . 
We show that triggering of CD40 signaling pathway ( s ) by CD40 ligands expressed on L cells led to strong activation of an NF-kappaB-controlled beta-globin reporter gene in primary B lymphocytes from transgenic mice . 
Analyses of nuclear translocation of individual members of Rel proteins after CD40 induction of primary B cells showed a strong and long-lasting accumulation of RelB and , less pronounced , of c-Rel . 
LPS stimulation did not give rise to a persistent nuclear accumulation of RelB and c-Rel , whereas nuclear c-Rel , but not RelB , accumulated after B cell receptor stimulation . 
CD40 induced not only nuclear translocation but also de novo synthesis of RelB RNA and protein . 
S107 plasmacytoma cells , which express CD40 but are defective for the nuclear appearance of p50\/p65-NF-kappaB , do not express RelB after CD40 stimulation . 
In S107 cells stably transfected with relB genes , stimulation of nuclear RelB translocation by CD40 was observed . 
These results indicate that stimulation of CD40 signaling pathways exerts a long-lasting stimulatory effect on both the transcription and nuclear translocation of RelB . 
Since LPS and anti-IgM were unable to activate RelB , CD40 appears to trigger a special program of gene expression involved in the proliferation and\/or differentiation of B lymphocytes . 
{ Cortisone-resistant bronchial asthma } 
There is general agreement on the inflammatory pathogenesis of bronchial asthma : an accumulation of activated eosinophils , degranulated mast cells , T lymphocytes and in very severe forms , granulocytes has constantly been found in the bronchial mucosa . 
In allergic bronchial asthma , inflammation seems to be orchestrated predominantly by a subset of T lymphocytes , with a phenotype similar to the Th2 subset able to produce IL-4 and IL-5 . 
Although corticosteroids are the most potent therapeutic agents used for this disease , their anti-inflammatory effect differs from patient to patient . 
Some criteria which can be used to define steroid-resistant bronchial asthma are listed here . 
This review analyzes various molecular alterations responsible for the deficient response to corticosteroid treatment observed in steroid-resistant bronchial asthmatic subjects . 
New knowledge on the mechanism of steroid resistance may have important implications for the treatment of chronic asthma and other diseases . 
Induction of the CD11b gene during activation of the monocytic cell line U937 requires a novel nuclear factor MS-2 { published erratum appears in J Immunol 1999 Jul 15 ; 163 ( 2 ) : 1091 } 
The differentiation of myeloid precursors into mature myelomonocytic cells is characterized by the induction of the gene encoding the beta2 integrin CD11b . 
The transcription factors Sp1 and PU.1 prime the CD11b promoter , but the nature of the factors responsible for its inducible expression are unknown . 
In addition to the CD11b gene , the homologous genes encoding CD11a and CD11c also exhibit inducible expression during myeloid differentiation . 
Therefore , we compared the nucleotide sequences of the CD11a , CD11b , and CD11c gene promoters to identify common elements that might contribute to inducible expression . 
This analysis identified one such element repeated four times within the CD11b promoter . 
Mutation of these elements indicated that two , MS-2beta and MS-2gamma , are critical to the induction of the CD11b gene during differentiation of the pro-monocytic cell line U937 . 
Electrophoretic mobility shift assays indicate that MS-2beta and MS-2gamma interact with nuclear factors that are induced during U937 differentiation . 
These factors are detected at the time 0 the CD11b promoter is activated . 
The molecular mass of these factors is approximately 28 kDa , and their DNA binding characteristics are indistinguishable from those of the novel nuclear factor MS-2 . 
Taken together , our data indicate that MS-2 mediates induction of the CD11b gene as cells of the monocytic lineage mature . 
The presence of multiple potential binding sites for MS-2 in the promoter regions of a wide range of genes expressed in mature myeloid cells suggests 0 this factor plays a general role in myeloid differentiation . 
Dexamethasone suppression test : corticosteroid receptors regulation in mononuclear leukocytes of young and aged subjects . 
The dexamethasone suppression test ( DST ) is considered an indicator of the function of the adrenal pituitary axis . 
The effect of the steroid is mediated by its binding to corticosteroid receptors . 
We previously suggested that the measurement of corticosteroid receptors in lymphocytes is an index of an analogous pattern in brain . 
In the present study , corticosteroid Type I and Type II receptors in mononuclear leukocytes were measured in 10 elderly subjects and in 9 young adults , before and after overnight DST ( 1 mg ) . 
Receptors were measured by radioreceptor assay . 
In all the subjects , dexamethasone was able to suppress plasma cortisol . 
The number of Type I and Type II receptors before the test was lower in elderly subjects than in adults . 
In the control group , dexamethasone produced a significant depression of Type I receptors ( from 267 +\/- 72 to 169 +\/- 71 receptors per cell ) , which can be interpreted as a primary involvement of Type I receptors in the response to dexamethasone ; Type II receptors decreased in half the subjects ( from 2849 +\/- 703 to 2345 +\/- 569 receptors per cell ) . 
In elderly healthy subjects , Type II receptors were also significantly decreased ( from 1796 +\/- 671 to 720 +\/- 345 ) . 
We suggest that in young subjects Type II receptors are initially up-regulated by dexamethasone , and then down-regulated , while in aged subjects an up-regulation can not be achieved , as suggested by the higher values of plasma cortisol usually found in aging subjects 
Octamer independent activation of transcription from the kappa immunoglobulin germline promoter . 
Previous analyses of immunoglobulin V region promoters has led to the discovery of a common octamer motif which is functionally important in the tissue-specific and developmentally regulated transcriptional activation of immunoglobulin genes . 
The germline promoters ( Ko ) located upstream of the J region gene segments of the kappa locus also contain an octamer motif ( containing a single base pair mutation and referred to as the variant octamer ) which has been shown previously to bind Oct-1 and Oct-2 transcription factors in vitro . 
To further elucidate the role of this variant octamer motif in the regulation of germline transcription from the unrearranged kappa locus , we have quantitated the relative binding affinity of Oct-1 and Oct-2 for the variant octamer motif and determined the functional role of this octamer motif in transcriptional activation . 
We find that , although the variant octamer motif binds Oct-1 and Oct-2 in vitro with 5-fold lower affinity than the consensus octamer motif , mutation of the variant octamer motif to either a consensus octamer or non-octamer motif has no effect on transcriptional activation from the germline promoter . 
We also find significant differences in activation of germline and V region promoters by kappa enhancers . 
Our results suggest that the germline promoters and V region promoters differ in their dependence on octamer for activation and respond differently to enhancer activation . 
These findings have important implications in regulation of germline transcription as well as concomitant activation of the V-J recombination of the kappa light chain locus . 
A novel SP-1 site in the human interleukin-1 beta promoter confers preferential transcriptional activity in keratinocytes . 
To investigate the mechanisms of transcriptional activation of interleukin-1beta ( IL-1beta ) in non-monocytic cells , we constructed a series of reporter plasmids with the bacterial chloramphenicol acetyltransferase gene linked to various parts of the human IL-1beta promoter and performed transient transfection experiments . 
We identified a promoter segment that activates transcription most efficiently in keratinocytes . 
Electrophoretic mobility shift assays ( EMSA ) with a 43-mer oligonucleotide derived from the functionally identified cis-acting element revealed specific complexes . 
By competition analysis with transcription factor consensus sequence oligonucleotides and by immunosupershift , transcription factor SP-1 or a closely related protein was shown to bind to this regulatory element . 
The closest match to the known SP-1 consensus sequence within the respective region is a TCCCCTCCCCT motif . 
Mutation of this motif almost completely , and specifically , abolished the binding of two low-mobility complexes and led to a 95 % decrease of constitutive transcriptional activation of a reporter construct IL-1beta ( -170\/+108 ) . 
Likewise , activation of this reporter construct by tumor necrosis factor-alpha depended on the SP-1 site . 
These observations suggest that a so-far-unrecognized SP-1 site in the human IL-1beta promoter may participate in the transcriptional regulation of this gene in keratinocytes . 
Stimulation of human peripheral blood mononuclear cells by zinc and related cations . 
Zinc is an important trace element for immune function . 
Here , we show that zinc addition in a serum- and lipopolysaccharide-free cell culture system leads to significantly enhanced levels of interleukin 1 beta ( IL-1 beta ) and tumour necrosis factor alpha ( TNF-alpha ) and to expression of the corresponding mRNA in human peripheral blood mononuclear cells ( PBMC ) . 
Structurally related divalent cations like cobalt , nickel , and mercury also partially increase monokine secretion but to a much lower and thus insignificant extent . 
They fail to induce mRNA of TNF-alpha after 3 h of culture . 
Therefore , monokine induction is a zinc-specific effect influenced by the physicochemical properties of the ion . 
Confirmation of the unique significance of zinc for immune function provides a better understanding of the mechanisms of specific zinc-mediated immune modulation . 
Identification of Bcd , a novel proto-oncogene expressed in B-cells . 
A novel B-cell derived ( Bcd ) oncogene has been isolated from the peripheral blood lymphocytes of one B-cell chronic lymphocytic leukemia ( B-CLL ) patient using DNA transfer and a mouse tumorigenicity assay . 
The Bcd proto-oncogene was activated by a truncation in the 5' UTR . 
It predicts for two open reading frames ( ORFs ) . 
ORF1 consists of 240 bp that would encode 80 amino acids , while the major ORF2 consists of 648 bp capable of coding for 216 amino acids . 
Predicted peptide sequence of ORF2 contained a zinc finger domain which showed significant homology to GC box binding proteins BTEB2 and SP1 . 
Transfection of an expression vector containing ORF2 but not full length cDNA was able to transform NIH3T3 cells and induce tumors in nude mice . 
Bcd mRNA transcripts of &lt; or = 2.6 kb were selectively expressed in PBL and testis of healthy individuals . 
Within the PBL , Bcd gene expression was restricted to CD19+ B-cells and absent from CD14+ monocytes and T-cells . 
Bcd transcripts were detected in all normal PBL samples tested but not in several malignant human B-cell lines and not in 50 % of B-cells from B-CLL patients . 
However , stimulation of B-cells from B-CLL patients under conditions which induced differentiation into plasma cells was associated with induction of Bcd gene expression . 
The Bcd gene may therefore play an important role in B-cell growth and development . 
Expression of erythroid-specific genes in megakaryoblastic disorders . 
Currently available data indicate that erythroid and megakaryocytic differentiation pathways are closely related to each other , and there may exist progenitor cells common to those two lineages may exist . 
Acute megakaryoblastic leukemia ( AML-M7 ) and transient myeloproliferative disorder in Down 's syndrome ( TMD ) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers . 
These blast cells express lineage-specific transcription factors such as GATA-1 common to these lineages and frequently express erythroid-specific mRNAs such as gamma-globin and erythroid delta-aminolevulinate synthase ( ALAS-E ) , indicating that most of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes . 
These results suggest that blasts in M7 and TMD may correspond to progenitors of both erythroid and megakaryocytic lineages . 
