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Biomedical subjects

R Sen

Publications and source records attributed to R Sen.

At least 19 recordsLinked to original sources
PubMed1999

Presence of pentoxifylline during T cell priming increases clonal frequencies in secondary proliferative responses and inhibits apoptosis.

Naive T cells appear to be primed by specific Ag to differentiate into either effectors or memory cells. We have been analyzing the factors involved in this differential commitment in the priming of alloresponsive human T cells in vitro and have shown that the presence of a phosphodiesterase inhibitor, pentoxifylline (POX), during priming results in a decrease in the primary response and enhancement in the secondary proliferative response. We now show that the POX-mediated effect can be mimicked by dibutyryl cAMP. The secondary response enhancement is due to the effects of POX on the T cells rather than the APCs, because even fixed APCs can prime T cells in the presence of POX. POX affects T cells directly by increasing clonal frequency rather than the burst size of the secondary responders. The known inhibition of IL-2 production by POX is not responsible for this effect, because exogenous IL-2 supplementation does not block it. The presence of POX during priming alters the outcome of T cell activation, resulting in a lower frequency of cells expressing IL-2R alpha (CD25) and a decrease in their subsequent apoptosis, and this antiapoptotic effect is consistent with the enhanced commitment of T cells to secondary responsiveness by POX.

Adjuvants, Immunologic↗
PubMed1999

A NF-kappa B/c-myc-dependent survival pathway is targeted by corticosteroids in immature thymocytes.

Glucocorticoid hormones modulate T cell maturation in vivo. While low levels of hormones are required for appropriate T cell development, high levels of glucocorticoid hormones target immature developing thymocytes for cell death during systemic stress. In this report, we propose a molecular mechanism for the induction of apoptosis in CD4+CD8+ double-positive thymocytes by dexamethasone in vivo. Dexamethasone injection induced the expression of IkappaBalpha and IkappaBbeta in thymocytes and down-regulated NF-kappaB DNA binding activated by intrathymic signals. Down-regulation of NF-kappaB DNA binding preceded cell death, suggesting that NF-kappaB may be important for the survival of immature thymocytes. In addition, ex vivo treatment of thymocyte single-cell suspension with dexamethasone accelerated p65/RelA down-regulation and cell death. Conversely, NF-kappaB induction diminished dexamethasone-induced death. Expression of the c-myc proto-oncogene, a NF-kappaB target, was also reduced in thymocytes of dexamethasone-treated animals, and ectopic transgenic expression of c-myc in mice provided partial rescue of double-positive thymocytes from dexamethasone mediated cell death. These observations suggest that viability of CD4+CD8+ thymocytes may be maintained by an NF-kappaB/c-myc-dependent pathway in vivo.

Animals↗
PubMed1999

Transcriptional activation by ETS and leucine zipper-containing basic helix-loop-helix proteins.

The immunoglobulin mu heavy-chain gene enhancer contains closely juxtaposed binding sites for ETS and leucine zipper-containing basic helix-loop-helix (bHLH-zip) proteins. To understand the mu enhancer function, we have investigated transcription activation by the combination of ETS and bHLH-zip proteins. The bHLH-zip protein TFE3, but not USF, cooperated with the ETS domain proteins PU.1 and Ets-1 to activate a tripartite domain of this enhancer. Deletion mutants were used to identify the domains of the proteins involved. Both TFE3 and USF enhanced Ets-1 DNA binding in vitro by relieving the influence of an autoinhibitory domain in Ets-1 by direct protein-protein associations. Several regions of Ets-1 were found to be necessary, whereas the bHLH-zip domain was sufficient for this effect. Our studies define novel interactions between ETS and bHLH-zip proteins that may regulate combinatorial transcription activation by these protein families.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1998

Reduction in abortive transcription from the lambdaPR promoter by mutations in region 3 of the sigma70 subunit of Escherichia coli RNA polymerase.

Transcription initiation by Escherichia coli RNA polymerase at most promoters is associated with a reiterative synthesis and release of short abortive RNA products. We have investigated the mechanism of abortive RNA synthesis by using holoenzymes containing mutant sigma70 subunits with changes in region 3 (S506F and P504L), which reduce the ratio of abortive to full-length products. Binary complexes formed by these mutant enzymes at a modified lambdaPR promoter contained a smaller fraction of open complexes than for normal polymerase, suggesting an involvement of region 3 in melting duplex DNA or in maintenance of the open complex. The half-lives of the majority of binary complexes formed by the mutant enzymes were less than 1 min, in contrast to 30 min for the wild-type complexes. The time courses of transcription and pulse-labeling assays showed that moribund complexes, which generate only abortive products (Kubori, T., and Shimamoto, N. (1996) J. Mol. Biol. 256, 449-457), were formed by the mutant enzymes. However, they accumulated to a lesser extent than for the wild-type enzyme, due both to faster dissociation and conversion into inactive complexes. This is the main cause of the low degree of abortive transcription displayed by the mutant enzymes on this promoter.

Bacteriophage lambda↗
PubMed1998

Pentoxifylline inhibits Ig kappa gene transcription and rearrangements in pre-B cells.

Pentoxifylline (PF) has been used in a wide variety of clinical situations; however, the molecular consequences of this drug are not well characterized. In this paper we assayed the effects of PF in two models of pre-B differentiation. In 70Z pre-B cells, transcriptional induction of rearranged Ig kappa-chain gene in response to LPS was suppressed by PF, without affecting the induction of Rel family proteins. In contrast, kappa induction by IFN-gamma was not suppressed by PF, indicating that the drug inhibited certain activation pathways. We also found that LPS-induced activation of germline kappa transcription and V kappa to J kappa recombination were inhibited by PF in the pre-B cell line 38B9. These observations suggest that PF may adversely affect B lymphopoiesis during chronic administration.

Animals↗
PubMed1998

Ion tolerance of Saccharomyces cerevisiae lacking the Ca2+/CaM-dependent phosphatase (calcineurin) is improved by mutations in URE2 or PMA1.

Calcineurin is a conserved, Ca2+/CaM-stimulated protein phosphatase required for Ca2+-dependent signaling in many cell types. In yeast, calcineurin is essential for growth in high concentrations of Na+, Li+, Mn2+, and OH-, and for maintaining viability during prolonged treatment with mating pheromone. In contrast, the growth of calcineurin-mutant yeast is better than that of wild-type cells in the presence of high concentrations of Ca2+. We identified mutations that suppress multiple growth defects of calcineurin-deficient yeast (cnb1Delta or cna1Delta cna2Delta). Mutations in URE2 suppress the sensitivity of calcineurin mutants to Na+, Li+, and Mn2+, and increase their survival during treatment with mating pheromone. ure2 mutations require both the transcription factor Gln3p and the Na+ ATPase Pmr2p to confer Na+ and Li+ tolerance. Mutations in PMA1, which encodes the yeast plasma membrane H+-ATPase, also suppress many growth defects of calcineurin mutants. pma1 mutants display growth phenotypes that are opposite to those of calcineurin mutants; they are resistant to Na+, Li+, and Mn2+, and sensitive to Ca2+. We also show that calcineurin mutants are sensitive to aminoglycoside antibiotics such as hygromycin B while pma1 mutants are more resistant than wild type. Furthermore, pma1 and calcineurin mutations have antagonistic effects on intracellular [Na+] and [Ca2+]. Finally, we show that yeast expressing a constitutively active allele of calcineurin display pma1-like phenotypes, and that membranes from these yeast have decreased levels of Pma1p activity. These studies further characterize the roles that URE2 and PMA1 play in regulating intracellular ion homeostasis.

Adenosine Triphosphatases↗
PubMed1998

Receptor-specific induction of NF-kappaB components in primary B cells.

The NF-kappaB transcription factor complex plays a key role in the expression of genes involved in immune responses. Nuclear NF-kappaB is induced in B lymphocytes by engagement of either the antigen receptor (sIg) or the CD40 receptor for a T cell activation antigen, although different intracellular pathways appear to be involved. In the present study the protein composition of NF-kappaB complexes triggered by sIg and CD40 was probed by electrophoretic mobility shift, supershift, shift-Western, and Western blot analyses. At the time of peak NF-kappaB induction (2 h), the NF-kappaB components detected in the complexes induced through sIg and through CD40 were the same. However, with continued stimulation RelB completely disappeared from anti-Ig-stimulated kappaB binding material, but remained a component of CD40L-induced NF-kappaB. The loss of DNA-binding RelB from anti-Ig-induced NF-kappaB did not result from depletion of RelB from B cell nuclei, suggesting specific regulation of RelB function which is not directly attributed to IkappaB function. These results indicate that NF-kappaB complexes may undergo protein-specific alterations in a time- and receptor-dependent fashion that may be associated with differences in the outcomes of B cell stimulation through sIg and CD40.

Animals↗
PubMed1998

Exploring functional redundancy in the immunoglobulin mu heavy-chain gene enhancer.

Immunoglobulin (Ig) mu heavy-chain gene enhancer activity is mediated by multiple DNA binding proteins. Mutations of several protein binding sites in the enhancer do not affect enhancer activity significantly. This feature, termed redundancy, is thought to be due to functional compensation of the mutated sites by other elements within the enhancer. In this study, we identified the elements that make the basic helix-loop-helix (bHLH) protein binding sites, muE2 and muE3, redundant. The major compensatory element is a binding site for interferon regulatory factors (IRFs) and not one of several other bHLH protein binding sites. These studies also provide the first evidence for a role of IRF proteins in Ig heavy-chain gene expression. In addition, we reconstituted the activity of a monomeric mu enhancer in nonlymphoid cells and defined the domains of the ETS gene required for function.

Animals↗
PubMed1998

ETS-core binding factor: a common composite motif in antigen receptor gene enhancers.

A tripartite domain of the murine immunoglobulin mu heavy-chain enhancer contains the muA and muB elements that bind ETS proteins and the muE3 element that binds leucine zipper-containing basic helix-loop-helix (bHLH-zip) factors. Analysis of the corresponding region of the human mu enhancer revealed high conservation of the muA and muB motifs but a striking absence of the muE3 element. Instead of bHLH-zip proteins, we found that the human enhancer bound core binding factor (CBF) between the muA and mu elements; CBF binding was shown to be a common feature of both murine and human enhancers. Furthermore, mutant enhancers that bound prototypic bHLH-zip proteins but not CBF did not activate transcription in B cells, and conversely, CBF transactivated the murine enhancer in nonlymphoid cells. Taking these data together with the earlier analysis of T-cell-specific enhancers, we propose that ETS-CBF is a common composite element in the regulation of antigen receptor genes. In addition, these studies identify the first B-cell target of CBF, a protein that has been implicated in the development of childhood pre-B-cell leukemias.

Animals↗
PubMed1998

ETS-mediated cooperation between basic helix-loop-helix motifs of the immunoglobulin mu heavy-chain gene enhancer.

The muE motifs of the immunoglobulin mu heavy-chain gene enhancer bind ubiquitously expressed proteins of the basic helix-loop-helix (bHLH) family. These elements work together with other, more tissue-restricted elements to produce B-cell-specific enhancer activity by presently undefined combinatorial mechanisms. We found that muE2 contributed to transcription activation in B cells only when the muE3 site was intact, providing the first evidence for functional interactions between bHLH proteins. In vitro assays showed that bHLH zipper proteins binding to muE3 enhanced Ets-1 binding to muA. One of the consequences of this protein-protein interaction was to facilitate binding of a second bHLH protein, E47, to the muE2 site, thereby generating a three-protein-DNA complex. Furthermore, transcriptional synergy between bHLH and bHLH zipper factors also required an intermediate ETS protein, which may bridge the transcription activation domains of the bHLH factors. Our observations define an unusual form of cooperation between bHLH and ETS proteins and suggest mechanisms by which tissue-restricted and ubiquitous factors combine to generate tissue-specific enhancer activity.

Animals↗
PubMed1997

A three-protein-DNA complex on a B cell-specific domain of the immunoglobulin mu heavy chain gene enhancer.

The lymphoid-specific immunoglobulin mu heavy chain gene intron enhancer (muE) contains multiple binding sites for trans-acting nuclear factors. We have used a combination of in vitro and in vivo assays to reconstruct protein-DNA interactions on a minimal B cell-specific mu enhancer that contains three motifs, muA, muB, and muE3. Using ETS-domain proteins that transactivate the minimal enhancer in non-lymphoid cells, we show that (i) PU.1 binds coordinately to both muA and muB sites in vitro and (ii) in the presence of Ets-1, this factor binds to the muA site and PU.1 to the muB site. Two factors, TFE3 and USF, bind to the muE3 element. When the ETS proteins are present together with muE3 binding proteins, a three-protein-DNA complex is generated. Furthermore, we provide evidence for protein-protein interactions between Ets-1 and PU.1 proteins that bind to muA and muB sites, and between Ets-1 and TFE3 bound to the muA and mu3 sites. We propose that this domain of the mu enhancer is assembled into a nucleoprotein complex that contains two tissue-restricted ETS domain proteins that recognize DNA from the same side of the helix and one ubiquitously expressed bHLH-leucine zipper protein that binds between them, recognizing its site from a different side of the helix.

3T3 Cells↗
PubMed1997

Differential interactions of the Mg2+ complexes of chromomycin A3 and mithramycin with poly(dG-dC) x poly(dC-dG) and poly(dG) x poly(dC).

The interaction of the two anticancer antibiotics, chromomycin A3 and mithramycin, with the polynucleotides poly(dG-dC) x poly(dC-dG), representative of B-DNA, and poly(dG) x poly(dC), representative of A-DNA, in the presence of Mg2+ is studied by spectroscopic techniques such as absorbance, fluorescence, and dircular dichroism (CD). The studies were done with both drug x Mg2+ complexes, I and II, having 1:1 and 2:1 stoichiometries with respect to drug and Mg2+, respectively [Aich, P., Sen, R., & Dasgupta, D. (1992) Biochemistry 31, 2988-2997]. The objective of the present work is 2-fold. First, an attempt is made to understand the structural basis of the ligand-DNA interaction, particularly the role of DNA backbone conformation with its groove size and the accessibility of the 2-amino group in the minor groove of guanosine. Second, the role of the antibiotic saccharide moieties in the association with DNA was studied. For this purpose, the spectroscopic characterization of the binding was done followed by the evaluation of binding parameters and associated thermodynamics. Analysis of the observed thermodynamics for the ligand-DNA interactions in terms of the different structures of the polynucleotides was done. The salient results are as follows. Complex I does not discriminate significantly among the A- and B-forms of DNA when it binds to them in an entropy-driven process. On the other hand, complex II for both drugs recognizes B- and A-forms of DNA in different ways. This observation implies that the sequence specificity shown by this complex is a sequel to the difference in the parameters such as groove size and accessibility of the guanosine amino group. Another important finding is that binding with the same polynucleotide is not comparable for the complex II of the two drugs. It emphasizes the involvement of the sugar moieties, when the drug x Mg2+ complex binds to DNA. The presence of an acetoxy group in the sugars of chromomycin A3 imparts some distinctive specific features of the association of the chromomycin dimer x Mg2+ complex with DNA. Finally, the results are compared with those available from NMR studies of different drug-oligonucleotide complexes under conditions where complex II is the ligand.

Chromomycin A3↗
PubMed1997

c-Rel is a target of pentoxifylline-mediated inhibition of T lymphocyte activation.

The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell-dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-kappaB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2R(alpha) chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo.

Animals↗
PubMed1997

Combinatorial determinants of tissue-specific transcription in B cells and macrophages.

A tripartite domain of the immunoglobulin mu heavy-chain gene enhancer that activates transcription in B cells contains binding sites for PU.1, Ets-1, and a leucine zipper-containing basic helix-loop-helix factor. Because PU.1 is expressed only in B cells and macrophages, we tested the activity of a minimal mu enhancer fragment in macrophages by transient transfections. The minimal mu enhancer activated transcription in macrophages, and the activity was dependent on all three sites. Analysis of mutated enhancers, in which spacing and orientation of the ETS protein binding sites had been changed, suggested that the mechanisms of enhancer activation were different in B cells and macrophages. Thus, ETS protein binding sites may be combined in different ways to generate tissue-specific transcription activators. Despite the activity of the minimal enhancer in macrophages, a larger mu enhancer fragment was inactive in these cells. We propose that formation of the nucleoprotein complex that is formed on the minimal enhancer in macrophages cannot be helped by the neighboring muE elements that are essential for activity of the monomeric enhancer.

Animals↗
PubMed1996

HMG box-activating factors 1 and 2, two HMG box transcription factors that bind the human Ig heavy chain enhancer.

We present the isolation of two cDNAs that encode distinct, yet related, proteins that bind the HE2 region of the human Ig heavy chain (IgH) enhancer. Designated HMG box-activating factors (HAF) 1 and 2, the two proteins are new members of the HMG box family of DNA binding proteins. Both are potent transcription activators when expressed 1) as GAL4 fusions targeted to promoters containing GAL4 operators, or 2) as intact proteins targeted to minimal promoters containing binding sites derived from the IgH enhancer. HAF-1 and HAF-2 mRNAs are apparently expressed in both B cells and non-B cells. However, activity generated by the isolated HE2 region in B cells is dependent on both an intact HAF-1/HAF-2 binding site and at least one additional site that has been reported previously to bind a B cell-restricted protein. Our results suggest a collaborative role for either or both HAF-1 and HAF-2 in establishing the B cell activity of the human IgH enhancer.

Amino Acid Sequence↗
PubMed1996

Context dependent transactivation domains activate the immunoglobulin mu heavy chain gene enhancer.

Enhancers and promoters nucleate the assembly of multiprotein complexes that are required for the transcriptional activation of eukaryotic genes. Although multimerized binding sites of individual transcription factors sometimes mimic the properties of an enhancer, the combinatorial use of factors is considered to be crucial for achieving biological specificity. The minimal B cell specific immunoglobulin mu heavy chain gene enhancer is activated by a combination of tissue-restricted ETS proteins and ubiquitously expressed basic helix-loop-helix transcription factors. Here we show that a domain of PU.1 that activates transcription from multimerized PU.1 binding sites is not required to activate the mu enhancer together with Ets-1. In contrast, a transactivation domain in Ets-1 is necessary to activate this enhancer synergistically with PU.1. Furthermore, the Ets-1 activation domain functions only when tethered to the muA site of the enhancer. These observations illuminate two forms of context dependence: first, all possible transcription activation domains may not be required to achieve combinatorial specificity; second, functional transcription activation domains may require appropriate positioning on DNA.

Enhancer Elements, Genetic↗