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R Sen

Publications and source records attributed to R Sen.

At least 37 records · Page 2Linked to original sources
PubMed1996

Differential regulation of c-Rel translocation in activated B and T cells.

c-Rel induction in activated lymphocytes is suppressed by the immunosuppressive drug, FK506. Here we show that FK506-suppressible, delayed c-Rel induction is similar in B and T cells and is regulated by mRNA production. In contrast, rapid nuclear translocation of pre-existing cytoplasmic c-Rel occurs only in B cells, but not in T cells. Analysis of I-kappaBalpha and -beta in these cells showed that both I-kappaBalpha and I-kappaBbeta were rapidly degraded in response to stimulation in B cells, but only I-kappaBalpha was affected in T cells. These observations suggest that 1) different Rel proteins in the same cell may be sequestered in the cytoplasm differently, 2) the sequestration mechanism is cell-type specific, and 3) differential sensitivities of I-kappaBalpha and beta in B and T cells may regulate, in part, the rapid response of family members. We propose that subunit-specific and cell-specific regulation of nuclear translocation may help determine the varied cellular responses to different stimuli.

Animals↗
PubMed1996

Intrathymic signals in thymocytes are mediated by p38 mitogen-activated protein kinase.

Thymocytes develop into mature functional T cells in the inductive environment of the thymus where thymocyte-stromal cell interactions and cytokines provide survival and differentiation signals as cues for thymocyte maturation. Disruption of the thymic microenvironment results in attenuation of T cell maturation, suggesting that intrathymic signals are essential for differentiation and repertoire selection. We have previously shown that several inducible nuclear factors such as AP-1, NF-AT, and NF-kappaB are activated in response to intrathymic signals. Here we demonstrate that in thymocytes p38 mitogen-activated protein (MAP) kinase, a member of the MAP kinase family of proteins that include the extracellular-signal regulated kinases and Jun aminoterminal kinases, is highly activated in response to intrathymic signals in vivo. These studies suggest a role for p38 MAP kinase in T cell survival and differentiation.

Animals↗
PubMed1996

Physical determinants of intermembrane protein transfer.

Intermembrane protein transfer between erythrocytes and phospholipid vesicles was examined under a variety of conditions to investigate physical factors governing this process. Human erythrocytes were incubated with sonicated dimyristoylphosphatidylcholine vesicles containing trace [14C]dipalmitoylphosphatidylcholine. Protein-vesicle complexes were separated from cells and from membrane fragments by density gradient centrifugation. The yield of isolated protein vesicles was determined from the 14C-vesicle marker; protein compositions were analyzed by SDS-polyacrylamide gel electrophoresis. Enzymatic removal of portions of the cytoplasmic or exoplasmic domains of cell membrane proteins had little effect on the extent of protein transfer. Membrane additives such as cholate produced a 2-fold increase in protein-vesicle yield. The selectivity of protein transfer from erythrocytes was influenced by the lipid composition of recipient vesicles: inclusion of cholesterol increased band 3 content while the presence of anionic phospholipids reduced transfer. Proteins transferred from 32P-labeled cells differed in specific radioactivity from bulk cell proteins: glycophorin, highly phosphorylated in the cell membrane, showed no detectable labeling in the corresponding protein-vesicle band. These observations suggest that cell-to-vesicle protein transfer is insensitive to bulk steric and electrostatic properties of cell membranes, but enhanced by membrane defects. Recipient membrane composition influences the selectivity of transferred proteins and may reveal subtle differences in the membrane association of protein subpopulations.

2,3-Diphosphoglycerate↗
PubMed1996

Conformational changes of E. coli RNA polymerase during transcription initiation.

Escherichia coli RNA polymerase-promoter complex undergoes a multistep process to initiate transcription. We have employed fluorescence spectroscopic approaches to detect the conformational states of the enzyme during this multistep process. A fluorescence assay based on the measurement of fluorescence of free and promoter-bound enzyme as a function of temperature within the range of 4 to 37 degrees C showed that, starting with initial 'closed complex', there are conformationally two distinct intermediate states of the polymerase till it attains the final form required for transcription initiation. The equilibrium from closed complex (RPc) to open complex (RPo) consists of at least the following two intermediate complexes: [formula: see text] Higher order structure of RNAP in each of these complexes was probed by means of measurement of accessibilities of the tryptophan fluorophores to the acrylamide. In the next part of the study, TbGTP, a fluorescent substrate, has been used to probe the state of active site in the enzyme for the complexes RPc, RPi1, RPi2 and RPo, respectively. From the comparison of changes in the parameters such as, fluorescence polarization anisotropy of TbGTP and its accessibility to the neutral quencher, acrylamide, in free and promoter-bound enzyme, we have further substantiated the first part of our results. Together these results suggest that formations of RPc and RPi1 do not involve radical conformational changes in the enzyme, while the enzyme undergoes major change in conformation in the steps RPil-->RPi2 and RPi2-->RPo. The strong tryptophan promoter cloned in plasmid pDR720 was chosen as a model promoter in these studies.

Binding Sites↗
PubMed1996

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.

Animals↗
PubMed1996

Peripheral odontogenic fibroma with chondroid differentiation.

An unusual case of peripheral odontogenic fibroma, presenting as a swelling on gingiva involving hard palate in a 3-year-old female child, is described. Even with cellular stroma and unencapsulation these tumors behave in a benign fashion. To the best of our knowledge, cartilagenous differentiation of stroma as observed in this case has not been reported in English literature so far.

Cartilage↗
PubMed1996

Demonstration of acid-fast bacilli in buffy coat and bone marrow smear--a diagnostic tool in pulmonary tuberculosis.

Buffy coat smears prepared from peripheral blood and bone marrow aspirate obtained from proved 60 untreated cases of pulmonary tuberculosis were stained with Ziehl-Neelsen method and thoroughly screened for presence of tubercle bacilli. Acid-fast bacilli were detected in 55% cases in buffy coat, 48.3% cases in bone marrow, 38.3% cases both in buffy coat and bone marrow and 65% cases either in buffy coat or in bone marrow or in both. Considering the fact that demonstration of acid-fast bacilli may not be possible in more than 25-50% of the suspected cases of tuberculosis, these techniques are recommended for evaluation of their utility in establishing diagnosis of tuberculosis, particularly in reference to sputum negative cases of pulmonary tuberculosis and tuberculosis of inaccessible extrapulmonary sites.

Bacteriological Techniques↗
PubMed1995

Activation of IL-2 receptor alpha-chain gene by individual members of the rel oncogene family in association with serum response factor.

Expression of the IL-2R alpha gene is regulated by members of the c-Rel/NF-kappa B family of transcription factors binding to the kappa B site in the promoter. Previous work has not defined the role of individual members of the c-Rel family in the activation of the IL-2R alpha gene. Using the COS cell system, we were able to reconstitute the regulation of the IL-2R alpha promoter by expressing cloned Rel family members with serum response factor (SRF). We found that c-rel alone activated the IL-2R alpha promoter only weakly but worked with the p50 subunit of NF-kappa B (NFKB1) to give a higher level of expression. We showed that c-rel heterodimerizes with p50 and the amount of this heterodimer correlated with the level of IL-2R alpha gene expression. Our results provide evidence that c-rel/p50 heterodimers activate gene expression in the context of a cellular promoter. We show that c-rel or p65 can cooperate with SRF in the activation of this promoter and the transactivation by c-rel with SRF was enhanced by p50. Synergistic activation required both kappa B and CArG sites, and binding studies show that these adjacent sites can be occupied simultaneously. The transactivation observed with cloned transcription factors mimics the physiologic induction of the IL-2R alpha gene since multiple sequence elements cooperate to give gene activation. The data support the model that c-rel/p50 or p65 can cooperate with SRF to specifically target the expression of the IL-2R alpha gene in activated T cells.

Amino Acid Sequence↗
PubMed1995

Expression and induction of nuclear factor-kappa B-related proteins in thymocytes.

Thymocytes mature in response to the cues from the thymic micro-environment, which regulate stage-specific gene expression during development. We find that several proteins that bind the kappa B sequence in vitro are constitutively activated in freshly isolated thymocytes. These include the rel-related p50 homodimers, p50/p65 heterodimers, low levels of c-rel, and two other factors that may be thymus specific. Disruption of the thymic micro-environment resulted in loss of DNA-binding, suggesting that lymphocyte-stromal cell interactions induce and maintain these proteins in a DNA-binding form. Phorbol ester and ionomycin treatment induced p50, p65, and p68 c-rel kappa B DNA-binding activity. Expression of p68 c-rel protein, but not p50 or p65, was suppressed by the immunosuppressive drug FK506. Because FK506 specifically inhibits the appearance of mature single-positive thymocytes, gene expression regulated by p68 c-rel may play a role in selection and maturational signals involved in the double-positive to single-positive transition.

Animals↗
PubMed1995

FK506 inhibits antigen receptor-mediated induction of c-rel in B and T lymphoid cells.

Stimulation of B and T cells via the antigen receptor, by phorbol ester or by phorbol ester and ionomycin, leads to nuclear translocation of the inducible transcription factor NF-kappa B, comprising the p50 and p65 rel-related polypeptides. In this report we show that c-rel is a component of the antigen receptor-induced kappa B binding proteins in both B and T cells. Whereas NF-kappa B can be induced by phorbol ester alone, optimal induction of c-rel requires stimulation by both phorbol ester and ionomycin, the dual signal that is necessary for proliferation of untransformed lymphocytes. Furthermore, c-rel induction is blocked by the immunosuppressive drug FK506 that is known to inhibit B and T cell activation. c-rel-dependent transactivation of the interleukin-2 receptor alpha chain (IL-2R alpha) promoter is augmented by coexpression of calcineurin, suggesting the involvement of a calcineurin-dependent intracellular pathway. Our results identify c-rel as a target of immunosuppressive agents and illustrate the similarity of activation pathways in both B and T cells.

Amino Acid Sequence↗
PubMed1995

Induction of the transcription factors NF-kappa B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor.

To address elements that might uniquely characterize CD40 mediated signaling, the nuclear expression of three transcription factors was evaluated following B cell stimulation by CD40L and by anti-Ig antibody. Cross-linked CD40L was found to induce nuclear expression of NF-kappa B, AP-1 and NF-AT with a time course and intensity similar to that produced by anti-Ig. Examination of NF-kappa B in more detail demonstrated that the CD40 mediated expression of DNA binding complexes correlated with induction of trans-activating activity which again attained similar levels following cross-linking of CD40 and slg. Despite the marked similarity in transcription factor induction triggered through CD40 and slg, differences in the intracellular signaling pathways utilized were apparent in that protein kinase C (PKC) depletion did not affect CD40 mediated induction of NF-kappa B even as induction by anti-Ig was abolished. These results suggest that a 'final common pathway' or convergence of transcription factor induction may exist for two distinct receptors, each of which is individually capable of triggering cell cycle progression, despite the use of separate intracellular signaling pathways that differ at the level of PKC. Although transcription factor induction was similar for CD40L and anti-Ig early on, subtle differences in expressed NF-kappa B and AP-1 nucleoprotein complexes were apparent at 24 h. Such differences may play a role in determining the variant effects on B cells of stimulation through these two receptors.

Animals↗
PubMed1995

Phase contrast microscopic evaluation of placental pathology in premature gestation.

Numerous representative samples taken from forty placentas immediately after delivery from mothers associated with prematurity (i.e. less than 38 weeks), were semiquantitatively studied by phase contrast microscopy. Twenty placentas delivered from mothers without any antenatal complication served as control. Hypoplasia of the syncytium, stromal edema, ischaemia were prominent findings on phase contrast microscopy in the study group. Increased basement membrane thickening and high villous edema scores observed on light microscopy were statistically significant in prematurity as compared to controls. Observations by phase contrast and light microscopy were found complementary to each other. Phase contrast microscopy provided quicker results without disadvantage of fixation artefact and was found distinctly superior over the conventional histological methods.

Case-Control Studies↗
PubMed1994

Nuclear factors that mediate intrathymic signals are developmentally regulated.

Thymocytes mature through several stages of development, defined by cell surface markers such as CD3, CD4, and CD8, in response to environmental cues. Signal transduction resulting from lymphocyte-stromal cell interactions is likely to activate inducible transcription factors which in turn govern stage-specific gene expression. In this report we show that inducible transcription factors such as AP-1 and NF-AT are constitutively nuclear, in response to intrathymic signals, in freshly isolated thymocytes at all stages of maturation. In CD4+CD8+ double positive (DP), but not in the more immature CD4-CD8- double negative (DN) thymocytes, constant stimulus from the thymic environment is required to maintain nuclear AP-1. Thus, disruption of the thymus and incubation of thymocytes at 37 degrees C downregulates DNA binding by nuclear factors AP-1 and NF-AT. Similar treatment of thymocytes has previously been shown to downregulate CD3 zeta chain phosphorylation and increase T cell receptor CD3 expression on DP thymocytes, which is a feature of repertoire selection. Since mature T cells maintain inducible nuclear factors in an inactive form until an encounter with antigen, we propose that downregulation of nuclear DNA binding proteins may reflect another feature of this stage of T cell maturation.

Aging↗
PubMed1994

PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter.

Expression of the low-affinity Fc receptor for IgG (murine Fc gamma RIIIA) is restricted to cells of myelomonocytic origin. We report here the promoter structure, the proximal DNA sequences responsible for transcription of Fc gamma RIIIA in macrophages and the protein factors which interact with these sequences. A 51 bp sequence, termed the myeloid restricted region (MRR), was both necessary and sufficient for conferring cell type-specific expression in macrophages. Reporter constructs containing mutations in this sequence result in the loss of MRR activity upon transfection into the macrophage cell line, RAW264.7. Two cis-acting elements have been identified and are required for full promoter function. These same elements analyzed by EMSA define two binding sites recognized by nuclear factors derived from macrophages. A 3' purine tract (-50 to -39) within the MRR binds the macrophage and B cell-specific factor, PU.1, and a second E box-like element, termed MyE, upstream of the PU.1 box (-88 to -78) binds the HLH factors TFE3 and USF. EMSA studies using RAW cell extracts suggest that both PU.1 and MyE factors may bind simultaneously to the MRR resulting in a ternary complex that is responsible, in part, for the myeloid-specific activity of the Fc gamma RIIIA promoter.

Amino Acid Sequence↗
PubMed1994

Intrinsic fluorescence of E. coli RNA polymerase as a probe for its conformational changes during transcription initiation.

A simple fluorimetric assay based on internal fluorescence of tryptophan residues of E. Coli RNA polymerase has been developed to ascertain the number of steps during conversion of closed complex of the polymerase-promoter (trp promoter cloned in plasmid pDR720) to open complex. Our results from measurement on relative ratio of fluorescence at 340 nm (lambda ex = 295 nm) for free and promoter-bound RNA polymerase as a function of temperature, within the range 4 degrees C to 37 degrees C, indicate following equilibria for the above conversion: R+P<-->RPc<-->RPi1<-->RPi2<-->RPo. Apart from detection of one more intermediate in terms of conformational states of the bound RNA polymerase, second feature of our studies is the examination of conformational state of the polymerase using accessibility of fluorophor, tryptophan residues, to a neutral quencher, acrylamide, as the probe. We observe that in terms of accessibility of tryptophan residues in protein, intermediate complex, RPi2, is conformationally most perturbed in comparison to free polymerase. Implications of these results are discussed and compared with the available reports from footprinting and gel retardation assays of RNA polymerase-promoter interactions.

Cloning, Molecular↗
PubMed1994

Cyclosporin A sensitivity of the NF-kappa B site of the IL2R alpha promoter in untransformed murine T cells.

We have investigated the characteristics of IL2R alpha gene induction in untransformed murine T cells. Induction of IL2R alpha mRNA by TCR/CD3 ligands in a murine T cell clone and in short-term splenic T cell cultures was inhibited by protein synthesis inhibitors and by CsA. This result was contrary to previous observations in JURKAT T leukemia cells and human peripheral blood T cells, suggesting a difference in the mechanisms of IL2R alpha gene induction in these different cell types. The CsA sensitivity of IL2R alpha mRNA induction represented a direct effect on the TCR/CD3 response, and was not due to CsA-sensitive release of the lymphokines IL2 or tumour necrosis factor alpha (TNF alpha) and consequent lymphokine-mediated induction of IL2R alpha mRNA. The NF-kappa B site of the IL2R alpha promoter was essential for gene induction through the TCR/CD3 complex, and the induction of reporter plasmids containing multimers of this site was significantly inhibited by CsA. Northern blotting analysis indicated that while the p65 subunit of NF-kappa B was constitutively expressed and not appreciably induced upon T cell activation, mRNA for the p105 precursor of p50 NF-kappa B was induced in response to TCR/CD3 stimulation and this induction was sensitive to CsA. Electrophoretic mobility shift assays and antiserum against the p50 subunit of NF-kappa B indicated that p50 was a component of the inducible nuclear complex that bound to the IL2R alpha kappa B site. Appearance of the kB-binding proteins was insensitive to CsA at early times after activation (approximately 15 min), but was partially sensitive to CsA at later times. Based on these results, we propose that the NF-kappa B site of the IL2R alpha promoter mediates at least part of the CsA sensitivity of IL2R alpha gene induction in untransformed T cells, possibly because de novo synthesis of p105 NF-kappa B is required for sustained IL2R alpha expression.

Animals↗