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Biomedical subjects

M Subramanyam

Publications and source records attributed to M Subramanyam.

16 recordsLinked to original sources
PubMed2005

Antimicrobial activities of bharangin from Premna herbaceae Roxb. and bharangin monoacetate.

The root nodules of Premna herbaceae, which are being used in ayurvedic system of medicine as gantubharangi for curing several ailments, have been studied for antimicrobial activities. The major compound responsible for the biological activity is bharangin, a yellow colored compound extractable with hexane. Bharangin monoacetate prepared by acetylation of bharangin has been investigated along with bharangin for their antimicrobial activity against gram positive and gram negative bacteria and fungi. In general bharangin monoacetate showed more activity with MIC of 3-6 microg/ml when compared with bharangin, which has MIC of 10-25 microg/ml. The enhanced antibacterial activity is attributed to the presence of acetoxyl group in place of hydroxyl group present in the structure of bharangin. The activity is compared with standard gentamycin for bacteria and nystatin for fungi.

Anti-Infective Agents↗
PubMed1998

Regulation of the epithelial Na+ channel by membrane tension.

The sensitivity of alphabetagamma rat epithelial Na+ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2-5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at -100 mV) from -3.42 +/- 0.34 to -2.02 +/- 0.23 microA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that alpha beta gamma rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

Actins↗
PubMed1998

Soluble human lymphocyte activation gene-3 modulates allospecific T cell responses.

Lymphocyte activation gene (LAG)-3, a member of the Ig superfamily, has been characterized as an activation antigen of T cells and NK cells. LAG-3 has been proposed as an alternate ligand for HLA class II due to some sequence homology and similarities in exon-intron organization with CD4. Here, we report the functional evaluation of a soluble Ig fusion molecule of human LAG-3 (LAG-3-Ig) in T cell activation assays. Cytofluorimetry studies revealed LAG-3-Ig binding predominantly to class II-expressing cells. In functional assays, inhibition of primary allogeneic mixed lymphocyte response (MLR) and murine-human xenogeneic MLR was observed in the presence of LAG-3-Ig. Effects of LAG-3-Ig addition were not observed on mitogen-, recall antigen- or superantigen-mediated stimulation. Cytotoxic T lymphocyte effector functions were also not affected by LAG-3-Ig. Inhibition of alloresponses by LAG-3-Ig occurred within the first 24 h of activation, resulting in a strong inhibition of IL-2 production. Unlike blockade of the CD28 receptor, however, LAG-3-Ig-mediated inhibition could not be reversed by exogenous IL-2 supplementation. Cytofluorimetric analysis of the phenotype of cells exposed to LAG-3-Ig in MLR cultures revealed a decrease in IL-2 receptor expression (CD25) on CD4+ cells in all donors tested. Based on the results from these studies, we conclude that LAG-3-Ig inhibits alloresponses of naive peripheral blood lymphocytes, by blocking the activation of a subpopulation of allo reactive cells.

Animals↗
PubMed1997

Cell swelling activates the K+ conductance and inhibits the Cl- conductance of the basolateral membrane of cells from a leaky epithelium.

Necturus gallbladder epithelial cells bathed in 10 mM HCO3/1% CO2 display sizable basolateral membrane conductances for Cl- (GClb) and K+ (GKb). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K+/Cl- selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca2+]i) increased transiently during hyposmotic swelling (peak at approximately 30 s, return to baseline within approximately 90 s), but chelation of cell Ca2+ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10-12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (Vcs) produced a significant increase in the change in Vcs elicited by lowering basolateral solution [Cl-], whereas hyperpolarization of Vcs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases GKb and decreases GClb. These two effects appear to be linked, i.e., the increase in GKb produces membrane hyperpolarization, which in turn reduces GClb. (b) Hyperosmotic shrinkage has the opposite effects on GKb and GClb. (c) Cell swelling causes a transient increase in [Ca2+]i, but this response may not be necessary for the increase in GKb during cell swelling.

Animals↗
PubMed1995

Effect of HCl on transmembrane potentials and intracellular pH in rabbit esophageal epithelium.

BACKGROUND/AIMS: Acidification of the basolateral membrane by adding HCl to the serosal solution of esophageal epithelium leads to more necrosis than acidification of the apical membrane by adding HCl to the luminal solution. The aim of this study was to examine the mechanism for this difference. METHODS: The effect of low extracellular pH (pHo) (HCl) on intracellular pH (pHi) and transmembrane potentials was examined in rabbit esophageal cells by impalement with intracellular microelectrodes. RESULTS: Lowering luminal pH to 3.0 had no effect on membrane voltage and/or pHi in either luminally or serosally impaled cells, although a decline in both parameters occurred at pH 1.5 in luminally impaled cells. In contrast, lowering serosal pH from 7.4 to 3.0 progressively reduced membrane voltage and/or pHi. Membrane depolarization at low pHo was inhibited by a high-potassium solution or barium and mimicked by lowering pHi (gassing with CO2) at neutral pHo. CONCLUSIONS: Basolateral, but not apical, membranes of esophageal epithelial cells are highly permeable to H+, accounting for the greater susceptibility to damage from exposure to serosal than luminal acid. Membrane depolarization at low pHo is mediated by low pHi through inhibition of basolateral membrane K+ conductance.

Animals↗
PubMed1994

Human immunodeficiency virus 1 Tat binds to dipeptidyl aminopeptidase IV (CD26): a possible mechanism for Tat's immunosuppressive activity.

The human immunodeficiency virus 1 (HIV-1) Tat protein suppresses antigen-induced, but not mitogen-induced, activation of human T cells when added to T-cell cultures [Viscidi, R. P., Mayur, K., Lederman, H. M. & Frankel, A. D. (1989) Science 246, 1606-1608]. This activity is potentially pertinent to the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Here we report that Tat binds with high affinity to the T-cell activation molecule dipeptidyl aminopeptidase IV (DP IV), also known as CD26. This molecule occurs on the surface of CD4+ cells responsible for the recall antigen response and appears to play an essential role in this response. Tat binds to both the cell surface and soluble forms of DP IV at physiological salt concentrations without inhibiting the protease activity of DP IV against small chromogenic substrates used to assay activity, but Tat markedly inhibits the activity of DP IV at lower salt concentrations. The kinetics of inhibition indicate the affinity of Tat for DP IV varies from 20 pM to 11 nM, and the activity of the Tat-DP IV complex varies from 13% to 100%, as the NaCl concentration varies from 0 to 140 mM. Cytofluorometry experiments demonstrate that Tat competes with anti-Ta1, a monoclonal antibody (mAb) specific for DP IV, for binding to cell surface DP IV, thus indicating that Tat binds DP IV at or near the Ta1 epitope. Moreover, the anti-Ta1 mAb blocks the immunosuppressive activity of Tat. The high affinity of Tat for DP IV, previous evidence implicating DP IV in antigen-specific T-cell activation events, and the ability of anti-Ta1 mAb to block the immunosuppressive effect of Tat make DP IV a plausible receptor for Tat's immunosuppressive activity.

Acquired Immunodeficiency Syndrome↗
PubMed1994

The role of superantigens in the immunobiology of retroviruses.

Murine mammary tumour viruses (MMTVs) are retroviruses that encode superantigens capable of stimulating T cells via superantigen-reactive T cell receptor V beta chains. MMTVs are transmitted to the suckling offspring via the milk. We have established that class II and B cell-deficient mice that were foster nursed by virus-secreting mice do not transfer infectious MMTVs to their offspring. No MMTV proviruses could be detected in the spleen and mammary tissue of these mice and there was no deletion of MMTV superantigen-reactive T cells. These results confirm that superantigen expression in the context of MHC class II molecules is required for MMTV transmission. We conclude that B cells are essential for the completion of the viral life cycle in vivo. This indicates that B cells are infected first and that viral amplification takes place only if infected B cells present the MMTV superantigen on their surface which, in turn, results in activation of T cells expressing the appropriate T cell receptor V beta chains. These activated T cells stimulate B cells which enables viral replication. Human T cells carry all the structural features required for an efficient response to murine retrovirally encoded superantigens. Superantigen-like stimulation of human T cells has been demonstrated in both infectious and autoimmune diseases. Human immunodeficiency virus may encode a superantigen but this has not been proven.

Animals↗
PubMed1994

Muscarinic stimulation of gallbladder epithelium. III. Antagonism of cAMP-mediated effects.

Elevation of adenosine 3',5'-cyclic monophosphate (cAMP) levels in Necturus gallbladder (NGB) epithelium activates an apical membrane Cl- conductance and decreases transepithelial fluid transport (Jv). Acetylcholine (ACh), which had no effects on Jv by itself, antagonized the electrophysiological effects of forskolin (FSK) and theophylline and the decrease in Jv produced by FSK. By itself, ACh had no effects on basal cAMP levels but antagonized the increases in cAMP induced by FSK and theophylline. ACh had no effect on phosphodiesterase activity and prevented both the electrophysiological response and the elevation in cAMP by theophylline. In conclusion, the effect of ACh is mediated by inhibition of adenylate cyclase. A pertussis toxin (PTX)-sensitive G protein may mediate inhibition of adenylate cyclase because pretreatment with PTX prevented the reversal of the electrophysiological effects of FSK by ACh, and PTX catalyzed the ribosylation of cell membranes from NGB epithelium. ACh could have a physiological role in modulating the effects of secretagogues that act via elevation of cAMP levels.

Acetylcholine↗
PubMed1993

Presentation of the Mls-1 superantigen by human HLA class II molecules to murine T cells.

Superantigens (SAG) presented in the context of MHC class II proteins stimulate a strong proliferative response in T cells expressing particular TCR V beta genes. Although this T-cell recognition is not MHC restricted, a strong hierarchy is observed in the ability of various MHC class II molecules to present SAG. Mls-1, encoded by the Murine Mammary Tumor Virus (MMTV) Mtv-7 sag gene, is the prototype of endogenous SAG. In the present study, we have analyzed whether this retroviral gene product can be presented in the context of human HLA class II proteins to murine T cells. Positive results were obtained with the DR isotype and in in vitro, as well as in vivo T-cell stimulation assays. However, the various DR beta alleles, expressed in combination with an identical DR alpha chain, differed in their Mls-1 presenting capacity, indicating that the MHC class II beta-chain contains the primary contact site for Mls-1. Interestingly, the same pattern of TCR V beta restriction was seen in response to Mls-1 presented in the context of human and mouse class II, suggesting that the TCR V beta specificity is uniquely determined by the retroviral SAG. Furthermore, Mls-1 presented in the context of the DQw1 and DPw2 isotypes did not elicit a T-cell response. The results from this study form the basis for further analysis of the exact region in the class II beta-chain that interacts with Mls-1.

Alleles↗
PubMed1993

Human T cells respond to mouse mammary tumor virus-encoded superantigen: V beta restriction and conserved evolutionary features.

Mouse mammary tumor virus (MMTV)-encoded superantigens (SAGs) influence the murine T cell repertoire and stimulate a strong mixed lymphocyte response in vitro. These SAGs are encoded by the open reading frame of the 3' long terminal repeat of MMTV, termed MMTV SAGs. The T cell response to MMTV SAGs is V beta restricted and requires expression of the class II molecules of the major histocompatibility complex (MHC) on the presenting cells. While human T cells respond to bacterial SAGs, it is not known if human T cells or human MHC class II molecules can interact with MMTV SAGs. A fibroblastic cell line expressing the human MHC class II molecule HLA-DR1 and the Mtv-7 sag gene encoding Mls-1 was used to stimulate human T cells. We show here that human T cells efficiently proliferate in response to Mls-1 presented by HLA-DR1. This T cell response was inhibited by mAbs directed against CD4 or MHC class II molecules but not by mAbs specific for CD8 or MHC class I molecules. Moreover, the response to Mls-1 was limited to human T cells expressing a restricted set of T cell receptor V beta chains. Human T cells expressing V beta 12, 13, 14, 15, and 23 were selectively amplified after Mtv-7 sag stimulation. Interestingly, these human V beta s share the highest degree of homology with the mouse V beta s interacting with Mls-1. These results show a strong evolutionary conservation of the structures required for the presentation and the response to retrovirally encoded endogenous SAGs, raising the possibility that similar elements operate in humans to shape the T cell repertoire.

Animals↗
PubMed1993

Mechanism of HIV-1 Tat induced inhibition of antigen-specific T cell responsiveness.

HIV-1 Tat has been shown to have an inhibitory effect on the Ag-specific responsiveness of human peripheral T cells. We have previously demonstrated that this retroviral protein binds to and partially inhibits the enzymatic activity of dipeptidyl aminopeptidase type IV (DP IV), also known as CD26, which is expressed on a variety of mammalian tissue, including T lymphocytes. A number of studies have implicated a role for DP IV in the activation of T lymphocytes. By utilizing HIV-1 Tat, as well as ProboroPro, a potent and specific boronic acid analog inhibitor of DP IV, we show here that blocking DP IV partially inactivates Ag and anti-CD3-mediated T cell proliferation. Neither mitogen nor anti-CD2 mediated proliferation of T lymphocytes, however, is impaired by blocking DP IV. The target molecule for the inhibition induced by both compounds was confirmed by the finding that soluble DP IV neutralized the reduced Ag responsiveness. The Ag-specific inhibition could be overcome by the addition of exogenous IL-2, suggesting that blocking or inactivation of DP IV results in a state of anergy, probably by interfering with the delivery or amplification of a signal necessary for IL-2 production. This is further substantiated by the finding that costimulation of human PBMC via the CD28 molecule, which initiates a non-TCR-dependent signaling pathway, overcomes the reduced Ag responsiveness induced by Tat and ProboroPro. The fact that ProboroPro has no impact on stimulation of T cells with PMA and ionomycin implies that blocking DP IV is influencing events before the activation of protein kinase C and Ca2+ flux. These results suggest that DP IV is necessary for amplification of signals generated by the engagement of the TCR-CD3 complex by nominal Ag.

Adult↗
PubMed1993

Production and characterization of an Mls-1-specific monoclonal antibody.

Superantigens (SAGs) represent a new class of antigens, characterized as T cell receptor (TCR) V beta-reactive elements. Bacterial toxins constitute the major group of exogenous SAGs, while the mouse mammary tumor virus (MMTV)-encoded Mls molecules represent the endogenous SAGs. Mls-1 is the prototype of the latter SAGs, because it elicits a very potent T cell stimulatory response in vitro in unprimed T cells expressing the TCR V beta 6 or 8.1 chains. In vivo, Mls-1 causes deletion of immature T cells bearing the V beta 6, 7, 8.1, or 9 chains. Although Mls-1 was functionally discovered > 20 yr ago, it has not been possible to raise antibodies against this molecule. We have previously cloned and sequenced the Mtv-7 sag gene, which encodes Mls-1. Sequence comparisons with other MMTV sag genes suggested that the polymorphic 3' end encodes the TCR V beta specificity of these SAGs. We have, therefore, immunized hamsters with a 14-amino acid peptide from the deduced COOH-terminal sequence of the Mtv-7 sag gene. We describe here the production of a monoclonal antibody (mAb), 3B12, which is peptide specific and reacts with a recombinant baculovirus product of Mtv-7 sag. This mAb blocks Mls-1-specific T cell recognition and detects the Mls-1 protein on the surface of the B cell hybridoma LBB.A, but not on LBB.11, which is an Mtv-7 loss variant of LBB.A. Transfection of the Mtv-7 sag gene into LBB.11 renders this cell functionally Mls-1+ as well as positive for 3B12 binding, confirming the specificity of this mAb. It is well documented that B cells and CD8+ T cells express T cell stimulatory Mls-1 determinants, and we show here that this functional profile correlates with the expression of MMTV-specific mRNA. However, primary lymphocytes derived from Mls-1+ mice do not stain with 3B12, even after in vitro activation with mitogens or phorbol ester.

Amino Acid Sequence↗
PubMed1993

Muscarinic stimulation of gallbladder epithelium. I. Electrophysiology and signaling mechanisms.

To understand the effects of acetylcholine (ACh) on fluid-absorbing epithelia, we carried out experiments on Necturus gallbladder epithelium. Binding studies with 1-quinuclidinyl[phenyl-4(N)-3H]benzilate (QNB) demonstrated that Necturus gallbladder epithelial cells express high-affinity muscarinic receptors. The effects of ACh and carbachol were exerted from the basolateral surface and consisted of a transient hyperpolarization of both cell membranes and a concomitant decrease in the apparent fractional resistance of the apical membrane. Atropine blocked both effects. ACh also elicited transient elevations of inositol 1,4,5-trisphosphate and intracellular free calcium ([Ca2+]i) levels, the latter by both release from intracellular stores and basolateral influx. The phospholipase C antagonist U-73122 inhibited the effects of ACh, whereas inhibition of prostaglandin and guanosine 3',5'-cyclic monophosphate synthesis with indomethacin or methylene blue, respectively, had no effect. In conclusion, Necturus gallbladder epithelium expresses muscarinic receptors in the basolateral membrane. Receptor activation stimulates phospholipase C and elevates cellular levels of inositol 1,4,5-trisphosphate and [Ca2+]i. The elevation in [Ca2+]i activates K+ channels but apparently not Cl- channels.

Acetylcholine↗
PubMed1993

Muscarinic stimulation of gallbladder epithelium. II. Fluid transport, cell volume, and ion permeabilities.

Activation of muscarinic receptors in the fluid-absorptive epithelium of the Necturus gallbladder elevates cytosolic Ca2+ concentration, transiently hyperpolarizes the cell membrane voltages, and decreases the apparent fractional resistance of the apical membrane [G. A. Altenberg, M. Subramanyam, J. S. Bergmann, K. M. Johnson, and L. Reuss. Am. J. Physiol. 265 (Cell Physiol. 34): C1604-C1612, 1993]. In these studies, we show that at the peak of the hyperpolarization both apical and basolateral membrane resistances (Ra and Rb, respectively) decreased, but in 2-3 min Ra returned to control values while Rb rose to a level approximately 60% higher than control. The acetylcholine (ACh)-induced decrease in Ra is caused by activation of apical membrane maxi K+ channels secondary to elevation of cytosolic Ca2+ concentration. The increase in Rb is due to decreases in K+ and Cl- conductances. ACh had no effects on cell KCl content or water volume, although K+ conductance transiently increased. These results can be explained by the changes in basolateral membrane conductances. ACh did not alter fluid absorption. In conclusion, ACh has complex time-dependent effects on K+ and Cl- electrodiffusive permeabilities without measurable changes in cell volume or in the rate of transepithelial fluid transport.

Acetylcholine↗