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M A Choudhry

Publications and source records attributed to M A Choudhry.

32 records · Page 2Linked to original sources
PubMed1998

Effect of CRF and related peptides on calcium signaling in human and rodent melanoma cells.

Corticotropin releasing factor (CRF) induces a rapid, within seconds, and dose-dependent increase in the intracellular Ca2+ in both human and hamster melanoma cells. This effect is inhibited by depletion of extracellular calcium using 3 mM EGTA and is attenuated by the CRF receptor antagonist, alpha-helical-CRF(9-41). Other peptides of the CRF superfamily, sauvagine and urocortin, also induce increases in cytoplasmic calcium concentration but at higher concentrations than CRF. We conclude that malignant melanocytes express CRF receptors, which are coupled to activation of plasma membrane calcium channels.

Amphibian Proteins↗
PubMed1998

Prostaglandin E2 modulation of p59fyn tyrosine kinase in T lymphocytes during sepsis.

Prostaglandin E2 (PGE2) has been implicated in the suppression of T cell IL-2 production and proliferation during burn and sepsis. The present study evaluated the potential intracellular mechanism of suppressed T cell responses by assessing the activation of p59fyn kinase in T cells from septic rats as well as the T cells incubated with PGE2. p59fyn is known to regulate T cell functions. Sepsis was induced in rats by implanting fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10(4) CFU) into the abdominal cavity. For the assessment of PGE2 role in sepsis, a group of septic rats were treated with indomethacin, which inhibits endogenous PGE2 synthesis. As assessed by immunoblotting or in vitro kinase assay, a more than 40% inhibition of p59fyn phosphorylation and kinase activity was observed in septic rat T cells compared with the T cells from sterile or control rats. A similar inhibition in p59fyn phosphorylation and kinase activity was observed in PGE2-treated T cells compared with the T cells incubated in the absence of PGE2. The septic-related suppression in p59fyn phosphorylation and kinase activity in T cells was prevented in rats treated with indomethacin. We observed that the inhibition in p59fyn activation in septic or PGE2-treated T cells was due primarily to a suppression in p59fyn phosphorylation and not due to alterations in p59fyn protein expression. These findings suggest that PGE2 released during sepsis could contribute to the sepsis-related suppression in T cell proliferation by attenuating p59fyn phosphorylation and its kinase activity.

Animals↗
PubMed1997

Transforming growth factor-beta negatively modulates T-cell responses in sepsis.

Sepsis is associated with depressed T-cell functions and increased circulating levels of immunosuppressive agents. TGF-beta is a potential anti-inflammatory cytokine that can modify T-cell growth and differentiation. The up-regulation of TGF-beta and the mechanism of its action on the T-cells during septic injury have not been resolved. We hypothesized that in sepsis TGF-beta produced by macrophages acts on T-cells in a paracrine manner to suppress interleukin (IL)-2 production and proliferation. In this study, we examined the circulating TGF-beta levels in a rat model of Gram-negative bacterial sepsis, and compared the abilities of adherent and non-adherent splenocytes to produce TGF-beta. Additionally, we investigated the causal relationships of hrTGF-beta to concanavalin A (ConA)-induced T-cell responses and the intracellular mechanism of the generation of these responses in normal splenic rat T-cells. Sepsis was induced in rats by intraabdominally implanting fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10000 CFU). Adherent and non-adherent splenocytes were isolated by differential adherence using Ficoll gradient centrifugation. T-cells were purified by use of Nylon wool columns. We observed a 3-6-fold increase in the circulating levels of TGF-beta in sepsis. Western blots and ELISA determinations revealed a 2.5-3-fold increase in cell-associated TGF-beta protein levels in adherent splenic cells. Northern analyses also showed a marked increase in TGF-beta mRNA expression in adherent cells during sepsis. On the other hand, a significant change was not observed in the TGF-beta protein and mRNA expression in non-adherent splenocytes. Pretreatment of control rat T-cells with hrTGF-beta decreased both ConA-induced proliferation (by 35-40%) and IL-2 mRNA expression (by > 50%). Further, whereas incubation of control rat T-cells with either ConA or TGF-beta for 24 h resulted in a 10-15-fold increase in cAMP generation, the addition of hrTGF-beta along with ConA resulted in a 50-60-fold increase in cAMP. These results suggest that in sepsis, TGF-beta produced by splenic macrophages can act in a paracrine manner on T-cells to depress their IL-2 mRNA expression, IL-2 production and proliferation after up-regulation of cAMP which can interfere with T-cell signaling for proliferation.

Animals↗
PubMed1997

The marine toxin okadaic acid reduces O2- generation and tyrosine phosphorylation in LPS-primed rat neutrophils.

Contrasting effects of okadaic acid (OKA) on neutrophil (PMN) superoxide anion (O2-) generation have been reported. In this study, we examined the effect of OKA on phorbol myristate acetate (PMA)-stimulated O2- generation in rat PMNs primed with LPS in vivo (LPS-PMN) and saline-treated rat PMNs (SAL-PMN). The following results were observed: (1) OKA, but neither genistein nor vanadate, markedly reduced O2- generation in a dose and time-dependent manner; (2) genistein, a tyrosine kinase inhibitor, as well as OKA, reduced tyrosine phosphorylation; (3) sodium orthovanadate, a tyrosine phosphatase inhibitor, potently enhanced tyrosine phosphorylation. Our studies suggest that OKA might reduce tyrosine phosphorylation by affecting the activity of tyrosine phosphatases regulated by serine-threonine phosphorylation.

Animals↗
PubMed1996

Ca2(+)-dependent and Ca2(+)-independent proteinase contents in the skeletal muscle in septic rats.

This study assessed sepsis-induced changes in the contents of calpain and cathepsin B in rat soleus muscle. Sepsis was induced in rats by intra-abdominally implanting fecal pellets containing Escherichia coil and Bacteroides fragilis. Intact soleus muscles were isolated from non-operated control rats, and from rats sacrificed 1 and 2 days after they were implanted with bacteria-free (sterile implanted) or bacteria-laden (septic implanted) pellets. Western blot analyses of muscle homogenates were performed to identify and quantitate these proteinases using specific antibodies. No significant differences in cathepsin B contents were observed between the septic and nonseptic animals on days 1 and 2, post-implantation. Among the three distinct bands recognized by anti-calpain, two prominent bands of 80 and 76 kDa, representing calpain subunits, did not seem to be altered in septic rats compared to the nonseptic groups. The content of the 45-kDa subunit decreased in both the septic and sterile groups compared with non-operated control. These results along with our previous observations suggest that although Gram-negative sepsis does not appear to have an effect on Ca2(+)-insensitive lysosomal cathepsin B content or activity, it upregulates the activity of the Ca2(+)-dependent calpain but not its content in the skeletal muscle during sepsis.

Animals↗
PubMed1996

Calcium signaling restitution prevents T-cell proliferative suppression by prostaglandin E2.

Previous studies from our laboratory have implicated a role for Ca2+ in prostaglandin E2 (PGE2)-induced suppression in T-cell proliferation during sepsis. The present study further elucidated the mechanism of PGE2-induced suppression in T-cell proliferation. We assessed whether prevention of the suppression in Ca2+ mobilization in PGE2-treated T-cells would restore proliferation. Rat splenic T-cell Ca2+ mobilization and proliferation were measured after stimulation of cells with concanavalin A (Con A) employing Fura-2 spectroscopy and cellular [3H]thymidine uptake techniques, respectively. PGE2 and other agents that directly up-regulate the PGE2-mediated cell signaling events (e.g., cholera toxin and forskolin), substantially suppressed both Con A-induced proliferation (p < .01) and Ca2+ mobilization in T-cells (p < .01). When stimulated with Con A plus ionomycin, [Ca2+]i in PGE2 treated T-cells (395 +/- 21, nM) was not significantly different (p > .05) from that observed in Con A-stimulated T-cells without the PGE2 exposure (351 +/- 8.6). The stimulation of PGE2-treated T-cells with ionomycin and Con A also significantly (p < .025), if not completely, prevented the PGE2-induced suppression in T-cell proliferation. These results suggest that the cross-talk between the TCR- and PGE2-mediated signaling in T-cells negatively modulates the TCR signals at the Ca2+ mobilization step and/or earlier to it.

Animals↗
PubMed1995

Role of Ca2+ in prostaglandin E2-induced T-lymphocyte proliferative suppression in sepsis.

Prostaglandin E2 (PGE2) has been known to modulate immune responses by inhibiting T-cell activation following hemorrhagic and traumatic injury. Recently, we documented a sepsis-related depression in concanavalin A (ConA)-induced T-cell proliferation and intracellular Ca2+ (Ca2+i) mobilization. The present study evaluated the potential role of PGE2 in the sepsis-related attenuation in Ca2+ signaling and proliferation in T cells. Sepsis was induced in rats by implanting into their abdomen fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10(4) CFU). A group of rats implanted with septic pellets were treated with indomethacin at three consecutive time points. Levels of PGE2 in blood were measured with a radioimmunoassay kit. ConA-induced [Ca2+]i mobilization in T cells obtained from indomethacin-treated and untreated rats was measured with Fura-2 and microfluorometry. We observed a 10-fold increase in PGE2 levels in the circulation of septic rats compared with levels in rats implanted with bacterium-free sterilized pellets. The proliferative response and Ca2+i mobilization were significantly depressed in T cells obtained from septic rats 48 h after implantations compared with those in rats implanted with sterile pellets. However, treatment of rats with the cyclooxygenase inhibitor indomethacin prevented the sepsis-related depression in ConA-induced T-cell Ca2+i mobilization as well as proliferation. Further, incubation of T cells from nonimplanted control rats with PGE2 resulted in a substantial depression in both T-cell proliferation and Ca2+i mobilization. The restoration of T-cell proliferation and Ca2+ signaling after indomethacin treatment of septic rats and the depression in the mitogen responsiveness in T cells previously exposed to PGE2 suggest that the PGE2 does play a significant role in the modulation of T-cell responses in septic rats and that such PGE2-induced suppression in T-cell activation is likely due to an attenuation in Ca2+ signaling.

Animals↗
PubMed1995

Quercetin, a bioflavonoid, inhibits the DNA synthesis of human leukemia cells.

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. It has been proposed that flavonoids may have potential as anticancer agents. To test an aspect of this hypothesis, we examined the effects of the flavonoid, quercetin, on the DNA synthesis of the human leukemia cell, HL-60. Quercetin induced a dose-dependent inhibition of DNA synthesis in the test range of 1 microM to 1 mM. The inhibitory effect on DNA synthesis was evident as early as 24 h after the addition of quercetin. At the concentrations of 10 microM, 100 microM and 1 mM, 50, 82 and 85% of DNA synthesis, respectively, was inhibited by quercetin as compared to the control. After 48 and 72 h incubation of the cells with 100 microM and 1 mM quercetin, DNA synthesis was almost completely abolished. These results suggest that the inhibitory effects of quercetin on HL-60 cell DNA synthesis is not due to a non-specific cytotoxic effect, since following removal of quercetin, the treated cells regrew normally.

DNA Replication↗
PubMed1994

Sepsis-induced myofibrillar protein catabolism in rat skeletal muscle.

This study evaluated sepsis-induced changes in myosin heavy chain (Mhc) protein breakdown and synthesis in rat soleus muscles. Rats were anesthetized and their external jugular veins were cannulated. After 12-16 h, rats were implanted intraabdominally with a sterile fecal pellet, or a pellet containing bacteria (Escherichia coli, 150 CFU and Bacteroides fragilis 10(4) CFU). Thirty hours after implantations, rats were infused with 14C-Leu (60 x 10(3) Bq/h) through the jugular cannula for 4 h. Protein fractional synthetic rate coefficient (FSRC) was determined in muscles of different rat groups. In separate experiments, intact soleus muscles were removed from the three rat groups on days 1 and 2 after implantations, and processed for their wet weight, total protein and Mhc contents. No mortality occurred in sterile-implanted rats. Approximately 40-45% of all septic-implanted rats died on days 1-3, post-implantation. Whereas an approximately 15% (P < 0.01, days 1 or 2) decrease occurred in Mhc content in sterile-implanted rats compared to unoperated controls, septic insult resulted in a greater Mhc loss (a 27% decrease, P < 0.001). Rats' body weight, soleus wet weight and tolat muscle protein changes with sepsis relative to controls were also greater than in the sterile groups. The FSRC value in the septic-implanted rats was significantly lower (P < 0.05) than in non-septic rat muscle. TNF-alpha administration to the septic animals or their treatment with diltiazem did not have a significant effect on FSRC. Overall, these results indicate myosin as a major muscle protein subjected to net catabolism during sepsis, and that the net catabolic response was related to a more pronounced increased in Mhc degradation than the decrease in Mhc synthesis.

Actins↗
PubMed1994

T-lymphocyte Ca2+ signalling and proliferative responses during sepsis.

Alterations in T-lymphocyte kinetics and/or activation have been implicated in burn and traumatic injury. This study evaluated concanavalin A (Con A) regulation of both intracellular Ca2+ (Cai2+) and proliferation in T-lymphocytes harvested from spleens of septic rats. Rats were implanted with fecal pellets containing Escherichia coli (150 colony forming units (CFU)) and Bacteroides fragilis (10(4) CFU). T-cell[Ca2+]i was measured before and after treatment of cells with ConA, using Fura-2 and microfluorophotometry. Splenic lymphocytes were cultured for 72 h with Con A to assess their proliferative response. As compared to sterile-implanted rats, the septic rat T-lymphocytes Cai2+ response to Con A significantly decreased on days 1 and 2 after implantation. A significant decrease in T-cell proliferative response to Con A, compared to sterile controls, was found in septic rats on day 2 but not on day 1. These results suggest that Con A-mediated proliferation in T-cells occurs secondarily to a decrease in cellular Ca2+ signalling. The depression in the T-cell proliferative response during sepsis could contribute to a decrease in host's resistance against sepsis.

Animals↗
PubMed1994

T-lymphocyte Ca2+ signalling and proliferative responses during sepsis.

Alterations in T-lymphocyte kinetics and/or activation have been implicated in burn and traumatic injury. This study evaluated concanavalin A (Con A) regulation of both intracellular Ca2+ (Ca2+i) and proliferation in T-lymphocytes harvested from spleens of septic rats. Rats were implanted with fecal pellets containing Escherichia coli (150 colony forming units (CFU)) and Bacteroides fragilis (10(4) CFU). T-cell [Ca2+]i was measured before and after treatment of cells with Con A, using Fura-2 and microfluorophotometry. Splenic lymphocytes were cultured for 72 h with Con A to assess their proliferative response. As compared to sterile-implanted rats, the septic rat T-lymphocytes Ca2+i response to ConA significantly decreased on days 1 and 2 after implantation. A significant decrease in T-cell proliferative response to Con A, compared to sterile controls, was found in septic rats on day 2 but not on day 1. These results suggest that Con A-mediated proliferation in T-cells occurs secondarily to a decrease in cellular Ca2+ signalling. The depression in the T-cell proliferative response during sepsis could contribute to a decrease in host's resistance against sepsis.

Animals↗
PubMed1991

Guinea-pig ileal loop assay: a better replacement of the suckling mouse assay for detection of heat-stable enterotoxins of Escherichia coli.

The suitability of guinea-pig ileal loop assay (GILA) for the assay of heat-stable (ST) enterotoxin was confirmed. Secretory response against Escherichia coli heat-stable enterotoxin in this model was determined in terms of dilatation index (DI). DI equal to 0.50 or more was considered as a positive secretory response. Kinetics of fluid accumulation and the titration of toxin in guinea-pig ileal loop suggest uniform secretory response throughout the small intestine and 31.5 microgram of crude ST toxin as the minimum effective dose to induce a DI of 0.5. Guinea-pig intestine was found sensitive to both methanol soluble (STa) and methanol insoluble (STb) toxins of E. coli and so was considered superior to the existing suckling mouse assay (SMA), which is known to be sensitive only to STa toxin. In addition, GILA was also found to be more suitable and economical as at least 10 strains together with the positive and negative controls can be tested in one animal, whereas in SMA, four suckling mice were needed to test a single strain. Hence, in SMA individual susceptibility among mice cannot be ruled out. GILA was considered to be an alternative to the presently available test, SMA, in the determination of ST toxin of E. coli.

Animals↗