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S T Reddy

Publications and source records attributed to S T Reddy.

At least 37 records · Page 2Linked to original sources
PubMed1997

Analysis of the secretory phospholipase A2 that mediates prostaglandin production in mast cells.

Prostaglandin D2 (PGD2) synthesis in activated mast cells occurs in two phases, an early phase that is dependent on prostaglandin synthase 1 and a delayed phase that is dependent on activation-induced prostaglandin synthase 2 gene expression. Early phase PGD2 synthesis in activated mast cells also requires the activity of a secretory phospholipase A2 (PLA2). It has been thought that the secretory PLA2 expressed in mast cells is group IIa PLA2, encoded by the Pla2 g2a gene. However, activated bone marrow-derived mast cells prepared from Pla2 g2a+/+ mice and mast cells prepared from mice with a mutation in the Pla2 g2a gene both demonstrate early phase PGD2 synthesis. Moreover, mast cells from both murine strains secrete PLA2 activity following activation. Northern and reverse transcriptase/polymerase chain reaction analyses demonstrate that mast cells from Pla2 g2a+/+ and Pla2 g2a-/- mice do not express group IIa PLA2 message. Instead, Northern and reverse transcriptase/polymerase chain reaction analyses demonstrate that both Pla2 g2a+/+ and Pla2 g2a-/- mast cells express mRNA for group V PLA2, encoded by the Pla2gV gene. An antisense oligonucleotide directed against group V PLA2 is also able to inhibit both the early phase of PGD2 production and the secretion of PLA2 activity by activated mast cells. Our data suggest that (i) group IIa PLA2 does not play a significant role in murine mast cell prostaglandin synthesis, (ii) group V PLA2 mediates early mast cell PGD2 production and transcellular PGE2 production in murine mast cells, and (iii) much of the data, based on studies with chemical inhibitors and antibodies, suggesting that group IIa PLA2 is responsible for arachidonic acid mobilization needs to be reevaluated.

Animals↗
PubMed1997

Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D2 in activated mast cells.

Aggregation of IgE cell surface receptors on MMC-34 cells, a murine mast cell line, induces the synthesis and secretion of prostaglandin D2 (PGD2). Synthesis and secretion of PGD2 in activated MMC-34 cells occurs in two stages, an early phase that is complete within 30 min after activation and a late phase that reaches a maximum about 6 h after activation. The early and late phases of PGD2 generation are mediated by prostaglandin synthase 1 (PGS1) and prostaglandin synthase 2 (PGS2), respectively. Arachidonic acid, the substrate for both PGS1 and PGS2, is released from membrane phospholipids by the activation of phospholipases. We now demonstrate that in activated mast cells (i) secretory phospholipase A2 (PLA2) mediates the release of arachidonic acid for early, PGS1-dependent synthesis of PGD2; (ii) secretory PLA2 does not play a role in the late, PGS2-dependent synthesis of PGD2; (iii) cytoplasmic PLA2 mediates the release of arachidonic acid for late, PGS2-dependent synthesis of PGD2; and (iv) a cytoplasmic PLA2-dependent step precedes secretory PLA2 activation and is necessary for optimal PGD2 production by the secretory PLA2/PGS1-dependent early pathway.

Animals↗
PubMed1997

Gastrin-releasing peptide-induced expression of prostaglandin synthase-2 in Swiss 3T3 cells.

Prostaglandins, produced in response to mitogens and cytokines, are potent modulators of gastrointestinal physiology and pathophysiology. We investigated modulation of Prostaglandin synthase 2 (PGS-2) expression by the gastrin-releasing peptide (GRP) receptor in Swiss 3T3 cells. PGS-2 mRNA expression in Swiss 3T3 cells was determined by Northern blot analysis. PGS-2 protein expression in Swiss 3T3 cells was measured by Western blot analysis. GRP caused a transient induction of PGS-2 mRNA in Swiss 3T3 cells that resulted in GRP-dependent expression of PGS-2 protein. Transcriptional activation of PGS-2 by GRP was independent of de novo protein synthesis and was not affected by pertussis toxin. Comparison of signaling pathways used by PMA or EGF to those used by GRP showed that PGS-2 induction by GRP increased under conditions that inhibit PKC activity. Dexamethasone, which blocks PMA and EGF induction of PGS-2, also inhibited GRP-induced accumulation of PGS-2 mRNA. These results show that PGS-2 expression in Swiss 3T3 cells is not only controlled by PKC and receptor tyrosine kinase pathways but also by G-protein coupled receptor signaling pathways.

3T3 Cells↗
PubMed1997

Intracellular proteases in sporulated Bacillus thuringiensis subsp. tenebrionis: detection and analysis by gelatin zymography.

Three intracellular proteases were identified from sporulated cultures of Bacillus thuringiensis subsp tenebrionis by fractionation with ammonium sulfate. In this study, we detected protease activities at M(r) 92 kDa, 81 kDa and 69 kDa employing gelatin zymography. The major proteolytic activity was due to the 81 kDa protease, which was identified as a metalloprotease being inhibited by both 1,10-phenanthroline and ethylenediamine tetraacetic acid. The proteases showed maximal azocasein hydrolytic activity at 60 degrees C and were heat-activated from 40 degrees C to 60 degrees C. The 69 kDa and 81 kDa proteases were thermo-inactivated at 70 degrees C and 80 degrees C respectively, while the 92 kDa protease was still active at 80 degrees C.

Bacillus thuringiensis↗
PubMed1996

Transcellular prostaglandin production following mast cell activation is mediated by proximal secretory phospholipase A2 and distal prostaglandin synthase 1.

Prostaglandins mediate many biological processes. Arachidonic acid, the common precursor for all prostaglandins, is released from membrane phospholipids by both secretory and cytoplasmic forms of phospholipase A2. Free arachidonate is converted to prostaglandin H2, the common precursor to all prostanoids, by prostaglandin synthase. Both mitogen-induced prostaglandin synthesis in fibroblasts and endotoxin-induced prostaglandin synthesis in macrophages require expression of the inducible prostaglandin synthase-2; arachidonate released in these contexts is unavailable to prostaglandin synthase-1 constitutively present in fibroblasts or macrophages. In contrast to the results for fibroblasts and macrophages, prostaglandin synthesis by activated mast cells is mediated by prostaglandin synthase-1. Mast cell activation also provokes release of secretory phospholipase A2 (sPLA2). We now demonstrate that sPLA2 released from activated mast cells can mobilize arachidonate from distal Swiss 3T3 cells. This arachidonate is then used by prostaglandin synthase-1 present in 3T3 cells for prostaglandin synthesis. We thus distinguish two pathways for prostaglandin synthesis: (i) an intracellular pathway by which arachidonate released following ligand stimulation is made available only to prostaglandin synthase-2, and (ii) a transcellular pathway by which sPLA2 of proximal cells mobilizes, in distal cells, arachidonate available to prostaglandin synthase-1. Molecular and pharmacologic approaches to modulating prostaglandin-mediated events will differ for these two pathways.

3T3 Cells↗
PubMed1995

Prostaglandin synthase 1 and prostaglandin synthase 2 both participate in activation-induced prostaglandin D2 production in mast cells.

Activation of MMC-34 cells, a murine mast cell line, or bone marrow-derived mast cells by aggregation of IgE cell surface receptors or addition of calcium ionophore stimulates prostaglandin (PG) D2 synthesis and secretion. An initial rapid burst of PGD2 synthesis, complete within 30 min, is followed by a slower subsequent production of PGD2 that reaches a maximum 4 to 8 h after activation in MMC-34 cells. PG synthase 1 (PGS-1) message and protein are expressed constitutively in MMC-34 cells and are not modulated by exposure to calcium ionophore or aggregation of IgE receptors. In contrast, activation of MMC-34 or bone marrow-derived mast cells induces expression of the PG synthase 2 (PGS-2) gene. PGS-2 induction following mast cell activation is blocked by dexamethasone. The initial PGD2 burst in activated MMC-34 cells is prevented by aspirin pretreatment, suggesting that constitutive PGS-1 present in mast cells before activation is responsible for the early PGD2 production in response to activation. In contrast, the later phase of PGD2 production is blocked by dexamethasone, cycloheximide, or NS-398, a PGS-2-specific nonsteroidal anti-inflammatory drug that inhibits PGS-2 enzyme activity but not PGS-1 activity. These data demonstrate that mast cell activation results 1) in the induction of PGS-2 gene expression, and 2) in both PGS-1-dependent PGD2 synthesis and PGD2 synthesis that is dependent on the activation-induced synthesis and activity of PGS-2.

Animals↗
PubMed1994

Ligand-induced prostaglandin synthesis requires expression of the TIS10/PGS-2 prostaglandin synthase gene in murine fibroblasts and macrophages.

Phospholipase activation has been considered rate-limiting for ligand-stimulated prostaglandin (PG) synthesis. Constitutive prostaglandin synthase EC 1.14.99.1 (PGS-1) has been thought to convert arachidonate released following ligand stimulation to PGH2. However, a second prostaglandin synthase, TIS10/PGS-2, is inducible in many cell types, including fibroblasts and macrophages. We have used antisense oligonucleotide inhibition of TIS10/PGS-2 induction to investigate the role of this enzyme in ligand-induced prostaglandin production in fibroblasts and macrophages. Antisense TIS10/PGS-2 oligonucleotides block mitogen-induced prostaglandin production and TIS10/PGS-2 protein synthesis in both Swiss 3T3 cells and murine embryo fibroblast cultures, without inhibiting either constitutive PGS-1 activity or mitogen-stimulated arachidonic acid release. TIS10/PGS-2 antisense oligonucleotides also block production of PGE2 from endogenous arachidonate following endotoxin stimulation of both RAW 264.7 macrophage cells and murine peritoneal macrophages. In contrast, the constitutive prostaglandin synthase present in macrophages is unable to convert arachidonate released following endotoxin stimulation into prostaglandin. TIS10/PGS-2 expression is necessary for both mitogen-induced prostaglandin production in murine fibroblasts and endotoxin-induced prostaglandin synthesis in macrophages. PGS-1 present in these cells cannot utilize arachidonic acid released in response to mitogen or endotoxin stimulation.

3T3 Cells↗
PubMed1994

Synthesis, degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3.

Murine TIS7 and TIS21 cDNAs were cloned from phorbol ester-induced Swiss 3T3 cells. The cognate rat cDNAs, PC4 and PC3, were cloned from nerve growth factor (NGF)-treated PC12 pheochromocytoma cells. The TIS7/PC4 and TIS21/PC3 primary response genes are rapidly and transiently induced in response to serum, phorbol esters, and polypeptide growth factors in quiescent Swiss 3T3 cells and by NGF and other ligands in PC12 cells. In both 3T3 and PC12 cells the appearance of the TIS21/PC3 message precedes that of TIS7/PC4 message following ligand stimulation, suggesting that the TIS21/PC3 protein is likely to be synthesized more rapidly than the TIS7/PC4 protein. Using antisera prepared against recombinant TIS21 and TIS7 proteins, we find that the TIS21/PC3 protein is, indeed, synthesized more rapidly than the TIS7/PC4 protein following stimulation in both 3T3 and PC12 cells. In addition, "pulse-chase" experiments demonstrate that the TIS21/PC3 protein is degraded much more rapidly than the TIS7/PC4 protein. The sequences of the predicted PC3 and PC4 proteins have lead to the speculation that these two proteins may both be secreted from cells following stimulation. The PC4 protein is reported to have some sequence similarity to interferons. The TIS21/PC3 protein contains a presumptive leader sequence. Using our antisera to the recombinant proteins, however, we cannot detect secretion of radiolabelled TIS7/PC4 or TIS21/PC3 protein. Immunohistochemical and subcellular fractionation experiments suggest that the TIS7 protein is a membrane associated, non-nuclear intracellular protein. The TIS21 protein, in contrast, is a non-nuclear, soluble intracellular protein.

3T3 Cells↗
PubMed1994

Transforming growth factor beta 1 augments mitogen-induced prostaglandin synthesis and expression of the TIS10/prostaglandin synthase 2 gene both in Swiss 3T3 cells and in murine embryo fibroblasts.

Transforming growth factor-beta (TGF-beta), a potent cytokine, modulates a wide variety of biological responses. Among its actions, TGF-beta can augment prostaglandin synthesis in several cell types. Although TGF-beta alone has no effect on prostaglandin production in Swiss 3T3 cells, we find that TGF-beta augments the ability of tetradecanoyl phorbol acetate (TPA) or serum to stimulate PGE2 production. The TIS10 gene is a primary response gene encoding a second form of prostaglandin synthase (PGS), the rate-limiting enzyme in the biosynthesis of prostaglandins, thromboxanes, and prostacyclins from arachidonic acid. TIS10/PGS-2 expression is induced by mitogens in Swiss 3T3 cells. TGF-beta also augments mitogen-induced synthesis and accumulation of TIS10/PGS-2 protein and induction of TIS10/PGS-2 message in Swiss 3T3 cells. In contrast, TGF-beta has little or no effect on the level of PGS-1 (EC1.14.99.1) message, either alone or in concert with TPA or serum. TGF-beta concentrations in the range of 0.01-0.10 ng/ml (0.4-4.0 pM) maximally enhance mitogen induction of TIS10/PGS-2 message. TPA-induced accumulation of unspliced TIS10/PGS-2 transcript is augmented by TGF-beta, suggesting that this cytokine exerts its effect on expression of the TIS10/PGS-2 gene by transcriptional regulation. TGF-beta also augments TPA-induced prostaglandin production, TIS10/PGS-2 antigen accumulation, and TIS10/PGS-2 message induction in primary cultures of mouse embryo fibroblasts. Dexamethasone attenuates TGF-beta enhancement of all these mitogen-induced responses: PGE2 accumulation, appearance of TIS10/PGS-2 protein and message, and accumulation of TIS10/PGS-2 unprocessed transcript.

3T3 Cells↗
PubMed1994

TGF-beta 1 inhibits both endotoxin-induced prostaglandin synthesis and expression of the TIS10/prostaglandin synthase 2 gene in murine macrophages.

Activated macrophages produce substantial quantities of paracrine mediators, including cytokines, nitric oxide, and prostaglandins. Transforming growth factor beta 1 (TGF-beta) is a potent modulator of immune function. TGF-beta inhibits the cytotoxic activity of endotoxin/lipopolysaccharide (LPS)-activated macrophage cell lines and primary macrophage cultures, reducing their expression of cytokines and nitric oxide. In this report we demonstrate that TGF-beta also attenuates the LPS-induced synthesis and secretion of prostaglandin E2 in murine RAW 264.7 macrophage cells. Macrophage activation also induces accumulation of the recently described ligand-responsive prostaglandin synthase (PGS) TIS10/PGS-2. While TGF-beta alone has no effect on expression from the TIS10/PGS-2 gene, this cytokine inhibits LPS-induced TIS10/PGS-2 protein accumulation and synthesis, as well as LPS-induced TIS10/PGS-2 message accumulation in RAW 264.7 cells. TGF-beta concentrations in the range of 0.1-1.0 ng/ml (4-40 pM) maximally inhibit LPS-induced TIS10/PGS-2 message accumulation. In contrast, neither LPS nor TGF-beta has any effect on the level of PGS-1 (EC 1.14.99.1) message. TGF-beta also attenuates LPS-induced accumulation of unspliced TIS10/PGS-2 transcripts in RAW 264.7 cells, suggesting that this cytokine exerts its effects on TIS10/PGS-2 expression at the transcriptional level. TGF-beta inhibits the LPS-induced accumulation of TIS10/PGS-2 protein and message in cultured murine peritoneal macrophages, as well as in macrophage cell lines.

Animals↗
PubMed1994

TGF-beta 1 augments expression of the TIS10/prostaglandin synthase-2 gene in intestinal epithelial cells.

Treatment of nontransformed rat intestinal crypt epithelial IEC-6 cells with tetradecanoyl phorbol acetate (TPA) + calcium ionophore (A23187) induces both the synthesis of prostacyclin and the expression of the TIS10/PGS-2 gene, a primary response gene encoding a second form of prostaglandin synthase (PGS). In addition to pharmacological induction by TPA + A23187, TIS10/PGS-2 message is also induced by the inflammatory cytokine interleukin 1-beta (IL-1 beta). Transforming growth factor beta 1 (TGF-beta), a potent cytokine known to modulate a variety of biological responses, does not by itself induce either prostanoid accumulation or TIS10/PGS-2 gene expression. TGF-beta does, however, augment both induced prostacyclin accumulation and the induced synthesis and accumulation of TIS10/PGS-2 protein and message in IEC-6 cells. TGF-beta concentrations in the range of 0.1-1.0 ng/ml (4.0-40 pM) maximally augment accumulation of TIS10/PGS-2 message. In contrast, dexamethasone attenuates prostacyclin production, TIS10/PGS-2 protein accumulation, and TIS10/PGS-2 message induction in IEC-6 cells. These results suggest that steroids and cytokines such as TGF-beta may (i) modulate intestinal epithelial cell growth and differentiation and (ii) influence gastrointestinal diseases such as gastric ulcers and colon cancer by modulating eicosanoid production.

6-Ketoprostaglandin F1 alpha↗
PubMed1994

In vivo glucocorticoids regulate cyclooxygenase-2 but not cyclooxygenase-1 in peritoneal macrophages.

Acute inflammatory stimuli elevate both the production of prostaglandins and the synthesis and activity of prostaglandin synthase/cyclooxygenase enzyme (COX) in murine peritoneal macrophages. Adrenalectomy also elevates prostaglandin production, COX synthesis and COX activity in these cells. We have utilized cDNA probes and antisera specific for the products of the prostaglandin synthase/cyclooxygenase-1 (COX-1) and TIS10/prostaglandin synthase-2/cyclooxygenase-2 (COX-2) genes to demonstrate that adrenalectomy causes elevation of mRNA and protein from the COX-2 gene, but not from the COX-1 gene, in peritoneal macrophages. Dexamethasone replacement suppressed the elevation of COX-2 mRNA message, COX-2 protein and the increased COX enzyme activity observed in adrenalectomized animals. In contrast, both COX-1 message and COX-1 protein levels were unaffected either by adrenalectomy or by dexamethasone administration. Thus, under normal physiological conditions, tonic glucocorticoid inhibition appears to play a major role in the in vivo regulation of the COX-2 gene. These data are consistent with COX-1 being the constitutive, housekeeping enzyme in macrophages in normal physiological conditions and with the enhanced prostaglandin synthesis seen after an inflammatory stimulus resulting from the rapid induction and activity of COX-2.

Adrenalectomy↗
PubMed1993

Expression of the protein product of the prostaglandin synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells.

TIS10/PGS-2 encodes a prostaglandin synthase (PGS) distinct from the previously described enzyme PGS-1 (EC 1.14.99.1). We have now generated antipeptide antisera, directed to an amino acid sequence unique to the murine TIS10/PGS-2 protein, which specifically recognize the TIS10/PGS-2 antigen. TIS10/PGS-2 protein was undetectable in quiescent Swiss 3T3 cells. The level of TIS10/PGS-2 protein peaked between 6 and 8 h following phorbol ester stimulation of cells, then declined to basal levels after 18-24 h. Synthesis of TIS10/PGS-2 protein was dramatically increased in the second hour following mitogen stimulation and remained elevated for several hours. The half-life of the TIS10/PGS-2 protein was 4 h. Immunofluorescence studies demonstrated a perinuclear and cytoplasmic localization of the TIS10/PGS-2 antigen. As expected, detection of induced TIS10/PGS-2 antigen was dependent on protein synthesis. Metabolically labeled TIS10/PGS-2 protein migrated as a 71/73-kDa doublet following immunoprecipitation. Dexamethasone blocked both the TPA- and serum-induced appearance of TIS10/PGS-2 antigen. These studies demonstrate the existence of a mitogen-inducible, glucocorticoid-inhibitable, immunologically distinct prostaglandin synthase protein.

3T3 Cells↗
PubMed1991

Expression of Rous sarcoma virus-derived retroviral vectors in the avian blastoderm: potential as stable genetic markers.

Retroviruses are valuable tools in studies of embryonic development, both as gene expression vectors and as cell lineage markers. In this study early chicken blastoderm cells are shown to be permissive for infection by Rous sarcoma virus and derivative replication-defective vectors, and, in contrast to previously published data, these cells will readily express viral genes. In cultured blastoderm cells, Rous sarcoma virus stably integrates and is transcribed efficiently, producing infectious virus particles. Using replication-defective vectors encoding the bacterial lacZ gene, we further show that blastoderms can be infected in culture and in ovo. In ovo, lacZ expression is seen within 24 hr of virus inoculation, and by 96 hr stably expressing clones of cells are observed in diverse tissues throughout the embryo, including epidermis, somites, and heart, as well as in extraembryonic membranes. Given the rapid onset of vector expression and the broad range of permissive cell types, it should be feasible to use Rous sarcoma virus-derived retroviruses as early lineage markers and expression vectors beginning at the blastoderm stage of avian embryogenesis.

Animals↗
PubMed1991

Platelet-activating factor (PAF) accumulation correlates with injury in the cornea.

This study reports the accumulation of platelet-activating factor (PAF) in corneas injured with either 0.1 N NaOH or 1 N NaOH. The degree of injury in corneas exposed to alkali for 5, 10, 20, or 60 sec was assessed by light microscopy and scanning electron microscopy. PAF accumulation in vivo increased with time (up to 24 hr) after injury and also according to the severity of the alkali injury. PAF was isolated by high-performance liquid chromatography (HPLC) and assayed by platelet aggregation of the HPLC fraction containing PAF. The specificity of the aggregating bioactivity was ascertained from inhibition of platelet aggregation by selective PAF antagonists. BN 50726, a new synthetic PAF antagonist, applied in vivo topically or subconjunctivally, was effective in inhibiting PAF formation. Because PAF is accumulated in vivo as soon as 30 min after corneal injury, this lipid mediator seems to be synthesized by corneal cells and not be recruited inflammatory cells, since they arrive later. Moreover, if the injury causes stromal edema and endothelial damage, the amount of PAF accumulated is even greater. Results from isolated corneas stimulated in vitro with calcium ionophore A23187 suggest that PAF synthesis is the result of stimulation of phospholipase A2 to form lyso-PAF and subsequent activation of an acetyltransferase.

Animals↗