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Biomedical subjects

S Raha

Publications and source records attributed to S Raha.

At least 37 records · Page 2Linked to original sources
PubMed1999

Thrombin releases calcium from internal stores of ultraviolet C-treated V79 fibroblasts independent of phosphatidylinositol bisphosphate hydrolysis: role of oxidative stress.

V79 fibroblasts were treated with ultraviolet (UV) C radiation alone as well as in conjunction with chronic oxidative stress. The effects of these treatments on calcium signaling were observed at 30 min post-irradiation. In the absence of extracellular calcium, thrombin released calcium from internal stores of UVC-irradiated V79 fibroblasts even after exposure to neomycin. In neomycin-treated control and chronic oxidative stress cells, no calcium release by thrombin was observed after chelation of external calcium. Calcium release by thrombin from internal stores of UV-irradiated and neomycin-treated cells was completely abolished by pretreatment with N-acetyl cysteine and dexamethasone. Cellular total soluble thiol content which is a good indicator of cellular reduced glutathione (GSH) level was significantly elevated 30 min after ultraviolet radiation, indicating an adaptive response after oxidative stress. Chronic oxidative stress alone resulted in a much smaller increase in GSH but chronic oxidative stress in conjunction with UVC produced a very prominent elevation in GSH levels. Our data suggest that thrombin can cause calcium release from internal stores of ultraviolet-irradiated fibroblasts which is independent of phosphatidylinositol bisphosphate hydrolysis and is directly related to the level of oxidative stress. Involvement of phospholipase A2 and a role for its products as possible mediators of calcium release from intracellular stores, is strongly indicated.

Animals↗
PubMed1998

Antibody to a cDNA-derived calreticulin protein from Amblyomma americanum as a biomarker of tick exposure in humans.

The antibody responses of human and animal hosts were studied to determine the utility of antibody against recombinant tick calreticulin (rTC), a cDNA-derived protein isolated from salivary glands of Amblyomma americanum L., as a biologic marker of tick exposure. Rabbits fed upon by either A. americanum or Dermacentor variabilis Say developed significant anti-rTC antibody responses, as measured by both ELISA and immunoblot assay. In contrast, gerbils exposed to Aedes aegypti did not develop anti-rTC antibodies, as measured by ELISA or immunoblot assay. The utility of the assay was next evaluated in humans at high risk for tick exposure. During April through September 1990, 192 military personnel who originated from either Fort Chaffee, Arkansas or Fort Wainwright, Alaska were studied during maneuvers in tick infested areas at Fort Chaffee. Study subjects completed a questionnaire and had pre- and post-maneuvers serum specimens analyzed for antibodies to rTC. In adjusted analysis (controlling for age, fort of origin, attached tick during maneuvers, and bed netting use), the use of bed netting and home station were associated with post-maneuvers anti-rTC antibody seropositivity by ELISA. Subjects from Fort Wainwright were more likely to be seropositive for anti-rTC antibody (adjusted odds ratio = 5.3, 95% confidence interval [CI] = 1.1-25.6). Personnel who did not report the use of bed netting were more likely to be anti-rTC seropositive (adjusted odds ratio = 6.8, 95% CI = 1.4-32.4). Immunoblot assays showed that humans had specific anti-rTC antibody responses. The animal experiments demonstrate that hosts exposed to naturally feeding ticks develop anti-rTC antibodies. The data also indicate that hosts exposed to Ae. aegypti saliva may not develop antibodies against rTC. Observations in tick-exposed humans support the hypothesis that anti-rTC antibody seropositivity is a biologic marker of tick exposure.

Adult↗
PubMed1997

Calreticulin, a potential vascular regulatory protein, reduces intimal hyperplasia after arterial injury.

Both thrombotic and inflammatory responses to arterial injury have been implicated in atherosclerotic plaque growth. Calreticulin is a ubiquitous calcium-binding protein with antithrombotic activity and, in addition, is associated with leukocyte activation. We are investigating calreticulin as a potential vascular regulatory protein. The development of intimal hyperplasia was studied at sites of balloon injury in iliofemoral arteries from 91 rats. Calreticulin was infused directly into the artery immediately before balloon injury, and plaque growth was then assessed at 4 weeks' follow-up. Parallel studies of the effects of each calreticulin domain as well as a related calcium-binding protein, calsequestrin, were examined. The effects of calreticulin on platelet activation, clot formation, and mononuclear cell migration were also studied. When infused before balloon injury in rat iliofemoral arteries, calreticulin, or its high-capacity Ca(2+)-binding C domain, significantly reduces plaque development, whereas calsequestrin, a related calcium-binding protein that lacks the multifunctional nature of calreticulin, does not decrease plaque area (saline: 0.037 +/- 0.007 mm2, calsequestrin: 0.042 +/- 0.021 mm2, calreticulin: 0.003 +/- 0.002 mm2, n = 46, P < .04). The N domain and more specifically the P domain, a low-capacity, high-affinity calcium-binding domain in calreticulin, do not reduce intimal hyperplasia (N + P domain: 0.038 +/- 0.012 mm2, C domain: 0.003 +/- 0.002 mm2, n = 45 rats, P < .0001). Calreticulin reduces macrophage and T cell staining in the arterial wall after injury but has no direct effect on monocyte migration in vitro (percent medial area staining positive for macrophage 24 hours after injury (N + P: 4.06 +/- 1.42, calreticulin: 0.273 +/- 0.02; n = 26, P < .009). Calreticulin does, however, reduce platelet-dependent whole blood clotting time, in vitro (baseline: 78.23 +/- 2.04 seconds, calreticulin: 113.5 +/- 1.95 seconds; n = 5, P < .002). We conclude that calreticulin significantly reduces intimal hyperplasia after arterial injury, potentially acting as a vascular regulatory protein.

Angioplasty, Balloon↗
PubMed1996

Relative importance of inositol (1,4,5)trisphosphate and inositol (1,3,4,5)tetrakisphosphate in Entamoeba histolytica.

[3H]Inositol tetrakisphosphate (Ins(1,3,4,5)P4) binding sites which were poorly displaced by unlabelled inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) were detected in membrane fractions of Entamoeba histolytica. Similarly, unlabelled Ins(1,3,4,5)P4 was 30-fold less efficient in displacing [3H]Ins(1,4,5)P3 binding. pH sensitivities of binding of the two isomers were markedly different. Scatchard analysis of the data revealed single binding sites and similar receptor densities for each of the two isomers. Formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in E. histolytica was also demonstrated. Calcium release studies showed that after treatment with a saturating dose of either Ins(1,4,5)P3 or Ins(1,3,4,5)P4 the other inositol polyphosphate could partially revive the response to a subsequent addition of the first inducer. Our data clearly demonstrate that Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are two equally important but independent second messengers in E. histolytica.

Animals↗
PubMed1996

Diagnosis of complex I deficiency in patients with lactic acidemia using skin fibroblast cultures.

The requirement for a rapid and easy method of preparing mitochondrial fractions from cultured skin fibroblasts led us to compare the results obtained from such a preparation with the more traditional methods of cellular fractionation. Values for NADH-cytochrome c reductase (rotenone sensitive) were compared for a series of three controls and nine patients with complex I (NADH-coenzyme Q reductase deficiency). Values obtained for deficient cell lines varied from 19 to 64% of the control values for the long mitochondrial preparation method and from 34 to 70% of control for the rapid preparation. Mean values were statistically significantly different from the lowest control cell line (P < 0.01) in all cases. The specific activity on the basis of activity per milligram of mitochondrial protein and of activity per unit of citrate synthase activity was lower in the rapid preparation of mitochondria by some 41%, indicating a lesser degree of mitochondrial purification. However, the overall result showed that this type of rapid preparation, which uses four 9-cm petri dishes of cultured cells, can be used to diagnose mitochondrial complex I deficiency. This method will find general use in the measurement of either mitochondrial enzymes of low specific activity or mitochondrial enzymes whose measurement is made difficult by contaminating nonmitochondrial enzymes.

Cells, Cultured↗
PubMed1995

Purine and pyrimidine nucleotides activate distinct signalling pathways in PC12 cells.

The role of extracellular nucleotides in intracellular signalling and neurosecretion was assessed in PC12 cells. Activation of phospholipase C and increased [Ca2+]i were mediated by purinoceptors with an agonist potency profile, ATP approximately UTP > 2-methylthioadenosine triphosphate (2-MeSATP), typical of P2U. ATP also evoked a rapid acidification followed by a more gradual alkalinization (measured with 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF)), while UTP induced only a gradual alkalinization. The amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride (EIPA) attenuated the alkalinization phase suggesting activation of the Na+/H+ exchanger by ATP and UTP. Using bisoxonol and [3H]tetraphenylphosphonium ([3H]TPP+) as potential-sensitive probes, we showed that while ATP rapidly depolarized PC12 cells in an Na(+)-dependent manner, UTP evoked a much reduced and delayed response. The potency profile (ATP approximately 2-MeSATP approximately adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) >> UTP, alpha, beta-methyleneATP) suggested involvement of a receptor subtype distinct from P2U. Secretion of endogenous dopamine was also assessed. Those nucleotides that induced depolarization (ATP, 2-MeSATP, ATP gamma S) were also the most potent secretagogues. UTP was ineffective. Our results suggest that ATP stimulates distinct purinoceptor subtypes and induces neurosecretion through the activation of multiple signalling pathways.

Adenosine Triphosphate↗
PubMed1995

Inositol(1,3,4,5) tetrakisphosphate plays an important role in calcium mobilization from Entamoeba histolytica.

Calcium release from internal stores of Entamoeba histolytica, a parasitic protozoan, was observed by measuring fluorescence of Fura-2. Emptying of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3)-sensitive calcium pools in permeabilized E. histolytica did not significantly affect subsequent calcium release by inositol(1,3,4,5)tetrakis-phosphate (Ins(1,3,4,5)P4). Similarly, prior depletion of Ins(1,3,4,5)P4-sensitive stores did not have any influence on subsequent calcium release by Ins(1,4,5)P3. The EC50 for calcium release was 0.15 microM with Ins(1,4,5)P3 and 0.68 microM with Ins(1,3,4,5)P4. In conclusion, the Ins(1,3,4,5)P4-sensitive calcium store in E. histolytica is separate and independent from the Ins(1,4,5)P3-sensitive pool.

Animals↗
PubMed1994

Myo-inositol trisphosphate-mediated calcium release from internal stores of Entamoeba histolytica.

Calcium mobilisation from internal stores of the parasitic protozoan Entamoeba histolytica was studied by fluorescence measurements of the calcium indicator quin 2 and 45Ca2+ incorporation studies in saponin-permeabilised amoebae. Prior energy-dependent calcium sequestration was found to be necessary for subsequent release of calcium by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Both Ins(1,4,5)P3 and inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) could release calcium equally well from permeabilised E. histolytica with similar EC50 (concentration which produced half maximal release) values for calcium release. Ins(1,4,5)P3-mediated calcium release occurred from a vesicular store, was sensitive to prior treatment by heparin and was attenuated by prior addition of a lower concentration of Ins(1,4,5)P3. cAMP failed to influence inositol trisphosphate induced calcium release, indicating the absence of control mechanisms through cAMP-dependent phosphorylation. GTP neither induced calcium release nor could potentiate inositol trisphosphate mediated calcium mobilisation. A saturating concentration of Ins(1,4,5)P3 could release 50% of radiolabelled calcium sequestered by energy-dependent mechanisms in E. histolytica. The energy-dependent calcium sequestration was inhibited by vanadate and the calcium antagonist Diltiazem but not by dicyclohexylcarbodiimide (DCCD), suggesting the involvement of an endoplasmic reticulum-like structure in calcium storage. Binding studies showed specific association of [3H]Ins(1,4,5)P3 to crude membrane fractions of E. histolytica, which was significantly inhibited by heparin in a dose-dependent manner. IC50 (concentration which produced half-maximal inhibition) values for displacement of radiolabelled Ins(1,4,5)P3 binding by unlabelled Ins(1,4,5)P3 and Ins(2,4,5)P3 were estimated to be 0.99 microM for both isomers. Our results suggested that Ins(1,4,5)P3-mediated calcium release from internal stores of E. histolytica most probably occurred in an inositol trisphosphate receptor-dependent manner.

Animals↗
PubMed1993

Plasma membrane properties in heterogeneous human blood platelet subfractions modulate the cellular response at the second messenger level.

Three subfractions of human blood platelets differing in density and function exhibited also differences when EPR (electron paramagnetic resonance) spin label measurements and fluorescence polarization were performed. The membrane anisotropy in the plasma membranes was higher in low density platelets as compared with that of platelets of intermediate and higher densities. This higher plasma membrane anisotropy correlated with a significantly higher cholesterol-phospholipid (C:P) molar ratio in the plasma membranes of low density platelets. As compared with the other platelet subfractions the low density platelets exhibited the smallest cAMP increase after activation with the aggregation inhibitor prostaglandin (PGE1), and the highest percentage of inositol phosphate accumulation after thrombin stimulation. The results suggest a high correlation between functional parameters and the plasma membrane architecture of human blood platelets.

Adenylyl Cyclases↗
PubMed1993

Sub-second oscillations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate during platelet activation by ADP and thrombin: lack of correlation with calcium kinetics.

The hypothesis that ADP and thrombin liberate Ins(1,4,5)P3 in blood platelets, with kinetics consistent for releasing Ca2+ within 2s, was tested by quenched-flow techniques. Both agonists stimulated transient and equal synthesis of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 near 200 ms and later short-lived peaks, which were not correlated with the slower steady increase in intracellular [Ca2+] between 0.5 to 2 s detected by Indo-1. Shear forces alone caused transient liberation of these inositol phosphates within 0.5 s and up to 4 s, yet failed to increase intracellular [Ca2+].

Adenosine Diphosphate↗
PubMed1993

Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells.

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i), as measured with the Ca(2+)-sensitive dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P1 receptors in mediating this response. The increase in [Ca2+]i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca2+]i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5'-[gamma-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) approximately adenosine 5'-[beta-thio]diphosphate >> adenosine 5'-[alpha beta-methylene] triphosphate, adenosine 5'[beta gamma-imido]triphosphate. This profile is not characteristic of either the P2X or the conventional P2Y receptors. The Ca2+ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P2Y nucleotide receptors (P2N) that mediate calcium influx and the accumulation of inositol phosphates.

Adenine Nucleotides↗