Search PubMed⌕ Search

Biomedical subjects

A Silva

Publications and source records attributed to A Silva.

At least 109 records · Page 6Linked to original sources
PubMed1995

Physical association and functional relationship between protein kinase C zeta and the actin cytoskeleton.

Protein kinase C (PKC) was initially identified as a serine/threonine protein kinase dependent on calcium and phospholipids and shown to be involved in intracellular signaling pathways. PKC isoforms have been classified into four groups: Ca(2+)-dependent conventional PKC alpha, beta I, beta II, gamma; Ca(2+)-independent, novel PKC delta, epsilon, eta, phi; atypical PKC zeta, lambda, iota which are not activated by Ca2+ or diacylglycerol, and the recently discovered PKCmu. We reported that activation of the zeta PKC isoform is an important step in interleukin-2 (IL-2)-mediated proliferation (Gómez, J., Pitton, C., García, A., Martínez, A., Silva, A. and Rebollo, A., Exp. Cell Res. 1995. 218: 105.). zeta PKC is also required for mitogenic activation of fibroblasts and for the maturation pathway activated by insulin and Ras. Contradictory results have been reported regarding the subcellular redistribution of zeta PKC upon activation. We report here, using confocal microscopy, that IL-2 induces expression, translocation and association of zeta PKC to a structure coincident with the actin cytoskeleton. Furthermore, we show that zeta PKC has a role in maintaining the integrity of the actin cytoskeletal structure in IL-2-stimulated cells. On the contrary, zeta PKC is not involved in the actin cytoskeleton organization when cells are maintained in IL-4, confirming our previous results showing that IL-4-induced signal transduction is PKC independent.

Actins↗
PubMed1995

Differential effect of rapamycin and cyclosporin A in proliferation in a murine T cell line expressing either intermediate or high affinity receptor for IL-2.

It has been reported that rapamycin (rap), cyclosporin A (CsA) and FK506 have immunosuppressive effect during the activation process of murine T cells. These drugs were investigated for their suppressive effect on a murine T cell line expressing intermediate (TS1 beta) or high (TS1 alpha beta) affinity IL-2R. Rap and CsA strongly inhibit the IL-2-dependent proliferation of TS1 alpha beta cells while they minimally affect the IL-2-mediated proliferation of TS1 beta cells. FK506 does not have any effect on the IL-2-driven proliferation of either TS1 beta or TS1 alpha beta cells. Simultaneous addition of Rap and CsA or Rap and FK506 inhibit the IL-2-mediated proliferation of TS1 beta and TS1 alpha beta cells and therefore FK506 does not revert the inhibition mediated by Rap in TS1 alpha beta cells. Neither Rap nor CsA affect IL-2R expression and internalization in TS1 alpha beta cells. CsA and Rap strongly inhibit the appearance of DNA binding activity of NF-AT and to a lesser extent NF-kappa B. Rap inhibits IL-2-stimulated phosphatidylinositol 3 (PI3) kinase activity in TS1 alpha beta cells. In TS1 beta cells, Rap activates PI3 kinase on its own, inhibiting the IL-2-stimulated PI3 kinase to a lesser extent. These results suggest that PI3 kinase is a target for Rap action. Our results strongly suggest that we have Rap and CsA sensitive and resistant activation pathways operating in TS1 beta and TS1 alpha beta cells.

Base Sequence↗
PubMed1995

The zeta isoform of protein kinase C controls interleukin-2-mediated proliferation in a murine T cell line: evidence for an additional role of protein kinase C epsilon and beta.

In order to address a role of protein kinase C in signal transduction through interleukin-2, interleukin-4, and interleukin-9 receptors, we took advantage of the availability of a selective protein kinase C inhibitor, GF109203X, and the availability of TS1 beta and TS1 alpha beta cell lines which can be maintained in interleukin-2, interleukin-4, or interleukin-9 independently. In this report we report that inhibition of protein kinase C activity by GF109203X does not block interleukin-4- or interleukin-9-dependent proliferation and, on the contrary, does block interleukin-2-dependent proliferation, suggesting that interleukin-4 and interleukin-9 do not use signal transduction pathways mediated by protein kinase C and that the common gamma chain of interleukin-2, interleukin-4, and interleukin-9 receptors is not responsible per se for the activation of protein kinase C through interleukin-2 receptor. Moreover, GF109203X induces apoptosis in cells cultured in interleukin-2 but not in interleukin-4 or interleukin-9. Using antisense oligonucleotides, we report that the zeta and epsilon protein kinase C isoforms are involved in signaling through high-affinity interleukin-2 receptor and beta and zeta are involved in signaling through intermediate-affinity interleukin-2 receptor. Taken together, our data indicate that activation of the zeta, beta, and epsilon protein kinase C isoforms is an important step in interleukin-2-mediated proliferation.

Animals↗
PubMed1995

Apoptosis induced by IL-2 withdrawal is associated with an intracellular acidification.

It is known that phorbol esters can protect IL-2-dependent lymphocytes against apoptosis induced by IL-2 withdrawal. However, the mechanism of this effect remains unclear. In this article we show that apoptosis induced by IL-2 withdrawal in the CTLL-2 cell line correlates with a decrease in intracellular pH (pHi). Supplementing the incubation medium with phorbol esters during IL-2 deprivation protects CTLL-2 cells against both apoptosis and intracellular acidification. Interestingly, IL-4 also supports short-term cell survival and maintenance of normal pHi. The protein kinase inhibitor staurosporine prevents the protective effects of IL-2, PMA, and IL-4 on apoptosis and intracellular acidification. In contrast, inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride reverts the protective effects of PMA and IL-4, but only weakly affects IL-2-mediated suppression of apoptosis. Taken together, these results indicate that intracellular acidification may be an important event during apoptosis induced by IL-2 deprivation in the CTLL-2 cell line. Moreover, they suggest a key role for protein kinase C activation both in the maintenance of pHi and in the suppression of apoptosis, through mechanisms which rely on the activation of the Na+/H+ antiporter to a different extent, depending on the rescuing factor employed.

Apoptosis↗
PubMed1995

Pharmacokinetic evaluation of two different formulations of megestrol acetate in patients with advanced malignancies.

The bioequivalence of two megestrol acetate formulations, 160-mg "tablets" and 160-mg "sachets," was investigated in a single-dose, open-label, balanced-for-sequence cross-over study involving 12 advanced-cancer patients. The observed plasma megestrol-acetate time course obtained with both formulations was consistent with the literature data. The main source of variability in the pharmacokinetic parameters was intersubject variability; drug formulation played only a minor (and nonsignificant) role. The width of the 90% confidence interval of the area-under-the-curve (AUC) ratio (sachets: tablets) computed according to Schuirmann (0.9-1.4) was mainly due to the presence of a single outlier, showing an AUC ratio of 2.7. The trend to higher bioavailability of the new formulation was not significant, especially as compared with the dose-response data reported in the literature.

Administration, Oral↗
PubMed1995

Regulation of synaptic strength at mixed synapses: effects of dopamine receptor blockade and protein kinase C activation.

Previous studies of the mixed excitatory synapses between eighth nerve afferents and the lateral dendrite of the goldfish Mauthner (M-) cell have shown that synaptic strength is enhanced for an hour or longer following either repeated brief tetanizations or local extracellular applications of dopamine. Both the initial electrotonic coupling potential, mediated via current flow through gap junctions, and the subsequent chemically mediated excitatory postsynaptic potentials (EPSPs) are potentiated. Different second messenger pathways are implicated in the postsynaptic induction of these potentiations, with a Ca2+ influx presumably triggering the activity dependent long-term potentiations (LTP) and dopamine acting via a cAMP dependent pathway. Experiments performed to determine whether the LTP involves a stimulus-induced release of dopamine or requires a background level of dopamine receptor activation suggest neither is the case, as tetanization in the presence of a D1 receptor antagonist, which blocks the dopamine effects, produced an LTP comparable to that in the absence of the blocker. The effects of Ca2+ are presumably not due to protein kinase C (PKC) activation, since phorbol esters had no effect on the mixed excitatory synaptic responses, although they did enhance the frequency of spontaneously occurring inhibitory PSPs.

Animals↗
PubMed1995

Vasorelaxant effect of the analgesic clonixin on rat aorta.

1. A novel vasorelaxant effect of clonixinate of L-lysine (Clx), analgesic and anti-inflammatory, was studied in rat aortic rings. 2. Clx completely relaxed aortic rings contracted by KCl 70 mM and together with its analog flunixin exhibited lesser potency but equal efficacy than verapamil. In comparison, indomethacin, which is a more potent cyclo-oxygenase inhibitor relaxed only about 40% of the maximal contraction of aortic rings. 3. Furthermore, Clx antagonized Ca2+ dependent aortic contraction and BAY K-8644 induced aortic contraction suggesting its calcium antagonist character. 4. From these results it can be concluded that the hypotensive effect seen in rats in vivo after Clx i.v. injection arises because of vasodilatory effect of Clx and gives further support to the proposal that the pharmacological mechanism of action of Clx should be calcium antagonism.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

A role for the intermediate affinity IL-2R in the protection against glucocorticoid-induced apoptosis.

Recent work has shown that T lymphocytes undergo apoptosis upon treatment with the glucocorticoid analogue dexamethasone. These cells can be protected from the effect of dexamethasone by interleukin-2 (IL-2) or IL-4. We were interested in analysing whether a transfected cell dependent on three different lymphokines could be protected by them from the effect of dexamethasone. In addition, we took advantage of our cellular system, in which we expressed intermediate- or high-affinity IL-2R independently, to analyse the role of these receptors in the protection from glucocorticoid-induced apoptosis. In this report we show that IL-2 rescues murine T cells expressing exogenous intermediate- (TS1 beta) or high-affinity (TS1 alpha beta) IL-2 receptor (IL-2R) from dexamethasone-induced apoptosis. This result suggests that intermediate-affinity IL-2R alone can replace high-affinity IL-2R for the protection from the effect of dexamethasone. In addition, IL-4 and IL-9 are rescue-factors, as well as IL-2, of glucocorticoid-treated TS1 beta and TS1 alpha beta cells. Our data suggest that the presence of the alpha-chain of the IL-2R is not required for rescue by IL-2 from the effect of dexamethasone. In addition, we show that proliferation is not required for preventing glucocorticoid-induced apoptosis. This result implies a new role for the intermediate-affinity IL-2R.

Animals↗
PubMed1995

The sound of the DNA language.

A new method is described for the study of DNA language employing the communicative strength of music. An algorithm was created to "translate" codons into musical notes. The analysis of the musical transcriptions of DNA sequences suggests the presence of some structural features of DNA language hidden in human and mouse interferon alpha 1 genes.

Amino Acid Sequence↗
PubMed1994

Implication of protein kinase C in IL-2-mediated proliferation and apoptosis in a murine T cell clone.

Several reports have suggested that protein kinase C plays an essential role in T cell activation and apoptosis. The recent synthesis of the selective protein kinase C inhibitor, GF109203X, has made it possible to test the relevance of protein kinase C in T cell proliferation and apoptosis. We report that the use of GF109203X, in concentrations that are below its toxicity limits, inhibits IL-2-dependent proliferation in murine T cells expressing intermediate or high-affinity IL-2R (TS1 beta and TS1 alpha beta). In addition, GF109203X reverts the suppression of apoptosis mediated by IL-2 or IL-2+ dexamethasone. The use of the phorbol ester PMA, a protein kinase C activator, allows a bypass of the IL-2/IL-2R interaction in the suppression of apoptosis mediated by dexamethasone or IL-2 withdrawal in TS1 beta cells but not in TS1 alpha beta cells. Taken together, our data indicate that activation of protein kinase C is an important step in IL-2-mediated proliferation and in suppression of apoptosis.

Animals↗
PubMed1994

Molecular variation in a novel polymorphic antigen associated with Plasmodium falciparum merozoites.

A cDNA clone encoding part of a novel polymorphic merozoite antigen from Plasmodium falciparum was isolated by screening a cDNA library with human immune serum from Papua New Guinea. Immunofluorescence microscopy and immunoblotting with affinity-purified antibodies recognized a highly polymorphic antigen, Ag956, present in schizonts and merozoites. Biosynthetic labeling and immunoprecipitation experiments demonstrated that Ag956 is proteolytically cleaved during merozoite maturation. The complete genomic sequence of Ag956 from the D10 clone of P. falciparum isolate FC27 encodes a secreted protein of calculated molecular mass 43,243 that is very hydrophilic and contains a region of unusual heptad repeats of the general structure AXXAXXX. This antigen has been named the secreted polymorphic antigen associated with merozoites (SPAM). The sequence of a second SPAM allele from the 3D7 clone of isolate NF54 reveals that the alanine heptad repeats and the hydrophilic C-terminal half of the protein are conserved. Variation among SPAM alleles is the result of deletions and amino acid substitutions in non-repetitive sequences within and flanking the alanine heptad-repeat domain. Heptad repeats in which the a and d position contain hydrophobic residues generate amphipathic alpha-helices which give rise to helical bundles or coiled-coil structures in proteins. Thus, SPAM is the first example of a P. falciparum antigen in which a repetitive sequence has features characteristic of a well-defined structural element.

Amino Acid Sequence↗
PubMed1994

Inhibition of nitric oxide synthase in experimental gram-negative sepsis.

Nitric oxide (NO) has been proposed as a mediator of hypotension in septic shock. The aim of this study was to determine whether an inhibitor of NO production, NG-monomethyl-L-arginine (L-NMMA), was able to protect against death in two murine models of experimental gram-negative sepsis. L-NMMA (3-300 mg kg-1) did not improve survival in intravenous or intraperitoneal models of sepsis. Seven h after intravenous infection, L-NMMA (100 mg/kg-1) reduced serum nitrite plus nitrate levels (NO breakdown products) from 774 microM in control-treated animals to 282 microM in L-NMMA-treated animals (P < .001). This compared to a level of 103 microM in uninfected mice. L-NMMA produced little change in bacterial load following infection and did not increase hepatic damage, as measured by serum levels of ornithine carbamoyltransferase. Thus, while L-NMMA may reverse the hyporesponsiveness of peripheral circulation in sepsis, it was unable to prevent death in these models of gram-negative septic shock.

Amino Acid Oxidoreductases↗
PubMed1994

[Thrombosis of mechanical tricuspid valve prosthesis: treatment by tissue-type plasminogen activator. Success after a 18 month-follow-up].

Four years after a triple valve replacement, a 51 year old woman presented with signs of thrombosis of her tricuspid Saint Jude valve prosthesis, confirmed at echocardiography and fluoroscopy. Treatment with tissue-type plasminogen activator was completely successful with a result which was sustained at 18 months. A review of the literature confirms the superiority of thrombolytic therapy of first intention in the treatment of thrombosis of tricuspid mechanical prosthetic valves.

Echocardiography, Doppler↗
PubMed1994

Yeast as an expression system for the production of bacterial and viral antigens.

To develop a yeast system that expresses bacterial and viral antigens in the future, we analyzed yeast secretion with a reporter system based on the cc1A gene of Clostridium thermocellum that encodes endoglucanase A. Sequences coding for the mature CelA protein were fused in frame to yeast promoters and secretion signals, introduced into different vectors and analyzed biochemically in yeast secretory mutants. Gene dosage had little effect, whereas expression from a weak promoter enhanced endoglucanase A secretion. In yeast secretory mutants, a major fraction of endoglucanase A piled up in the endoplasmic reticulum as a core glycosylated protein.

Antigens, Bacterial↗
PubMed1994

Toxic effects caused by stainless steel corrosion products on mouse seminiferous cells.

Stainless steel type AISI 316L is widely used in orthopaedic surgery. The toxic effects of its corrosion products on the morphology of mouse seminiferous cells were investigated. Chemical elements from stainless steel were released into a physiological medium using an electrochemical method. This metallic solution was injected subcutaneously into male Charles River mice at 72 h intervals for 10 days. Electron microscopic observations of seminiferous tubule thin sections showed that the metallic suspension caused tissue vacuolation, cell degeneration, and multinucleated cell formation. This apparent tissue toxicity induced by stainless steel corrosion products suggests that long term implantation of such biomaterials may impair spermatogenesis.

Animals↗