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Biomedical subjects

A Azzi

Publications and source records attributed to A Azzi.

At least 19 recordsLinked to original sources
PubMed1995

Stimulation of protein kinase C activity by compactin in vascular smooth muscle cells.

The effect of compactin (a lovastatin analogue) on vascular smooth muscle cells was studied at the level of cell proliferation and protein kinase C. It was observed: a) an inhibition of cell proliferation by compactin at a micromolar range, which was prevented by simultaneous addition of mevalonate; b) a stimulation of DNA synthesis with a shift in the cell cycle kinetics, either in the presence or absence of fetal calf serum and c) an increase in protein kinase C activity in compactin-treated cells in the G1 phase of the cycle. This increase was similar to the one elicited by calyculin A, an inhibitor of protein phosphatases of type PP-1 and PP-2A. It is suggested that compactin behaves as a PP-1/PP-2A protein phosphatase inhibitor, inhibiting proliferation of smooth muscle cells by a block of the cell cycle after the S-phase.

Animals↗
PubMed1995

Hydrogen peroxide-and fetal bovine serum-induced DNA synthesis in vascular smooth muscle cells: positive and negative regulation by protein kinase C isoforms.

Hydrogen peroxide and fetal bovine serum stimulate DNA synthesis in growth-arrested smooth muscle cells with remarkably similar kinetics and cell density dependence. However, while stimulation with fetal bovine serum results in cell proliferation, that by H2O2 is followed by cell death. Depletion of conventional and novel protein kinase C isoforms, resulting from a long treatment with phorbol-12-myristate-13-acetate, further increases H2O2-induced DNA synthesis. On the other hand, the specific protein kinase C inhibitor calphostin C abolished the increased DNA synthesis promoted by fetal bovine serum or H2O2. H2O2 increases protein kinase C activity in smooth muscle cells. This effect is markedly reduced, but not abolished, by down-regulation of the alpha, delta and epsilon protein kinase C isoforms. Thus, the zeta isoform of protein kinase C, which is not down-regulated, may be responsible for the residual H2O2 stimulation of protein kinase C. In conclusion, the results obtained show that H2O2 stimulates protein kinase C activity and DNA synthesis in growth-arrested smooth muscle cells: these events are not followed by cell proliferation but rather by cell death. This H2O2 stimulated DNA synthesis appears to be negatively controlled by alpha, delta and epsilon isoforms and positively controlled by the zeta isoform of protein kinase C.

Cell Count↗
PubMed1995

Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-alpha-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain.

Cellular signalling by the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) has been suggested to involve generation of low levels of reactive oxygen species (ROS). Certain antioxidants and metal chelators can inhibit cytotoxicity and gene expression in response to TNF alpha in numerous cell types. However, neither the source nor function of TNF alpha-induced oxidant generation is known. Using specific inhibitors, we ruled out involvement of several oxidant-generating enzymes [cyclo-oxygenase (indomethacin), cytochrome P-450 (metyrapone), nitric oxide synthase (NG-methyl-L-arginine), NADPH oxidase (iodonium diphenyl), xanthine oxidase (allopurinol), ribonucleotide reductase (hydroxyurea)] in TNF alpha-mediated apoptosis of the murine fibrosarcoma line, L929. We also demonstrated no role for mitochondrial-derived radicals/respiratory chain in the lytic pathway using specific inhibitors/uncouplers (rotenone, KCN, carboxin, fluoroacetate, antimycin, malonate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and chloramphenicol-derived respiration-deficient cells. Significant ROS (H2O2, O2-.) generation was not observed in response to TNF alpha in L929 cells using four separate assays. Also, prevention of intracellular H2O2 removal by inhibition of catalase did not potentiate TNF alpha-mediated cell death. These data suggest that neither H2O2 nor O2-. plays a direct role in TNF alpha cytotoxicity. Finally, we suggest a central role for lipoxygenase in TNF alpha-mediated lysis. Three inhibitors of this radical-generating signalling pathway, including an arachidonate analogue (5,8,11,14-eicosatetraynoic acid), could protect cells against TNF alpha. The inhibitor nordihydroguaiaretic acid is also a radical scavenger, but it could not protect cells from ROS toxicity at concentrations that effectively prevented TNF alpha killing. Therefore protection by nordihydroguaiaretic acid cannot be due to scavenging of cytotoxic H2O or O2-.. The lipoxygenase product, (12S)-hydroxyeicosatetraenoic acid, was also significantly protective. As this analogue can act as a substrate for certain lipoxygenases, this effect may be due to prevention of generation of physiological products.

Animals↗
PubMed1995

Vitamin E: a sensor and an information transducer of the cell oxidation state.

We studied the effects of RRR-alpha-tocopherol and RRR-beta-tocopherol in smooth muscle cells from rat (line A7r5) and human aortas. RRR-alpha-Tocopherol, but not RRR-beta-tocopherol, inhibited smooth muscle cell proliferation in a dose-dependent manner at concentrations in the range from 10 to 50 mumol/L. RRR-beta-Tocopherol added simultaneously with RRR-alpha-tocopherol prevented growth inhibition. The earliest event brought about by RRR-alpha-tocopherol in the signal transduction cascade controlling receptor-mediated cell growth was the activation of the transcription factor AP-1. RRR-beta-tocopherol alone was without effect but in combination with RRR-alpha-tocopherol prevented the AP-1 activating effect of the latter. Protein kinase C was inhibited by RRR-alpha-tocopherol and not by RRR-beta-tocopherol, which also in this case prevented the effect of RRR-alpha-tocopherol. Calyculin A, a protein phosphatase inhibitor, prevented the effect of RRR-alpha-tocopherol on protein kinase C. The data can be rationalized by a model in which a tocopherol-binding protein discriminates between RRR-alpha-tocopherol and RRR-beta-tocopherol and initiates a cascade of events at the level of cell signal transduction that leads to the inhibition of cell proliferation.

Animals↗
PubMed1993

[Fresh sections of the flexor tendons of the fingers and thumb. New therapeutic trends. Apropos of a clinical series of 77 tendon lesions].

In flexor tendon surgery, the main concern of hand surgeons in the last two decades has been to find an effective and reproducible means to avoid post-operative adhesions. For most authors, these adhesions were responsible for the bad results. Since 1960, a constant progress has been achieved with the progress in operative procedures and the better understanding of tendon healing process. Post-operative rehabilitation, especially Kleinert's and Duran's active and passive methods, have radically transformed the prognosis of fresh tendon lesions. In spite of all this progress, zone II tendon injuries are still a difficult problem. In this clinical study, we wanted to introduce two new orientations in order to improve the overall results, the use of human fibrin sealant instead of the epitendinous running suture and an improvement of Duran's technique, developed by the Bichat rehabilitation team since 1987. Seventy-seven tendon lesions treated according to our technique (55 fingers and 22 thumbs) between 1987 and 1991, were reviewed. All the lesions studied were in zone II and T II. The mean follow-up is 14.4 months. The evaluation is based on the International Federation of Hand Surgery score for fingers, and the Tubiana score for thumbs. 74% of fingers and 86% of thumbs were scored as good and excellent.

Adult↗
PubMed1992

Isolation of tocopherol-binding proteins from the cytosol of smooth muscle A7r5 cells.

Cultured smooth muscle A7r5 cells were able to take up alpha-tocopherol (32 +/- 1.2 nmol/mg protein) the largest part of which (60%) was present in the cytosolic fraction. Using a tocopherol-based affinity chromatography and alpha-, beta-, gamma-, and delta-tocopherols as eluants, three polypeptides of molecular masses 81, 58 and 31 kDa were eluted. This preparation had alpha-[3H]tocopherol binding capability. The 58-kDa polypeptide could also be eluted by chromanol and the 81-kDa polypeptide could be eluted also by phytol. The 81-kDa polypeptide had the unique P-E-E-D-Q-X-Q-Y N-terminal sequence.

Amino Acid Sequence↗
PubMed1992

The protein kinase C family.

Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn-1,2-diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor-promoting activity. The cellular localization of the different calcium-activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.

Amino Acid Sequence↗
PubMed1992

The activity of pyruvate carrier in a reconstituted system: substrate specificity and inhibitor sensitivity.

The pyruvate carrier, of molecular mass 34 kDa, was purified from mitochondria isolated from rat liver, rat brain, and bovine heart, by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate. Its activity after reconstitution in phosphatidylcholine vesicles was measured either as uptake of [1-14C]pyruvate or as exchange with different 2-oxoacids. All preparations exhibited similar apparent Km values for pyruvate, but somewhat different V(max) values. The ability to exchange different anions of physiological significance, including branched-chain 2-oxoacids, confirmed the known substrate specificity described for the pyruvate carrier in mitochondria. The sensitivity of pyruvate transport toward phenylglyoxal suggested an important role of arginyl residues in the transport activity, while a role of lysyl and histidyl residues was not confirmed.

Acetoacetates↗
PubMed1992

Human parvovirus B19 infection in hemophiliacs first infused with two high-purity, virally attenuated factor VIII concentrates.

Human parvovirus B19 can be transmitted by coagulation factor concentrates and is highly resistant to virucidal methods. To evaluate whether the additional removal of virus by chromatographic methods during the manufacture of high-purity concentrates reduces the risk of B19 transmission, we have prospectively evaluated the rate of anti-B19 seroconversion in two groups of susceptible (anti-B19 negative) hemophiliacs infused with high-purity, heated (pasteurized) or solvent-detergent-treated factor VIII concentrates. Both products infected a relatively high proportion of patients (nine of 20).

Detergents↗
PubMed1992

Molecular basis of alpha-tocopherol inhibition of smooth muscle cell proliferation in vitro.

The molecular events responsible for the inhibition of cell proliferation by alpha-tocopherol have been investigated. Smooth muscle cells in vitro have been shown to be specifically inhibited by alpha-tocopherol with a concomitant inhibition of protein kinase C activity. beta-Tocopherol was inactive, despite its similar radical scavenging activity. The point of inhibition of alpha-tocopherol relative to the cell cycle was localized in the late G1 phase. A second effect of alpha-tocopherol observed with smooth muscle cells was the stimulation of protein kinase C biosynthesis in both the S and G2 phases of the cell cycle. The implications of these findings for the onset of arteriosclerosis are discussed.

3T3 Cells↗
PubMed1992

Human parvovirus B19 in clotting factor concentrates: B19 DNA detection by the nested polymerase chain reaction.

The presence of B19 parvovirus in plasma from blood donors is seldom demonstrable, but clotting factor concentrates, prepared from large plasma pools, may be able to transmit B19 virus infection, and the effectiveness of different chemical and physical treatment to inactivate this virus is not yet known. In this study we report on the detection of B19 DNA in 25 clotting factor concentrates, prepared by a variety of procedures of purification and inactivation; dot blot hybridization and Southern blot hybridization assays, as well as a 'nested' polymerase chain reaction (PCR) have been employed. Nine out of 25 products were B19 DNA positive by PCR, whereas only two gave positive results by hybridization techniques. B19 DNA positive concentrates have been found in 'untreated' products but also in some solvent/detergent or steam-treated products and even in monoclonal purified concentrates. PCR may be useful for the screening of blood products to be used in immunocompromised haemophiliacs, particularly in HIV positive subjects, at risk of severe chronic anaemia following B19 infection.

Base Sequence↗
PubMed1991

Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae).

Isolated yeast mitochondria were subjected to solubilization by Triton X-114 and the detergent extract was subsequently chromatrographed on dry hydroxyapatite. Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nałecz K.A. and Azzi, A. (1989) J. Biol. Chem. 264 18024-18030). The final preparation contained two polypeptides of apparent molecular mass 26 and 50 kDa. The yeast carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes. The carrier was able to catalyse the pyruvate / pyruvate and pyruvate / acetoacetate exchange reactions, both reactions being sensitive to cyanocinnamate and its derivatives, to phenylpyruvate and to mersalyl and p-chloromercuribenzoate. In the pyruvate / acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyruvate was found to be 0.8 mM and the Vmax 135 mumol/min per mg protein. Among other substrates of the yeast carrier, all transported with similar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate. Lactate was not translocated by this carrier with a measurable rate, neither were di- or tricarboxylates.

Animals↗
PubMed1991

Inhibition of cell proliferation by alpha-tocopherol. Role of protein kinase C.

The effect of alpha-tocopherol (vitamin E) on the proliferation of vascular smooth muscle cells (A7r5), human osteosarcoma cells (Saos-2), fibroblasts (Balb/3T3), and neuroblastoma cells (NB2A) has been studied. The proliferation of vascular smooth muscle cells was inhibited by physiologically relevant concentrations of alpha-tocopherol, neuroblastoma cells were only sensitive to higher alpha-tocopherol concentrations, and proliferation of the other cell lines was not inhibited. The inhibition of smooth muscle cell proliferation was specific for alpha-tocopherol. Trolox, phytol, and alpha-tocopherol esters had no effect. Proliferation of smooth muscle cells stimulated by platelet-derived growth factor or endothelin was completely sensitive to alpha-tocopherol. If smooth muscle cells were stimulated by fetal calf serum, proliferation was 50% inhibited by alpha-tocopherol. No effect of alpha-tocopherol was observed when proliferation of smooth muscle cells was stimulated by bombesin and lysophosphatidic acid. The possibility of an involvement of protein kinase C in the cell response to alpha-tocopherol was suggested by experiments with the isolated enzyme and supported by the 2- to 3-fold stimulation of phorbol ester binding induced by alpha-tocopherol in sensitive cells. Moreover, alpha-tocopherol also caused inhibition of protein kinase C translocation induced by phorbol esters and inhibition of the phosphorylation of its 80-kDa protein substrate in smooth muscle cells. A model is discussed by which alpha-tocopherol inhibits cell proliferation by interacting with the cytosolic protein kinase C, thus preventing its membrane translocation and activation.

Animals↗
PubMed1991

Azido derivative of tricarboxylic acid for photoaffinity labeling.

A new photoaffinity probe, 5-(1-hydroxy-4-azidophenylazo)-1,2,3-benzenetricarboxylic acid, was synthesized and characterized. This reagent can be potentially used in photoaffinity labeling of the mitochondrial tricarboxylate carrier, as well as of enzymes interacting with tricarboxylic acids. Inhibition and labeling of the mitochondrial tricarboxylate carrier is presented.

Affinity Labels↗
PubMed1991

Cytochrome c oxidase metal centers: location and function.

Cytochrome c oxidase of Paracoccus denitrificans is spectroscopically and functionally very similar to the mammalian enzyme. However, it has a very much simpler quaternary structure, consisting of only three subunits instead of the 13 of the bovine enzyme. The known primary structure of the Paracoccus denitrificans subunits, the knowledge of a large number of sequences from other species, and data on the controlled proteolytic digestion of the enzyme allow structural restrictions to be placed on the models describing the binding of the active metal centers to the polypeptide structure.

Amino Acid Sequence↗
PubMed1991

Alpha-tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity.

Alpha-Tocopherol (vitamin E) protects against free radical damage, which has been implicated in aging, cancer initiation, and atherosclerosis. We have found that physiological concentrations of alpha-tocopherol specifically inhibited aorta smooth muscle cell (VSMC, line A7r5) proliferation and protein kinase C (PKC) activity. Other water and lipid soluble antioxidants were inactive. alpha-Tocopherol inhibition of PKC and of VSMC proliferation may represent a physiological mechanism, relevant to the onset of diseased states such as atherosclerosis.

Adenosine Triphosphate↗