Biomedical subjects
Publications and source records attributed to A Azzi.
3-Aminobenzamide, a known inhibitor of poly-(ADP-ribose)-polymerase has been found in the cell line U-937 to inhibit protein kinase C at the same concentration as poly-(ADP-ribose)-polymerase. 3-Aminobenzamide was not able, however, to inhibit the isolated enzyme. An indirect mechanism of protein kinase C inhibition is proposed.
The sensitivity of PKC alpha to two protein phosphatases (PP1 and PP2A) has been studied. The results show that both phosphatases reversibly inhibit PKC alpha activity suggesting an effect at PKC autophosphorylation sites and not at transphosphorylation sites. Moreover, PP1 has been found at low concentration to activate PKC alpha implying the existence of an inhibitory phosphorylation site. Further, PKC alpha has been shown to phosphorylate PP2A at its regulatory subunit B.
The preventive effect of vitamin E and Probucol against atherosclerosis in rabbits were compared. Atherosclerosis was induced by a 2% cholesterol-containing vitamin E-poor diet (5-10 ppm). Six groups of five rabbits each were studied. Group I (control) was fed on a vitamin E-poor diet. The other groups had the following supplements: group II, 50 mg/kg vitamin E i.m.; group III, 2% cholesterol; group IV, 2% cholesterol plus 50 mg/kg vitamin E i.m., group V, 2% cholesterol plus 1% Probucol; group VI, 2% cholesterol + 1% Probucol plus 50 mg/kg vitamin E i.m. After 4 weeks, aortas were removed and analyzed by light and scanning electron microscopy for atherosclerotic lesions. Samples of the media were analyzed for protein kinase C activity. The aortas of cholesterol-fed rabbits showed typical atherosclerotic lesions, detected by microscopic examination, their media smooth muscle cells exhibited an increase in protein kinase C activity. Vitamin E fully prevented cholesterol-induced atherosclerotic lesions and the induction of protein kinase C activity. Probucol was not effective in preventing either cholesterol-induced atherosclerotic lesions or the induction of protein kinase C activity. These results show that the protective effect of vitamin E against hypercholesterolemic atherosclerosis is not produced by an other antioxidant such as Probucol, and therefore, may not be linked to the antioxidant properties of this vitamin. The effects observed at the level of smooth muscle cells ex vivo suggest an involvement of signal transduction events in the protective effect of vitamin E against atherosclerosis.
Rat and human vascular smooth muscle cell proliferation is specifically sensitive to alpha-tocopherol, but not beta-tocopherol. The former, but not the latter, is capable of limiting proliferation and inhibiting protein kinase C activity in a dose-dependent manner. The phenomenon occurs at concentrations in the range 10-50 microM. beta-tocopherol addition together with alpha-tocopherol, prevents both cell growth and protein kinase C inhibition. alpha-tocopherol increases de novo synthesis of protein kinase C molecules. The enzyme specific activity, however, is diminished, due to a decreased phosphorylation of protein kinase C, occurring in the presence of alpha-tocopherol. Experiments with protein kinase C isoform-specific inhibitors and precipitating antibodies show that the only isoform affected by alpha-tocopherol is protein kinase C-alpha. The effect of alpha-tocopherol is prevented by okadaic acid indicating a phosphatase of the PP2A type as responsible for protein kinase C-alpha dephosphorylation produced in the presence of alpha-tocopherol. At a gene level alpha-tocopherol but not beta-tocopherol induces a transient activation of alpha-tropomyosin gene transcription and protein expression. It is proposed that, by inhibiting protein kinase C activity via an activation of a phosphatase PP2A, alpha-tocopherol controls smooth muscle cell proliferation through changes in gene expression.
European isolates of parvovirus B19 were analyzed by restriction enzyme analysis of PCR products of the VP1/2 coding region and sequencing of the same amplified region, five cloned fragments from each PCR product. Two main groupings were found based on three perfectly linked point deviations. On the assumption that identical point deviations causing the various restriction patterns regardless of time and origin of virus isolation were unlikely to emerge independently in different evolutionary lineages, traits of evolutionary lineages were identified, suggesting a clonal population structure of global circulating B19 strains. However, combinations of markers from different evolutionary lineages were also found, particularly in a strain derived from an individual chronically infected with B19 for more than 7 years. As chronically infected individuals might be subject to superinfections due to contacts or possibly due to blood transfusions or the administration of gamma-globulin, it is suggested that coexistence of, and recombination between variants of B19 of different phylogenetic origin incidentally occur in such individuals.
RRR-alpha-Tocopherol, but not RRR-beta-tocopherol, negative regulates proliferation of vascular smooth muscle cells at physiological concentrations. At the same concentrations RRR-alpha-tocopherol inhibits protein kinase C activity, whereas RRR-beta-tocopherol is ineffective. Furthermore, RRR-beta-tocopherol prevents the inhibition of cell growth and of protein kinase C activity caused by RRR-alpha-tocopherol. The negative regulation by RRR-alpha-tocopherol of protein kinase C activity appears to be the cause of smooth muscle cell growth inhibition. RRR-alpha-Tocopherol does not act by binding to protein kinase C directly but presumably by preventing protein kinase C activation. A second RRR-alpha-tocopherol effect has been found at the level of AP 1, the latter becoming activated by RRR-alpha-tocopherol under condition of protein kinase C inhibition or down regulation. AP-1 inhibition by RRR-alpha-tocopherol is seen, however, under condition of protein kinase C stimulation. Compositional changes of AP-1 have been found to be at the basis of the RRR-alpha-tocopherol effects. RRR-beta-tocopherol, provided with similar antioxidant properties, not only it does not affect AP 1 but it prevents the effects of RRR-alpha-tocopherol. Moreover, it has been observed that RRR-alpha-tocopherol is able to affect TRE regulated gene transcription. It is concluded that RRR-alpha-tocopherol acts specifically in vascular smooth muscle cells, by controlling a signal transduction pathway leading to cell proliferation by a non-antioxidant mechanism.
U937 cell growth in the presence of either chloramphenicol or ethidium bromide rapidly leads to respiratory deficiency. The novel finding of this report is that this response is paralleled by a specific increase in Se-dependent and independent glutathione peroxidase activities as well as of glutathione peroxidase and heme oxygenase mRNAs. Under the same experimental conditions, catalase activity and catalase mRNA do not show appreciable changes. These results can be explained by an increased formation of H2O2 at the early times of development of respiratory deficiency followed by induction of antioxidant enzymes.
Previous work had established that, in smooth muscle cells, alpha-tocopherol negatively regulates protein kinase C by preventing its activation [Tasinato, A., Boscoboinik, D., Bartoli, G. M., Maroni, P. & Azzi, A. (1995) Proc. Natl Acad. Sci. USA 92, 12190-12194]. In this study, the mechanism by which this event takes place has been analyzed. The regulation by alpha-tocopherol of protein kinase C expression, activity and phosphorylation has been followed during the synthesis of protein kinase C after its down-regulation by phorbol 12-myristate 13-acetate. The data show that protein kinase C isoenzyme alpha is synthesised significantly more (30% 72 h after down-regulation) in the presence of alpha-tocopherol. However, its activity is significantly less (45% diminution) and its phosphorylation state is also decreased (60% diminution). The effect of alpha-tocopherol appears not to be shared by the analogue beta-tocopherol, provided with similar radical-scavenging properties. The data are interpreted in terms of a diminution of protein kinase C phosphorylation, specifically caused by alpha-tocopherol, resulting in a decreased enzyme specific activity.
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A large spectrum of pro-oxidant agents, including molecules with lipoperoxidative effect, can modulate gene expression through modification of the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B). In this study the effect on these redox-sensitive factors by 4-hydroxy-2-nonenal (HNE), a major aldehydic product of lipid peroxidation, was examined in two cell lines of the macrophage type. Incubation in the presence of microM concentrations of the aldehyde led to a rapid increase of AP-1 binding with a transient maximum 30 min from HNE addition to the culture medium in both cell lines. On the contrary, HNE did not stimulate nuclear translocation of NF-kappa B. The diverging effect of HNE on the two transcription factors is likely related to the demonstrated differential activation pathway of AP-1 and NF-kappa B in macrophages. The HNE-induced activation of AP-1 suggests the aldehyde's involvement in the regulatory mechanisms of cell proliferation and differentiation.
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The effects of alpha-tocopherol and beta-tocopherol have been studied in rat and human aortic smooth muscle cells. Alpha-tocopherol, but not beta-tocopherol, inhibited smooth muscle cell proliferation and protein kinase C in a dose-dependent manner, at concentrations ranging from 10 to 50 microM. Beta-tocopherol added simultaneously with alpha-tocopherol prevented both proliferation and protein kinase C inhibition. Protein kinase C inhibition was cell cycle-dependent and it was prevented by okadaic acid, a protein phosphatase inhibitor. Protein kinase C activity measured from aortas of cholesterol-fed rabbits was also inhibited by alpha-tocopherol. By using protein kinase C (PKC) isoform-specific inhibitors and immunoprecipitation reactions it was found that PKC-alpha was selectively inhibited by alpha-tocopherol. Further, an activation of protein phosphatase 2A by alpha-tocopherol was found, which caused PKC-alpha dephosphorylation and inhibition. Ultimately, this cascade of events at the level of cell signal transduction leads to the inhibition of smooth muscle cell proliferation.
BACKGROUND: Double inactivation by solvent/detergent treatment plus heating at 100 degrees C for 30 minutes after lyophilization has been adopted to improve viral safety of factor VIII and factor IX concentrates, particularly with respect to non-lipid-enveloped viruses. The aim of this study was to evaluate the safety of concentrates exposed to these virucidal methods. STUDY DESIGN AND METHODS: Twenty-six previously untreated hemophiliacs, 19 with factor VIII deficiency and 7 with factor IX deficiency, were investigated in a prospective multicenter study over a 12-month follow-up period by the use of serologic and virologic markers for lipid- and non-lipid-enveloped viruses (human immunodeficiency virus types 1 and 2; hepatitis A, B, and C viruses; B19 parvovirus antibodies; and B19 DNA). Overall, 270,000 U of factor VIII and 102,000 U of factor IX concentrate were administered during the study period. RESULTS: None of the 26 patients seroconverted for human immunodeficiency virus or hepatitis C virus. Hepatitis B virus markers remained negative in the 10 unvaccinated hemophiliacs. No hepatitis A virus seroconversion occurred among 17 susceptible patients. B19 seroconversion (IgM) and B19 viremia were observed within 2 weeks of the first concentrate infusion in 8 of 15 susceptible patients, 5 of 11 treated with factor VIII and 3 of 4 with factor IX concentrate. CONCLUSION: This prospective study indicates that very high temperatures applied to lyophilized concentrates appear to prevent the transmission of hepatitis A virus to hemophiliacs. However, B19 parvovirus still contaminates concentrates despite the use of this robust virucidal method.
alpha-Tocopherol, but not beta-tocopherol, negatively regulates proliferation of A7r5 vascular smooth muscle cells at physiological concentration. The HeLa cell line was not affected whereas the Chinese hamster ovary cell line (CHO) was slightly inhibited by both alpha-tocopherol and beta-tocopherol. In A7r5 cells alpha-tocopherol inhibited protein kinase C activity, and this correlated with inhibition of proliferation. beta-Tocopherol did not inhibit either protein kinase C or proliferation. In HeLa cells no inhibition by alpha-tocopherol or beta-tocopherol of protein kinase C activity and cell proliferation was observed. In Chinese hamster ovary cells both tocopherols inhibited protein kinase C activity but not proliferation. Thus in the latter cells proliferation was not protein kinase C-dependent. In A7r5 cells alpha-tocopherol but not beta-tocopherol activated AP-1-mediated gene expression. In HeLa cells no change in gene expression was observed in agreement with the finding that also protein kinase C was not affected. In CHO cells gene-expression was activated by both alpha-tocopherol and beta-tocopherol. In this case also a positive correlation was found with similar inhibition of protein kinase C activity. In these cells, however, the changes at the level of protein kinase C activity and gene expression did not result in proliferation changes. The effect of alpha-tocopherol and beta-tocopherol on protein kinase C activity and gene expression suggest a cause-to-effect relationship. Inhibition of proliferation, however, correlates in the case of A7r5 and HeLa cells but not in the case of CHO suggesting a different proliferation pathway for these cells.
Hydrogen peroxide (H2O2) or 4-hydroxy-2,3-trans-nonenal (HNE) treatment of rat vascular smooth muscle cells (A7r5) caused induction of aldose reductase mRNA. Induction was dose (10-100 microM H2O2, 1-10 microM HNE) and time dependent, reaching a maximum (three- to fourfold) after 7-12 h. Treatment of cells with actinomycin D confirmed de novo synthesis of aldose reductase mRNA. H2O2-induced expression was prevented by catalase but unaffected by Desferal, indicating that metal catalyzed degradation of peroxide was not involved. Induction of enzymatically active aldose reductase by H2O2 and HNE was confirmed using Western blotting and enzyme assays. Aldose reductase can metabolize several aldehyde compounds including HNE, a major toxic product of lipid peroxidation. Inclusion of Sorbinil, an aldose reductase inhibitor, in toxicity assays resulted in a significant (twofold) enhancement of HNE-mediated killing of A7r5 cells, suggesting a protective role of aldose reductase against HNE-induced cell death. These data indicate that the induction of aldose reductase during oxidative stress might represent an important cellular antioxidant defense mechanism.
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alpha-Tocopherol but not beta-tocopherol, activates protein phosphatase 2A, decreases protein kinase C activity and attenuates smooth muscle cell proliferation at physiological concentrations. beta-Tocopherol prevents the effects of alpha-tocopherol. Inhibition of protein kinase C alpha, but not of the other isoforms, by the inhibitor Gö6976 prevents the effect of alpha-tocopherol. Protein kinase C alpha, immunoprecipitated from alpha-tocopherol treated cells, is less phosphorylated and inactive. It is proposed that the specific activation of protein phosphatase 2A by alpha-tocopherol results in dephosphorylation and inactivation of protein kinase C alpha. Finally, this cascade of events leads to smooth muscle cell proliferation inhibition.