Biomedical subjects

M R Ciriolo

Publications and source records attributed to M R Ciriolo.

At least 37 records · Page 2Linked to original sources

PubMed1997

Antiretroviral effect of combined zidovudine and reduced glutathione therapy in murine AIDS.

A combination of antiretroviral drugs acting at different points in the virus replication cycle was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). Intramuscular administration of high doses of reduced glutathione (GSH, 100 mg/mouse/day) and AZT (0.25 mg/ml in drinking water) was found to reduce lymphoadenopathy (92%), splenomegaly (80%), and hypergammaglobulinemia (90%) significantly more than AZT alone. Combined treatment resulted in a reduction in proviral DNA content of 69, 66, and 60%, respectively, in lymph nodes, spleen, and bone marrow. Furthermore, the stimulation index of B cells was also significantly higher in animals receiving GSH and AZT whereas additional responses were not observed in the T cell stimulation index and blood lymphocyte phenotype analyses. In conclusion, the administration of high doses of GSH and AZT, a new combination of antiviral drugs, seems to provide additional advantages compared to single-agent therapy.

Animals↗

PubMed1997

Decrease of immunoreactive catalase protein in specific areas of ageing rat brain.

The activity of catalase, the main enzyme responsible for detoxification against hydrogen peroxide, decreases in specific brain areas of aged rats. The reduction of enzyme activity appears to be the consequence of a decreased protein expression rather than an impaired function of the native enzyme. In fact, diminution of the immunoreactive protein parallels enzyme activity decrease. Since the extent of decrease of both activity and protein content was observed to be area dependent, we hypothesise that this phenomenon may underlie, at least in part, the increased susceptibility of specific brain regions to oxidative insults observed in pathological situations related to ageing.

Aging↗

PubMed1997

Loss of GSH, oxidative stress, and decrease of intracellular pH as sequential steps in viral infection.

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

Adsorption↗

PubMed1997

Intracellular GSH content and HIV replication in human macrophages.

In vitro HIV-1 infection induced a significant decrease in intracellular reduced glutathione (GSH) in human macrophages. Such a decrease was observed at the time of infection corresponding to maximum release of virus from infected cells and was not related to cell cytotoxicity. GSH los was not related to its oxidation or leakage through the cell membrane. Inhibition of intracellular GSH synthesis by buthionine sulfoximine (BSO) did not further decrease GSH levels with respect to the decrease caused by HIV alone. However, treatment of macrophages with BSO significantly increased the HIV yield in the supernatant. Exogenous GSH strongly suppressed the production of p24 gag protein as well as the virus infectivity. Previous observations with other RNA and DNA viruses consistently showed that GSH antiviral effect occurred at late stages of virus replication and was related to the selective decrease of specific glycoproteins, such as gp120, which are particularly rich in disulfide bonds.

Buthionine Sulfoximine↗

PubMed1997

Purification of a fully metal-depleted Cu, Zn superoxide dismutase from copper-deficient rat liver.

A copper-deprived form of the enzyme Cu, Zn superoxide dismutase was identified in the liver of rats made copper-deficient by dietary restriction. In homogenates of such livers Cu, Zn superoxide dismutase presents a dis-homogeneous electrophoretic profile with respect to the native enzyme. When rat liver extracts were treated with exogenous copper an electrophoretic pattern resembling the native one was observed. Enzyme purified by chromatography on DE-52 resin shows two major components, one corresponding to genuine, native enzyme and another one, eluting at higher ionic strength. The latter protein (Fraction II) consists of several isoforms which show the same characteristics of the native superoxide dismutase as far as immunoreactivity and molecular weight are concerned, but with decreased contents of copper and zinc. Its catalytic constant, referring to copper content, was 15 times lower than that obtained for the native enzyme. Moreover, the catalytic power of purified Fraction II was not regained upon incubation with copper. The occurrence of a superoxide dismutase void of metals confirms the hypothesis that this protein plays a dual physiological role: in metal metabolism and in superoxide anion dismutation.

Animals↗

PubMed1996

Cocaine increases Sendai virus replication in cultured epithelial cells: critical role of the intracellular redox status.

Cocaine was found to increase parainfluenza-1 Sendai virus (SV) replication in Madin Darby canine kidney (MDCK) cells. Its effect was maximal when it was added before SV infection, while practically no effect was observed when cocaine was added at the time of or after infection. Enhanced SV replication was associated with increased viral protein expression. Cocaine also greatly reduced the intracellular level of glutathione (GSH), namely the most abundant cell thiol with antioxidant functions, recently proposed as an important factor influencing viral infection. Support for this view was provided in the present study by the reversal of cocaine-induced enhancement of SV replication when the intracellular content of GSH was restored by addition of exogenous GSH.

Animals↗

PubMed1996

Inhibition of murine AIDS by reduced glutathione.

The imbalance of the redox state in cells and body fluids in HIV-1-infected patients may result in progression of the disease as well as in immunologic disfuctions. In this report, we have evaluated whether the direct administration of high doses of reduced glutathione (GSH) exerts any antiviral activity and/or improves immune functions in a murine immunodeficiency animal model. Intramuscular administration of 50 or 100 mg GSH/mouse for five consecutive days weekly to LP-BM5-infected mice did not show local or systemic signs of acute toxicity. During the first 3 weeks from infection, a period in which clinical signs of disease were not yet detectable, GSH significantly reduced the viral load in lymph nodes and spleen as evaluated by a PCR semiquantitative assay of the proviral DNA content. At 10 weeks a GSH concentration-dependent reduction of splenomegaly, lymphadenopathy and hypergammaglobulinemia was evident in all treated mice. Evaluation of proviral DNA content showed that GSH was effective in inhibiting LP-BM5 infectivity in lymph nodes, spleen, and bone marrow at 100 mg/day, while it was less effective when administered at 50 mg/day. At 10 weeks some animals receiving the highest GSH dose died, thus only the mice receiving 50 mg GSH were followed up to 15 weeks without signs of toxicity. In this case, almost not significant differences among infected untreated or treated animals were observed. Thus, GSH is effective in reducing the proviral DNA load in the first period of infection. These data and the failure of sulfhydril supplementation to further counteract the progression of disease after 10 weeks of infection suggest that combinations of GSH and other antiviral agents may be useful for improving current antiviral therapies.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Non-oxidative loss of glutathione in apoptosis via GSH extrusion.

Reduced glutathione (GSH) has been hypothesized to play a role in the rescue of cells from apoptosis, by buffering an endogenously induced oxidative stress. We correlated GSH levels and apoptosis in U937 human monocytic cells induced to apoptosis by different agents. All treatments led to depletion of GSH concomitant with the onset of apoptosis. The loss was due to extrusion of GSH outside the cell, while GSSG was not accumulated in the apoptosing cells, nor was it found in the extracellular medium. Modulation of intracellular GSH level did not influence the overall extent of apoptosis. We conclude that glutathione loss in apoptosis is not necessarily preceded by an oxidative stress, and that GSH depletion alone is not sufficient to lead cells to apoptosis.

Antimetabolites, Antineoplastic↗

PubMed1995

Evidence for antiviral activity of glutathione: in vitro inhibition of herpes simplex virus type 1 replication.

The role of glutathione (GSH) in the in vitro infection and replication of human herpes simplex virus type 1 (HSV-1) was investigated. Intracellular endogenous GSH levels dramatically decreased in the first 24 h after virus adsorption, starting immediately after virus challenge. The addition of exogenous GSH was not only able to restore its intracellular levels almost up to those found in uninfected cells, but also to inhibit > 99% the replication of HSV-1. This inhibition was concentration-dependent, not related to toxic effects on host cells and also maintained if the exogenous GSH was added as late as 24 h after virus challenge, i.e. when virus infection was fully established. Electron microscopic examination of HSV-1-infected cells showed that GSH dramatically reduced the number of extracellular and intracytoplasmic virus particles, whereas some complete nucleocapsids were still detected within the nuclei of GSH-treated cells. Consistent with this observation, immunoblot analysis showed that the expression of HSV-1-glycoprotein B, crucial for the release and the infectivity of virus particles, was significantly decreased. Data suggest that exogenous GSH inhibits the replication of HSV-1 by interfering with very late stages of the virus life cycle, without affecting cellular metabolism.

Animals↗

PubMed1994

Purification and characterization of Ag,Zn-superoxide dismutase from Saccharomyces cerevisiae exposed to silver.

Cu,Zn-superoxide dismutase plays an important role in protecting cells from oxygen toxicity by catalyzing the dismutation of superoxide anion into hydrogen peroxide and oxygen. In Saccharomyces cerevisiae Cu,Zn-superoxide dismutase is coregulated with copper-thionein by copper via the transcription factor ACE 1. We demonstrate here that presence of AgNO3 in the culture medium leads to a five times increase of Cu,Zn-superoxide dismutase mRNA, with a concomitant six times decrease of the enzyme activity. Susceptibility of yeast to silver was apparently inversely related to Cu,Zn-superoxide dismutase activity. From silver-treated yeast a Cu,Zn-superoxide dismutase with impaired dismutase function was purified and was shown to contain silver, which was located to the copper site. These data suggest that Cu,Zn-superoxide dismutase may play an additional direct role in the defense of S. cerevisiae against metal stress by functioning as metal chelator.

Cell Division↗

PubMed1994

Differential decrease of copper content and of copper binding to superoxide dismutase in liver, heart and brain of copper-deficient rats.

Dietary copper-deficiency in rats produced a organ-specific decrease of copper content. This was paralleled by a decrease of the activity of the copper-enzyme superoxide dismutase. In liver such a decrease is partially due to the existence of an apo-form of superoxide dismutase, which can be reactivated by addition of exogenous copper to tissue extracts. These results demonstrate in vivo that superoxide dismutase is post-translationally modulated by copper in higher vertebrates as previously found for yeast and mammalian cells in culture.

Animals↗

PubMed1993

Transduction of reducing power across the plasma membrane by reduced glutathione. A 1H-NMR spin-echo study of intact human erythrocytes.

The NMR signal of reduced glutathione (GSH) was monitored in intact human erythrocytes by the 1H spin-echo Carr-Purcell-Meiboom-Gill pulse sequence. Addition of GSH, which was unable to cross the erythrocyte membrane, produced an approximate twofold increase of the GSH signal in glucose-depleted cells. Addition of oxidised glutathione (GSSG), did not affect the signal, and addition of GSH to hemolysates gave a much smaller increase. Reduction of internal GSSG by NADPH-dependent enzymes was excluded by experiments with glucose-supplied or glucose-6-phosphate dehydrogenase deficient cells. Involvement of external thiol groups of the erythrocyte membrane was shown by the lack of effect in cells treated with an impermeable thiol-blocking compound. Involvement of spectrin was indicated by the proportional loss of the effect in erythrocytes with variable genetic deficiency of spectrin. Protein-glutathione mixed disulfides appeared to be the source of the NMR response since an increase of their content, by diamide treatment or aging procedures, produced a higher GSH signal, while their reduction by permeable reductants gave the opposite effect. It is concluded that GSH can transduce its reducing power by a thiol/disulfide exchange mechanism that sequentially involves sulfur-rich proteins spanning across the erythrocyte membrane.

Adult↗

PubMed1993

Copper(I) transfer into metallothionein mediated by glutathione.

Rabbit liver metallothionein depleted of Cd(II) and Zn(II) was fully reconstituted using a Cu(I)-GSH complex under strictly anaerobic conditions. Anaerobic fluorescence titration, using an emission band at 625 nm which is diagnostic of the correct insertion of Cu(I) into the thiolate clusters of metallothionein, showed that the fluorescence maximum was obtained on addition of as many Cu(I) equivalents as the available Cu(I)-binding sites in the protein (i.e. 12). Binding was nearly complete within 1 min, and Cu(I)-GSH was much more efficient than Cu(I)-thiourea or Cu(I)-acetonitrile in metallothionein reconstitution. In air, full reconstitution was obtained with stoichiometric copper only when an excess of GSH was present in the reaction mixture. Cu(I)-GSH was also able to displace Zn(II) and Cd(II) from natural metallized thionein. It is concluded that: (a) Cu(I)-GSH is a potential physiological Cu(I) carrier, not only for Cu2+/Zn2+ superoxide dismutase [Ciriolo, Desideri, Paci and Rotilio (1990) J. Biol. Chem. 265, 11030-11034] but also for metallothionein; (b) in the case of metallothionein, physiological concentrations of GSH protect the protein from autoxidation in air and facilitate Cu(I)-thiolate exchange; (c) the natural metal composition of metallothionein may be related to metal bioavailability rather than to evolutionary changes in protein structure.

Anaerobiosis↗

PubMed1993

An X-ray absorption study of the reconstitution process of bovine Cu,Zn superoxide dismutase by Cu(I)-glutathione complex.

The Cu(I)GSH complex has recently been shown to be a good candidate for delivering copper to the active site of Cu-free Cu,Zn superoxide dismutase both in vivo and in vitro. In this work X-ray absorption spectroscopy has been used to characterize the Cu(I)GSH complex and to follow in vitro the reconstitution of Cu,Zn superoxide dismutase from the copper-free protein and this complex. The results obtained indicate that the copper is directly transferred as Cu(I) from the GSH complex into the empty copper binding site. No evidence has been obtained for a ternary complex in which the metal is bound to both GSH and the protein.

Animals↗

PubMed1993

Electrostatic recognition in redox copper proteins: a 1H NMR study of the protonation behavior of His 19 in oxidized and reduced Cu,Zn superoxide dismutase.

The pK shift of the His 19 residue in bovine Cu,Zn superoxide dismutase was carefully measured by nuclear magnetic resonance spectroscopy as a function of the change of the copper oxidation state. The measured pK values were identical within experimental error, at variance with results obtained with other copper proteins under similar conditions. The DelPhi program, based on a macroscopic dielectric model for the electrostatic interactions in proteins, was used to calculate pK shifts, as a function of charge perturbation introduced by metal oxidation, between the oxidized and the reduced enzyme, which are assumed to have the same structure on the basis of previous spectroscopic data. A nice fit with the experimental pK values was obtained protonating the imidazole of the copper-zinc bridging His 61, which is known to release the copper in the reduced enzyme. Protonation of His 61 in the reduced state gives rise to an electrostatic potential distribution around the protein almost identical to that observed in the oxidized one. These results suggest that a major role for the bridging histidine in Cu,Zn superoxide dismutase is to provide identical electrostatic steering of the substrate in the two oxidation states of the enzyme by redox-linked protonation-deprotonation processes. This property is discussed in comparison with the effect of copper reduction on the distribution of the electric field in small blue copper proteins.

Acids↗

PubMed1992

Glutathione inhibits replication and expression of viral proteins in cultured cells infected with Sendai virus.

Addition of reduced glutathione inhibited the production of Sendai virus in African green monkey kidney (AGMK) cells. This result could be accounted for by a direct action of GSH on viral replication. The inhibitory action was associated to an increase of the GSH intracellular level, while the host cell metabolism was unaffected. The antiviral effect was related to decrease and inactivation of the hemagglutinin-neuraminidase (HN) virus glycoprotein.

Animals↗

PubMed1992

NGF restores decrease in catalase activity and increases superoxide dismutase and glutathione peroxidase activity in the brain of aged rats.

The effects of ageing on the activity of copper-zinc superoxide dismutase (SOD), selenium-dependent and independent glutathione peroxidase (GSH-Px) and catalase in several areas of the brain in 3-, 12-, and 24-month-old rats were studied. In addition, the effects of a subacute intracerebroventricular treatment of NGF (1 microgram daily for 28 consecutive days) on SOD, GSH-Px, and catalase activity in the same areas of the brain were assessed. The effects of ageing on the activities of antioxidant enzymes varied considerably in the different brain areas studied. Copper-zinc SOD was alone in being unaffected by ageing. Intraventricular infusion of NGF significantly increased SOD activity in the prefrontal cortex, hypothalamus, caudate nucleus, and mesencephalon of 24-month-old rats. Selenium-dependent GSH-Px activity did not significantly change in 12-month-old rats but it increased in the lower brain stem of 24-month-old animals. In comparison to vehicle-treated rats, NGF significantly increased selenium-dependent GSH-Px activity in all brain areas studied in 12- and 24-month-old rats. Catalase activity decreased significantly in the majority of the brain areas studied in 12- and 24-month-old rats. NGF completely restored the fall in catalase activity in 12- and 24-month-old animals to levels similar to those occurring in young rats. In conclusion, the present experiments show, for the first time, that long-term intraventricular administration of NGF significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of superoxide radicals and hydrogen peroxide.

Aging↗

PubMed1991

Age-related changes in Cu,Zn superoxide dismutase, Se-dependent and -independent glutathione peroxidase and catalase activities in specific areas of rat brain.

Oxidative injury of tissues involves both accumulation of damage due to persistent oxidative stress and loss of the proper balance of antioxidative enzymes. These events may produce a faster rate of tissue senescence. In this regard, we have assayed the antioxidative enzyme activities (Cu,Zn superoxide dismutase, glutathione peroxidase and catalase), in various areas of rat brain (prefrontal cortex, parietal cortex, hippocampus, hypothalamus, caudate nucleus, mesencephalon and lower brain stem) for the age groups of 3, 6, 12, 24 months. The results obtained show that the levels of antioxidant enzyme activities differed considerably in the various brain parts studied. Furthermore, changes in the specific activities of superoxide dismutase, catalase, and glutathione peroxidase did not follow the same pattern as a function of aging. In particular, in prefrontal cortex and caudate nucleus, superoxide dismutase and glutathione peroxidase activities did not change, while catalase activity decreased. In parietal cortex and mesencephalon, superoxide dismutase and glutathione peroxidase activities increased, but the catalase activity decreased in parietal cortex and did not change in mesencephalon. In lower brain stem, the activities of glutathione peroxidase and catalase decreased in 3-12-month-old rats. The activity of glutathione peroxidase was increased in the hippocampus and was decreased in hypothalamus during aging. In this area the catalase activity was also significantly diminished.

Aging↗