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G Bellomo

Publications and source records attributed to G Bellomo.

At least 127 records · Page 7Linked to original sources
PubMed1990

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells. I. Biochemical and immunocytochemical features.

Cytoskeletal abnormalities occurring during oxidative stress generated by the metabolism of the redox cycling compound 2-methyl-1,4-naphtoquinone (menadione) have been investigated in different mammalian cells in culture. Extraction of the whole cytoskeleton as well as the intermediate filament- and the microtubule-enriched fractions from menadione-treated cells revealed a marked depletion of protein sulfhydryl groups. The analysis of the whole cytoskeletal fraction by PAGE showed a menadione-dependent and thiol-sensitive oxidation of actin, leading to the formation of high-molecular-weight aggregates. In addition, the extraction of this fraction with high concentrations of KCl entailed only a partial solubilization of actin. The comparative cytochemical analysis performed on treated cells showed a menadione-dependent clustering of actin microfilaments. The metabolism of menadione induced microtubule depolymerization and inhibition of GTP-induced microtubule assembly from soluble cytosolic components. The latter phenomenon was prevented by previously treating the cytosolic fraction with thiol reductants such as dithiothreitol. Menadione increased the protein content of the intermediate-size filament fraction, partially purified by one or more cycles of disassembly/assembly, and particularly enriched in polypeptides reacting with antikeratin antibodies. Furthermore, a reversible and oxidation-dependent change of the electrophoretic mobility of some polypeptides in this fraction was detected. The immunocytochemical investigation of intermediate-size filament distribution in menadione-treated cells, however, revealed only minor modifications mainly consisting of perinuclear condensation of cytokeratin structures. These findings suggest that cytoskeletal structures (actin microfilaments, microtubules, and intermediate-size filaments) are actually significant targets in quinone-induced oxidative stress.

Actin Cytoskeleton↗
PubMed1990

1,2-Dichloropropane (DCP) toxicity is correlated with DCP-induced glutathione (GSH) depletion and is modulated by factors affecting intracellular GSH.

Acute 1,2-dichloropropane (DCP) poisoning in humans is relatively frequent in Italy, where DCP is widely diffused as a constituent of commercial solvents and dry cleaners. In this study we have investigated the effects of DCP on intracellular glutathione (GSH) content in main target tissues of male Wistar rats, i.e. liver, kidney and blood, in order to establish if a correlation between DCP-induced GSH depletion and tissue damage exists. Administration of DCP (2 ml/kg body weight orally) caused a dramatic loss of tissue GSH occurring 24 h after DCP intoxication, followed by a slow restoration approaching physiological levels after 96 h. GSH depletion was associated with a marked increase in serum GOT, GPT, 5'-nucleotidase, gamma-glutamyl transpeptidase, alkaline phosphatase, urea and creatinine, and a significant degree of hemolysis. When animals were pretreated with a GSH depleting agent, buthionine-sulfoximine (BSO) (0.5 g/kg body weight) i.p. 4 h before DCP intoxication, an increase of overall mortality was found, significantly different from the group of animals treated with DCP alone. On the contrary, the administration of a GSH precursor, N-acetylcysteine (NAC) i.p. (250 mg/kg body weight) 2 and 16 h after DCCP intoxication prevented the dramatic loss of cellular GSH and reduced the extent of injury in target tissues, as demonstrated by laboratory indices. Furthermore, statistical analysis of the data revealed a correlation between: (1) depletion of liver GSH and increase in serum GOT, GPT, 5'-nucleotidase, (2) depletion of kidney GSH and increase in serum urea and creatinine and (3) depletion of blood GSH and the occurrence of hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗
PubMed1990

Stable angina pectoris and controlled ischemia: what causes the abnormalities in whole blood filterability?

The determinants of the altered whole blood filterability observed during coronary ischemia are still under discussion. Since no studies have been carried out to date on what exactly causes these alterations during the early stages of controlled ischemia in coronary heart disease, a model was set up using a bicycle ergometer test (with a 25 W increase every 2 minutes). Blood samples were taken from 48 stable angina pectoris patients and from a group of 28 matched controls before and immediately after exercise and 8 minutes later. Plasma viscosity, the filterability (through 5 microns diameter pore filters) of whole blood, erythrocytes, and polymorphonuclear and mononuclear leukocytes (separated by density gradient) were monitored. Alterations in whole blood filterability could be linked only to an impairment in polymorphonuclear cell filterability in those stable angina pectoris patients who reported chest pain and/or whose ST segment depression was greater than or equal to 2 mm.

Angina Pectoris↗
PubMed1990

Temporal arteritis presenting as an extrapyramidal disorder.

A case of temporal arteritis presenting with extrapyramidal symptoms (tremor, rigidity and extrapyramidal hypertonus) unresponsive to conventional treatment is here described. The onset of headache and laboratory abnormalities suggestive of temporal arteritis prompted a temporal artery biopsy which confirmed the diagnosis; the administration of corticosteroids led to the resolution of all symptoms.

Aged↗
PubMed1990

The cytoskeleton as a target in quinone toxicity.

The exposure of mammalian cells to toxic concentrations of redox cycling and alkylating quinones causes marked changes in cell surface structure known as plasma membrane blebbing. These alterations are associated with the redistribution of plasma membrane proteins and the disruption of the normal organization of the cytoskeletal microfilaments which appears to be due mainly to actin cross-linking and dissociation of alpha-actinin from the actin network. The major biochemical mechanisms responsible for these effects seem to involve the depletion of cytoskeletal protein sulfhydryl groups and the increase in cytosolic Ca2+ concentration following the alkylation/oxidation of free sulfhydryl groups in several Ca2+ transport systems. Depletion of intracellular ATP is also associated with quinone-induced plasma membrane blebbing. However, ATP depletion occurs well after the onset of the morphological changes, and thus it does not seem to be causatively related to their appearance. Thiol reductants, such as dithiothreitol, efficiently prevent the oxidation of cytoskeletal protein thiols, the increase in cytosolic free Ca2+ concentration and cell blebbing induced by redox cycling, but not alkylating, quinones. These results demonstrate that alkylating and redox cycling quinones cause similar structural and biochemical modifications of the cytoskeleton by means of different mechanisms, namely alkylation and oxidation of critical sulfhydryl groups.

Actinin↗
PubMed1989

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca2+ release in rat liver microsomal fractions.

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.

Animals↗
PubMed1989

Effects of carbon tetrachloride on calcium homeostasis. A critical reconsideration.

The incubation of isolated rat hepatocytes with 0.172 mM carbon tetrachloride caused a rapid decrease in the calcium content of both mitochondrial and extramitochondrial compartments. However, the release of Ca2+ from the intracellular stores was not associated with an increase in the cytosolic Ca2+ levels as measured by activation of phosphorylase alpha or by Quin-2 fluorescence. A rapid rise in hepatocyte free calcium was only observed with concentrations of CCl4 higher than 0.172 mM. The lack of activation of phosphorylase alpha was not due to the inhibition of the enzyme by CCl4, since in CCl4-treated hepatocytes the phosphorylase activity could be stimulated by glucagon, butyryl--cAMP or by the increase of cell calcium induced by the addition of A23187. Ca2+-dependent ATPase of plasma membranes was only slightly affected in the early phases of poisoning with CCl4 when both mitochondrial and extramitochondrial calcium pools were already lowered. This led to the conclusion that calcium released from intracellular organelles could be extruded from the cells in sufficient amounts to prevent the increase of the cytosolic levels. A rise in hepatocyte free calcium was observed during the second hour of incubation with CCl4, concomitantly with the appearance of both LDH leakage and plasma membrane blebbing. The addition of EGTA to the medium prevented both the increase in cytosolic Ca2+ and the blebbing suggesting that they were a consequence of an influx of calcium into the cells. However, neither EGTA nor the addition of inhibitors of calcium-dependent phospholipase A2 or non-lysosomal proteases were able to protect against cell death. These latter results suggested that the alterations of calcium distribution induced by CCl4 in isolated hepatocytes were not a primary cause of the toxic effects, although they did not exclude that a sustained rise in cytosolic Ca2+ could contribute in the progression of cell injury.

Animals↗
PubMed1989

On the role of mitochondria in cell injury caused by vanadate-induced Ca2+ overload.

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca2+ in the intracellular compartments. Vanadate- induced Ca2+ accumulation was related to inhibition of the plasma membrane Ca2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca2+ or the enhanced operation of a putative Na+/Ca2+ exchanger. After an initial rise in the cytosolic free Ca2+ concentration, as revealed by phosphorylase activation, Ca2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca2+ overload.

Adenosine Triphosphate↗
PubMed1989

Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity.

Exposure of cultured Chinese hamster ovary (CHO) cells to hydrogen peroxide results in the production of extensive DNA breakage which can be prevented by the intracellular calcium chelator Quin 2. This effect occurs at Quin 2 AM concentrations as low as 0.1 microM and is maximal at 1 microM. Addition of the extracellular calcium chelator, EGTA, does not affect the level of DNA breakage generated by H2O2. Quin 2 also significantly reduces cellular toxicity caused by the oxidant. Experiments with spin-trapping techniques demonstrate that Quin 2 does not affect the formation of hydroxyl radicals generated by the action of Fe2+ on H2O2. Quin 2 at high concentrations, similar to those reached within the cell, actually enhanced generation of hydroxyl radical in the absence of other iron chelators under our experimental conditions. These results suggest that H2O2 or H2O2-derived radicals do not directly induce DNA strand breakage in intact mammalian cells; rather, these radicals may disturb intracellular Ca2+ homeostasis which results in secondary reactions ultimately leading to DNA strand breakage. In addition to strand breakage, membrane and protein oxidation probably contribute to the cytotoxic effect of H2O2.

Aminoquinolines↗
PubMed1989

Cytoskeletal alterations in human platelets exposed to oxidative stress are mediated by oxidative and Ca2+-dependent mechanisms.

The metabolism of the redox-active quinone, menadione (2-methyl-1,4-naphthoquinone), in human platelets was associated with superoxide anion production, oxidation and depletion of intracellular glutathione, and modification of protein thiols. The cytoskeletal fraction extracted from menadione-treated platelets exhibited a dose-dependent increase in the amount of cytoskeleton-associated protein and a concomitant loss of protein thiols. These alterations were associated with oxidative modifications of actin, including beta-mercaptoethanol-sensitive crosslinking of actin to form dimers, trimers, and high-molecular-weight aggregates which also contained other cytoskeletal proteins, i.e., alpha-actinin and actin-binding protein. In addition, analysis of the cytoskeletal fraction from platelets treated with high concentrations (greater than or equal to 100 microM) of menadione by polyacrylamide gel electrophoresis under reducing conditions revealed a net decrease in the relative abundance of the individual cytoskeletal polypeptides. Under the same incubation conditions the platelets exhibited a sustained increase in cytosolic Ca2+ concentration. The presence of glucose, or the omission of Ca2+ from the incubation medium, prevented both the increase in cytosolic Ca2+ and the decrease in the relative amounts of cytoskeletal proteins. The latter effect was also largely prevented in platelets loaded with Quin-2 tetraacetoxymethyl ester to buffer the menadione-induced elevation of cytosolic Ca2+. Finally, the presence of a protease inhibitor, leupeptin, in the incubation medium prevented the menadione-induced decrease in the amount of actin-binding protein but not the decrease in the other cytoskeletal proteins. Our findings demonstrate that the multiple effects of oxidative stress on the platelet cytoskeleton are mediated by oxidative as well as by Ca2+-dependent mechanisms.

Actins↗
PubMed1989

Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca2+ concentration.

Glucocorticoid hormones kill immature thymocytes through the induction of a suicide process commonly referred to as "apoptosis." A characteristic marker for this process is the stimulation of endogenous endonuclease activity which results in the extensive cleavage of cell chromatin. In an attempt to characterize the biochemical events involved in this process, we studied the role of Ca2+ in glucocorticoid-induced DNA fragmentation and cell killing in thymocytes. Treatment of thymocytes from immature rats with the synthetic glucocorticoid methylprednisolone resulted in extensive DNA fragmentation which was preceded by an early, sustained increase in cytosolic Ca2+ concentration. This increase in Ca2+ level was blocked by cycloheximide and actinomycin D, inhibitors of de novo protein and mRNA synthesis, respectively. Prevention of the Ca2+ increase by buffering cytosolic Ca2+ with quin-2, or through incubation of the thymocytes in a "Ca2+-free" medium, prevented endonuclease activation and cell killing. Inhibitors of calmodulin also prevented DNA fragmentation without inhibiting the glucocorticoid-stimulated elevation of cytosolic Ca2+ concentration. The Ca2+ increase appeared to be due to the action of a heat-labile cytosolic factor, synthesized in response to glucocorticoids, which facilitated the influx of extracellular Ca2+. Our findings suggest that glucocorticoids induce thymocyte suicide through an elevation of cytosolic Ca2+ concentration resulting in endonuclease activation, DNA fragmentation, and cell death.

Animals↗
PubMed1989

Role of Ca2+ in toxic cell killing.

Recent work has shown that a sustained increase in cytosolic Ca2+ concentration is often linked to the onset of cytotoxicity. Sten Orrenius and colleagues describe several biochemical mechanisms that are stimulated by such a Ca2+ increase and can directly mediate cell death by causing disruption of the cytoskeleton, DNA fragmentation and extensive damage to other cell components.

Animals↗
PubMed1989

Leucocyte rheology in controlled coronary ischaemia.

Since no studies have been carried out on the exact origin of the alterations in white blood cell rheology during the early stages of controlled ischaemia in coronary arterial disease, a model was set up using a cycle ergometer test (with a 25 watts increase every 2 minutes). Blood samples were taken (before and after exercise and again 8 minutes later at recovery) from 18 patients with stable angina pectoris and a group of 22 matched controls. The filterability (through 5 micrometer diameter pore filters) of the polymorphonuclear leucocyte sub-population (separated by density gradient), the monocyte and lymphocyte sub-fractions (separated by adhesion to Petri dishes) as well as leucocyte activation (observed under a light microscope) were monitored. Our results showed that the total leucocyte count in patients and controls rose after exercise and was accompanied by a differential shift from the polymorphonuclear to the lymphocyte cells. The polymorphonuclear filterability rate increased significantly in patients when compared to their basal values at rest, and to the controls after exercise (+ 19.58%; P less than 0.002 vs basal values at rest; + 18.72%; P less than 0.002 vs controls). This increase persisted throughout the recovery period (+ 19.86%; P less than 0.002 vs basal values; and + 23.52% P less than 0.001 vs controls), indicating that a reduced polymorphonuclear leucocyte filterability can be associated with the first signs of ischaemia.

Aged↗
PubMed1989

Non-invasive cardiovascular monitoring in Shy-Drager syndrome.

The pattern of arterial blood pressure and heart rate were investigated in a patient with progressive autonomic dysfunction and nineteen healthy age-matched controls. A marked postprandial reduction of blood pressure was observed in the patient; this phenomenon was not noted in the control group; besides, the rhythmicity of blood pressure and heart rate were altered with peak values shifted approximately eight hours later with respect to the controls.

Adult↗
PubMed1988

Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress.

To investigate the relationship between alterations of cytosolic Ca2+ concentration and development of cytotoxicity, isolated rat hepatocytes were loaded with the fluorescent indicator Quin-2 AM and then incubated with non-toxic or toxic levels of menadione (2-methyl-1,4-naphthoquinone) or tert-butyl hydroperoxide (t-BH). The resulting changes in cytosolic Ca2+ concentration were compared to those seen upon exposure of the hepatocytes to an alpha 1-adrenergic agonist, phenylephrine, as well as to those induced by menadione and t-BH in hepatocytes pretreated with agents that modify their toxicity. Exposure of hepatocytes to phenylephrine or non-toxic levels of menadione caused a moderate and transient increase in cytosolic Ca2+ (less than or equal to 0.7 microM), whereas a toxic concentration of menadione produced a marked, sustained increase in Ca2+ which fully saturated the binding capacity of Quin-2 (greater than 1.5 microM). Treatment of the hepatocytes with the protective agent, dithiothreitol, prevented both the increase in cytosolic Ca2+ and the cytotoxicity induced by menadione. On the other hand, pretreatment of cells with diethylmaleate to deplete intracellular glutathione made otherwise non-toxic concentrations of menadione cause both a sustained increase in cytosolic Ca2+ and cytotoxicity. Similarly, toxic concentrations of t-BH also caused a sustained increase in cytosolic Ca2+. The iron chelator, desferrioxamine, and dithiothreitol (DTT), which protected the cells from t-BH toxicity, also prevented the sustained elevation of cytosolic Ca2+. Our findings provide further support for the hypothesis that a perturbation of intracellular Ca2+ homeostasis is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress.

Animals↗
PubMed1988

Alterations in hepatocyte cytoskeleton caused by redox cycling and alkylating quinones.

Quinones may induce toxicity by a number of mechanisms, including alkylation and oxidative stress following redox cycling. The metabolism of quinones by isolated rat hepatocytes is associated with cytoskeletal alterations, plasma membrane blebbing, and subsequent cytotoxicity. The different mechanisms underlying the effects of alkylating (p-benzoquinone), redox cycling (2,3-dimethoxy-1,4-naphthoquinone), and mixed redox cycling/alkylating (2-methyl-1,4-naphthoquinone) quinones on hepatocyte cytoskeleton have been investigated in detail in this study. Analysis of the cytoskeletal fraction extracted from quinone-treated cells revealed a concentration-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols, irrespective of the quinone employed. In the case of redox cycling quinones, these alterations were associated with an oxidation-dependent actin crosslinking (sensitive to the thiol reductant dithiothreitol). In contrast, with alkylating quinones an oxidation-independent cytoskeletal protein crosslinking (insensitive to thiol reductants) was observed. In addition to these changes, a dose-dependent increase in the relative abundance of F-actin was detected as a consequence of the metabolism of oxidizing quinones in hepatocytes. Addition of dithiothreitol solubilized a considerable amount of polypeptides from the cytoskeletal fraction isolated from hepatocytes exposed to redox cycling but not alkylating quinones. Our findings indicate that the hepatocyte cytoskeleton is an important target for the toxic effects of different quinones. However, the mechanisms underlying cytoskeletal damage differ depending on whether the quinone acts primarily by oxidative stress or alkylation.

Alkylation↗
PubMed1988

Alterations of surface morphology caused by the metabolism of menadione in mammalian cells are associated with the oxidation of critical sulfhydryl groups in cytoskeletal proteins.

Incubation of freshly-isolated (rat hepatocytes) or cultured (HeLa, GH3, and McCoy) mammalian cells with menadione (2-methyl-1,4-naphthoquinone) resulted in the appearance of numerous cell surface protrusions. The perturbation of surface structure was associated with an increase in the amount of cytoskeletal protein and the oxidation of sulfhydryl groups in actin, leading to the formation of high-molecular weight aggregates sensitive to treatment with thiol reductants. Our findings indicate that the oxidation of thiol groups in cytoskeletal proteins may be responsible for menadione-induced cell surface abnormalities in mammalian cells.

Animals↗