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Biomedical subjects

U M Marinari

Publications and source records attributed to U M Marinari.

At least 19 recordsLinked to original sources
PubMed2001

Induction of heme oxygenase 1 in liver of spontaneously diabetic rats.

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified beta-actin mRNA. The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.

Animals↗
PubMed2000

Glutathione depletion induces apoptosis of rat hepatocytes through activation of protein kinase C novel isoforms and dependent increase in AP-1 nuclear binding.

Treatment of isolated rat hepatocytes with the glutathione depleting agents L-buthionine-S,R-sulfoximine or diethylmaleate reproduced various cellular conditions of glutathione depletion, from moderate to severe, similar to those occurring in a wide spectrum of human liver diseases. To evaluate molecular changes and possible cellular dysfunction and damage consequent to a pathophysiologic level of GSH depletion, the effects of this condition on protein kinase C (PKC) isoforms were investigated, since these are involved in the intracellular specific regulatory processes and are potentially sensitive to redox changes. Moreover, a moderate perturbation of cellular redox state was found to activate novel PKC isoforms, and a clear relationship was shown between novel kinase activation and nuclear binding of the redox-sensitive transcription factor, activator protein-1 (AP-1). Apoptotic death of a significant number of cells, confirmed in terms of internucleosomal DNA fragmentation was a possible effect of these molecular reactions, and was triggered by a condition of glutathione depletion usually detected in human liver diseases. Finally, the inhibition of novel PKC enzymatic activity in cells co-treated with rottlerin, a selective novel kinase inhibitor, prevented glutathione-dependent novel PKC up-regulation, markedly moderated AP-1 activation, and protected cells against apoptotic death. Taken together, these findings indicate the existence of an apoptotic pathway dependent on glutathione depletion, which occurs through the up-regulation of novel PKCs and AP-1.

Adenosine Triphosphate↗
PubMed2000

Oxidative stress induces increase in intracellular amyloid beta-protein production and selective activation of betaI and betaII PKCs in NT2 cells.

Amyloid beta-protein (Abeta) aggregation produces an oxidative stress in neuronal cells that, in turn, may induce an amyloidogenic shift of neuronal metabolism. To investigate this hypothesis, we analyzed intra- and extracellular Abeta content in NT2 differentiated cells incubated with 4-hydroxy-2,3-nonenal (HNE), a major product of lipid peroxidation. In parallel, we evaluated protein kinase C (PKC) isoenzymes activity, a signaling system suspected to modulate amyloid precursor protein (APP) processing. Low HNE concentrations (0.1-1 microM) induced a 2-6 fold increase of intracellular Abeta production that was concomitant with selective activation of betaI and betaII PKC isoforms, without affecting either cell viability or APP full-length expression. Selective activation of the same PKC isoforms was observed following NT2 differentiation. Our findings suggest that PKC beta isoenzymes are part of cellular mechanisms that regulate production of the intracellular Abeta pool. Moreover, they indicate that lipid peroxidation fosters intracellular Abeta accumulation, creating a vicious neurodegenerative loop.

Amyloid beta-Peptides↗
PubMed1999

Regulation of rat hepatocyte protein kinase C beta isoenzymes by the lipid peroxidation product 4-hydroxy-2,3-nonenal: A signaling pathway to modulate vesicular transport of glycoproteins.

A major aldehydic end product of the peroxidation of arachidonic acid, 4-hydroxy-2,3-nonenal (HNE), has recently been considered for its potential involvement in a variety of cell functions. Here we report on the differential regulation of rat hepatocyte protein kinase C (PKC) isoforms by concentrations of HNE actually detectable in specific biological fluids or tissues. PKC betaI and, to a much greater extent, PKC betaII activities were markedly increased by 0.1 micromol/L HNE (final concentration in cell medium) whereas they were unaffected or even inhibited by 1 to 10 micromol/L HNE. On the contrary, the calcium independent PKC delta activity was inhibited by 0.1 micromol/L and increased by 1 and 10 micromol/L. Further, we show here that HNE-induced stimulation of PKC betaI and betaII activities, both in cytosolic and in membrane fractions, is paralleled by a marked stimulation of the anterograde transport of a lysosomal enzyme within the central vacuolar system. In fact, the treatment with 0.1 micromol/L HNE accelerated the PKC-dependent transport of lysosomal procathepsin D from the trans-Golgi network to the endosomal-lysosomal compartment and, in addition, increased the exocytosis of mature cathepsin D (CD) from these compartments. On the other hand, hepatocyte cotreatment with a selective inhibitor of classic PKCs prevented the aldehyde-induced activation of CD transport. These results support the possible involvement of HNE in the PKC-dependent regulation of the traffic of secretory glycoproteins, and point to remarkable implications of this aldehyde in the pathophysiology of various exocytic processes including hepatocyte lipoprotein secretion.

Aldehydes↗
PubMed1999

Levels of carbonyl groups in plasma proteins of type 2 diabetes mellitus subjects.

Carbonyl groups result from protein oxidation and their level in tissues and plasma is a relatively stable marker of oxidative damage. Carbonyl content of plasma proteins in 43 type 2 diabetic subjects, 30-87 years of age (25 males and 18 females) and in 20 age-matched healthy controls (31-89 years of age, 12 males and 8 females) was evaluated with 2,4-dinitro-phenyl-hydrazine method. In both groups, lipids, tocopherols (HPLC) and glycated hemoglobin (HPLC) were studied. Fasting blood glucose, glycated hemoglobin and lipids were significantly higher in the diabetic group; carbonyl content and alpha-tocopherol were slightly, but not significantly higher in the diabetic group (1.06 +/- 0.03 vs. 0.97 +/- 0.04 nmol/mg protein, 27. 07 +/- 2.82 vs. 31.55 +/- 2.11 micromol/l, respectively). Significant relationships between age and lipids, alpha-tocopherol and proteins were found in controls, but not in diabetics. Alpha-tocopherol correlated with lipids in both groups; glycated hemoglobin, a marker of glycemic control, was related to lipids, alpha-tocopherol and protein carbonyl groups in diabetics, while only the correlation with carbonyls was found in controls. These results suggest that impaired glycemic control is connected to protein oxidation. Glycation cascade also releases free radicals, becoming responsible for further oxidative attacks. In conclusion, increased oxidative stress, if any, in the diabetic group, is doubtlessly induced by hyperglycemia, and the tocopherols are not seriously affected by a worsening of the metabolic control.

Adult↗
PubMed1999

Lipoperoxidation in hepatic subcellular compartments of diabetic rats.

It is known that an accumulation of lipoperoxidative aldehydes malondialdehyde (MDA) and 4-hydroxynonenal (HNE) takes place in liver mitochondria during aging. The existence and role of an increased extra- and intra-cellular oxidative stress in diabetes, an aging-accelerating disease, is currently under discussion. This report offers evidence that lipoperoxidative aldehydes accumulate in liver microsomes and mitochondria at a higher rate in spontaneously diabetic BB/WOR rats than in control non-diabetic animals (HNE content, diabetes vs. control: microsomes 80.6+/-19.9 vs. 25.75+/-3.6 pmol/mg prot, p = .024; mitochondria 77.4+/-15.4 vs. 26.5+/-3.5 pmol/mg prot, p = .0103). Liver subcellular fractions from diabetic rats, when exposed to the peroxidative stimulus ADP/Fe, developed more lipoperoxidative aldehydes than those from non diabetic rats (HNE amount, diabetes vs. control: microsomes 3.60+/-0.37 vs. 2.33+/-0.22 nmol/mg prot, p = .014; mitochondria 3.62+/-0.26 vs. 2.30+/-0.17 nmol/mg prot, p = .0009). Liver subcellular fractions of diabetic rats developed more fluorescent chromolipids related to HNE-phospholipid adducts, either after in vitro peroxidation (microsomes: p = .0045; mitochondria: p = .0023) or by exposure to exogenous HNE (microsomes: p = .049; mitochondria: p = .0338). This higher susceptibility of diabetic liver membranes to the non-enzymatic attack of HNE may be due to an altered phospholipid composition. Moreover, a decreased activity of the HNE-metabolizing systems can be involved: diabetic liver mitochondria and microsomes were unable to consume exogenous HNE at the same rate as non-diabetic membranes; the difference was already significant after 5' incubation (microsomes p<.001; mitochondria p<.001). These data show an increased oxidative stress inside the hepatocytes of diabetic rats; the impairment of the HNE-metabolizing systems can play a key role in the maintenance and propagation of the damage.

Aldehydes↗
PubMed1999

Dietary nucleotide supplementation raises erythrocyte 2, 3-diphosphoglycerate concentration in neonatal rats.

The present study was designed to test if dietary intake of nucleotides increases erythrocyte 2,3-diphosphoglycerate (2,3-DPG) in neonatal rats. To this end, rat pups were fed a nucleotide-supplemented formula (S, n = 14) from d 9 until d 16 after birth. The results were compared with those obtained from a group of breast-fed pups (C, n = 14) and a group of pups artificially fed with nucleotide-free formula (NS, n = 14). Neonatal weight, 2,3-DPG concentration, hematocrit (Hct) and hemoglobin concentration (Hb) were determined before the experiment (d 9) and after 7 d of treatment (d 16). In all groups, 2,3-DPG concentration was greater at d 16 than d 9, and the increase was greater in the S group than in the NS group. Alterations in neonatal weight, Hct and Hb concentration did not differ among the groups. On d 16 the 2, 3-DPG/Hb ratio, reflecting the affinity of hemoglobin for oxygen, was significantly higher in the C and S groups than in the NS group. We conclude that in neonatal rats, dietary nucleotides increase erythrocyte 2,3-DPG concentration. Studies need to be conducted in humans to assess the effect of this increase on both neonatal peripheral hemodynamics and metabolism in this species.

2,3-Diphosphoglycerate↗
PubMed1998

Ethanol-induced effects on expression level, activity, and distribution of protein kinase C isoforms in rat liver Golgi apparatus.

Acute ethanol administration induces significant modifications both in secretive and formative membranes of rat liver Golgi apparatus. The decrease in glycolipoprotein secretion and their retention into the hepatocyte contribute to the pathogenesis of alcohol-induced fatty liver. Molecular and cellular mechanisms behind the ethanol-induced injury of the liver secretory pathway are not yet completely defined. In this study on intact livers from ethanol-treated rats, the involvement of the Golgi compartment in the impairment of hepatic glycolipoprotein secretion has been correlated with changes in the expression level, subcellular distribution and enzymatic activity of protein kinase C (PKC) isoforms. Acute ethanol exposure determined a translocation of classic PKCs and delta isoform from the cytosol to cis and trans Golgi membranes, the site of glycolipoprotein retention in the hepatic cell. A marked stimulation of cytosolic epsilon PKC activity was observed throughout the period of treatment. The presence of activated PKC isozymes at the Golgi compartment of alcohol-treated rat livers may play a role in hepatic secretion and protein accumulation. Direct and indirect effects of ethanol consumption on PKC isozymes and Golgi function are discussed.

Animals↗
PubMed1998

Immunological evidence for increased oxidative stress in diabetic rats.

The role of oxidative stress in aging and diabetes mellitus is currently under discussion. We previously showed age-dependent accumulations of fluorescent protein adducts with lipoperoxidative aldehydes, (malondialdehyde (MDA), and hydroxynonenal (HNE)) in rat skin collagen with diabetic BB rats exhibiting faster accumulation. Modified proteins have been shown to be immunogenic: antibody titres against rat serum albumin modified by MDA and HNE (MDA-RSA and HNE-RSA) or oxidized by reactive oxygen species were measured by ELISA as markers of oxidative damage in BB diabetic and non-diabetic rats. Each tested antibody titre was significantly higher in the diabetic than in the non-diabetic rats. A significant correlation existed between anti-MDA-RSA and anti-HNE-RSA antibody titers. Only the anti-HNE-RSA antibody titre increased significantly with age (p=0.052) in diabetic animals, while no titres increased significantly in non-diabetic animals. A major factor which correlated with the development of these antibodies was diabetes duration: this was significant (p=0.032) for anti-HNE-RSA antibody titre and slightly significant (p=0.05) for anti-MDA-RSA antibody titre. Thus, chronic hyperglycaemia is probably fundamental in the increase of oxidative stress. There is correlation between anti-aldehyde-RSA antibody titres and the corresponding aldehyde-related collagen-linked fluorescence: modified collagen may play a part in the observed immune response. Our data indicate a stronger immune response of diabetic rats against proteins modified by lipoperoxidative aldehydes and oxygen free radicals, and they support the hypothesis of increased oxidative damage in diabetes.

Aging↗
PubMed1998

Mechanisms of inactivation of hepatocyte protein kinase C isoforms following acute ethanol treatment.

Acute ethanol exposure of rat isolated hepatocytes leads to a significant decrease (-30%) in cytosolic enzymatic activity of classic protein kinase C (PKC) isoforms, while immunoreactive protein level measured by Western Blot remains unaffected. The inactivation of classic cytosolic isoforms appears dependent on the modification of the enzyme function, probably due to ethanol metabolism. In fact, pretreatment with 4-methylpyrazole (4MP), an inhibitor of alcohol dehydrogenase, fully prevented such damage. After ethanol treatment, a decrease of about 40% in both enzymatic activity and immunoreactive protein level of novel PKC isoforms was evident both in the soluble and particulate fractions. Even if 4MP cell pre-treatment afforded protection in this case too, the inhibitory action of ethanol on novel PKC hepatocyte isoforms involves a proteolytic mechanism as shown by Western Blot analysis. The reproduction of PKC inactivation by ethanol in hepatocyte lysate excluded a role of peroxisomal hydrogen peroxide in the pathogenesis of the damage investigated. This damage was not reduced by addition of catalase to the lysate model system.

Animals↗
PubMed1998

Basal synthesis of heat shock protein 70 increases with age in rat kidneys.

The heat shock protein (HSP) system is a mechanism of cell defense induced by stress, constitutively expressed during basal conditions and essential to the maintenance of cellular integrity. Acutely induced HSP synthesis decreases with aging, but the effect of age on the basal expression of HSP70 has not been specifically addressed so far. The aim of this work is to study the age-dependent basal concentrations of HSP70 mRNA in rat kidneys. In 8 young (2-3 months), 6 adult (6-11 months) and 6 old male Wistar rats (22-27 months), steady-state concentrations of HSP70 and gamma-actin mRNA and of rRNA were measured. Pentosidine was measured by HPLC. The basal, unstimulated HSP70 mRNA is increased in young and old rats compared with adult subjects [young: 182% of adult levels (100-299), old: 167% of adults (142-209); p < 0.005]. The amount of pentosidine increases with age (young: 0.6 +/- 0.1; adult: 1.65 +/- 0.15; old: 2.3 +/- 0.3 pmol/mg of protein; p < 0.0001). It seems likely that different mechanisms are responsible for the increased HSP70 basal synthesis in both the young and old animals. The prevalence of anabolic activity can trigger the increased basal production of HSP70 in young rats. The accumulation of posttranslational modified proteins, documented by pentosidine, can chronically enhance HSP70 synthesis in aged animals. The suppression of the synthesis of other proteins accompanying HSP-selective production might contribute to the impairment of specific cell functions in aging.

Actins↗
PubMed1998

Effect of ethanol administration on the level of dolichol in rat liver microsomes and Golgi apparatus.

Data obtained in our laboratory had suggested that acute ethanol administration (6 g/kg body weight) selectively and rapidly affects the intracellular system of protein glycosylation at the level of the Golgi apparatus. Dolichols are important membrane components, and dolichyl phosphate is a glycosyl sugar carrier for N-glycosylation of proteins in endoplasmic reticulum and is considered rate-limiting for this process. In this study, modifications in the concentration and distribution of liver microsomal dolichols after acute ethanol administration were investigated. Between 3 and 24 hr after ethanol administration, the microsomal dolichyl phosphate concentration was significantly lower than in control animals. The highest reduction was observed at 12 hr (-52%). An earlier and more marked reduction of total dolichol was observed in the Golgi apparatus, and, in particular, in the secretory fraction F1 (-70% at 6 hr). Ethanol treatment of isolated hepatocytes led to a significant reduction of the de novo synthesis of both dolichyl phosphate and free dolichol. Moreover, in vitro experiments have demonstrated that pro-oxidant agents lead to a significant decrease of both free dolichol and dolichyl phosphate. Our results suggest that acute ethanol administration induces a marked decrease of dolichols, probably by increasing the degradation and impairing the biosynthetic pathway of these molecules.

Animals↗
PubMed1997

Mutual interaction between glycation and oxidation during non-enzymatic protein modification.

Aging pathogenesis involves non-enzymatic modifications of proteins; protein oxidation, glycation and their interactions have aroused a particular interest. Possible interrelations between oxidation and glycation have been evaluated in vitro: bovine serum albumin was oxidized by gamma-irradiation and then exposed to in vitro glycation. Fluorescence modifications induced by radiolytic oxidation and glycation were similar and tended to be additive. Both non-enzymatic processes provoked a loss of free sulfhydryl groups and a strong increment of protein carbonyl content: this supports that glycation can act through oxidative mechanisms. The observed rearrangement of amino groups after irradiation could predispose proteins to glycation attacks. Protein peroxides generated during irradiation appear able to give birth to further protein modifications leading to the generation of carbonyl groups and to interact with monosaccharides, probably stimulating their autoxidation and in turn glycative protein damage. Glycation increases the oxidation-mediated structural damage revealed by SDS-PAGE. Therefore our data support the hypothesis of mutual enhancement between oxidation and glycation of proteins and suggest possible molecular mechanisms of interactions.

Analysis of Variance↗
PubMed1997

Different efficacy of iodoacetic acid and N-ethylmaleimide in high-performance liquid chromatographic measurement of liver glutathione.

The widely used high-performance liquid chromatography (HPLC) procedure to determine glutathione in biological samples utilizing iodoacetic acid as thiol quenching agent and 1-fluoro-2,4-dinitrobenzene for derivatization has been modified regarding tissue sample processing and storage of the working solutions. The modified procedure compared with the original method reduces artifactual oxidation in rat liver glutathione measurement (1.47+/-0.8% vs. 2.84+/-0.69%, respectively). In both HPLC procedures, an increase in artifactual oxidation was found in both standard glutathione solutions and hepatic samples when N-ethylmaleimide instead of iodoacetic acid was used for thiol trapping.

Animals↗
PubMed1997

Effects of acute glutathione depletion induced by L-buthionine-(S,R)-sulfoximine on rat liver glucose-6-phosphatase activity.

The effect of acute glutathione (GSH) depletion induced by GSH-depleting agent L-buthionine-(S,R)-sulfoximine (BSO) on hepatic microsomal glucose-6-phosphatase (G6Pase) activity in male Wistar rats was investigated. Liver GSH evaluated in high-performance liquid chromatography after administration of 4 mmol.Kg-1 BSO i.p. was decreased by 19% and 50% at the time-points of 1.5 h and 3 h, respectively. In these conditions, a significant decrease in Vmax and an increasing trend in K(m) of hepatic G6Pase activity were observed, especially in 3 h BSO-rats. Alterations in kinetic parameters of G6Pase were calculated in both intact and detergent-treated microsomes, using glucose-6-phosphate and pyrophosphate as substrate. A little increase in thiobarbituric acid-reactive substances and a limited decrease in 5,5'-dithiobis(2-nitrobenzoate)-reactive protein thiols were also noted. The results of this study show that acute GSH depletion induced by BSO is able to affect hepatic microsomal G6Pase activity. A possible explanation to account for the effect of BSO-induced GSH depletion on hepatic G6Pase system is discussed.

Animals↗
PubMed1996

Effects of ethanol metabolism on PKC activity in isolated rat hepatocytes.

Isolated rat hepatocytes were exposed to increasing concentrations of ethanol. During exposure of cells to ethanol a moderate but significant modification in the level of hepatic PKC c-isoforms has been observed. The ethanol-induced effect on liver protein kinase C was reversed by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, indicating that the conversion of ethanol to acetaldehyde may be involved in the enzyme inactivation. The involvement of the alcohol metabolite in PKC modifications was confirmed by the exposure of hepatocytes or partially purified liver enzyme to acetaldehyde concentrations of pathological interest.

Acetaldehyde↗