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C Guerri

Publications and source records attributed to C Guerri.

At least 55 records · Page 3Linked to original sources
PubMed1993

Analysis of the endocytic-lysosomal system (vacuolar apparatus) in astrocytes during proliferation and differentiation in primary culture.

The endocytic-lysosomal system of proliferating and differentiated astrocytes in primary culture was investigated using a combination of cytochemical, immunocytochemical and biochemical procedures. These included impregnation with osmium tetroxide and potassium iodide, phosphotungstic acid staining, cytochemical demonstration of acid phosphatase and thiamine pyrophosphatase activities and incorporation of cationized ferritin. The acid phosphatase activity was also analyzed using biochemical techniques. Our results indicate that while all astrocytes in primary culture have a developed endocytic-lysosomal system, this system is different in proliferating cells from that in differentiated astrocytes. Whereas in proliferating astrocytes it appears to be composed mainly of a variety of vacuoles and vesicles displaying a heterogeneous osmium tetroxide staining pattern, differentiated cells are characterized by the presence of small size vesicles showing an intense reaction. Both types of astrocyte showed abundant lysosomes, including multivesicular bodies, which presented an intense phosphatase acid activity. Biochemical analyses demonstrated that this activity increase during the proliferation period, reaching a maximum at 15 days of culture. Incorporation of cationized ferritin revealed that lysosomes and endosomes constitute separate systems. Finally, we have also found that the activity of thiamine pyrophosphatase, a marker for the Golgi complex, increases throughout the culture period. These results indicate that astrocytes could play an important role in regulating the macromolecular composition of the extracellular space.

Acid Phosphatase↗
PubMed1993

Derangement of astrogliogenesis as a possible mechanism involved in alcohol-induced alterations of central nervous system development.

We have postulated that the brain alterations observed after "in utero" alcohol exposure could be due, in part, to a direct effect of ethanol on astrogliogenesis. In the present work we summarize the main findings concerning the effect of prenatal exposure to alcohol on astrocytes in primary culture. We have found that this treatment alters astrocyte growth and the content and synthesis of DNA and proteins. However, "in utero" exposure to ethanol also causes important alterations in the astrocyte cytoskeleton which could be related to an alteration in the amount and activity of several plasma membrane glycoproteins. These results agree with ones obtained "in vivo" and suggest that ethanol-induced alterations in astrogliogenesis could be an important mechanism underlying the CNS disfunctions observed after prenatal exposure to ethanol.

Animals↗
PubMed1992

Alcohol dehydrogenase isoenzymes in rat development. Effect of maternal ethanol consumption.

The alcohol dehydrogenase (ADH) isoenzymes (alcohol:NAD oxidoreductase, EC 1.1.1.1) of classes I, III and IV were investigated by activity and starch gel electrophoresis analyses during rat ontogeny. Class I was studied in the liver, class III in the brain and class IV in the stomach and eyes. Classes I and IV exhibited very low activity during the fetal period, reaching 12% and 3%, respectively, of the adult value at birth. Class III was relatively more active in the fetus, with 38% of the adult activity at birth. In the three cases, activity increased after birth and adult values were found around day 20 (classes I and III), day 39 (stomach class IV) and after day 91 (eye class IV). The very low activity of the isoenzymes responsible for ethanol oxidation, i.e. liver class I and stomach class IV, in the fetus demonstrates that metabolism of ethanol during gestation is essentially performed by the maternal tissues. Development of ADH isoenzymes were also studied in the offspring of rats exposed to an alcoholic liquid diet. Activities of liver class I and stomach class IV were severely reduced: they were only 30% and 50%, respectively, of the control values. In contrast, eye class IV activity did not change and brain class III showed a 30% increase. Moreover, the concentration of liver soluble protein exhibited a 1.3-1.5-fold increase with respect to control animals. The effects on activities and liver protein were more pronounced in the adult than in the perinatal period, and they seem irreversible since normal values were not recovered after 6 weeks of feeding with a non-alcoholic diet. The low activities of the alcohol-oxidizing isoenzymes indicate tht maternal ethanol consumption results in an impaired ethanol metabolism of the offspring.

Alcohol Dehydrogenase↗
PubMed1992

Prenatal exposure to ethanol alters plasma membrane glycoproteins of astrocytes during development in primary culture as revealed by concanavalin A binding and 5'-nucleotidase activity.

We have investigated the effect of prenatal exposure to ethanol on the extent of binding and surface distribution of the lectin concanavalin A (con A) on rat cortical astrocytes during the periods of proliferation and differentiation in primary culture. The enzymatic activity of the plasma membrane glycoprotein 5'-nucleotidase was also assessed. The cells were obtained from control fetuses (no exposure to ethanol) and from fetuses prenatally exposed to ethanol. The main findings were: 1) both proliferating and differentiating control astrocytes showed two distinct types of surface con A receptors that could correspond to high- and low-affinity binding sites; 2) the extent of con A binding was greater in mature than in proliferating control cells; 3) the distribution of con A on cell surface components changed with differentiation; 4) the activity of 5'-nucleotidase showed a substantial increment during the period of differentiation; and 5) prenatal exposure to ethanol clearly decreased the ability of astrocytes to bind con A, altered the surface distribution of the receptors for this lectin, and decreased the activity of 5'-nucleotidase. These effects were more marked in proliferating cells. In conclusion, it is shown that the extent of con A labeling and the activity of 5'-nucleotidase in astrocytes are dependent on the stage of cell differentiation and that prenatal exposure to ethanol alters the plasma membrane structure of these cells during development.

5'-Nucleotidase↗
PubMed1992

Chronic ethanol consumption induces accumulation of proteins in the liver Golgi apparatus and decreases galactosyltransferase activity.

The effect of chronic ethanol consumption on labeled glycoprotein secretion and galactosyltransferase activity has been analyzed in cis- and trans-Golgi apparatus fractions isolated from rat liver. Rats were injected intraperitoneally with 3H-leucine, after different chase periods (30, 60, 180 min), and the radioactivity of the different subcellular fractions as well as of the isolated Golgi apparatus was measured. Chronic alcohol treatment induces an increase in liver weight as well as an enhancement of total liver protein. Ethanol treatment produces a significant accumulation of labeled proteins in isolated Golgi apparatus fractions after a 60- and 180-min chase. An accumulation of labeled proteins in the cytosolic fraction was observed only after 180 min. The alcohol treatment also induces a significant decrease in the activity of galactosyltransferase in both liver homogenate and Golgi apparatus fractions. These results suggest that an impairment of Golgi apparatus functions, including glycosylation and glycoprotein trafficking, could be one of the mechanisms involved in the accumulation of hepatic protein and thus in the pathogenesis of alcohol-induced injury in the liver of chronic ethanol-consuming animals.

Alcoholism↗
PubMed1992

Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus.

Alcohol dehydrogenase (ADH), the major enzyme catalyzing the biological oxidation of ethanol in mammals, includes four classes with very different capacities for ethanol oxidation. Class III ADH is present in all the tissues and is well conserved throughout evolution. This enzyme has a low activity with ethanol, is specific for the glutathione-dependent oxidation of formaldehyde, and is therefore a formaldehyde dehydrogenase (FALDH). Until now there have been few and conflicting studies concerning its intracellular distribution, which is important for the understanding of its role in cell function. In the present work we used biochemical and immunocytochemical methods to assess the distribution of FALDH in rat hepatocytes and astroglial cells. With the glutathione-dependent formaldehyde dehydrogenase assay, we found the highest activity in the cytosol of hepatocytes and brain cells (12 and 2.6 mU/mg protein, respectively), but nuclei also exhibited significant activity (1.16 and 2.1 mU/mg protein, respectively). The immunocytochemical results showed the presence of FALDH binding sites in both the cytoplasm and the nucleus of the different cell types studied. Whereas no specific gold particle labeling was seen associated with any cytoplasmic component, in the nucleus the particles were found mainly over condensed chromatin and interchromatin regions. Finally, the gold particle density over both the nucleus and cytoplasm was greater in differentiated than in proliferating astrocytes in primary culture. In contrast, class I ADH, primarily responsible for ethanol metabolism, was found only in the cytoplasm of hepatocytes. We propose that one of the functions of FALDH is to protect cell structures, including DNA, from the toxic effects of endogenous formaldehyde, which is an intermediate in many metabolic process.

Aldehyde Oxidoreductases↗
PubMed1992

Cytochemical and stereological analysis of rat cortical astrocytes during development in primary culture. Effect of prenatal exposure to ethanol.

This study has investigated the effect of prenatal alcohol exposure on the qualitative and quantitative ultrastructure of proliferating and differentiated astrocytes in primary cultures as well as on the cytochemical activity of several subcellular phosphatase markers, including acid phosphatase, uridine diphosphatase, thiamine pyrophosphatase, 5'-nucleotidase and glucose-6-phosphatase. The astrocytes were obtained from 21-day-fetuses of both control and alcohol-fed rats. Our results show that several cell components, such as mitochondria, rough endoplasmic reticulum and lysosomes, exhibit qualitative and/or quantitative ultrastructural changes during the process of astrocyte maturation. In some cases these morphological changes are accompanied by variations in the cytochemical activity of enzymes located in these and other cell components, suggesting that these enzymes, and therefore the functional state of these organelles, are modulated during astrocyte development. When prenatally exposed to ethanol, both proliferating and differentiated astrocytes showed striking ultrastructural alterations compared with controls, including an increment of lysosomes as well as a decrease in the values of stereological parameters relative to mitochondria, rough endoplasmic reticulum and Golgi apparatus. Cytochemical analysis of these cells indicates that prenatal exposure to ethanol decreased the activities of all the enzymes tested, except for acid phosphatase, which was increased in both groups of treated astrocytes. These results suggest that prenatal exposure to ethanol could affect astrocytes during development in two different but probably complementary ways: a) by causing a delay in astrocyte maturation and, b) by inducing a direct toxic effect on these cells.

5'-Nucleotidase↗
PubMed1991

Evolution of several cytoskeletal proteins of astrocytes in primary culture: effect of prenatal alcohol exposure.

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein alpha-tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of alpha-tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti alpha- and anti-beta tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin. In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed "in vivo".

Aging↗
PubMed1990

Ethanol alters astrocyte development: a study of critical periods using primary cultures.

Using astrocytes obtained from 21-day-old rat fetuses, in primary culture, we have analyzed the effect of prenatal alcohol consumption on DNA and protein synthesis of astrocytes during their development. The variation in sensitivity of astrocytes to ethanol "in vitro" during the proliferation and maturation periods was also assessed. Control astrocytes showed peaks of DNA and protein synthesis at 8 and 15 days, respectively. A significant decrease in both DNA and protein synthesis was found in astrocytes from fetuses prenatally exposed to ethanol. This effect on DNA synthesis was also observed when control astrocytes were exposed to ethanol (100 mM) "in vitro" during the entire culture period. The effects on astrocytes of short term (48h) exposure to ethanol during the proliferation or differentiation periods on the above mentioned parameters and on the cell cycle as well as the possible recovery from these effects were also evaluated. Decreases in DNA and protein synthesis were found in both periods. However, DNA synthesis and content were more affected in astrocytes exposed to ethanol during the proliferation period. This effect correlates with an accumulation of cells in the G0/G1 phase of the cell cycle. On the other hand, when cells exposed to ethanol were cultured in alcohol-free medium to assess recovery, only cells exposed to ethanol during days 4 to 6 still showed DNA ethanol-induced effects at 21 days. In conclusion, our results show that ethanol consumption during gestation induces serious damage to cortical astrocyte progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The role of liquid diet formulation in the postnatal ethanol exposure of rats via mother's milk.

The nutritional adequacy of three liquid diets containing ethanol to support lactation was studied in rats. Diets 1 and 2 provided 18 and 25% kcal, respectively, as protein with 36% of total calories as ethanol, while in diet 3 alcohol provided 28% and protein 25% of total calories. Three series of isoenergetically pair-fed rats, as well as an ad libitum group fed a solid diet, were studied. A primary maternal malnutrition was evident in rats fed diet 1. With respect to diet 2, the 96% postnatal mortality which occurred may have been due to an inhibition of milk production mediated by exaggerated blood alcohol concentrations present in the lactating dams. Diet 3 seemed to be nutritionally adequate for the extra requirements for lactation and a direct effect of ethanol was observed in the sucklings. High blood alcohol levels (25-50 mmol/1) were obtained in dams fed ethanol diets 1 and 3; however only 1% of maternal blood alcohol appeared in the blood of sucklings, demonstrating a low transfer of ethanol from mother to offspring through the milk. Finally the model for postnatal exposure to alcohol via mother's milk is discussed in terms of other indirect alcohol-related factors which make it difficult to evaluate the direct impact of ethanol per se in the normal development of the suckling pups.

Animals↗
PubMed1989

Prenatal exposure to ethanol alters the synthesis and glycosylation of proteins in fetal hepatocytes.

In the present work we have studied the effect of prenatal exposure to alcohol on the synthesis, glycosylation, and transport of proteins in fetal hepatocytes isolated from 21-day-old fetuses derived from control and chronic alcoholic rats. Protein synthesis was evaluated both in a cell-free system and in hepatocytes after (35S)methionine and (3H)leucine incorporation, respectively. Glycosylation was assessed using (3H)mannose and (3H)galactose as precursors. Protein synthesis was significantly decreased in treated hepatocytes. In control hepatocytes, quantitative electron microscope autoradiography showed that both (3H)leucine and (3H)mannose incorporation occur first in the rough endoplasmic reticulum (rER). Later the silver grains appeared over the Golgi apparatus, and, finally there was a transport towards the cell periphery. After pulse, silver grains corresponding to (3H)galactose incorporation appeared over the Golgi apparatus. The label then moved to the hepatocyte periphery. Alcohol treated hepatocytes showed a retention of grains over the Golgi apparatus with a diminution in the label at the cell periphery. These results indicate that prenatal exposure to alcohol induces a decrease in the synthesis of proteins in the hepatocyte as well as an alteration in the process of glycosylation and/or transport of secretory proteins.

Animals↗
PubMed1989

Effects of prolonged ethanol exposure on the glial fibrillary acidic protein-containing intermediate filaments of astrocytes in primary culture: a quantitative immunofluorescence and immunogold electron microscopic study.

We investigated the effects of ethanol exposure on the shape of the cell and the morphology of intermediate filaments (IF) of cortical astrocytes in primary culture. The content and distribution of glial fibrillary acidic protein (GFAP), the major component of glial IF, was assessed using an anti-GFAP monoclonal antibody and fluorescence scanning densitometry together with quantitative pre- and post-embedding immunogold electron microscopy. The astrocytes were from 21-day-old fetuses obtained from both control and chronic alcoholic rats and were cultured for 28 days in the absence or presence of ethanol (25 mM). The main findings were: (a) ethanol-exposed astrocytes failed to develop processes or to acquire a filamentous IF distribution pattern; (b) these cells showed less GFAP than astrocytes without alcohol; (c) ethanol interfered with the reorganization of the anti-GFAP binding sites from clustered to random; and (d) astrocytes from alcohol-exposed fetuses cultured in the absence of ethanol also showed these alterations, suggesting initial damage to astrocyte precursor cells. Since the glial filaments play a crucial role in creating a scaffolding that guides neuronal migration, the effect of ethanol on astrocyte IF may possibly be correlated with the mechanisms underlying mental retardation and motor dysfunction which are characteristics of fetal alcohol syndrome.

Animals↗
PubMed1989

Effect of prenatal exposure to alcohol on membrane-bound enzymes during astrocyte development in vivo and in primary culture.

In the present work we have analyzed the effect of prenatal ethanol exposure on the activity of several glial marker and functional enzymes during the development of astrocytes isolated from rat brain as well as in primary culture. The activity of marker enzymes glutamine synthetase and butylcholinesterase showed no differences between isolated astrocytes from 15 and 70 day old control rats. However, the activity of the membrane-bound enzymes (Na+K)ATPase and 5'-nucleotidase was higher in astrocytes from 70 day old control rats than in those from 15 day old animals. Although the pattern found in astrocytes from alcohol-exposed rats was similar to that of controls, the levels of activity of the enzymes were lower in alcoholic than in control animals. When control astrocytes in primary culture were used, the activity of (Na+K)ATPase and 5'-nucleotidase increased throughout the entire culture period. In contrast, the maximal activity of glutamine synthetase was found at 7 days of culture. Ethanol also induced a decrease in the activity of all enzymes, which was more evident at the end of the culture period. These results indicate that the activity of the enzyme markers analyzed increased mainly during the first weeks of life and remained constant after this period. By contrast, the membrane-bound enzymes studied showed a progressive increase with age. In conclusion, since these astrocyte enzymes are important in the regulation of several neuronal functions through the control of the composition of extracellular fluid, the effect of ethanol on their activities could explain some of the neuronal alterations reported in children and animals exposed to ethanol during development.

Animals↗
PubMed1988

Effects of ethanol on rat brain (Na + K)ATPase from native and delipidized synaptic membranes.

The role of lipids in the effect of ethanol on synaptosomal (Na + K)ATPase was studied using native and partially delipidized synaptosomal membranes from control and alcoholic rats. A biphasic effect of alcohol was observed with the (N + K)ATPase from control membranes. Ethanol at low concentrations (less than 100 mM) appears to enhance the enzyme activity, but at higher concentrations (greater than 300 mM) was inhibitory. The biphasic response to ethanol was also observed with the (Na + K)ATPase isolated from alcoholic animals; however, in this case the enzyme showed a resistance to the inhibitory effect of ethanol. Delipidization of synaptic membranes with Lubrol WX or phospholipase A practically abolishes the effects of alcohol on (Na + K)ATPase from both control and alcoholic animals. It thus seems that the effects of ethanol are due mainly to their interaction with the lipids surrounding the enzyme. Furthermore, addition of ethanol to native membranes did not change the Vmax and Km for K+. However, when ethanol at the same concentration was added to delipidized membranes, a decrease in Km with no change in Vmax was observed. Ethanol under these conditions apparently interacts also with the enzyme protein. On the other hand, chronic ethanol intake produces an increase of both Vmax and Km for K+. However, when alcohol was added in vitro, there were no changes in the kinetic parameters of either native or delipidized membranes. These data indicate that although the effects of ethanol on synaptosomal (Na + K)ATPase are mainly due to its interaction with the lipid microenvironment of the enzyme, a direct ethanol action on the enzyme protein also occurs. Our data further suggest that chronic ethanol treatment alters enzyme sensitivity to the effect of ethanol which may be related to the membrane-lipid composition and/or to changes in the conformation of the enzyme protein.

Animals↗
PubMed1988

Thyroid hormone levels in rats exposed to alcohol during development.

Maternal ingestion of alcohol appears to cause a pattern of congenital anomalies with a reduction of pre- and postnatal growth in the offspring. In order to study the possible implication of thyroid function in the effects of pre- and/or postnatal exposure to alcohol, we have studied serum thyroxine (T4) and triiodothyronine (T3) levels in rats from alcohol-fed mothers during the postnatal period (0-50 days). Blood alcohol levels of ethanol-treated pregnant rats were approximately equal to 20-25 mM and their serum T4 levels were decreased, compared with the pair-fed controls, at 15 and 21 days of gestation. No significant changes were observed in T3 levels. Prenatal alcohol exposure was associated with a decrease in both T4 and T3 levels in pups at birth. Although T4 levels continued reduced in the 40-50 days of the postnatal period, no clear effects were observed on T3 levels during this time. Moreover, the more marked alterations were obtained when the offspring were postnatally and pre + postnatally exposed to alcohol. Significant decreases were found in both T4 and T3 levels following postnatal exposure, except at the 20-25th day when a marked but transient increase in T4 levels was observed. These results indicate that alcohol exposure disturbs the hypothalamo-pituitary-thyroid axis, as measured by T3 and T4 hormone levels, mainly when the rats are exposed during the postnatal period.

Aging↗
PubMed1988

Effect of short and long-term ethanol feeding on the extent of metabolic tolerance in female rats.

The effects of feeding an ethanol liquid diet for short (4-6 weeks) and long (48-50 weeks) periods on the extent of metabolic tolerance to the drug were studied in female rats. Appropriate pair-fed and solid-diet controls were also included. At the end of both periods of treatment, the rats (4 or 14-month-old), received an acute dose of ethanol (2 g/kg body wt) and pharmacokinetic parameters of ethanol elimination were studied using Widmark's lineal model. Although there were differences in the patterns of ethanol kinetics for solid and liquid diet controls, the rate of ethanol metabolism was clearly enhanced by both short and long-term ethanol intake. Moreover, longer alcohol intake produced higher metabolic tolerance (congruent to 37.5%) than a shorter ethanol feeding period (congruent to 21.5%). The effect of aging on ethanol clearance in the groups fed the solid-diet showed that the rate of ethanol metabolism decreased with increasing age.

Age Factors↗