RESUMO
Retinal ganglion cell axons grow towards the optic fissure in close contact with the basal membrane, an excellent growth substratum. One of the ligands of receptor tyrosine phosphatase CRYPalpha is located on the retinal and tectal basal membranes. To analyze the role of this RPTP and its ligand in intraretinal growth and guidance of ganglion cell axons, we disrupted ligand- receptor interactions on the retinal basal membrane in culture. Antibodies against CRYPalpha strongly reduced retinal axon growth on the basal membrane, and induced a dramatic change in morphology of retinal growth cones, reducing the size of growth cone lamellipodia. A similar effect was observed by blocking the ligand with a CRYPalpha ectodomain fusion protein. These effects did not occur, or were much reduced, when axons were grown either on laminin-1, on matrigel or on basal membranes with glial endfeet removed. This indicates that a ligand for CRYPalpha is located on glial endfeet. These results show for the first time in vertebrates that the interaction of a receptor tyrosine phosphatase with its ligand is crucial not only for promotion of retinal axon growth but also for maintenance of retinal growth cone lamellipodia on basal membranes.
Assuntos
Proteínas Aviárias , Axônios/ultraestrutura , Proteínas Tirosina Fosfatases/fisiologia , Células Ganglionares da Retina/fisiologia , Células Ganglionares da Retina/ultraestrutura , Animais , Axônios/fisiologia , Moléculas de Adesão Celular/fisiologia , Comunicação Celular , Células Cultivadas , Laminina/fisiologia , Ligantes , Proteínas Tirosina Fosfatases Semelhantes a Receptores , Transdução de Sinais/fisiologiaRESUMO
We have identified the yeast gene STM1 in an overexpression screen for new proteasomal substrates. Stm1 is unstable in wild-type cells and stabilized in cells with defective proteasomal activity and thus a bona fide substrate of the proteasome. It is localized in the perinuclear region and is required for growth in the presence of mutagens. Overexpression in cells with impaired proteasomal degradation leads to cell death accompanied with cytological markers of apoptosis: loss of plasma membrane asymmetry, chromatin condensation, and DNA cleavage. Cells lacking Stm1 display deficiency in the apoptosis-like cell death process induced by treatment with low concentrations of H(2)O(2). We suggest that Stm1 is involved in the control of the apoptosis-like cell death in yeast. Survival is increased when Stm1 is completely missing from the cells or when inhibition of Stm1 synthesis permits proteasomal degradation to decrease its amount in the cell. Conversely, Stm1 accumulation induces cell death. In addition we identified five other genes whose overexpression in proteasomal mutants caused similar apoptotic phenotypes.
Assuntos
Cisteína Endopeptidases/genética , Proteínas Fúngicas/metabolismo , Complexos Multienzimáticos/genética , Fatores de Iniciação de Peptídeos , RNA Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Cafeína/farmacologia , Morte Celular , Cromatina/metabolismo , Cisteína Endopeptidases/metabolismo , Fatores de Iniciação em Eucariotos , Proteínas Fúngicas/genética , Biblioteca Gênica , Peróxido de Hidrogênio/farmacologia , Marcação In Situ das Extremidades Cortadas , Microscopia de Fluorescência , Complexos Multienzimáticos/metabolismo , Oxidantes/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Complexo de Endopeptidases do Proteassoma , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Raios UltravioletaRESUMO
Phosphopeptide and phosphoprotein phosphorylation was studied in rat brain microsomes and rat brain slices which were incubated in the presence of [gamma-32 P] ATP under various experimental conditions. Radioactive phosphoserine was isolated from phosphopeptides and phosphoproteins. Naplus, K+, Mg2+ and cyclic AMP had a stimulating effect on the labelling of phosphopeptides. Ouabain and Ca2+ lowered the level of 32P incorporation into the phosphopeptides. The phosphoproteins behaved similarly to the phosphopeptides except for the potassium effect. Chase experiments showed a faster decrease in the labelling of phosphopeptides than in phosphoproteins. We suggest that both compounds may be involved in active transport phenomena.
Assuntos
Trifosfato de Adenosina/metabolismo , Córtex Cerebral/metabolismo , Microssomos/metabolismo , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Animais , Transporte Biológico Ativo , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Compostos Organofosforados/metabolismo , Ouabaína/farmacologia , Fosforilação Oxidativa , Radioisótopos de Fósforo , Ratos , Serina/metabolismoRESUMO
ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was shown in the soluble fraction of rat liver micochondria. Two molecular forms (ATPase 1 and 2) were isolated. ATPase 1 has already been studied. The present paper deals with the purification method of ATPase 2 which was achieved by the following steps: (NH4)2SO4 precipitation. DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G100 filtration and AMP-Sepharose affinity chromatography. The purified protein was characterized by bidimensional polyacrylamide gel electrophoresis. Molecular weight evaluated by SDS-polyacrylamide gel electrophoresis and Sephadex G100 gel filtration was found to be 61 500 +/- 3000.
Assuntos
Adenosina Trifosfatases/isolamento & purificação , Mitocôndrias Hepáticas/enzimologia , Monofosfato de Adenosina , Animais , Cromatografia de Afinidade , Métodos , Peso Molecular , Ratos , SefaroseRESUMO
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.
Assuntos
Astrócitos/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Etanol/toxicidade , Feto/efeitos dos fármacos , Pirofosfatases , 5'-Nucleotidase/análise , Fosfatase Ácida/análise , Animais , Astrócitos/enzimologia , Astrócitos/ultraestrutura , Células Cultivadas/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Idade Gestacional , Glucose-6-Fosfatase/análise , Complexo de Golgi/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/análise , Gravidez , Ratos , Ratos Endogâmicos , Tiamina Pirofosfatase/análiseRESUMO
We determined the phosphopeptide (PP) level in several tissues and compared the values to the phosphoprotein and phosphatide level. We studied whole organs and subcellular fractions isolated from these organs. The results showed that the PP were preferentially localised in membranes. Among all the organs analyzed, the electrical organ of "Torpedo marmorata" had the highest PP level. The phosphoprotein distribution was different from the PP one; the highest level of phosphorproteins was found in nuclei. There was also an increase in phosphoprotein level comparing whole tissues and membranes isolated from the same tissue. The preferential localisation of PP in membranes is also suggested by the parallelism between PP and phosphatide level, which is also higher in membranes than in whole organs. The peculiar polyanionic structure, as well as the active metabolism and the membranous localisation of PP support the hypothesis that these compounds might be good intermediates in active transport mechanisms.
Assuntos
Membranas/análise , Fosfopeptídeos/análise , Anguilla , Animais , Transporte Biológico Ativo , Química Encefálica , Bovinos , Galinhas , Órgão Elétrico/análise , Eritrócitos/análise , Peixes , Humanos , Fígado/análise , Especificidade de Órgãos , Fosfatos/análise , Fosfolipídeos/análise , Fosfoproteínas/análise , Ratos , Retina/análise , Especificidade da Espécie , Frações Subcelulares/análiseRESUMO
We describe a method of extraction and partial purification of phosphopeptides isolated from pig brain or from the electrical organ of Torpedo marmorata. The extraction of the phosphopeptides was achieved by alcoholic 0,04 N potassium hydroxyde solution or by 10(-1) M KCL containing 10(-3) M EDTA and 10(-4) M DTT. After having tried various fractionation methods like ion exchange chromatography or gel filtration we chose chromatography on DEAE Sephadex followed by purification of the isolated fractions by Sephadex G 25 filtration. The most important phosphate fractions (one was purified to about 90 per cent) were characterized by the determination of the N/P ratio which was different from one phosphopeptide to another. The amino acid composition showed a high glycin, serine and "acid" amino acid content. The presence of phosphoserine was shown by electrophoresis and chromatography of a partial hydrolysate of in vivo 32P labelled phosphopeptides isolated from rat liver. The polyanionic structure of these phosphopeptides allow them to act as real ion exchangers which might be involved during active transport mechanisms in cellular membranes.
Assuntos
Química Encefálica , Órgão Elétrico/análise , Fosfopeptídeos/isolamento & purificação , Aminoácidos/análise , Animais , Peixes , Ratos , OvinosRESUMO
A method for isolation of a soluble ATPase from rat liver mitochondria after freeze thaw cycling is described. Two enzymatically active fractions were separated by DEAE-cellulose chromatography (ATPase 1 and ATPase 2). ATPase 1 has been purified 300 fold. ATPase 1 was homogenous as judged by polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8-6.0 and the optimum temperature was 45 degrees C. The enzyme follows Michaelis-Menten kinetics: Km (9 X 10(-4) M), Vmax (23,6 mumoles Pi released X min -1 X mg protein -1). The enzyme hydrolysed nucleoside triphosphates, but was inactive upon nucleoside di and monophosphates, glucose 6-phosphate, phosphoserine, pyrophosphate and glycerol 2-phosphate. In contrast to membrane bound ATPase, cations have no effect on the enzyme activity. Nucleoside di and mono phosphates and glycerol 2 phosphate inhibited competitively the enzyme. The enzyme was not affected by oligomycin, but was stimulated by lactate, 2-mercaptoethanol and dithiothreitol.
Assuntos
Adenosina Trifosfatases , Mitocôndrias Hepáticas/enzimologia , Monofosfato de Adenosina/farmacologia , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Animais , Ligação Competitiva , Cromatografia de Afinidade , Cromatografia em Gel , Ditiotreitol/farmacologia , Congelamento , Glicerofosfatos/farmacologia , Lactatos/farmacologia , Masculino , Mercaptoetanol/farmacologia , Ratos , Solubilidade , Frações Subcelulares/enzimologiaRESUMO
We described previously the existence of a soluble ATPase activity in rat liver mitochondria [1]. The purification and catalytic properties have been described [2]. In a continuation of these experiments, we have studied the immunologic and structural properties of one molecular form of this enzyme : ATPase I. We have prepared the antiserum anti-ATPase I and demonstrated the purity of our enzyme preparation by immunodiffusion and immunoelectrophoresis. An immunohistochemical method also confirmed the localization of ATPase I in the soluble fraction of mitochondria. The molecular weight of ATPase I was measured by G 100 Sephadex gel filtration and was found to be 18,400; electrophoresis on polyacrylamide gels gave a value of 18,600. The pHi of ATPase I was found to be 7,2. Amino acid analysis showed high amounts of aspartic acid, glutamic acid, serine and glycine. The molecular weight calculated from the total amino acid residues was found to be 17,000. Alanine is the NH2 terminal amino acid. The peptide maps obtained after degrading ATPase I with cyanogen bromide or trypsin are in accordance with the methionine, lysine and arginine residues we found in the ATPase I molecule. ATPase I does not appear to be a glycoprotein.
Assuntos
Adenosina Trifosfatases/análise , Mitocôndrias Hepáticas/enzimologia , Adenosina Trifosfatases/imunologia , Aminoácidos/análise , Animais , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Histocitoquímica , Imunodifusão , Imunoeletroforese , Ponto Isoelétrico , Peso Molecular , RatosRESUMO
The effect of paternal alcohol exposure on neurochemical and behavioral parameters was investigated using as a model system glial cells derived from newborn rat brain and cultured for 4 weeks. The total brain neurochemical parameters from rats born to mothers sired by an alcohol treated father were also investigated. Enzymatic markers of nerve cell development (enolase isoenzymes and glutamine synthetase) and the defense system (superoxide dismutase) against free radicals formed during alcohol degradation were measured in order to evaluate nerve cell damage. Behavioral locomotor tests (open-field, novelty-seeking, light/dark) were carried out to show long-lasting effects of paternal alcoholization on the offspring. Behavioral and developmental alterations were found until 1 year of age in the offspring and a significant growth retardation was observed in the males. Our results suggest that paternal alcohol exposure produces developmental and behavioral effects in the offspring. The consequence of either alcohol withdrawal during stage one spermatogenesis, or maternal diet supplementation with manganese during pregnancy were investigated. It was observed that some of the effects of paternal alcohol exposure on the offspring may be reversed by these treatments.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Neuroglia/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Encéfalo/enzimologia , Células Cultivadas , Pai , Feminino , Glutamato-Amônia Ligase/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Neuroglia/enzimologia , Fosfopiruvato Hidratase/metabolismo , Gravidez , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
The effect of various factors affecting non-neuronal enolase (NNE) and neuron-specific enolase (NSE) was investigated in developing brain of two different chick strains, in primary cultures of pure neurons and of mixed cultures of neuronal and glial cells. NNE and NSE activities reached their maximum at an earlier stage of brain development in the fast growing Hybro strain than in the Leghorn strain. In pure neurons cultured during 6 days, NNE was stimulated by hydrocortisone in presence or in absence of serum. Dibutyryl cyclic AMP (diBcAMP) stimulated NNE only in serum-free medium. NSE activity was increased by glial cell-conditioned medium in presence of serum and by removal of serum from the medium. Hydrocortisone and diBcAMP had no effect on NSE. In mixed cultures of neurons and glial cells both enolase activities were raised in absence of serum. Hydrocortisone and diBcAMP had no effect. Steroid hormones, insulin and serum albumin also modify both enolase activities in pure neurons and in mixed cultures of neurons and glial cells. Our results suggest that NNE and NSE are regulated separately by various factors involved in nerve cell maturation.
Assuntos
Encéfalo/enzimologia , Fosfopiruvato Hidratase/metabolismo , Animais , Química Encefálica , Bucladesina/farmacologia , Células Cultivadas , Embrião de Galinha , DNA/análise , Hidrocortisona/farmacologia , Soros Imunes/farmacologia , Proteínas do Tecido Nervoso/análise , Neuroglia/enzimologia , Neuroglia/fisiologiaRESUMO
Developmental changes of the neuron-specific enolase (NSE) and the non-neuronal enolase (NNE) were determined in pure neuronal, or mixed cultures of neuronal and glial cells, prepared from brains of 8-day-old chick embryos, as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain during the development of the nervous tissue in the embryo and during the post-hatching period. In the whole brain, the total enolase activity increases progressively until the adult level, the NSE appears only 4 days before hatching. The total enolase activity of pure neurons increases until 8 days of culture but the NSE never appears during this period. Mixed cultures of neurons and glial cells present an increase of total activity until 14 days of culture and neuron-specific form of the enzyme can be observed after 6 days of culture. Our results suggest that NSE appears only in the functional brain and is expressed in cultivated neurons only when glial cells are present simultaneously.
Assuntos
Diferenciação Celular , Córtex Cerebral/citologia , Fosfopiruvato Hidratase/metabolismo , Animais , Embrião de Galinha , Técnicas de Cultura , Neuroglia/citologia , Neurônios/citologiaRESUMO
Superoxide dismutase in nervous system protects readily oxidizable compounds such as catecholamines against toxic effects of oxygen. We investigated superoxide dismutase activity during development in 5 brain regions selected for a wide range of catecholamine concentration and turnover: cerebellum, neocortex, striatum, hypothalamus and medulla-pons. The cytosolic and the particulate enzyme were measured from birth to 6 months of age. In cerebellum the cytosolic enzyme shows considerable activity on the first postnatal days; the particulate enzyme is less active, both reach a maximum at 3 months. In cortex and striatum both activities were low during the postnatal days and reach a plateau at 3 months. In hypothalamus both activities are higher during the postnatal days and reach a maximum at 3 months. In medulla-pons the values are 2 times higher than in all other regions; the cytosolic enzyme reaches a maximum at 2 months whereas the particulate enzyme reaches a plateau at 3 months. Thus our results show an increase of superoxide dismutase activity during development in all brain regions; the highest activities were found in regions with high catecholamine content.
Assuntos
Encéfalo/crescimento & desenvolvimento , Superóxido Dismutase/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Encéfalo/enzimologia , Citosol/enzimologia , Cinética , Masculino , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
Glutaminase activity was determined in pure cultures of neurons, glial cells and in mixed cultures obtained from chick embryo brain. The development of this enzyme was observed periodically over time and compared to its evolution in corresponding cerebral hemispheres during embryonic and postnatal development. The specific activity of brain glutaminase increased between the twelfth and sixteenth day of embryogenesis. A similar increase was observed in cultures of neuroblasts during the corresponding period of time, although the activity in culture was about one-third lower than in vivo. In contrast to neurons, there was no significant increase of glutaminase activity in glial cells before the fifteenth day of culture. The enzyme level in glial cells between the thirteenth and fifteenth days of culture was approximately 25% of that in 7- and 8-day-old neurons. The different development of glutaminase activity in neurons and glial cells was demonstrated in both pure and mixed cultures. The results support the hypothesis that there is a glutamine shunt from glial cells to neurons.
Assuntos
Córtex Cerebral/embriologia , Glutaminase/metabolismo , Neuroglia/enzimologia , Neurônios/enzimologia , Animais , Células Cultivadas , Córtex Cerebral/enzimologia , Embrião de Galinha , HistocitoquímicaRESUMO
Primary cultures of astroglial cells and of neurons obtained from chick embryos were grown in culture medium with and without serum added. The expression of glutamine synthetase (GS) in the cultured nerve cells was investigated immunocytochemically and biochemically. The cellular localization of GS in cerebellar tissue sections and in cerebral cortex of chick embryos was investigated by immunohistochemical staining. In tissue sections the enzyme is only present in astrocytes and their processes; neurons and their structures do not express the enzyme. In contrast, in pure neuronal primary cultures, a high level of GS was detected by biochemical and immunochemical methods. Thus, our results clearly indicate the presence of GS in pure neuronal cell cultures and its absence in this type of cells in vivo. Removal of serum from the culture medium enhanced GS levels in primary astrocyte cultures, but was without effect on GS activity in neurons. Addition of calf serum to the culture medium induces a two-fold increase of cellular lactate dehydrogenase (LDH) activity in neurons by increasing specifically the M subunit containing isoenzymes. The sensitivity of chick astroglial cells and neurons toward the GS inducing effect of hydrocortisone and modulation of its effect by serum was also investigated. Differences in the sensitivity of the two types of nerve cells in culture toward the GS inducing effect of hydrocortisone, and the effect of serum could be demonstrated.
Assuntos
Astrócitos/metabolismo , Sangue , Metabolismo Energético , Glutamato-Amônia Ligase/metabolismo , Hidrocortisona/farmacologia , Neurônios/metabolismo , Animais , Astrócitos/enzimologia , Células Cultivadas , Embrião de Galinha , Glutamato Desidrogenase/metabolismo , Histocitoquímica , Imunoquímica , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Neurônios/enzimologiaRESUMO
The effect of acetaldehyde on astrocytes have been investigated because not only do they play an important role in brain maturation but also recent reports have shown their delayed proliferation following both 'in vivo' and 'in vitro' ethanol exposure. Biochemical parameters related to apoptotic and necrotic processes were examined in primary cultures of rat astrocytes exposed for 4 days to acetaldehyde generated from ethanol by co-cultured alcohol dehydrogenase-transfected Chinese hamster ovary cells. Acetaldehyde levels in the culture media attained concentrations of approximately 450 microM. To study ethanol effects, alcohol oxidation was inhibited by 4-methylpyrazole (an inhibitor of alcohol dehydrogenase). Acetaldehyde but not ethanol increased intracellular calcium levels by 155%. Moreover, significant DNA fragmentation was detected using a random oligonucleotide primed synthesis assay, by flow cytometry and when using agar gel electrophoresis. Transglutaminase activity was elevated in the cells treated with acetaldehyde but when acetaldehyde formation was inhibited by 4-methylpyrazole the enzyme activity was unaffected. Nitrate levels in the culture media were unchanged. Additionally, microscopic examination of cell nuclei revealed chromatin condensation in astrocytes exposed to acetaldehyde. It can be concluded, that in 'in vitro' acetaldehyde exposed rat astrocytes apoptotic pathways are activated.
Assuntos
Acetaldeído/toxicidade , Apoptose/efeitos dos fármacos , Astrócitos/citologia , Depsipeptídeos , Peptídeos , Álcool Desidrogenase/genética , Animais , Animais Recém-Nascidos , Antibacterianos/farmacologia , Astrócitos/química , Astrócitos/enzimologia , Células CHO , Cálcio/análise , Técnicas de Cultura de Células/métodos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Depressores do Sistema Nervoso Central/farmacologia , Cromatografia Líquida de Alta Pressão , Cricetinae , Meios de Cultura/química , Fragmentação do DNA , Etanol/farmacologia , Citometria de Fluxo , Expressão Gênica , Nitritos/análise , Nitritos/metabolismo , Ratos , Transfecção , Transglutaminases/metabolismoRESUMO
We report the presence and distribution of alpha (ubiquitous) and gamma (neuron-specific) subunits of the dimeric glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase) in cultured neural cells. The gamma gamma enolase is found in vivo at high levels only in neurons and neuroendocrine cells. Neuronal cells in culture also contain relatively high levels of alpha gamma and gamma gamma enolase. Here we show, by enzymatic and immunological techniques, that the gamma subunit also is expressed in cultured rat astrocytes and meningeal fibroblasts and, as we previously reported, in oligodendrocytes. Both neuron-specific isoforms alpha gamma and gamma gamma are expressed in all these cells, but the alpha alpha isoform accounts for the major part of total enolase activity. The sum of alpha gamma and gamma gamma enolase activities increases with time in culture. i.e. maturation processes, reaching the highest level in oligodendrocytes (40% of total enolase activity) and 15 and 10% of total enzymatic activity in astrocytes and fibroblasts, respectively. The gamma enolase transcripts were found not only in cultured neuronal cells but also in cultured oligodendrocytes astrocytes, and meningeal fibroblasts. Our data indicate that neuron-specific enolase should be used with caution as a specific marker for neuronal cell differentiation.
Assuntos
Astrócitos/enzimologia , Isoenzimas/metabolismo , Meninges/enzimologia , Neurônios/enzimologia , Oligodendroglia/enzimologia , Fosfopiruvato Hidratase/metabolismo , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Fibroblastos/enzimologia , Imuno-Histoquímica , Meninges/citologia , Fosfopiruvato Hidratase/genética , Ratos , Distribuição TecidualRESUMO
Rats were exposed to mercury vapors (30 mg/m3) for either 1 or 2 h. Histological lesions like alveolar oedema, hyaline membranes and sometimes fibrosis were observed. The lesions were more significant after 2 h of exposure, with about 50% of the animals dying within 2 weeks. The mercury level and the superoxide dismutase activity in the blood and the lungs demonstrated differences according to the time of exposure. In the animals exposed for 2 h to mercury vapors, N-acetylcysteine treatment increased survival time and the percentage of living animals. The lung superoxide dismutase was lower than in the non-treated animals indicating an antioxidant effect. Mercury levels were decreased in blood and lung, suggesting some chelating effect of N-acetylcysteine. The exact mechanism of its action must be further elucidated.
Assuntos
Acetilcisteína/uso terapêutico , Pulmão/enzimologia , Mercúrio/toxicidade , Superóxido Dismutase/sangue , Administração por Inalação , Animais , Pulmão/efeitos dos fármacos , Masculino , Mercúrio/antagonistas & inibidores , Mercúrio/sangue , Ratos , Ratos Endogâmicos , Superóxido Dismutase/metabolismoRESUMO
The effect of ethanol on membrane enzymes (Na+, K+ and Mg2+ ATPases, 5'-nucleotidase, adenylate cyclase) alcohol dehydrogenase, aldehyde dehydrogenase and superoxide dismutase were studied in nerve cells (established cell lines, primary cultures of chick and rat brain) cultured in the presence of 100 mM ethanol, and in total rat brain, following various ethanol treatments of the rats (20% ethanol as the sole liquid source, intraperitoneal injection). The results show a difference between neuronal and glial cells. Most of the observed changes in enzymatic activities returned rapidly to control values when ethanol was withdrawn from the culture medium or from the diet. Alcohol dehydrogenase was more stimulated by ethanol than aldehyde dehydrogenase; therefore acetaldehyde may be accumulated. The inhibition of superoxide dismutase activity may allow an accumulation of cytotoxic O2- radicals in nervous tissue and may explain the polymorphism of lesions brought about by alcohol intoxication.
Assuntos
Etanol/farmacologia , Neurônios/metabolismo , Adenosina Trifosfatases , Adenilil Ciclases/metabolismo , Oxirredutases do Álcool/metabolismo , Aldeído Oxirredutases/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Camundongos , Nucleotidases/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
The effect of maternal alcohol exposure before mating was investigated in the offspring over a period of 6 months concerning some specific aspects of energy metabolism in the brain and the liver. The following biochemical parameters were analyzed: superoxide dismutase (involved in elimination of free radicals produced during ethanol oxidation), enolase isoenzymes (markers of nerve cell maturation), and alcohol and aldehyde dehydrogenase (the main alcohol degradating enzymes). These enzymatic activities were measured at their subcellular level. In these animals never directly exposed to alcohol, superoxide dismutase activity was decreased mainly in the liver cytosol. Only the nonneuronal form of enolase activity was modified. Alcohol dehydrogenase was decreased in the liver as well as in the brain. Aldehyde dehydrogenase was also decreased in the liver and in the brain, mainly in the mitochondria. Behavioral observations showed decreased emotional reactivity as well as an increase in locomotor activity. Our results suggest that long-lasting biochemical and behavioral effects of alcohol may occur in the offspring starting at the earliest stage of development.