RESUMO
Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.
Assuntos
Apoproteínas/uso terapêutico , Cuprizona , Doenças Desmielinizantes/tratamento farmacológico , Recuperação de Função Fisiológica/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Transferrina/uso terapêutico , Análise de Variância , Animais , Animais Recém-Nascidos , Antígenos/metabolismo , Apoproteínas/farmacologia , Encéfalo/patologia , Bromodesoxiuridina/farmacocinética , Antígeno CD11b/metabolismo , Contagem de Células/métodos , Proteínas do Citoesqueleto/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/fisiopatologia , Interações Medicamentosas , Galactolipídeos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Indóis , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Regeneração/fisiologia , Fatores de Tempo , Transferrina/farmacologiaRESUMO
Growth-associated protein-43 (GAP-43) is a phosphoprotein whose expression in neurons is related to the initial establishment and remodeling of neural connections. GAP-43 gene expression is known to be regulated at both the transcriptional and the postranscriptional levels. However, very little is known about the cellular mechanism involved in the degradation of this protein. Ubiquitin (Ub) is well known for its role in targeting cytoplasmic proteins for degradation by the 26S proteasome. The ubiquitin-proteasome system (UPS) consists of a conserved cascade of three enzymatic components that attach Ub covalently to various substrates and control the degradation of protein involved in several important cellular processes. In this study, we investigated the degradation of GAP-43 in transfected NIH 3T3 cells and neuronal cultures. We found that the proteasome inhibitors, lactacystin and MG132 increased the cellular GAP-43 level, leading to the accumulation of polyubiquitinated forms of this protein in transfected cells and that the Ub-proteasome pathway is also involved in the turnover of this protein in neurons. We conclude based on our findings that GAP-43 is a substrate of the UPS.
Assuntos
Acetilcisteína/análogos & derivados , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Acetilcisteína/farmacologia , Animais , Western Blotting/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Proteína GAP-43/genética , Vetores Genéticos/fisiologia , Proteínas de Fluorescência Verde/biossíntese , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Leupeptinas/farmacologia , Masculino , Camundongos , Mutagênese/fisiologia , Células NIH 3T3 , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Gravidez , Ratos , Ratos Wistar , Transfecção/métodosRESUMO
2,4-Dichlorophenoxyacetic acid (2,4-D) and derivatives are herbicides widely used in Argentina and other parts of the world. Exposure to 2,4-D, its ester and salt formulations, have been associated with a range of adverse health effects in humans and different animal species, from embryotoxicity and teratogenicity to neurotoxicity. In this work, we demonstrate that after 24 hs of treatment with 1 and 2 mM 2,4-D there is an induction of apoptosis in cerebellar granule cells (CGC) in culture. However, with 2 mM 2,4-D one population of CGC developed features of apoptosis while another appeared to die by necrosis. This process is associated with an increase in caspase-3 activity after 12 hs of treatment with the herbicide, which is preceded by cytochrome c release from the mitochondria. Treatment of CGC with 2,4-D appears to induce apoptosis by a direct effect on mitochondria producing cytochrome c release and consequently activation of caspase-3, being mitochondrial damage sufficient for triggering the events that may cause apoptosis.
Assuntos
Ácido 2,4-Diclorofenoxiacético/farmacologia , Apoptose/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Herbicidas/farmacologia , Animais , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cerebelo/citologia , Cerebelo/enzimologia , Grupo dos Citocromos c/metabolismo , Grânulos Citoplasmáticos/enzimologia , Ativação Enzimática , Ratos , Ratos WistarRESUMO
We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.
Assuntos
Proteínas de Membrana/genética , Proteínas do Tecido Nervoso , Neurônios/citologia , Proteínas Repressoras , Transativadores/genética , Proteínas de Xenopus , Xenopus laevis/embriologia , Secretases da Proteína Precursora do Amiloide , Animais , Apoptose , Ácido Aspártico Endopeptidases , Diferenciação Celular , Divisão Celular , Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endopeptidases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Hibridização In Situ , Fatores de Transcrição Kruppel-Like , Proteínas de Membrana/fisiologia , Mutagênese Sítio-Dirigida , Oligonucleotídeos Antissenso , Presenilina-1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transativadores/fisiologia , Fatores de Transcrição/genética , Tretinoína/farmacologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Proteína Gli3 com Dedos de ZincoRESUMO
We have recently shown that sustained neonatal hyperthyroidism in the rat activates apoptosis of oligodendroglial cells (OLGc) and that inhibition of the proteasome-ubiquitin (Ub) pathway by lactacystin produces increased apoptosis in cerebellar granule cells (CGC). In the present study we have analyzed the relationship between the activation of the Ub-dependent pathway, the expression of the Ub genes and programmed cell death in neurons of the rat cerebellum and cerebral cortex and in OLGc. This study was carried out in normal animals, in rats submitted to sustained neonatal hyperthyroidism and in cell cultures treated with an excess of thyroid hormones. In neurons of the cerebral cortex, thyroid hormone produces an increase of Ub-protein conjugates, an enhancement in the expression of the Ub genes and an increase in apoptosis, while the opposite results are obtained in CGC. These results indicate that in neurons, the changes in the cell death program produced by thyroid hormone run in parallel with those occurring in the Ub-dependent pathway. In OLGc, thyroid hormone increases apoptosis but does not produce changes in the Ub pathway. Preliminary studies indicate that in coincidence with what occurs in optic nerves, the sciatic nerves both in controls and in hyperthyroid animals are unable to form Ub-protein conjugates. These results indicate that in cells of the CNS such as neurons, in which the Ub-dependent pathway is actively expressed, it appears to be closely correlated with apoptosis.
Assuntos
Apoptose/fisiologia , Encéfalo/fisiologia , Oligodendroglia/fisiologia , Nervo Isquiático/fisiologia , Tri-Iodotironina/farmacologia , Ubiquitinas/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Tronco Encefálico/citologia , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/fisiologia , Ciclo Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Feminino , Masculino , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Wistar , Nervo Isquiático/citologia , Nervo Isquiático/efeitos dos fármacos , Transcrição Gênica , Ubiquitinas/genéticaRESUMO
The multicatalytic protease complex or proteasome is a fundamental nonlysosomal tool that the cell uses to process or degrade proteins at a fast rate through the ubiquitin and ATP-dependent proteolytic pathway. Examples of these important proteins include the tumor suppressor protein p53, various cyclins, the cyclin-dependent kinase inhibitor p27, NFkappaB, IkappaB, c-fos, and c-jun. The activation of proteolytic enzymes, including certain cystein-proteases of the ced-3/ICE (interleukin-1beta-converting enzyme) family, is a characteristic feature of the apoptotic program. However, the role of the multicatalytic protease complex in apoptosis is not well known. In order to obtain further information regarding the participation of the ubiquitin-mediated pathway in the decision of the cell to execute the cell death program, we have used a specific inhibitor of the multicatalytic protease complex, lactacystin, in cultured cerebellar granule cells. Cells were obtained from the cerebellum of 6- to 8-day-old Wistar rats and cultured in Neurobasal medium supplemented with B-27. Addition of lactacystin to the cultures induced apoptosis of the granule cells in a time-dependent fashion. The morphological changes produced by the proteasome inhibitor included nuclear condensation and DNA fragmentation measured by the diphenylamine test, as well as a positive labeling by the TUNEL (terminal deoxynucleotidyltransferase mediated-dUTP nick end labeling) assay, all of them typical features of apoptosis. Concomitant with apoptosis, there were changes in the expression of the ubiquitin mRNA, a progressive depletion in the free ubiquitin pool, and an increase in the high molecular weight ubiquitin-protein conjugates. Caspase-3, a member of the ced-3/ICE family of cystein-proteases, showed a marked increase in activity in the lactacystin-treated cells. In flow cytometry studies, the amount of cells in the S phase of the cell cycle was smaller in the lactacystin-treated cells than in controls, suggesting that apoptosis could be due, in part, to an alteration of the cell cycle.
Assuntos
Acetilcisteína/análogos & derivados , Apoptose , Caspases/metabolismo , Cerebelo/fisiologia , Cisteína Endopeptidases/efeitos dos fármacos , Complexos Multienzimáticos/efeitos dos fármacos , Neurônios/fisiologia , Acetilcisteína/farmacologia , Animais , Caspase 3 , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/enzimologia , Cisteína Endopeptidases/fisiologia , Ativação Enzimática , Expressão Gênica/efeitos dos fármacos , Complexos Multienzimáticos/fisiologia , Neurônios/citologia , Neurônios/enzimologia , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos Wistar , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Ubiquitin (Ub) modification of different proteins plays an important role in many cellular processes. However, the best studied function of Ub is the labeling of proteins committed to rapid degradation, by an ATP-dependent pathway. We previously found that this pathway is operative in the central nervous system (CNS) of adult rats (Adamo et al. [1994] J. Neurosci. Res. 38:358-364). In the present study, we examined the changes in the capacity to form high-molecular-weight Ub protein conjugates (UbPC) and the changes in the production of 2-thiobarbituric acid-reactive substances (TBARS), in the content of protein-associated carbonyl groups (PAC), and in the activity of glutamine synthetase produced by in vitro peroxidation of the cell cytosolic proteins and of the mitochondrial fraction isolated from rat brain. Under these experimental conditions, there was an increase in PAC and TBARS in the cytosol, indicating that damage to certain cellular components had occurred. Simultaneously there was a marked increase in UbPC in comparison with the nonoxidized controls. These conjugates showed an active turnover and accumulated when Ub-mediated proteolysis was inhibited. In vitro peroxidation of the mitochondrial fractions resulted in an increase in the production of PAC and in an enhancement in the formation of UbPC. These results demonstrate that the oxidized proteins can be recognized by the ubiquitylating system and that in the CNS the Ub-dependent proteolytic pathway is one of the possible mechanisms involved in the removal of cytosolic and mitochondrial fraction damaged proteins.
Assuntos
Encéfalo/metabolismo , Estresse Oxidativo/fisiologia , Proteínas/metabolismo , Ubiquitinas/metabolismo , Animais , Cisteína Endopeptidases/metabolismo , Citosol/metabolismo , Feminino , Técnicas In Vitro , Masculino , Mitocôndrias/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredução , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos WistarRESUMO
We have carried out a study of the effects of sustained neonatal hyperthyroidism on myelin and on the oligodendroglial cells, in an effort to obtain further insight into the molecular mechanisms underlying the action of thyroid hormones on the central nervous system (CNS). Expression of the mRNAs of myelin basic protein (MBP) myelin proteolipid protein (PLP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), transferrin, and c-Jun was investigated in 10- and 17-day-old normal and hyperthyroid rats, using Northern blot analysis. At 10 days of age, the levels of all the explored mRNAs were markedly higher in the experimental animals. The mRNA of transferrin showed a ninefold increase over control values, suggesting the possibility that this putative trophic factor might act as one of the mediators in the action of thyroid hormones. At 17 days of age on the other hand, the levels of all the mRNAs decreased markedly, reaching values below control, except for c-Jun, which remained higher than in normals. At 70 days of age, hyperthyroid rats showed clear evidence of myelin deficit, in agreement with previous results of our laboratories (Pasquini et al.: J Neurochem 57: Suppl S124, 1991). Immunocytochemistry of 70-day-old rat brain tissue sections showed a substantial reduction in the amount of MBP-reacting structures and a marked decrease in the number of oligodendroglial cells. Although the above-mentioned results could be the consequence, as proposed by Barres et al. (Development 120:1097-1108, 1994) and Baas et al. (Glia 19:324-332, 1997) of a premature arrest in oligodendroglial cell proliferation followed by early differentiation, the persistent high levels of expression of c-Jun, together with the dramatic decrease in the number of oligodendrocytes, suggested the possibility that prolonged hyperthyroidism could activate apoptotic mechanisms in the myelin forming cells. Using propidium iodide-labeled isolated oligodendroglial cells, we found, by flow cytometry, a significant increase in the number of apoptotic/hypo-diploid propidium iodide-positive cells. These results indicate that one of the actions of sustained levels of thyroid hormones in the neonate rat is to increase oligodendroglial cell death by apoptosis.
Assuntos
Animais Recém-Nascidos/fisiologia , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/patologia , Hipertireoidismo/patologia , Bainha de Mielina/fisiologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Northern Blotting , Western Blotting , Sondas de DNA , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Masculino , Proteína Básica da Mielina/metabolismo , Ratos , Ratos Wistar , Tri-Iodotironina/sangueRESUMO
Using in situ hybridization techniques with an RNA probe coding for approximately 3.5 repeats of ubiquitin, corresponding to the polyubiquitin genes, we were able to demonstrate that under normal conditions the expression of the ubiquitin genes predominates specially in regions CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus and in Purkinje cells of the cerebellum, being less prominent in neuronal cell bodies of the cerebral cortex. When the animals were submitted to an acute oxidative stress by injection of Fe/Dextran, the hybridization signal was apparently increased in the above mentioned regions of the hippocampus and in the cerebral cortex. On the other hand, the animals chronically injected with Fe/Dextran showed a highly intense gene expression in the cerebral cortex and in the cerebellum, particularly in the granular cell layer of this structure. The hybridization signal of the transcripts was absent in the Purkinje cells. The results suggest that the expression of the ubiquitin genes by CNS neurons depends on the anatomical location of the cells and that it increases as a consequence of the oxidative stress conditions to which they are submitted.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Dextranos/farmacologia , Expressão Gênica , Ferro/farmacologia , Estresse Oxidativo , Ubiquitinas/genética , Animais , Córtex Cerebral/metabolismo , Giro Denteado/metabolismo , Feminino , Hipocampo/metabolismo , Hibridização In Situ , Masculino , Células de Purkinje/metabolismo , Sondas RNA , RNA Antissenso , RatosRESUMO
The capacity to form ubiquitin (Ub)-protein conjugates was investigated in the cytosol of different structures of the rat central nervous system (CNS) in order to confirm the presence of this extralysosomal, adenosine triphosphate (ATP)-dependent, protein degradation system as well as its structural localization. Using 125I-Ub, we found that in the presence of ATP, the cytosol obtained from whole brains was able to form high molecular weight Ub-protein conjugates. These conjugates could be detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. The formation of these conjugates was much higher in the cerebral cortex than in the brain stem, which is mainly constituted by white matter, being intermediate in the cytosol isolated from whole brain total homogenates. These results suggested to us that under normal conditions the capacity to form Ub-protein conjugates was mainly located in structures containing neuronal cell bodies. Strong support for this contention was obtained when the cytosol isolated from rat optic nerves or from oligodendroglial cells isolated from whole brain was found to be totally unable to form Ub-protein conjugates. The inability of certain CNS structures to form conjugates with Ub could be attributed, among other reasons, to the lack of enzymes catalyzing the various steps of the Ub degradation system, to the absence of short half-life (target) proteins in those structures, or to the lack of activity of the enzymes catalyzing the reaction due to regulatory control mechanisms operating under normal conditions.
Assuntos
Sistema Nervoso Central/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitinas/metabolismo , Animais , Autorradiografia , Sistema Nervoso Central/citologia , Radioisótopos do Iodo , Neurônios/metabolismo , Oligodendroglia/metabolismo , Nervo Óptico/metabolismo , Prostaglandinas E , Ratos , Frações Subcelulares/metabolismoRESUMO
Thyroid hormones have a significant influence on the development and maturation of the central nervous system. Among their actions, T3 and T4 have effects on the differentiation of various cell types in the rat brain and cerebellum as well as on the process of myelination. Recently, several investigators have shown effects of thyroid hormones on myelin protein gene expression. Thyroid hormones seem to have a regulatory role with regard to life span. Hyperthyroid animals appear to have a shorter life and, at advanced age, show a myelin deficit. This may be due to the damage produced by the oxidative stress generated by an excess of thyroid hormones.
Assuntos
Encéfalo/fisiologia , Tiroxina/fisiologia , Tri-Iodotironina/fisiologia , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Cerebelo/fisiologia , Expressão Gênica , Homeostase , Hipertireoidismo/fisiopatologia , Hipotireoidismo/fisiopatologia , Proteínas da Mielina/biossíntese , Bainha de Mielina/fisiologia , Estresse Oxidativo , Ratos , Tiroxina/farmacologia , Tri-Iodotironina/farmacologiaRESUMO
The effect of neonatal hyperthyroidism produced by injection of tri-iodothyronine (T3) on myelination and on the microperoxisomal population of the brain was studied in young rats. Data on the lipid composition of myelin show that myelinogenesis starts earlier in treated animals. In coincidence with the early appearance of myelin, there is an increase in the population of brain microperoxisomes, indicated by the increase in the activity of two enzymes that have been shown to be located in these organelles: catalase and acyl CoA-dihydroxyacetone phosphate acyl transferase. Double-label experiments using (1,2,3-3H) and (2-3H) glycerol to study the synthesis of glycerophospholipids through the dihydroxyacetone phosphate (DHAP) pathway give further support to the above-mentioned findings and suggest that there is an active participation of microperoxisomes in the synthesis of myelin lipids during the period of myelin formation.
Assuntos
Aciltransferases/metabolismo , Encéfalo/enzimologia , Catalase/metabolismo , Hipertireoidismo/enzimologia , Microcorpos/enzimologia , Bainha de Mielina/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/fisiopatologia , Feminino , Hipertireoidismo/fisiopatologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
Newborn Wistar rats were made hyperthyroid by injection of tri-iodothyronine and assayed for survival, brain oxygen uptake, brain chemiluminescence and activity of antioxidant enzymes. Brain chemiluminescence was measured (1) by removing the parietal bones or (2) through the translucid parietal bones. Control animals showed a brain chemiluminescence of 130 +/- 12 c.p.s./cm2 and 99 +/- 10 c.p.s./cm2 for procedures (1) and (2) respectively. Hyperthyroid rats showed increases in the spontaneous brain photoemission of 46 and 70% compared with controls, measured by procedures 1 and 2 respectively. The hyperthyroid state did not modify the oxygen-dependent chemiluminescence of brain homogenates. The hyperthyroid animals showed a 30% increase in the oxygen uptake of brain slices and a dramatic shortening of life-span to about 16 weeks. Superoxide dismutase (the Cu-Zn enzyme), catalase and Se-dependent glutathione peroxidase activities of brain homogenates were increased by 18, 36 and 30% respectively in the hyperthyroid animals. Isolated brain mitochondria produced 0.18-0.20 nmol of H2O2/min per mg of protein in state 4 in the presence of succinate as substrate. No difference was observed between control and hyperthyroid animals. It is concluded that hyperthyroidism leads to hypermetabolism and oxidative stress in the brain. The increased levels of oxygen and peroxyl radicals may contribute to premature ageing in these animals.
Assuntos
Encéfalo/metabolismo , Hipertireoidismo/metabolismo , Oxigênio/metabolismo , Animais , Encéfalo/enzimologia , Catalase/antagonistas & inibidores , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Peróxido de Hidrogênio/metabolismo , Hipertireoidismo/enzimologia , Medições Luminescentes , Mitocôndrias/metabolismo , NADP/metabolismo , Ratos , Ratos Endogâmicos , Superóxido Dismutase/metabolismoRESUMO
Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr.
Assuntos
Cisteína/análise , Cistina/análise , Tecido Nervoso/análise , Proteolipídeos , Animais , Bovinos , Dicroísmo Circular , Dissulfetos/análise , Ácido Ditionitrobenzoico , Formiatos , Iodoacetatos , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
Using catalase activity as a marker enzyme of microperoxisomes we determined the presence of these organelles in rat cerebral cortex (grey matter) and in brain stem (white matter) throughout development. While in grey matter the values of specific activity of catalase remained constant during all the period studied, in white matter the values increased up to 17 days of age, remained constant up to 31 days and decreased thereafter. Studies carried out in isolated oligodendroglial cells confirmed the results obtained in white matter. The results give support to the hypothesis of a possible relationship between the increase in the population of microperoxisomes and the appearance of myelin.