RESUMO
The present study describes, for the first time, a temporal and spatial cellular expression of erythropoietin (Epo) and Epo receptor (Epo-R) with the evolution of a cerebral infarct after focal permanent ischemia in mice. In addition to a basal expression of Epo in neurons and astrocytes, a postischemic Epo expression has been localized specifically to endothelial cells (1 day), microglia/macrophage-like cells (3 days), and reactive astrocytes (7 days after occlusion). Under these conditions, the Epo-R expression always precedes that of Epo for each cell type. These results support the hypothesis that there is a continuous formation of Epo, with its corresponding receptor, during the active evolution of a focal cerebral infarct and that the Epo/Epo-R system might be implicated in the processes of neuroprotection and restructuring (such as angiogenesis and gliosis) after ischemia. To support this hypothesis, a significant reduction in infarct volume (47%; P < 0.0002) was found in mice treated with recombinant Epo 24 hours before induction of cerebral ischemia. Based on the above, we propose that the Epo/Epo-R system is an endogenous mechanism that protects the brain against damages consequent to a reduction in blood flow, a mechanism that can be amplified by the intracerebroventricular application of exogenous recombinant Epo.
Assuntos
Isquemia Encefálica/metabolismo , Eritropoetina/biossíntese , Eritropoetina/farmacologia , Receptores da Eritropoetina/biossíntese , Animais , Astrócitos/efeitos dos fármacos , Western Blotting , Encéfalo/citologia , Química Encefálica/fisiologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Infarto Cerebral/tratamento farmacológico , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Eritropoetina/administração & dosagem , Imuno-Histoquímica , Hibridização In Situ , Injeções Intraventriculares , Camundongos , Neurônios/efeitos dos fármacosRESUMO
There has been an increasing interest in recent years in the evaluation of the neuronal and glial responses to ischemic insult. Some cytokines, including transforming growth factor-beta (TGF-beta), that are overexpressed after experimental stroke in rodents are thought to be implicated in the neuronal processes that lead to necrosis. Thus, such cytokines could predict tissue fate after stroke in humans, although data are currently sparse for gyrencephalic species. The current study addressed the expression pattern of TGF-beta1 in a nonhuman primate model of middle cerebral artery occlusion. Focal permanent ischemia was induced for 1 or 7 days in 6 baboons and the following investigations were undertaken: cerebral oxygen metabolism (CMRO2) positron emission tomography studies, magnetic resonance imaging, postmortem histology, and reverse transcription-polymerase chain reaction. The aim of the current study was to correlate the expression of TGF-beta1 to the underlying metabolic and histologic state of the threatened cerebral parenchyma. The authors evidenced increased TGF-beta1 mRNA levels (up to 25-fold) in those regions displaying a moderate (20% to 49%) reduction in CMRO2. The current findings suggest that the greatly enhanced expression of TGF-beta1 in the penumbral zones that surround tissue destined to infarction may represent a robust index of potentially salvageable brain. The current investigation, in the nonhuman primate, strengthens the authors' hypothesis, derived from rodent models, that TGF-beta1 may be involved in the physiopathology of human stroke.
Assuntos
Biomarcadores , Isquemia Encefálica/metabolismo , Expressão Gênica , Neurônios/fisiologia , Fator de Crescimento Transformador beta/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Imageamento por Ressonância Magnética , Masculino , Artéria Cerebral Média/cirurgia , Consumo de Oxigênio , Papio , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tomografia Computadorizada de EmissãoRESUMO
NADPH-cytochrome c (P-450) reductases from horse placenta and rat liver were purified and their biological activities compared using cytochrome c as substrate. Rat liver reductase was purified to electrophoretic homogeneity in one chromatographic step on 2',5'-ADP agarose, and had a relative mass of 85,000 Da as estimated by SDS-PAGE. Equine placental reductase was separated from cytochrome P-450 on aminohexyl-Sepharose 4B and further purified on 2',5-ADP agarose; this preparation exhibited two bands, one of 85,000 and one of 80,000 Da, on SDS-PAGE. The lower molecular weight form was assumed to be a proteolytic product of the higher molecular weight form. A high retention of activity was obtained in both preparations. Equine placenta and rat liver enzymes were found to exhibit very similar Vmax and Km, suggesting that they are not species specific.
Assuntos
Aromatase/metabolismo , Cavalos/metabolismo , Microssomos Hepáticos/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Placenta/enzimologia , Animais , Cromatografia , Eletroforese em Gel de Poliacrilamida , Feminino , Masculino , Microssomos/enzimologia , Peso Molecular , NADPH-Ferri-Hemoproteína Redutase/isolamento & purificação , Gravidez , Ratos , Ratos EndogâmicosRESUMO
There is increasing evidence that oxygen free radicals (OFR) are involved in cerebral ischaemia-reperfusion injury, possibly via a modulation of Na+,K(+)-ATPase activity, one of the major membrane pumps responsible for ionic homeostasis. We measured OFR-mediated modulation of this enzymatic activity and examined the roles of lipid and/or protein alterations. Using mouse brain microsomes exposed to UV-C irradiation, our results show a good correlation between activity inhibition and lipoperoxidation estimated by PUFA loss as well as malondialdehyde production. The protective effect of thiourea (OH scavenger) and the lack of effect noted with DTT (thiol protector) suggest that the functionality of the Na+,K(+)-ATPase is altered by perturbation of membrane integrity rather than by a structural alteration of the protein itself.
Assuntos
Trifosfato de Adenosina/farmacologia , Encéfalo/enzimologia , Microssomos/enzimologia , Espécies Reativas de Oxigênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/efeitos da radiação , Encéfalo/ultraestrutura , Ditiotreitol/farmacologia , Sequestradores de Radicais Livres/farmacologia , Radicais Livres , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/efeitos da radiação , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Oxirredução , Tioureia/farmacologia , Raios UltravioletaRESUMO
Oxygen-reactive species are being described as agents responsible for cell degeneration mechanisms resulting from membrane, enzyme, and nuclear alterations. Lipid peroxidation on its own is considered to be one of the consequences of the free radicals attack, and among the different reactive aldehydes that can be formed from the decomposition of lipid peroxides, the most extensively assayed have been malondialdehyde (MDA). However, the different techniques currently used for MDA assay (HPLC, GLC) are barely sensitive enough to follow its production at the cellular level. In order to develop an immunofluorescent technique able to detect cellular damages provoked by lipoperoxidation, polyclonal antibodies against lysozyme modified by MDA treatment have been raised in rabbits. We show that this immunserum recognizes specifically all the MDA-treated proteins tested, but not the intact proteins or the proteins treated by other aldehydes. Moreover, we demonstrate using an ELISA technique that the amount of immunoreactive proteins in MDA-treated membrane erythrocytes is proportional to the concentration of MDA applied, suggesting that this assay may represent a quantitative method of determination of lipoperoxidative alterations. In addition, when coupled to an indirect fluorophore antibody (FITC), the immunserum allows a precise location of these modified proteins within the membranes of erythrocytes in which lipid peroxidation was initiated by far UV irradiation. In summary, the interest of this work is to provide an immunological probe that can precociously detect membrane damages induced by MDA, regardless of the cell type and pro-oxidant (physiological or pathological) conditions.
Assuntos
Membrana Eritrocítica/fisiologia , Peroxidação de Lipídeos/efeitos da radiação , Malondialdeído/análise , Proteínas de Membrana/sangue , Raios Ultravioleta , Animais , Anticorpos , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática/métodos , Membrana Eritrocítica/efeitos da radiação , Membrana Eritrocítica/ultraestrutura , Eritrócitos/fisiologia , Eritrócitos/efeitos da radiação , Eritrócitos/ultraestrutura , Imunofluorescência , Humanos , Immunoblotting , Proteínas de Membrana/análise , Proteínas de Membrana/efeitos da radiação , Muramidase/análise , Muramidase/imunologia , Estresse Oxidativo , Coelhos/imunologia , Sensibilidade e EspecificidadeRESUMO
A complete study of pityriasis rosea (Gibert) has been performed; this study includes: --a pathological and ultrastructural examination of the skin biopsies; --an immunological study: direct and indirect immunofluorescence (seric anti-cutaneous antibodies); --a virological investigation: ultrastructural examination of the specimens, inoculation of homogenized-skin specimens to 3 cellular cultures; --serological investigations for influenza A, B, parainfluenza 1, 2, 3, adenovirus, respiratory syncitial virus, Mycoplasma pneumoniae, ornithosis-psittacosis, Q-fever, herpes-virus, herpes-virus varicellae, cytomegalovirus, Epstein-Barr virus. All viral investigations had a negative result. Virus or virus-like particle has were not detected on the ultrastructural examinations. A viral infective agent cannot be incriminated as the cause of PRG. However, a large number of PRG patients had antibodies for the Epstein-Barr virus early antigen. The authors are carrying on with this particular investigation.
Assuntos
Pitiríase/patologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Imunofluorescência , Herpesvirus Humano 4/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Pitiríase/imunologia , Pitiríase/microbiologia , Pele/ultraestruturaRESUMO
In order to develop an immunofluorescent technique able to detect the cellular damage induced by lipid peroxidation, we produced rabbit polyclonal antibodies (AbAIP) against malondialdehyde (MDA)-modified lysozyme. These antibodies specifically recognized all the MDA-treated proteins tested, but not native proteins or proteins treated by other aldehydes. Moreover, we showed by an enzyme-linked immunosorbent assay (ELISA) that the amount of immunoreactive protein in MDA-treated erythrocytes was proportional to the concentration of MDA added, suggesting that this ELISA measurement may represent a quantitative determination of peroxidative protein alterations. In addition, when coupled to an indirect fluorochrome antibody conjugate [fluorescein isothiocyanate (FITC)], these antibodies permitted precise localization of the modified proteins within the membranes of peroxidized cells.
Assuntos
Anticorpos/imunologia , Membrana Eritrocítica/metabolismo , Peroxidação de Lipídeos/fisiologia , Malondialdeído/farmacologia , Proteínas de Membrana/metabolismo , Muramidase/metabolismo , Animais , Anticorpos/metabolismo , Galinhas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Membrana Eritrocítica/imunologia , Membrana Eritrocítica/efeitos da radiação , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Microscopia de Fluorescência , Muramidase/química , Muramidase/imunologia , Estresse OxidativoRESUMO
The peptides of the transforming growth factor-beta (TGF-beta) family transduce their signal through ligand-induced heteromeric complexes that consist of type I and type II serine/threonine kinases. Both TGF-beta receptors are abundant in many peripheral tissues, but clear evidence of their expression in cortical astrocytes and neurons has not been published so far. In this study, we investigated the expression of type I and type II TGF-beta receptors and their potential ligands (TGF-beta1, TGF-beta2, and TGF-beta3) in the CNS by using RT-PCR and immunohistochemistry. Moreover, to further the study of those cell types that exhibit TGF-beta isoforms and related receptors, we examined through the use of RT-PCR whether cortical neurons and astrocytes in culture express the mRNAs for TGF-betas and their receptors. We show that the three TGF-beta isoform mRNAs are present in the CNS. However, although astrocytes in culture display all three isoforms, neurons in culture express only TGF-beta2. We have demonstrated that both type I and type II TGF-beta receptor mRNAs and proteins are present in the CNS and in cultures of cortical neurons and astrocytes. Thus, TGF-betas may act as autocrine and paracrine signals in the CNS between both neurons and astrocytes via the same receptor systems as those found in peripheral tissues. TGF-beta1 has been shown to be induced following hypoxic-ischemic brain injury and may play a critical role in the pathophysiology of degenerative processes in the CNS. In the present investigation, we confirmed that the expression of TGF-beta1 was increased markedly up until 24 h and thereafter was stable over the first 3 days following permanent occlusion of the middle cerebral artery in mice. However, whereas the expression of the type I TGF-beta receptor was not altered by the ischemic insult, the pattern of the type II TGF-beta receptors was modified dramatically in the ischemic area 3 days after the occlusion. These data show that, even if ligands are present, they may not be able to transduce their signal. Finally, the present study clearly demonstrates that a knowledge of the expression of ligand-specific receptors following brain injury is a fundamental step in clarifying the involvement of cytokines in neurodegenerative diseases.