RESUMO
Perinatal asphyxia (PA) is one of the most frequent risk factors for several neurodevelopmental disorders (NDDs) of presumed multifactorial etiology. Dysfunction of neuronal connectivity is thought to play a central role in the pathophysiology of NDDs. Because underlying causes of some NDDs begin before/during birth, we asked whether this clinical condition might affect accurate establishment of neural circuits in the hippocampus as a consequence of disturbed brain plasticity. We used a murine model that mimics the pathophysiological processes of perinatal asphyxia. Histological analyses of neurons (NeuN), dendrites (MAP-2), neurofilaments (NF-M/Hp) and correlative electron microscopy studies of dendritic spines were performed in Stratum radiatum of the hippocampal CA1 area after postnatal ontogenesis. Protein and mRNA analyses were achieved by Western blot and RT-qPCR. Behavioral tests were also carried out. NeuN abnormal staining and spine density were increased. RT-qPCR assays revealed a ß-actin mRNA over-expression, while Western blot analysis showed higher ß-actin protein levels in synaptosomal fractions in experimental group. M6a expression, protein involved in filopodium formation and synaptogenesis, was also increased. Furthermore, we found that PI3K/Akt/GSK3 pathway signaling, which is involved in synaptogenesis, was activated. Moreover, asphyctic animals showed habituation memory changes in the open field test. Our results suggest that abnormal synaptogenesis induced by PA as a consequence of excessive brain plasticity during brain development may contribute to the etiology of the NDDs. Consequences of this altered synaptic maturation can underlie some of the later behavioral deficits observed in NDDs.
Assuntos
Asfixia/patologia , Hipocampo/fisiopatologia , Plasticidade Neuronal/fisiologia , Análise de Variância , Animais , Asfixia/fisiopatologia , Aprendizagem da Esquiva/fisiologia , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Espinhas Dendríticas/ultraestrutura , Comportamento Exploratório/fisiologia , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/ultraestrutura , Microscopia Eletrônica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Gravidez , Células Piramidais/metabolismo , Células Piramidais/patologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestruturaRESUMO
Disruption/denudation of the ependymal lining has been associated with the pathogenesis of various human CNS disorders, including hydrocephalus, spina bifida aperta, and periventricular heterotopia. It has been traditionally considered that ependymal denudation is a consequence of mechanical forces such as ventricular enlargement. New evidence indicates that ependymal disruption can precede ventricular dilation, but the cellular and molecular mechanisms involved in the onset of ependymal denudation are unknown. Here, we present a novel model to study ependymal cell pathophysiology and demonstrate that selective disruption of N-cadherin-based adherens junctions is sufficient to provoke progressive ependymal denudation. Blocking N-cadherin function using specific peptides that interfere with the histidine-alanine-valine extracellular homophilic interaction domain caused early pathologic changes characterized by disruption of zonula adherens and abnormal intracellular accumulation of N-cadherin. These changes then triggered massive apoptosis of ependymal cells and denudation of brain ventricular walls. Because no typical extrinsic mechanical factors such as elevated pressure or stretching forces are involved in this model, the critical role of N-cadherin-based adherens junctions in ependymal survival/physiology is highlighted. Furthermore, the results suggest that abnormal adherens junctions between ependymal cells should be considered as key components of the pathogenesis of CNS disorders associated with ependymal denudation.
Assuntos
Junções Aderentes/metabolismo , Antígenos CD/metabolismo , Apoptose/fisiologia , Encéfalo/citologia , Caderinas/metabolismo , Epêndima/metabolismo , Junções Aderentes/efeitos dos fármacos , Análise de Variância , Animais , Anticorpos/farmacologia , Antígenos CD/química , Antígenos CD/imunologia , Apoptose/efeitos dos fármacos , Caderinas/química , Caderinas/imunologia , Bovinos , Relação Dose-Resposta a Droga , Impedância Elétrica , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Epêndima/citologia , Epêndima/ultraestrutura , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica de Transmissão , Técnicas de Cultura de Órgãos , Peptídeo Hidrolases/imunologia , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Fatores de TempoRESUMO
Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the alpha5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.
Assuntos
Complexo de Endopeptidases do Proteassoma , Subunidades Proteicas , Frações Subcelulares/enzimologia , Trypanosoma cruzi , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Núcleo Celular/enzimologia , Centrifugação com Gradiente de Concentração , Citoplasma/enzimologia , Imunofluorescência , Imuno-Histoquímica , Estágios do Ciclo de Vida , Microscopia Confocal , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Frações Subcelulares/ultraestrutura , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestruturaRESUMO
Synaptic dysfunction has been associated with neuronal cell death following hypoxia. The lack of knowledge on the mechanisms underlying this dysfunction prompted us to investigate the morphological changes in the postsynaptic densities (PSDs) induced by hypoxia. The results presented here demonstrate that PSDs of the rat neostriatum are highly modified and ubiquitinated 6 months after induction of hypoxia in a model of perinatal asphyxia. Using both two dimensional (2D) and three dimensional (3D) electron microscopic analyses of synapses stained with ethanolic phosphotungstic acid (E-PTA), we observed an increment of PSD thickness dependent on the duration and severity of the hypoxic insult. The PSDs showed clear signs of damage and intense staining for ubiquitin. These morphological and molecular changes were effectively blocked by hypothermia treatment, one of the most effective strategies for hypoxia-induced brain injury available today. Our data suggest that synaptic dysfunction following hypoxia may be caused by long-term misfolding and aggregation of proteins in the PSD.
Assuntos
Hipotermia Induzida/métodos , Hipóxia Encefálica , Neostriado/metabolismo , Sinapses/metabolismo , Ubiquitinas/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Calbindinas , Modelos Animais de Doenças , Tomografia com Microscopia Eletrônica/métodos , Feminino , Hipóxia Encefálica/metabolismo , Hipóxia Encefálica/patologia , Hipóxia Encefálica/terapia , Masculino , Microscopia Imunoeletrônica/métodos , Neostriado/patologia , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Gravidez , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/metabolismo , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Sinapses/ultraestrutura , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismoRESUMO
Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC-MS/MS analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles.
Assuntos
Cromatografia Líquida , Vesículas Citoplasmáticas/química , Espectrometria de Massas , Proteínas de Protozoários/análise , Frações Subcelulares/química , Trypanosoma cruzi/química , Animais , Fracionamento Celular , Vesículas Citoplasmáticas/ultraestrutura , Metabolismo dos Lipídeos , Microscopia Eletrônica de Transmissão , Proteômica/métodos , Frações Subcelulares/ultraestrutura , Trypanosoma cruzi/ultraestruturaRESUMO
Anp32e/Cpd1, a member of the acidic nuclear phosphoprotein (Anp)32 family, is characterized by the presence of an amino terminal domain containing four leucine-rich repeats and a carboxyl-terminal low-compositional complexity acidic region. In previous studies performed to understand the biological role of Anp32e/Cpd1, we showed a predominant presence of Anp32e/Cpd1 in the nucleus. However, when Anp32e/Cpd1 is in the cytoplasm, it co-localizes spatially with protein phosphatase 2A (PP2A) near cell membranes, far from the synapses. In the present work, we show that Anp32e/Cpd1 is also present as a membrane-bound 74/76-kDa protein with a widespread distribution in the brain. We reveal that the expression, synthesis and half-life of this high-molecular-weight form of Anp32e/Cpd1 are spatially and temporally correlated with the cerebellar synaptogenesis period. We demonstrate that synaptic Anp32e/Cpd1 co-localizes, interacts and inhibits PP2A activity, and that phosphorylation of Anp32/Cpd1 is required for the Anp32e-PP2A interaction. Also, subcellular localization was shown with electronic microscopy. Finally, we examine Anp32e/Cpd1 and PP2A distribution in two ataxic mutant models, weaver and staggerer, and show that their co-localization in Purkinje cell dendrites depends on parallel fibre/Purkinje cell contacts. Based on these observations, we propose that Anp32e/Cpd1 mediates synaptogenesis process by modulating PP2A activity.
Assuntos
Encéfalo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Sinapses/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Encéfalo/citologia , Encéfalo/metabolismo , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Microscopia Imunoeletrônica/métodos , Chaperonas Moleculares , Peso Molecular , Organogênese , Isoformas de Proteínas/metabolismo , Proteína Fosfatase 2 , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Sinapses/ultraestruturaRESUMO
The kidneys play a pivotal role in the pathogenesis of essential hypertension because of a primary defect in renal hemodynamics and/or tubule hydro-saline handling that results in the retention of fluid and electrolytes. Previous studies have shown that increasing the renal pelvic pressure increased ipsilateral afferent renal nerve activity (ARNA), the ipsilateral renal pelvic release of substance P (SP) and the contralateral urinary sodium excretion in Wistar--Kyoto rats (WKy). However, spontaneously hypertensive rats (SHR) present an impaired renorenal reflex activity associated, partly, with a peripheral defect at the level of the sensory receptors in the renal pelvis. Furthermore, the renal pelvic administration of SP failed to increase ARNA in most of SHR at concentrations that produced marked increases in WKy. Since we have assessed the expression and localization of NK(1) receptor (NK(1)R), SP and calcitonin gene-related peptide (CGRP) in different dorsal root ganglia (DRG) cell subtypes and renal pelvis of 7- and 14-week-old SHR. The results of this study show increased SP and CGRP expression in the dorsal ganglia root cells of SHR compared to WKy rats. Additionally, there was a progressive, significant, age-dependent, decrease in NK(1)R expression on the membrane surface in SHR DRG cells and in the renal pelvis. In conclusion, the results of the present study suggest that the impaired activation of renal sensory neurons in SHR may be related to changes in the expression of neuropeptides and/or to a decreased presence of NK(1)R in DRG cells. Such abnormalities could contribute to the enhanced sodium retention and elevation of blood pressure seen in SHR.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Gânglios Espinais/citologia , Neurônios/metabolismo , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Fatores Etários , Análise de Variância , Animais , Western Blotting/métodos , Regulação da Expressão Gênica , Imuno-Histoquímica/métodos , Masculino , Microscopia Imunoeletrônica/métodos , Neurônios/ultraestrutura , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestruturaRESUMO
Reservosomes are acidic compartments present at the posterior region of epimastigote forms of Trypanosoma cruzi that store proteins and lipids. During metacyclogenesis, they consume their contents and disappear. Reservosomes are rich in cruzipain, the main proteolytic enzyme of this parasite. By centrifugation in a sucrose gradient, we have obtained a highly purified subcellular fraction containing reservosomes from 5-day-old Y strain epimastigotes. Transmission electron microscopy showed that the fraction contained well-preserved organelles. The protein profile of the organelle analyzed by SDS-PAGE depicted a wide range of protein bands, predominating those corresponding to a triplet of 60-51 kDa and a doublet of 25-23 kDa. Protease activity in substrate-containing gels, in the presence or absence of protease inhibitors, showed that cysteine proteinase is enriched and very active in the purified fraction. Enzymatic assays demonstrated the absence of pyrophosphatase, an acidocalcisome marker, and succinate cytochrome c reductase, a mitochondrial marker, although these enzymes were active in other regions of the purification sucrose gradient. Thin layer chromatographic neutral lipid analysis of purified reservosomes demonstrated that the organelle stores large amounts of ergosterol and esterified cholesterol. Phospholipid analysis indicated phosphatidylcholine and phosphatidylethanolamine as the major constituents of reservosome membranes.
Assuntos
Endossomos , Frações Subcelulares , Trypanosoma cruzi/ultraestrutura , Animais , Centrifugação com Gradiente de Concentração/métodos , Endossomos/química , Endossomos/ultraestrutura , Lipídeos/análise , Microscopia Eletrônica , Proteínas/análise , Frações Subcelulares/química , Frações Subcelulares/ultraestrutura , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The paraflagellar rod (PFR) is a component of the flagellar cytoskeleton of trypanosomatid protozoa, representing a filamentous structure that runs alongside the common 9 + 2 microtubular axoneme. The high degree of ultrastructural complexity and organization of the PFR suggests that it might be formed by numerous biochemical components. However, biochemical analysis of the PFR has revealed, to date, a modest degree of complexity in what concerns both major and minor PFR proteins. In this paper the preparation of purified PFR fractions by a combination of conventional cell-fractionation procedures, non-ionic detergent treatment and limited proteolysis is described. Comparative SDS-PAGE analysis of the different purification steps indicates that the purified PFR fractions possess high amounts of the well-known major PFR proteins (77 and 83 kDa). Also, bands of 147, 139, 129 and 122 kDa are clearly enriched in such fractions and may correspond to minor PFR components. A slight enrichment in a specific fraction of a doublet of bands of 181/188 kDa suggest the participation of these proteins in the composition of the bridges between the PFR and the axoneme.
Assuntos
Citoesqueleto/ultraestrutura , Flagelos/ultraestrutura , Trypanosomatina/ultraestrutura , Animais , Fracionamento Celular , Frações Subcelulares/ultraestruturaRESUMO
The enzyme oxalate oxidase was identified in mycelial extracts of the basidiomycete Ceriporiopsis subvermispora and thereafter purified to homogeneity. The purification procedure included only three steps: Q-Sepharose chromatography, precipitation at pH 3.0, and phosphocellulose chromatography. The enzyme is a 400-kDa homohexamer, as determined by gel permeation in Sephadex G-200 and SDS-polyacrylamide gel electrophoresis. Isoelectrofocusing revealed a pI of 4.2. Optimal activity was obtained at pH 3.5 and at 45 degrees C. The purified enzyme has Km and kcat values of 0.1 mM and 88 s-1, respectively. It is highly specific for oxalate, although it is inhibited at concentrations of this substrate above 2.5 mM. Hystochemistry studies conducted over mycelium slices showed reactions products in both endocellular and periplasmic associated elements. A possible connection between the intracellular metabolism of oxalate and the extracellular ligninolytic activity of the fungus is proposed.
Assuntos
Oxirredutases/química , Oxirredutases/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Polyporaceae/enzimologia , Ativação Enzimática , Histocitoquímica , Microscopia Eletrônica , Oxirredutases/fisiologia , Proteínas de Plantas/fisiologia , Polyporaceae/ultraestrutura , Frações Subcelulares/enzimologia , Frações Subcelulares/ultraestruturaRESUMO
The liver was previously shown to be able to hydrolyze dithiothreitol tetraacetate (DTTAC) to dithiothreitol (DTT) via a DTTAC-S-acetyl esterase (DTTACEST). In the present studies the intracellular distribution of DTTACEST activity and its characteristics are reported. Enzyme specific activity was: microsomes > > mitochondria > > nuclei and was absent in the cytosolic fraction. The Km of the DTTACEST in each fraction was: mitochondria > microsomes > nuclei and the Vmax was microsomes > mitochondria > nuclei. The results were analyzed in relation to the previously established antioxidative stress and free radical trapping properties of DTTAC and DTT and the preventive effects exerted by DTTAC against carbon tetrachloride induced liver necrosis or cancer.
Assuntos
Acetilesterase/metabolismo , Fígado/enzimologia , Estresse Oxidativo , Frações Subcelulares/enzimologia , Animais , Cinética , Fígado/ultraestrutura , Masculino , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/ultraestruturaRESUMO
An effective methodology to isolate and characterize the Golgi complex of Tritrichomonas foetus is described in this work. Using sucrose density gradient centrifugation, two highly enriched Golgi fractions (GF1 and GF2) were obtained. Enzymatic assays of GF1 and GF2 showed a strong enrichment in galactosyltransferase activity (20- and 7-fold, respectively), with minimal contamination with other organelles. The GF fraction was further subfractionated by alkaline treatment, which resulted in the production of Golgi content and membrane subfractions. Electron microscopic observations of intact cells or Golgi fractions fixed in solutions containing glutaraldehyde and tannic acid, as well as of deep-etched replicas of isolated fractions, revealed the presence of discrete bridges only between closely apposed cisternae.
Assuntos
Complexo de Golgi/ultraestrutura , Tritrichomonas foetus/ultraestrutura , 5'-Nucleotidase/análise , Animais , Biomarcadores , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Técnica de Congelamento e Réplica , Galactosiltransferases/análise , Complexo de Golgi/enzimologia , Membranas Intracelulares/ultraestrutura , Malato Desidrogenase/análise , Malato Desidrogenase (NADP+) , Microscopia Eletrônica , NADPH-Ferri-Hemoproteína Redutase/análise , Frações Subcelulares/ultraestrutura , Tritrichomonas foetus/enzimologia , UltracentrifugaçãoRESUMO
A comparative morphometrical analysis was carried out on secretory cells from Bothrops jararacussu venom glands, before manual extraction of the venom (milking) and 4 and 8 days after milking. At the 8th day after milking, the cytoplasmic volume increased by 160%. The rough endoplasmic reticulum (RER) volume density increase, up to the 8th day after milking, is mainly due to widening of the intra-scisternal space. The total volume and membrane surface of the RER. Golgi apparatus and subcomponents, secretory vesicles and mitochondria, increased during the experimental period while the volume and surface densities of these organelles, with the exception of the RER, did not vary. The numerical density of Golgi-associated microvesicles per Golgi volume unit also increased. The greatest relative increments in these parameters occurred within the first 4 days. These results are compatible with an increased rate of membrane synthesis and transport in the milked glands and suggest that the membrane biogenesis, degradation and circulation that takes place in the first week after milking is achieved through coordinated cellular mechanisms that maintain the rate between total membrane surface and total cytoplasmic volume unaltered.
Assuntos
Venenos de Crotalídeos/metabolismo , Glândulas Exócrinas/citologia , Serpentes/fisiologia , Animais , Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Glândulas Exócrinas/ultraestrutura , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Serpentes/anatomia & histologia , Frações Subcelulares/ultraestruturaRESUMO
A Ca2+-stimulated, Mg2+-dependent ATPase activity was found in subcellular fractions from Schistosoma mansoni. Its specific and relative activities were higher in the heterogeneous cuticle fraction and in the microsomal fraction. The K0.5 for ATPase activation by free Ca2+ was 0.2-0.5 microM. This is the first description of an ATPase activity stimulated by Ca2+ in the micromolar range in S. mansoni.