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1.
J Histochem Cytochem ; 66(3): 189-202, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29303644

RESUMO

Glutamate transport activities have been identified not only in the brain, but also in the liver, kidney, and intestine. Although glutamate transporter distributions in the central nervous system are fairly well known, there are still uncertainties with respect to the distribution of these transporters in peripheral organs. Quantitative information is mostly lacking, and few of the studies have included genetically modified animals as specificity controls. The present study provides validated qualitative and semi-quantitative data on the excitatory amino acid transporter (EAAT)1-3 subtypes in the mouse liver, kidney, and intestine. In agreement with the current view, we found high EAAT3 protein levels in the brush borders of both the distal small intestine and the renal proximal tubules. Neither EAAT1 nor EAAT2 was detected at significant levels in murine kidney or intestine. In contrast, the liver only expressed EAAT2 (but 2 C-terminal splice variants). EAAT2 was detected in the plasma membranes of perivenous hepatocytes. These cells also expressed glutamine synthetase. Conditional deletion of hepatic EAAT2 did neither lead to overt neurological disturbances nor development of fatty liver.


Assuntos
Transportador 1 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/análise , Transportador 3 de Aminoácido Excitatório/análise , Intestinos/ultraestrutura , Rim/ultraestrutura , Fígado/ultraestrutura , Animais , Immunoblotting , Imuno-Histoquímica , Intestinos/química , Rim/química , Fígado/química , Camundongos , Coloração e Rotulagem
2.
J Exp Med ; 214(2): 547-563, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28028152

RESUMO

The glutamate transporter GLT-1 is critical for the maintenance of low interstitial glutamate concentrations. Loss of GLT-1 is commonly observed in neurological disorders, including temporal lobe epilepsy (TLE). Despite the hypothesis that targeting the mechanisms of GLT-1 deficiency may be a novel strategy for treating drug-resistant epilepsy, the underlying molecular cascade remains largely unknown. Here, we show that Hsp90ß is up-regulated in reactive astrocytes of the epileptic hippocampus in patients with TLE and mouse models of epilepsy. Inhibition of Hsp90, but not Hsp70, increased GLT-1 levels. Mechanistically, Hsp90ß recruits GLT-1 to the 20S proteasome, thereby promoting GLT-1 degradation. Hsp90 inhibitor prevents GLT-1 degradation by disrupting the association between Hsp90ß and GLT-1. Using a model of TLE, we demonstrated that long-term systemic administration of 17AAG dramatically suppressed spontaneous recurrent seizures and ameliorated astrogliosis. Overall, these results suggest that up-regulation of GLT-1 by inhibiting Hsp90ß in reactive astrocytes may be a potential therapeutic target for the treatment of epilepsy and excitotoxicity.


Assuntos
Epilepsia/tratamento farmacológico , Transportador 2 de Aminoácido Excitatório/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Animais , Astrócitos/metabolismo , Benzoquinonas/farmacologia , Células Cultivadas , Epilepsia/metabolismo , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/metabolismo , Transportador 2 de Aminoácido Excitatório/análise , Ácido Glutâmico/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Lactamas Macrocíclicas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Convulsões Febris/tratamento farmacológico , Convulsões Febris/metabolismo
3.
Biochim Biophys Acta ; 1862(6): 1074-83, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26976331

RESUMO

Lafora disease (LD, OMIM 254780) is a fatal rare disorder characterized by epilepsy and neurodegeneration. Although in recent years a lot of information has been gained on the molecular basis of the neurodegeneration that accompanies LD, the molecular basis of epilepsy is poorly understood. Here, we present evidence indicating that the homeostasis of glutamate transporter GLT-1 (EAAT2) is compromised in mouse models of LD. Our results indicate that primary astrocytes from LD mice have reduced capacity of glutamate transport, probably because they present a reduction in the levels of the glutamate transporter at the plasma membrane. On the other hand, the overexpression in cellular models of laforin and malin, the two proteins related to LD, results in an accumulation of GLT-1 (EAAT2) at the plasma membrane and in a severe reduction of the ubiquitination of the transporter. All these results suggest that the laforin/malin complex slows down the endocytic recycling of the GLT-1 (EAAT2) transporter. Since, defects in the function of this transporter lead to excitotoxicity and epilepsy, we suggest that the epilepsy that accompanies LD could be due, at least in part, to deficiencies in the function of the GLT-1 (EAAT2) transporter.


Assuntos
Astrócitos/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Doença de Lafora/metabolismo , Animais , Astrócitos/patologia , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Fosfatases de Especificidade Dupla/análise , Fosfatases de Especificidade Dupla/metabolismo , Endocitose , Transportador 2 de Aminoácido Excitatório/análise , Homeostase , Humanos , Doença de Lafora/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ubiquitinação
4.
PLoS One ; 11(2): e0150290, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26919701

RESUMO

The isolation and study of cell-specific populations in the central nervous system (CNS) has gained significant interest in the neuroscience community. The ability to examine cell-specific gene and protein expression patterns in healthy and pathological tissue is critical for our understanding of CNS function. Several techniques currently exist to isolate cell-specific populations, each having their own inherent advantages and shortcomings. Isolation of distinct cell populations using magnetic sorting is a technique which has been available for nearly 3 decades, although rarely used in adult whole CNS tissue homogenate. In the current study we demonstrate that distinct cell populations can be isolated in rodents from early postnatal development through adulthood. We found this technique to be amendable to customization using commercially available membrane-targeted antibodies, allowing for cell-specific isolation across development and animal species. This technique yields RNA which can be utilized for downstream applications-including quantitative PCR and RNA sequencing-at relatively low cost and without the need for specialized equipment or fluorescently labeled cells. Adding to its utility, we demonstrate that cells can be isolated largely intact, retaining their processes, enabling analysis of extrasomatic proteins. We propose that magnetic cell sorting will prove to be a highly useful technique for the examination of cell specific CNS populations.


Assuntos
Córtex Cerebral/citologia , Expressão Gênica , Separação Imunomagnética , Proteínas do Tecido Nervoso/análise , Animais , Astrócitos/metabolismo , Biomarcadores , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/imunologia , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real
5.
J Neurochem ; 134(5): 857-64, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26083406

RESUMO

Besides the well-described inflammatory and dysfunction effects on the respiratory tract, accumulating evidence indicates that ozone (O3 ) exposure also affects central nervous system functions. However, the mechanisms through which O3 exerts toxic effects on the brain remain poorly understood. We previously showed that O3 exposure caused a neuronal activation in regions of the rat nucleus tractus solitarii (NTS) overlapping terminal fields of vagal lung afferents. Knowing that O3 exposure can impact astrocytic protein expression, we decided to investigate whether it may induce astroglial cellular alterations in the NTS. Using electron microscopy and immunoblot techniques, we showed that in O3 -exposed animals, the astrocytic coverage of NTS glutamatergic synapses was 19% increased while the astrocyte volume fraction and membrane density were not modified. Moreover, the expression of glial fibrillary acidic protein and S100ß, which are known to be increased in reactive astroglia, did not change. These results indicate that O3 inhalation induces a glial plasticity that is restricted to the peri-synaptic coverage without overall astroglial activation. Taken together, these findings, along with our previous observations, support the conclusion that O3 -induced pulmonary inflammation results in a specific activation of vagal lung afferents rather than non-specific overall brain alterations mediated by blood-borne agents. Exposure to ozone, a major atmospheric pollutant, induces an increase in the glial coverage of neurons that is restricted to peri-synaptic compartments. This observation does not support the view that the ozone-induced neuronal disorders are related to non-specific overall brain alterations. It rather argues for a specific activation of the vagus nerve in response to pulmonary inflammation.


Assuntos
Poluentes Atmosféricos/toxicidade , Astrócitos/fisiologia , Ácido Glutâmico/farmacologia , Ozônio/toxicidade , Núcleo Solitário/patologia , Sinapses/patologia , Administração por Inalação , Animais , Astrócitos/química , Biomarcadores , Proteínas do Citoesqueleto/análise , Transportador 2 de Aminoácido Excitatório/análise , Proteína Glial Fibrilar Ácida/análise , Glutamato-Amônia Ligase/análise , Masculino , Proteínas do Tecido Nervoso/análise , Plasticidade Neuronal , Ozônio/administração & dosagem , Ratos , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100/análise , Núcleo Solitário/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura
6.
J Histochem Cytochem ; 60(11): 811-21, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22859703

RESUMO

Glutamate transporters (GLT-1, GLAST, EAAC1) limit the actions of excitatory amino acids. Because a disturbed transporter operation can cause or aggravate neurological diseases, transporters are of considerable neuropathological interest. Human samples, however, are seldom obtained fresh. Here, we used mice brains to study how fast glutamate transporters are degraded after death. Immunoblots showed that terminal GLT-1 epitopes (within residues 1-26 and 518-573) had mostly disappeared after 24 hr. GLAST termini (1-25 and 522-543) degraded slightly slower. In contrast, epitopes within central parts of GLT-1 (493-508) and the EAAC1 C-terminus (510-523) were readily detectable after 72 hr. The decline in immunoreactivity of the GLT-1 and GLAST termini was also seen in tissue sections, but proteolysis did not happen synchronously in all cells. At 24 hr, scattered cells remained strongly immunopositive, while the majority of cells were completely immunonegative. GLAST and GLT-1 co-localized in neocortical tissue, but at 12 hr, many GLAST-positive cells had lost the GLT-1 termini. The uneven disappearance of labeling was not observed with the antibodies to GLT-1 residues 493-508. The immunoreactivity to this epitope correlated better with the reported glutamate uptake activity. Thus, postmortem delay may affect epitopes differently, possibly causing erroneous conclusions about relative expression levels.


Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Encéfalo/patologia , Mudanças Depois da Morte , Sistema X-AG de Transporte de Aminoácidos/análise , Animais , Western Blotting , Encéfalo/citologia , Transportador 1 de Aminoácido Excitatório/análise , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteólise
7.
Toxicology ; 300(1-2): 12-8, 2012 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-22627295

RESUMO

Aroclor 1254, a commercially produced mixture of polychlorinated biphenyls, is known to cause many adverse conditions, including neurotoxicity. It has been recently postulated that upregulation of N-methyl-d-aspartate receptors (NMDARs) and enhanced glutamate signalling which leads to excitotoxicity, is the mechanism of Aroclor-induced neurotoxicity. To obtain insights into the mechanisms underlying glutamatergic overstimulation, we investigated the function and expression of sodium-dependent glutamate transporters which are known to regulate extracellular glutamate concentrations in the brain. Exposure to Aroclor 1254 was found to significantly lower the uptake of radioactive glutamate into gliosomal fractions obtained from adult rat brains. It also markedly decreased the expression of both protein and mRNA of GLT-1, the main glial glutamate transporter. This indicates that downregulation of GLT-1 may potentially lead to disturbances in glutamate clearance. The expression of GLAST, another astroglial glutamate transporter, was unchanged under conditions of Aroclor toxicity. Conversely, we observed enhanced glutamate uptake into nerve-endings fractions paralleled by increased EAAC1 protein expression. This may reflect the induction of protective mechanisms.


Assuntos
/efeitos adversos , Transportador 2 de Aminoácido Excitatório/antagonistas & inibidores , Neuroglia/efeitos dos fármacos , Prosencéfalo/efeitos dos fármacos , Animais , Cerebelo/química , Cerebelo/efeitos dos fármacos , Transportador 1 de Aminoácido Excitatório/análise , Transportador 1 de Aminoácido Excitatório/antagonistas & inibidores , Transportador 1 de Aminoácido Excitatório/biossíntese , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/biossíntese , Expressão Gênica/efeitos dos fármacos , Masculino , Neuroglia/química , Prosencéfalo/química , RNA Mensageiro/análise , Ratos , Ratos Wistar
8.
J Comp Neurol ; 520(17): 3912-32, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22522966

RESUMO

The major regulators of synaptic glutamate in the cerebral cortex are the excitatory amino acid transporters 1-3 (EAAT1-3). In this study, we determined the cellular and temporal expression of EAAT1-3 in the developing human cerebral cortex. We applied single- and double-label immunocytochemistry to normative frontal or parietal (associative) cortex samples from 14 cases ranging in age from 23 gestational weeks to 2.5 postnatal years. The most striking finding was the transient expression of EAAT2 in layer V pyramidal neuronal cell bodies up until 8 postnatal months prior to its expression in protoplasmic astrocytes at 41 postconceptional weeks onward. EAAT2 was also expressed in neurons in layer I (presumed Cajal-Retzius cells), and white matter (interstitial) neurons. This expression in neurons in the developing human cortex contrasts with findings by others of transient expression exclusively in axon tracts in the developing sheep and rodent brain. With western blotting, we found that EAAT2 was expressed as a single band until 2 postnatal months, after which it was expressed as two bands. The expression of EAAT2 in pyramidal neurons during human brain development may contribute to cortical vulnerability to excitotoxicity during the critical period for perinatal hypoxic-ischemic encephalopathy. In addition, by studying the expression of EAAT1 and EAAT2 glutamate transporters, it was possible to document the development of protoplasmic astrocytes.


Assuntos
Astrócitos/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Transportador 2 de Aminoácido Excitatório/biossíntese , Neurônios/metabolismo , Western Blotting , Córtex Cerebral/embriologia , Pré-Escolar , Transportador 2 de Aminoácido Excitatório/análise , Feto , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Neurogênese/fisiologia
9.
J Histochem Cytochem ; 60(3): 174-87, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22215633

RESUMO

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.


Assuntos
Anticorpos/imunologia , Especificidade de Anticorpos , Imuno-Histoquímica/métodos , Adsorção , Animais , Anticorpos/metabolismo , Afinidade de Anticorpos , Antígenos/imunologia , Artefatos , Western Blotting , Reações Cruzadas , Epitopos , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/imunologia , Transportador 3 de Aminoácido Excitatório/análise , Transportador 3 de Aminoácido Excitatório/imunologia , Proteínas da Membrana Plasmática de Transporte de GABA/análise , Proteínas da Membrana Plasmática de Transporte de GABA/imunologia , Soros Imunes/imunologia , Camundongos , Camundongos Knockout , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Soluções
10.
Vet Immunol Immunopathol ; 145(1-2): 395-401, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22240145

RESUMO

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6h, 12h and 24h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.


Assuntos
Doenças do Pé/veterinária , Produtos Finais de Glicação Avançada/análise , Casco e Garras/química , Doenças dos Cavalos/imunologia , Animais , Sequência de Bases , Clonagem Molecular , Transportador 2 de Aminoácido Excitatório/análise , Doenças do Pé/imunologia , Doenças do Pé/metabolismo , Regulação da Expressão Gênica/imunologia , Transportador de Glucose Tipo 4/análise , Casco e Garras/imunologia , Doenças dos Cavalos/metabolismo , Cavalos/genética , Cavalos/imunologia , Cavalos/metabolismo , Peroxidação de Lipídeos/imunologia , Dados de Sequência Molecular , Estresse Oxidativo/imunologia , Reação em Cadeia da Polimerase/veterinária , Espécies Reativas de Oxigênio/análise , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/análise , Receptores Imunológicos/genética
11.
Pathol Oncol Res ; 17(1): 61-6, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20512538

RESUMO

GLUT-1 is a transmembrane glucose transport protein that allows the facilitated transport of glucose into cells, normally expressed in tissues which depend mainly on glucose metabolism. Enhanced expression of GLUT-1 can also be found in a large spectrum of carcinomas. This study aimed to investigate GLUT-1 expression in gallbladder tissue: from normal tissue samples, hyperplasias, low-grade and high-grade dysplasias to gallbladder carcinomas. In all, 115 archived samples of gallbladder tissue from 68 patients, presented after cholecystectomy, were immunohistochemically stained for GLUT-1. According to the intensity of GLUT-1 immunoreactivity, samples were divided into negative (stained 0-10% of cells stained), positive with weak to moderate (10-50%) and positive with strong (>50%) GLUT-1 expression. The GLUT-1 immunoreactivity of the samples showed a characteristic increase from premalignant lesions to carcinomas. Normal gallbladder tissue samples did not express GLUT-1 (100%). Weak expression was shown only focally in hyperplasias, but to a greater extent with low-grade dysplasias (20%), high-grade dysplasias (40%) and carcinomas (51.8%). Normal gallbladder tissue is GLUT-1 negative. GLUT-1 expression in carcinoma tissue is significantly higher than in dysplastic lesions. Strong GLUT-1 expression indicates 100% specificity for detecting gallbladder carcinomas. Therefore, GLUT-1 is a candidate as a diagnostic as well as a tissue prognostic marker in gallbladder carcinoma patients.


Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/análise , Transportador 2 de Aminoácido Excitatório/biossíntese , Neoplasias da Vesícula Biliar/metabolismo , Lesões Pré-Cancerosas/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Transportador 2 de Aminoácido Excitatório/análise , Feminino , Neoplasias da Vesícula Biliar/patologia , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/patologia , Prognóstico
12.
Hepatology ; 52(1): 256-65, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20583283

RESUMO

UNLABELLED: Cell culture studies and animal models point to an important role of oxidative/nitrosative stress in the pathogenesis of cerebral ammonia toxicity. However, it is unknown whether oxidative/nitrosative stress in the brain is also characteristic of hepatic encephalopathy (HE) in humans. We therefore analyzed post mortem cortical brain tissue samples from patients with cirrhosis dying with or without HE in comparison with brains from patients without liver disease. Significantly elevated levels of protein tyrosine-nitrated proteins, heat shock protein-27, and 8-hydroxyguanosine as a marker for RNA oxidation were found in the cerebral cortex of HE patients, but not of patients with cirrhosis but without HE. Glutamine synthetase (GS) activity was significantly decreased, whereas GS protein expression was not significantly affected. Protein expression of the glutamate/aspartate cotransporter was up-regulated in HE, whereas protein expression of neuronal and inducible nitric oxide synthases, manganese-dependent and copper/zinc-dependent superoxide dismutase, and glial glutamate transporter-1 were not significantly increased. CONCLUSION: These data indicate that HE in patients with cirrhosis is associated with oxidative/nitrosative stress, protein tyrosine nitration, and RNA oxidation, suggesting a role of oxidative stress in the pathogenesis of HE in patients with cirrhosis.


Assuntos
Córtex Cerebral/metabolismo , Encefalopatia Hepática/metabolismo , Cirrose Hepática/complicações , Nitratos/metabolismo , Estresse Oxidativo , Tirosina/metabolismo , Adulto , Idoso , Sistema X-AG de Transporte de Aminoácidos/análise , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Córtex Cerebral/química , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/metabolismo , Feminino , Glutamato-Amônia Ligase/análise , Glutamato-Amônia Ligase/metabolismo , Guanosina/análogos & derivados , Guanosina/análise , Guanosina/metabolismo , Proteínas de Choque Térmico HSP27/análise , Proteínas de Choque Térmico HSP27/metabolismo , Encefalopatia Hepática/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Nitratos/análise , RNA/análise , RNA/metabolismo , Tirosina/análise
13.
Cancer Cytopathol ; 118(2): 90-6, 2010 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-20209622

RESUMO

BACKGROUND: The distinction of benign from malignant mesothelial proliferations in cytologic specimens can be problematic. In this study, the authors investigated the utility of immunohistochemical (IHC) markers in making this distinction. METHODS: Archival paraffin-embedded cell blocks of pleural and peritoneal fluids from 52 patients with malignant mesothelioma (MM) and 64 patients with reactive mesothelial hyperplasia (MH) were retrieved. IHC stains included desmin, epithelial membrane antigen (EMA), glucose-transport protein 1 (GLUT-1), Ki67, and p53. RESULTS: Desmin was positive in 84% (54 of 64) cases of reactive MH and in 6% (3 of 52) of MM cases (P < .001). EMA was positive in 9% (6 of 64) of benign and 100% (52 of 52) of malignant cases (P < .001). GLUT-1 was positive in 12% (5 of 43) of benign and 47% (7 of 15) of malignant cases. Ki67 showed strong nuclear positivity in >40% of mesothelial cells in 9% (6 of 64) of benign and 16% (8 of 49) of malignant cases (P = .38). p53 showed strong nuclear positivity in 2% (1 of 46) of benign and 47% (7 of 15) of malignant cases (P < .001). EMA positivity and desmin negativity were found in 2% (1 of 64) of reactive MH cases and 98% (49 of 52) of MM cases (P < .001). EMA negativity and desmin positivity were found in 86% (55 of 64) of reactive MH cases and 0% of MM cases. CONCLUSIONS: The combination of positive EMA and negative desmin strongly favors MM; conversely, a combination of negative EMA and positive desmin favors a reactive process. Likewise, strong membranous positivity for GLUT-1 and/or strong nuclear staining for p53 favors a mesothelioma. Ki67 proliferative index showed no significant difference between reactive MH and MM cases.


Assuntos
Líquido Ascítico/metabolismo , Epitélio/patologia , Imuno-Histoquímica , Mesotelioma/diagnóstico , Derrame Pleural/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Desmina/análise , Transportador 2 de Aminoácido Excitatório/análise , Feminino , Humanos , Hiperplasia/patologia , Masculino , Pessoa de Meia-Idade , Mucina-1/análise , Proteína Supressora de Tumor p53/análise
14.
Gastroenterology ; 138(7): 2418-25, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20226190

RESUMO

BACKGROUND & AIMS: The molecular basis underlying visceral hypersensitivity in functional irritable bowel syndrome remains elusive, resulting in poor treatment effectiveness. Because alterations in spinal non-neuronal (astrocytic) glutamate reuptake are suspected to participate in chronic pain, we asked whether such processes occur in visceral hypersensitivity. METHODS: Visceral hypersensitivity was induced in Sprague-Dawley rats by maternal separation. Separated adults were given a systemic administration of riluzole (5 mg/kg), an approved neuroprotective agent activating glutamate reuptake. Visceral hypersensitivity was assessed using colorectal distension (40 mm Hg). Somatic nociception was quantified using Hot Plate, Randall-Sellito, and Hargreaves tests. Spinal proteins were quantified using immunofluorescence and Western blot. The dependence of visceral sensory function upon spinal glutamate transport was evaluated by intrathecal injection of glutamate transport antagonist DL-threo-beta-benzyloxyaspartate (TBOA). For in vitro testing of riluzole and TBOA, primary cultures of astrocytes were used. RESULTS: We show that riluzole counteracts stress-induced visceral hypersensitivity without affecting visceral response in nonseparated rats or altering nociceptive responses to somatic pain stimulation. In addition, maternal separation produces a reduction in glial excitatory amino acid transporter (EAAT)-1 with no change in EAAT-2 or gamma-amino butyric acid transporters. Stress was not associated with changes in glial fibrillary acidic protein or astrocytic morphology per se. Furthermore, visceral normosensitivity relies on spinal EAAT, as intrathecal TBOA is sufficient to induce hypersensitivity in normal rats. CONCLUSIONS: We identify spinal EAAT as a therapeutic target, and establish riluzole as a candidate to counteract gastrointestinal hypersensitivity in disorders such as irritable bowel syndrome.


Assuntos
Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Ácido Glutâmico/metabolismo , Síndrome do Intestino Irritável/tratamento farmacológico , Riluzol/uso terapêutico , Medula Espinal/metabolismo , Estresse Psicológico/complicações , Animais , Ácido Aspártico/farmacologia , Transportador 1 de Aminoácido Excitatório/análise , Transportador 1 de Aminoácido Excitatório/fisiologia , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/fisiologia , Proteína Glial Fibrilar Ácida/análise , Masculino , Privação Materna , Atividade Motora , Ratos , Ratos Sprague-Dawley
15.
Neurobiol Aging ; 31(4): 578-90, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18586353

RESUMO

Astrocyte pathology occurs in association with Alzheimer's disease (AD) and in brain ageing, but is poorly characterised. We sought to define the detailed cellular pathology of astrocytes, the extent of population variation and the relationship to Alzheimer-type changes in a population-based cohort. Three staining patterns were associated with GFAP and excitatory amino acid transporter 2 (EAAT2): minimal, moderate or extensive immunoreactivity. GFAP and EAAT2 expression were inversely related (p=0.015), with trends to increased expression of GFAP (p=0.019) and decreased expression of EAAT2 (p=ns) with increasing Braak stage. GFAP and EAAT2 correlated incompletely with beta-amyloid and tau immunoreactivity. However, gliosis increased with increasing burden of neuritic (p=0.011), but not diffuse (p=ns), plaques. Double-staining revealed distinct subsets of astrocytes; GFAP(+)EAAT(-), GFAP(-)EAAT(+), or GFAP(+)EAAT(+). In contrast to the variation in GFAP and EAAT2, levels of EAAT1 and S100B showed consistent staining patterns. Alzheimer-type pathology only partially explains the variation in gliosis and astrocyte functional markers, suggesting that other factors contribute to the population variance in astrocyte pathology.


Assuntos
Doença de Alzheimer/patologia , Astrócitos/patologia , Encéfalo/patologia , Gliose/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Estudos de Coortes , Transportador 1 de Aminoácido Excitatório/análise , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Gliose/fisiopatologia , Humanos , Imuno-Histoquímica , Estudos Longitudinais , Masculino , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Valor Preditivo dos Testes , Estudos Prospectivos , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/análise , Proteínas S100/metabolismo , Índice de Gravidade de Doença , Proteínas tau/análise , Proteínas tau/metabolismo
16.
Acta Neurochir (Wien) ; 152(5): 845-53, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19859653

RESUMO

PURPOSE: An abnormal increase in the extracellular glutamate is thought to play a crucial role in the initiation, spread, and maintenance of seizure activity.In normal conditions, the majority of this excess glutamate is cleared via glial glutamate transporters (EAAT-1 and EAAT-2). We aimed to examine the immunohistochemical expression of these transporters in the dysplastic tissues of patients with focal cortical dysplasia (FCD). METHODS: The parafin-embedded dysplastic tissues of 33 patients who were operated on due to medically intractable epilepsy and histopathologically diagnosed with FCD between 2001 and 2006 were stained immunohistochemically with appropriate antibodies, and the distribution and intensity of immunoreactivity (IR) of EAAT-1 and EAAT-2 were examined.The findings were compared with the histologically normal tissues of five patients who underwent temporal lobectomy for epilepsy surgery and 10 fresh postmortem cases. RESULTS: In the majority of the patients, the EAAT-1 and EAAT-2 IR were decreased, their astrocytic expression were lower, and the pattern of distribution were more diffused when compared to the control groups.Analyzing these findings according to the types of FCD revealed that as the severity of the dysplasia increased, the IR and astrocytic expression of both transporters are decreased and their distribution tend to be more "diffused." CONCLUSION: The results of this study suggest a relationship between the decreased glutamate transporter expressions in dysplastic tissues which,in turn, may cause increased extracellular concentrations of glutamate and FCD pathophysiology.Further studies with larger patient populations,investigating the expression of glutamate transporters at mRNA and protein levels, are required to clarify their roles in the pathophysiology of FCD.


Assuntos
Astrócitos/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/metabolismo , Ácido Glutâmico/metabolismo , Malformações do Desenvolvimento Cortical/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Adulto , Biomarcadores/análise , Biomarcadores/metabolismo , Córtex Cerebral/fisiopatologia , Transportador 1 de Aminoácido Excitatório/análise , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Malformações do Desenvolvimento Cortical/patologia , Malformações do Desenvolvimento Cortical/fisiopatologia , Pessoa de Meia-Idade
17.
Int J Oral Maxillofac Surg ; 38(10): 1066-70, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19574025

RESUMO

Vascular anomalies often affect the soft tissues and primary intraosseous lesions are uncommon, with only 33 cases reported in the zygoma. Intraosseous vascular anomalies of the zygoma have traditionally been designated 'haemangiomas' with little attempt to clarify the specific type of the lesion. Recently, specific immunohistochemical markers such as erythrocyte type glucose transporter protein 1 (GLUT-1) have been described to differentiate haemangiomas and vascular malformations. The authors report a case of intraosseous venous malformation of the zygoma and provide supporting evidence to justify the nomenclature. The literature on zygomatic 'haemangiomas' is critically reviewed and the basis for their proper designation as venous malformations highlighted.


Assuntos
Hemangioma/classificação , Terminologia como Assunto , Malformações Vasculares/classificação , Zigoma/irrigação sanguínea , Idoso , Transportador 2 de Aminoácido Excitatório/análise , Feminino , Humanos , Imuno-Histoquímica , Veias/anormalidades
18.
Mol Pain ; 5: 36, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-19570219

RESUMO

BACKGROUND: Removing and sequestering synaptically released glutamate from the extracellular space is carried out by specific plasma membrane transporters that are primarily located in astrocytes. Glial glutamate transporter function can be monitored by recording the currents that are produced by co-transportation of Na+ ions with the uptake of glutamate. The goal of this study was to characterize glutamate transporter function in astrocytes of the spinal cord dorsal horn in real time by recording synaptically evoked glutamate transporter currents. RESULTS: Whole-cell patch clamp recordings were obtained from astrocytes in the spinal substantia gelatinosa (SG) area in spinal slices of young adult rats. Glutamate transporter currents were evoked in these cells by electrical stimulation at the spinal dorsal root entry zone in the presence of bicuculline, strychnine, DNQX and D-AP5. Transporter currents were abolished when synaptic transmission was blocked by TTX or Cd2+. Pharmacological studies identified two subtypes of glutamate transporters in spinal astrocytes, GLAST and GLT-1. Glutamate transporter currents were graded with stimulus intensity, reaching peak responses at 4 to 5 times activation threshold, but were reduced following low-frequency (0.1 - 1 Hz) repetitive stimulation. CONCLUSION: These results suggest that glutamate transporters of spinal astrocytes could be activated by synaptic activation, and recording glutamate transporter currents may provide a means of examining the real time physiological responses of glial cells in spinal sensory processing, sensitization, hyperalgesia and chronic pain.


Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/fisiologia , Células do Corno Posterior/fisiologia , Transmissão Sináptica , Sistema X-AG de Transporte de Aminoácidos/análise , Animais , Eletrofisiologia , Transportador 1 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/análise , Masculino , Neuroglia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
19.
Ann Surg Oncol ; 16(10): 2834-9, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19603235

RESUMO

INTRODUCTION: Approximately 23% of melanoma patients will eventually develop pulmonary metastases and have a median survival of only about 7-11 months. Because pulmonary metastasectomy can improve this statistic, we investigated clinicopathologic features and biological correlates that might be used to identify surgical candidates. METHODS: Archived operative specimens and clinical records were retrieved for 20 melanoma patients who underwent resection of isolated pulmonary metastases at the John Wayne Cancer Institute, Saint John's Health Center. Five-year postmetastasectomy survival (PMS) rate was correlated with age, number of pulmonary metastases, tumor doubling time (TDT), tumor necrosis, and immunohistochemical expressions of four biological markers: Ki-67, glucose transporter-1 (Glut-1), caspase-3, and CD31. RESULTS: Median TDT was 61 days. On multivariate analysis, TDT (P = 0.008), Glut-1 intensity (P = 0.04), and CD31 expression (P = 0.004) were the significant predictors of PMS. Age, number of pulmonary metastases, tumor necrosis, and expression of Ki-67 or caspase-3 did not significantly impact survival. Median TDT was 56 days with Glut-1 expression versus 165 days without Glut-1 expression (P = 0.002), and Glut-1 staining intensity independently affected TDT (P = 0.012). CONCLUSIONS: Surgical resection may be preferable to toxic systemic therapies in melanoma patients whose isolated pulmonary metastases have a long TDT (> or = 61 days) and no biopsy evidence of Glut-1 expression.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Pulmonares/química , Neoplasias Pulmonares/mortalidade , Melanoma/química , Melanoma/mortalidade , Adulto , Idoso , Apoptose , Caspase 3/análise , Proliferação de Células , Transportador 2 de Aminoácido Excitatório/análise , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Antígeno Ki-67/análise , Neoplasias Pulmonares/secundário , Masculino , Melanoma/secundário , Pessoa de Meia-Idade , Necrose , Estadiamento de Neoplasias , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Prognóstico , Fatores de Risco , Taxa de Sobrevida , Resultado do Tratamento
20.
FEBS Lett ; 583(13): 2194-200, 2009 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-19527721

RESUMO

Phosphorylated tau (p-tau) is the principal component of neurofibrillary tangles, a pathological hallmark, and likely plays a neurotoxic role in tauopathies including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). We subjected brains from autopsy cases of AD, PSP, and CBD to a variety of immunohistochemical, immunoblotting, and pull-down assays. In this study, we show that excitatory amino acid transporter 2 (EAAT2) preferentially interacted with phosphorylated tau and was localized in neurofibrillary tangles in the brains of such patients. These results strongly indicate that EAAT2 acts in tauopathy-related neurodegeneration, and abnormalities in glutamate transport play an important role in the pathogenesis of tauopathies.


Assuntos
Encéfalo/metabolismo , Transportador 2 de Aminoácido Excitatório/análise , Transportador 2 de Aminoácido Excitatório/metabolismo , Emaranhados Neurofibrilares/metabolismo , Tauopatias/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Encéfalo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Tauopatias/patologia , Proteínas tau/análise
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