RESUMO
STEP (striatal-enriched phosphatase) is a non-receptor tyrosine phosphatase that is specifically expressed in the neurons of the central nervous system. STEP regulates the activity of several effector molecules involved in synaptic plasticity and neuronal cell survival, including MAPKs (mitogen-activated protein kinases), Src family kinases and NMDA (N-methyl-D-aspartic acid) receptors. The critical role of STEP in regulating these effectors requires that its activity be tightly regulated. Previous studies have demonstrated that the activity of STEP is regulated through reversible phosphorylation of a serine residue within the KIM (kinase-interacting motif), by cAMP-dependent PKA (protein kinase A). In the present paper we show that STEP is endogenously phosphorylated at two additional sites located within the KISs (kinase-specificity sequences). The basal activity of ERK (extracellular-signal-regulated kinase) and p38 MAPKs plays an important role in the phosphorylation of these two sites. Dephosphorylation of these two sites leads to polyubiquitination and proteolytic degradation of STEP. Conversely, the proteasome inhibitors MG-132 and epoxomicin can stabilize STEP. The active form of STEP is more susceptible to degradation than the inactive form. Taken together the results of the present paper establish that ubiquitin-dependent proteolysis could be a novel mechanism for irreversibly terminating the activity of STEP.
Assuntos
Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Ubiquitina/metabolismo , Animais , Células COS , Domínio Catalítico , Linhagem Celular , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Camundongos , Estrutura Terciária de ProteínaRESUMO
The present study examines the role of a neuron-specific tyrosine phosphatase (STEP, striatal-enriched tyrosine phosphatase) in excitotoxic cell death. Our findings demonstrate that p38 MAPK, a stress-activated kinase that is known to play a role in the etiology of excitotoxic cell death is a substrate of STEP. Glutamate-mediated NMDA receptor stimulation leads to rapid but transient activation of p38 MAPK, which is primarily dependent on NR2A-NMDA receptor activation. Conversely, activation of NR2B-NMDA receptors leads to dephosphorylation and subsequent activation of STEP, which in turn leads to inactivation of p38 MAPK. Thus, during transient NMDA receptor stimulation, increases in STEP activity appears to limit the duration of activation of p38 MAPK and improves neuronal survival. However, if NR2B-NMDA receptor stimulation is sustained, protective effects of STEP activation are lost, as these stimuli cause significant degradation of active STEP, leading to secondary activation of p38 MAPK. Consistent with this observation, a cell transducible TAT-STEP peptide that constitutively binds to p38 MAPK attenuated neuronal cell death caused by sustained NMDA receptor stimulation. The findings imply that the activation and levels of STEP are dependent on the duration and magnitude of NR2B-NMDA receptor stimulation and STEP serves as a modulator of NMDA receptor dependent neuronal injury, through its regulation of p38 MAPK.
Assuntos
Ácido Glutâmico/toxicidade , Neurônios/enzimologia , Proteínas Tirosina Fosfatases não Receptoras/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Morte Celular/fisiologia , Células Cultivadas , Feminino , Células HeLa , Humanos , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Hemophilia A is an X-linked recessive bleeding disorder caused by defects in factor VIII gene (F8). Our study examines variations of single nucleotide polymorphism (SNP) in F8 in the Indian population and establishes the utility of a combination of SNP and microsatellite markers for the successful identification of carriers in the affected families.
Assuntos
Hemofilia A/genética , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , Estudos de Avaliação como Assunto , Feminino , Triagem de Portadores Genéticos , Marcadores Genéticos , Humanos , Índia , MasculinoRESUMO
PURPOSE: Myocilin, a 57 kDa glycoprotein, has been of much interest because of its association with primary open-angle glaucoma, lack of understanding of its biological function, and sequence homology of its N- and C-termini with two distinctly different proteins, myosin and olfactomedin, respectively. In that context, the molecular evolution of myocilin was investigated. METHODS: The human myocilin protein was used as query in sequence alignment program and the similar protein sequences were searched in the protein databases for different species. The secondary structure analysis of human myocilin and the prediction of disulfide bonded cysteine residues in the protein were done using PSIPRED and CYSPRED software, respectively. Presence of putative motifs in the protein sequences was determined using the ScanProsite tool with the option of including patterns with the high probability of occurrence. The phylogenetic analyses of human, mouse, rat, and bovine myocilin were done at the DNA Data Bank of Japan (DDBJ) server. RESULTS: It was observed that while two different protein sequences from Drosophila melanogaster contained significant homology with either C-terminal or N-terminal of myocilin, a single protein from Xenopus laevis showed homology covering entire C-terminal and most of the N-terminal region of myocilin. These observations are noteworthy in the context of the previously reported homology of N-terminal domain of myocilin with a single protein (non-muscle myosin) in Dictyostelium discoideum, and C-terminal domain with a single protein (olfactomedin-like) in Caenorhabditis elegans, both representing lower organisms. Further, specific amino acids and putative functional motifs were observed to be conserved between the two sets of proteins having homology to two ends (N- and C-termini) of myocilin. At the secondary structure level, myocilin shows two distinctly different domains: (a) the N-terminal region is primarily a-helical type, and (b) the C-terminal region consists mostly of beta-sheet and turn. CONCLUSIONS: These observations led to the hypothesis that during evolution myocilin might have resulted from fusion of genes for at least two different proteins with functional implications relevant to mammals. It is noteworthy that mutations in the myocilin gene, causal to Primary Open Angle Glaucoma, have only been detected in the first exon (corresponding to myosin like region) and the last exon (corresponding to olfactomedin domain) but not the region (exon 2) between the two domains. Phylogenetic analysis of mammalian myocilin revealed that rat and mouse myocilins demonstrate a closer relationship compared to its human or bovine homologues.
Assuntos
Evolução Molecular , Proteínas do Olho/genética , Glicoproteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Bovinos , Proteínas do Citoesqueleto , Bases de Dados Factuais , Dictyostelium/genética , Drosophila melanogaster/genética , Proteínas da Matriz Extracelular/genética , Glaucoma de Ângulo Aberto/genética , Humanos , Zíper de Leucina/genética , Camundongos , Dados de Sequência Molecular , Miosinas/genética , Estrutura Secundária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Xenopus laevis/genéticaRESUMO
PURPOSE: Glaucoma is the second leading cause of blindness worldwide after cataract. Defects in the myocilin gene (MYOC) have been shown to be associated with Primary Open Angle Glaucoma (POAG), the most common form of the disease, especially in its juvenile form. Most of the reported mutations in MYOC are in POAG patients of Caucasian origin. A few studies have been reported on Asian patients (such as Chinese, Japanese, and Koreans) but none from the Indian subcontinent. The purpose of this study was to investigate the molecular basis of POAG among Indians, using MYOC as the candidate gene, and broaden our understanding on the pathogenesis caused by MYOC. METHODS: Fifty-six unrelated POAG patients, comprising 39 sporadic cases and 17 patients having familial history for POAG were enrolled in this study. The coding sequence of the gene was amplified by polymerase chain reaction (PCR) using genomic DNA from 30 POAG patients, followed by sequencing of the PCR products. Nucleotide changes were detected by identifying double peaks in the chromatogram due to heterozygosity and pairwise BLAST analysis of the sequence output data against the normal copy of the MYOC cDNA. Alteration of restriction sites due to nucleotide changes was identified. Twenty-six patients (not sequenced) and controls were screened for nucleotide changes by allele specific restriction digestion of the PCR products followed by separation of the digested DNA fragments by polyacrylamide gel electrophoresis. RESULTS: From a pool of 56 unrelated POAG patients two mutations were identified. A putative novel mutation (144 G->T; Gln48His) of a conserved amino acid was detected in the exon 1 of MYOC from three unrelated patients but none in the 51 control samples examined. The other mutation (1109 C->T; Pro370Leu), located in exon 3 and detected in a family affected with POAG, cosegregated with the disease and was not present in control samples. Two single nucleotide polymorphisms (SNPs) were identified; one in the promoter region (-83 G->A) and the other in the coding sequence (227 G->A; Arg76Lys). These two SNPs were found to be highly heterozygous both in the control (0.480) and the patient (0.477) populations, and were observed to be in linkage disequilibrium. CONCLUSIONS: The presence of a novel non-conservative change in codon 48 of MYOC in 3 POAG patients, but none in the healthy controls, suggests a causal association of the mutation with the disease, either singly or in combination with other genetic loci. The other point mutation (Pro370Leu) detected in the members of an affected POAG family represents a hotspot of mutation in the gene. Two identified SNPs (-83 G->A and 227 G->A) are not associated with the disease phenotype but could be used as highly informative markers in POAG affected families to determine any causal association of MYOC with the disease, and for identification of presymptomatic carriers in the family, where applicable. A comparison of our data with other studies revealed that these two polymorphisms are in complete linkage disequilibrium among Asians, but not among other ethnic groups studied so far.
Assuntos
Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Mutação Puntual , Adolescente , Adulto , Idoso , Criança , Proteínas do Citoesqueleto , Análise Mutacional de DNA , Eletroforese em Gel de Poliacrilamida , Feminino , Glaucoma de Ângulo Aberto/etnologia , Humanos , Índia/epidemiologia , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Although colorectal cancer is a major cause of concern in the western population, recent studies are showing the incidence and mortality of colorectal cancer to be rapidly rising in Asia. The present study is an insight into the epidemiological profile of colorectal cancer of a representative Eastern Indian population. Over a period of three years, all histologically proved patients with colorectal cancer were assessed for age, sex, body mass index, dietary habits, socioeconomic status and stage of disease. Of a total of 168 patients male to female ratio was 1.7:1.The mean age of presentation was 47.01 years. Although colorectal cancer has been known as a disease of sedentary obese men, 41.66% of the patients were from a low socioeconomic rural set-up and 40.47% were involved in heavy physical labour with only 15% of being obese; 62% patients were harbouring a locally advanced disease at the time of presentation. The epidemiological pattern of colorectal cancer in India is different from that of the west as regards to earlier age of presentation, prevalence in low socio economic class with low fat diet and scanty meat intake.
Assuntos
Carcinoma/epidemiologia , Neoplasias Colorretais/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Dieta , Feminino , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Atividade Motora , Obesidade/epidemiologia , Prevalência , Fatores de Risco , População Rural/estatística & dados numéricos , Classe Social , População Urbana/estatística & dados numéricos , Adulto JovemRESUMO
Internal herniations constitute one of the relatively uncommon surgical emergencies. Among them double omental hernia with bowel strangulation is very rare and is a major diagnostic challenge. A case of a strangulated double omental hernia in a 42-year-old female patient is reported. The patient presented with a painful tender epigastric lump.There was a diagnostic dilemma. CT scan was followed by laparotomy which revealed a strangulated double omental hernia.
Assuntos
Hérnia/diagnóstico , Íleo/cirurgia , Omento/cirurgia , Adulto , Feminino , Hérnia/diagnóstico por imagem , Herniorrafia , Humanos , Laparotomia , RadiografiaRESUMO
The profile and pattern of abdominal trauma is changing with progressing civilisation. We are lacking epidemiological data from most parts of the world. This study was conducted to prepare a database in our set up and look into the pattern of abdominal trauma, make an aetiological correlation of abdominal trauma with the types of injuries, identify the preventable factors causing delay in intervention and, compare the data with the other available national and international data. This prospective, observational study was done in a teaching hospital in a metropolitan city of eastern India. Records of patients with abdominal trauma were collected in predesigned forms, from admission to discharge. Data were analysed applying standard statistical techniques. Males (87.3%) predominated with the age range between 21 and 30 years, and the majority (73.5%) had blunt abdominal trauma. Compression injury (57.3%) commonly caused blunt trauma and stab injuries caused majority of penetrating trauma. The commonest organ injured both in blunt and penetrating trauma was small bowel (30.7% and 33.3% respectively). It was found that prehospital trauma care is virtually non-existent in this region. We are lacking a uniform protocol for the management of abdominal trauma across the hospitals. With the availability of better investigational modalities we are moving more towards a conservative approach to the abdominal trauma patients, especially the blunt abdominal trauma patients with solid organ injuries.
Assuntos
Traumatismos Abdominais/epidemiologia , População Urbana/estatística & dados numéricos , Ferimentos não Penetrantes/epidemiologia , Ferimentos Penetrantes/epidemiologia , Adolescente , Adulto , Idoso , Pré-Escolar , Feminino , Hospitais de Ensino/estatística & dados numéricos , Humanos , Índia , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
Parathyroid carcinoma is a very rare endocrine malignancy, which usually presents with features of hypercalcaemia and a neck mass. Here a case of parathyroid carcinoma, whose only presenting feature was multiple pathological fractures, without any palpable neck mass is reported. En masse resection of the parathyroid mass along with ipsilateral hemithyroidectomy was performed, while the fractures were treated conservatively. At three years follow-up the patient does not have any recurrence or any evidence of metastasis.