Acoplamento das unidades catalítica e regulatória do proteassomo e a funcionalidade mitocondrial em leveduras após mutações sítio-específicas na unidade catalítica do proteassomo

Em leveduras da espécie S. cerevisiae, foi descrito uma modificação redox pós-traducional denominada S-glutationilação em resíduos Cys da subunidade α5 da unidade catalítica 20S do proteassomo, especificamente o resíduo α5-C76, posteriormente mutada para α5-C76S. A linhagem carregando essa mutação apresentou como alteração fenotípica um menor tempo de vida cronológico (CLS: chronological life span) e uma maior frequência da conformação fechada da câmara catalítica da unidade 20S. Uma dupla mutação randômica na subunidade α5 (α5-S35P/C221S) criou uma linhagem duplo-mutante (DM), a qual induziu a abertura da câmera catalítica do 20S e também aumentou o CLS da célula. O presente estudo teve como objetivo estudar o grau de acoplamento entre as unidades catalítica e regulatória do proteassomo nas linhagens de levedura (C76S, WT e DM), bem como avaliar a funcionalidade mitocondrial em todas elas. A análise do acoplamento foi feita com eletroforese em gel nativo. Para determinar a funcionalidade mitocondrial, foi medida a atividade da enzima citrato sintase a partir da reação entre DTNB e CoA-SH. Foi observado que na linhagem C76S com CLS reduzido havia um menor grau de acoplamento entre as unidades catalítica 20S e regulatória 19S, além de uma atividade mitocondrial diminuída. Portanto, um menor grau de acoplamento entre as unidades do proteassomo muito provavelmente provocou uma disfunção mitocondrial devido a um importe de proteínas mitocondriais defeituoso, resultando em uma CLS reduzida, fato que pode melhor elucidar a nossa compreensão acerca do processo de envelhecimento e da morte celular prematura vista em doenças degenerativas causadas por acumulação proteica.A post-translational redox modification called S-glutathionylation in S. cerevisiae was described at Cys residues of the α5 subunit of the 20S catalytic unit of the proteasome, specifically α5-C76, posteriorly mutated to α5-C76S. The α5-C76S strain presented, as a phenotypic alteration, a higher frequency of the closed conformation of the catalytic chamber of the 20S unit and a shorter chronological life span (CLS: chronological life span). A double random mutation (DM: double mutated) in the α5 subunit (α5-S35P/C221S) induced the opening of the catalytic chamber and also increased CLS. This project aimed to assess the coupling between the catalytic and the regulatory units of the proteasome in some yeast strains (C76S, WT and DM) and to further evaluate their mitochondrial functionality. The study of the coupling of 20S-19S units was carried out in native gel electrophoresis. To determine mitochondrial functionality, the activity of citrate synthase was measured by the reaction between DTNB with CoA-SH. It was observed that in the C76S strain there was a lower degree of coupling between the 20S catalytic and 19S regulatory units, in addition to a decreased citrate synthase activity. Therefore, less coupling between the proteasomal units triggers mitochondrial dysfunction most likely due to deficient mitochondrial protein import, ultimately leading to decreased CLS, which can better our understanding in regards to the process of aging and premature cell death seen in degenerative disorders caused by protein accumulation.

Adherence and virulence genes of Escherichia colifrom children diarrhoea in the Brazilian Amazon

The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli. Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli. Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpAgene. EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg, aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea (P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC

Rotavirus genotyping in gastroenteritis cases of an infantile population from Western Brazilian Amazonia

INTRODUCTION: During the period from 2000 to 2002, 79 rotavirus-positive stool samples were collected from children presenting diarrhea in the Western Brazilian Amazon. METHODS: Molecular characterization of the G and P genotypes was performed using RT-PCR and electropherotyping analysis by polyacrylamide gel electrophoresis. RESULTS: A total of 59 samples were confirmed as group A rotavirus. A long electrophoretic profile was exhibited by the G1P[8], G3P[8], and G4P[8] genotypes. The G1P[8] genotype was found in greater proportion. The short electropherotype was exhibited only by G2 genotype strains. CONCLUSIONS: The proportion of the rotavirus genotypes observed was not different from that in other areas of Brazil. This study is the first genotyping of rotavirus in the Western Brazilian Amazon

The prevalence of norovirus, astrovirus and adenovirus infections among hospitalised children with acute gastroenteritis in Porto Velho, state of Rondônia, western Brazilian Amazon

Although viruses are well-established causes of acute gastroenteritis, few data on the circulation of these pathogens in Porto Velho, state of Rondônia, Brazil, are available. Thus, faecal samples from hospitalised diarrhoeic children, under six years of age, were collected and tested for the presence of norovirus (NoV), adenovirus (AdV) and astrovirus (AstV) from February 2010-February 2012. Specimens were screened by reverse-transcription polymerase chain reaction and viruses were found in 10.7% (63/591) of the cases. NoV, AdV and AstV were detected in 7.8%, 2% and 0.8% of the samples, respectively. NoV infection was observed at all ages and was most prevalent in zero-18-month-old children (84.7%; p = 0.002). A higher incidence of NoV was detected from February-April 2010, when it was found in 52.2% of the cases. Co-infections involving these viruses, rotavirus and enteropathogenic bacteria were detected in 44.4% (28/63) of the children with viral diarrhoea. Nosocomial infections were demonstrated in 28.6% (18/63) of the cases in which viruses were detected. The present paper reports, for the first time, the circulation of NoV and AstV among the paediatric population of Porto Velho and it contributes to our understanding of the roles of these pathogens in gastrointestinal infections

The prevalence of norovirus, astrovirus and adenovirus infections among hospitalised children with acute gastroenteritis in Porto Velho, state of Rondônia, western Brazilian Amazon

Submitted by EMERSON LEAL ([email protected]) on 2016-01-25T14:30:30Z No. of bitstreams: 1 The prevalence of norovirus, astrovirus and adenovirus infections.pdf: 493123 bytes, checksum: 57ff81944791914472827805bc4bcbb2 (MD5)Approved for entry into archive by EMERSON LEAL ([email protected]) on 2016-01-25T15:07:49Z (GMT) No. of bitstreams: 1 The prevalence of norovirus, astrovirus and adenovirus infections.pdf: 493123 bytes, checksum: 57ff81944791914472827805bc4bcbb2 (MD5)Made available in DSpace on 2016-01-25T15:07:49Z (GMT). No. of bitstreams: 1 The prevalence of norovirus, astrovirus and adenovirus infections.pdf: 493123 bytes, checksum: 57ff81944791914472827805bc4bcbb2 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Secretaria de Estado da Saúde. Hospital Infantil Cosme e Damião. Porto Velho, RO, Brasil.Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Instituto Evandro Chagas. Belém, PA, Brasil.Instituto Evandro Chagas. Belém, PA, Brasil.Instituto Evandro Chagas. Belém, PA, Brasil.Fundação Oswaldo Cruz. Porto Velho, RO, Brasil.Although viruses are well-established causes of acute gastroenteritis, few data on the circulation of these pathogens in Porto Velho, state of Rondônia, Brazil, are available. Thus, faecal samples from hospitalised diarrhoeic children, under six years of age, were collected and tested for the presence of norovirus (NoV), adenovirus (AdV) and astrovirus (AstV) from February 2010-February 2012. Specimens were screened by reverse-transcription poly¬merase chain reaction and viruses were found in 10.7% (63/591) of the cases. NoV, AdV and AstV were detected in 7.8%, 2% and 0.8% of the samples, respectively. NoV infection was observed at all ages and was most prevalent in zero-18-month-old children (84.7%; p = 0.002). A higher incidence of NoV was detected from February-April 2010, when it was found in 52.2% of the cases. Co-infections involving these viruses, rotavirus and enteropathogenic bacteria were detected in 44.4% (28/63) of the children with viral diarrhoea. Nosocomial infections were demonstrated in 28.6% (18/63) of the cases in which viruses were detected. The present paper reports, for the first time, the circulation of NoV and AstV among the paediatric population of Porto Velho and it contributes to our understanding of the roles of these pathogens in gastrointestinal infections