O infarto do miocárdio (IM), uma das principais causas de morte nas sociedades industrializadas, é sempre acompanhado por uma notável alteração da modulação autonômica, caracterizada por hiperatividade simpática e diminuição do tono parassimpático ao coração. O bloqueio da atuação do simpático cardíaco tem sido amplamente utilizado como estratégia terapêutica eficaz para redução da morbi-mortalidade em pacientes com IM. Entretanto, há evidências de que o restabelecimento da função parassimpática ao coração pode ser igualmente benéfica, uma vez que a diminuição do parassimpático cardíaco é um fator de risco independente de morte súbita. Assim, o objetivo do presente estudo foi avaliar a influência da inibição da acetilconinesterase plasmática (AChE), por meio da administração endovenosa do brometo de piridostigmina (PIR), sobre o eletrocardiograma (ECG), hemodinâmica e modulação autonômica apos o IM agudo em ratos. Ratos foram anestesiados com uretana e mantidos a uma temperatura de 36-37 ºC. Tiveram eletrodos subcutâneos para registro do ECG implantados, e a artéria e veia femoral cateterizadas para medida direta de PA e administração de drogas, respectivamente. Experimentos preliminares foram realizados para determinação de uma dose de PIR que não causasse grande repercussão hemodinâmica. A atividade da acetilcolinesterase plasmática, bem como o tono autonômico cardíaco também foram avaliados em ratos normais. Em outro protocolo, ratos anestesiados, sob registro contínuo do ECG e PA, tiveram o ramo descendente anterior da artéria coronária esquerda ligado para provocar um extenso IM e, após 10 min, foram tratados com PIR (0,25 mg/kg, i.v) ou salina (solução fisiológica 0,9%), e os registros foram acompanhados por 4 h. Ratos controles tiveram o tórax aberto, mas a artéria coronária foi mantida intacta. Ao final, os ratos tiveram o coração retirado para avaliação da extensão da isquemia miocárdica e para o estudo da conexina 43. A administração endovenosa de PIR foi efetiva em reduzir a atividade da AChE e provocou uma discreta redução da FC (438±8 para 387±10 bpm), sem alteração da PA. Ratos tratados com PIR tiveram menor tono simpático e maior tono vagal cardíaco que os ratos que receberam salina. O tratamento com PIR diminuiu a incidência de arritmias nos animais com IM e aumentou a porcentagem de ratos que sobreviveram até a 4ª hora após o infarto (72 vs 58% nos não tratados). A PIR também preveniu o aumento do intervalo QTc, observado após o IM em ratos não tratados (=-2±4, vs 33±13 ms). A quantidade de conexina 43 foi marcadamente reduzida pelo IM em ratos não tratados (0,7±0,1 vs 2,2±0,4 ua), redução esta que não ocorreu nos ratos com IM tratados com PIR (1,3±0,3 ua). Por fim, foi realizado um ensaio, in vitro, em cardiomiócitos da linhagem H9c2 em cultura, e foi observado que a PIR preveniu a degradação da Cx43 induzida por meio isquêmico durante 4 horas. Portanto, a administração aguda de PIR provocou uma bradicardia pouco intensa, sem repercussões hemodinâmicas importantes, aumentou o tono vagal cardíaco, preveniu o prolongamento do intervalo QTc, diminuiu a incidência de arritmias, e preveniu a degradação da Cx43 nos corações dos ratos infartados. / Myocardial infarction (MI), a leading cause of death in industrialized societies, is always accompanied by a remarkable change in cardiovascular autonomic modulation, characterized by sympathetic overactivity and decreased vagal tone to the heart. The blockade of the cardiac sympathetic activity has been widely used as an effective therapeutic strategy to reduce morbidity and mortality in patients with MI. However, there is evidence that improvement of parasympathetic function can also be beneficial since reduction of cardiac vagal function is an independent risk factor for sudden death. The aim of this study was to evaluate the influence of inhibition of plasma acetilconinesterase (AChE), by intravenous administration of pyridostigmine bromide (PYR) on the electrocardiogram (ECG), hemodynamics and cardiovascular autonomic modulation shortly after MI in rats. Rats were anesthetized with urethane and maintained on a heating pad. Subcutaneous electrodes to record the ECG were installed and catheters were inserted into femoral artery and vein for measurement of blood pressure and drug administration, respectively. Preliminary experiments were performed to determine a dose of PYR that would not cause major hemodynamic consequences. Plasma AChE activity, and cardiac autonomic tone were also evaluated in normal rats. Then, anesthetized rats under continuous recording of ECG and BP, had the anterior descending branch of the left coronary artery ligated to elicit extensive MI and, after 10 min, were treated with PYR (0.25 mg/kg, iv) or saline (0.9% NaCl) and monitored for the next 4 h. Control rats had the chest open, but coronary artery was kept intact. At the end, the rats had the heart removed to determine the size of myocardial isquemia and to study connexin 43. Intravenous administration of PYR was effective in reducing AChE activity of and caused a mild reduction in HR (438±8 to 387±10 bpm) with no change in BP. Also, rats treated with PYR had lower sympathetic and higher cardiac vagal tone as compared to untreated rats. The treatment with PYR decreased the incidence of arrhythmias after MI and increased the percentage of rats that survived until the 4th hour after infarction (72 vs 58 % in untreated). PYR also prevented the increase in QTc interval observed after MI in untreated rats (=-2±4 vs 33±13 ms). The amount of connexin 43 was markedly reduced by MI in untreated rats (2.24±0.46 vs 0.72±0.14 au), nevertheless, this reduction was not observed in infarcted rats that received PYR (1.31±0.29 au). Finally, an in vitro assay was performed on the line H9c2 cardiomyocytes in culture, and it was observed that PYR prevented the degradation of Cx43 induced by ischemic medium for 4 hours. Therefore, the acute administration of PYR caused mild bradycardia without hemodynamic repercussions, increased vagal tone, prevented the prolongation of the QTc interval and decreased the incidence of arrhythmias and prevented the degradation of Cx43 in infarcted rat hearts.

Os canais de junções comunicantes (gap junctions, JC) são formados por subunidades chamadas de conexinas (Cx). Estas proteínas têm papel relevante no acoplamento celular, participando da condutância de células nervosas ou gliais e modulando vários processos fisiológicos e fisiopatológicos. O objetivo deste trabalho é avaliar o envolvimento das conexinas na nocicepção aguda, por meio de ensaios comportamentais e estudos de mapeamento da sua expressão em células do gânglio da raiz dorsal de ratos. Para tanto, foi analisado o efeito de carbenoxolone (CBX) e quinina (bloqueadores de JCs), assim como de oligonucleotídeos antisense para conexinas 36 e 43 na indução e manutenção de hiperalgesia induzida por carragenina em ratos. Os resultados mostraram que a carragenina induz uma diminuição do limiar nociceptivo em ratos e que esse efeito hiperalgésico da carragenina foi bloqueado pelo tratamento com carbenoxolone (nas doses 20-50mg) e significantemente inibido por quinina (nas doses 20-50mg), sugerindo uma participação das junções comunicantes (JC) no processo. / Gap junctions channels (GJ) are formed by proteic subunits called connexins (Cx). These proteins have an important role in cellular coupling, participating in the conductance of glial and nerve cells or modulating various physiological and pathophysiological processes. The aim of this study is to evaluate Cx36 and Cx43 involvement in acute nociception through behavioral assays, mapping studies of its expression in rat dorsal root ganglion cells. For this purpose, we analyzed the effect of intrathecal treatment with carbenoxolone (CBX) and quinine (GJs blockers), as well as antisense oligonucleotides for connexins 36 and 43 in the induction and maintenance of carrageenan-induced hyperalgesia in rats. The results show that carrageenan induces a nociceptive threshold decrease in rats. The hyperalgesic effect was blocked by treatment with carbenoxolone (20-50mg doses), Cx43 antisense and inhibited significantly by quinine (at doses 20 -50mg) but no with Cx36 antisense, suggesting an involvement of gap junctions (JC) in the process.

As junções comunicantes são canais protéicos formados entre células adjacentes que permitem a passagem de moléculas e íons menores do que 1kDa; conexinas são proteínas que formam estas junções. Vêm sendo demonstradas na literatura a diminuição da capacidade de comunicação celular e alterações na expressão e/ou localização das conexinas em neoplasias. Este estudo foi realizado com o intuito de se verificar a influência da deleção de um dos alelos da Cx43 na carcinogênese pulmonar. Para tanto, camundongos geneticamente manipulados heterozigotos (Cx43± ou selvagens (Cx43±) de ambos os sexos receberam 3g/kg de uretana aos 15 e 17 dias de idade, e foram sacrificados após 25 semanas. As quantificações macro e microscópicas das lesões revelaram que os camundongos Cx43± apresentaram maior multiplicidade de adenomas pulmonares. Estes apresentavam também maior taxa de proliferação celular, avaliada pela quantificação de núcleos positivos para o PCNA. As expressões das Cxs 26, 32, 43 e 46, presentes no epitélio pulmonar, foram avaliadas por PCR em tempo real (RT-PCR) e por imunoistoquímica. A expressão da Cx43 revelou-se cerca de 50% menor em camundongos Cx43± quando comparada à dos correspondentes Cx43+/+, como esperado. Estudos in vitro mostraram que os pneumócitos de tipo II (APTII) extraídos de camundongos Cx43±, apresentaram capacidade de comunicação menor do que os APTII de camundongos Cx43+/+. Quando submetidos ao tratamento com uretana, a expressão de Cx 43 aumentou em 100% no tecido pulmonar. As demais Cxs tiveram a expressão reduzida pelo tratamento e não foram evidenciadas no epitélio pulmonar livre de lesões após o tratamento com uretana. Não foi detectada a expressão da Cx43 e da Cx32 nos adenomas provenientes dos camundongos Cx43±. A expressão das Cxs 26 e 46 foi correlacionada com o fenótipo papilífero das lesões. Constatou-se que a Cx32 acumulava-se no citoplasma das células epiteliais pulmonares e teve sua expressão, juntamente com a da Cx43, associada ao sexo, provavelmente contribuindo para a menor susceptibilidade das fêmeas aos adenomas induzidos pela uretana. Em conclusão, a redução da expressão da Cx43 conferiu maior susceptibilidade ao desenvolvimento de adenomas pulmonares pela uretana. Este foi o primeiro estudo in vivo mostrando a influência da deleção de um único alelo da Cx43 na carcinogênese / Gap junctions are communicating protein channels formed between adjacent cells that allow the exchange of molecules and ions smaller than 1kDa; connexins are proteins that form these junctions. Studies in the literature have been showing the lower level of cell communication capacity and alterations in the expression and/or localization of connexins in neoplasia. This study was performed to verify the influence of the deletion of one allele of Cx43 on lung carcinogenesis. Genetically manipulated heterozygous (Cx43Gap junctions are communicating protein channels formed between adjacent cells that allow the exchange of molecules and ions smaller than 1kDa; connexins are proteins that form these junctions. Studies in the literature have been showing the lower level of cell communication capacity and alterations in the expression and/or localization of connexins in neoplasia. This study was performed to verify the influence of the deletion of one allele of Cx43 on lung carcinogenesis. Genetically manipulated heterozygous (Cx43± or wild type mice (Cx43+/+) were injected with 3g/kg of at the age of 15 and 17 days and were euthanized after 25-weeks. Macroscopic and microscopic quantification of pulmonary lesions revealed that Cx43± mice presented higher multiplicity of pulmonary adenomas. These presented also a higher cell proliferation index, as evaluated by counting PCNA positive nuclei. Cxs 26, 32, 43 and 46 expressions in the pulmonary epithelium were investigated by Real-Time PCR and by immunohistochemistry. Cx43 expression was about 50% lower in Cx43± mice, in comparison to Cx43+/+ mice, as expected. In vitro studies showed that the APTII cells extracted from Cx43± mice presented a reduced communication capacity. When treated with urethane, the expression of Cx43 was increased by 100%. Other Cxs were down-regulated after the treatment with urethane, and were not observed lung areas devoid of adenomas after the treatment with urethane. Cx43 and Cx32 were not detected in Cx43± mouse adenomas. However, Cx26 and Cx46 were correlated with papillary lesions. Cx32 was cumulated in the cytoplasm of the lung epithelial cells and its expression, together Cx43, were associated with the sex, maybe contributing to the lower susceptibility of the female mice to urethane. In conclusion, the reduced expression of Cx43 determines a higher susceptibility to the development of pulmonary adenomas by urethane. This study was the first in vivo showing the influence of the deletion of one allele of Cx43± or wild type mice (Cx43+/+) were injected with 3g/kg of at the age of 15 and 17 days and were euthanized after 25-weeks. Macroscopic and microscopic quantification of pulmonary lesions revealed that Cx43± mice presented higher multiplicity of pulmonary adenomas. These presented also a higher cell proliferation index, as evaluated by counting PCNA positive nuclei. Cxs 26, 32, 43 and 46 expressions in the pulmonary epithelium were investigated by Real-Time PCR and by immunohistochemistry. Cx43 expression was about 50% lower in Cx43± mice, in comparison to Cx43+/+ mice, as expected. In vitro studies showed that the APTII cells extracted from Cx43± mice presented a reduced communication capacity. When treated with urethane, the expression of Cx43 was increased by 100%. Other Cxs were down-regulated after the treatment with urethane, and were not observed lung areas devoid of adenomas after the treatment with urethane. Cx43 and Cx32 were not detected in Cx43± mouse adenomas. However, Cx26 and Cx46 were correlated with papillary lesions. Cx32 was cumulated in the cytoplasm of the lung epithelial cells and its expression, together Cx43, were associated with the sex, maybe contributing to the lower susceptibility of the female mice to urethane. In conclusion, the reduced expression of Cx43 determines a higher susceptibility to the development of pulmonary adenomas by urethane. This study was the first in vivo showing the influence of the deletion of one allele of Cx43 in carcinogenesis

As junções comunicantes são canais protéicos formados entre células adjacentes que permitem a passagem de moléculas e íons menores do que 1kDa; conexinas são proteínas que formam estas junções. Vêm sendo demonstradas na literatura a diminuição da capacidade de comunicação celular e alterações na expressão e/ou localização das conexinas em neoplasias. Este estudo foi realizado com o intuito de se verificar a influência da deleção de um dos alelos da Cx43 na carcinogênese pulmonar. Para tanto, camundongos geneticamente manipulados heterozigotos (Cx43± ou selvagens (Cx43±) de ambos os sexos receberam 3g/kg de uretana aos 15 e 17 dias de idade, e foram sacrificados após 25 semanas. As quantificações macro e microscópicas das lesões revelaram que os camundongos Cx43± apresentaram maior multiplicidade de adenomas pulmonares. Estes apresentavam também maior taxa de proliferação celular, avaliada pela quantificação de núcleos positivos para o PCNA. As expressões das Cxs 26, 32, 43 e 46, presentes no epitélio pulmonar, foram avaliadas por PCR em tempo real (RT-PCR) e por imunoistoquímica. A expressão da Cx43 revelou-se cerca de 50% menor em camundongos Cx43± quando comparada à dos correspondentes Cx43+/+, como esperado. Estudos in vitro mostraram que os pneumócitos de tipo II (APTII) extraídos de camundongos Cx43±, apresentaram capacidade de comunicação menor do que os APTII de camundongos Cx43+/+. Quando submetidos ao tratamento com uretana, a expressão de Cx 43 aumentou em 100% no tecido pulmonar. As demais Cxs tiveram a expressão reduzida pelo tratamento e não foram evidenciadas no epitélio pulmonar livre de lesões após o tratamento com uretana. Não foi detectada a expressão da Cx43 e da Cx32 nos adenomas provenientes dos camundongos Cx43±. A expressão das Cxs 26 e 46 foi correlacionada com o fenótipo papilífero das lesões. Constatou-se que a Cx32 acumulava-se no citoplasma das células epiteliais pulmonares e teve sua expressão, juntamente com a da Cx43, associada ao sexo, provavelmente contribuindo para a menor susceptibilidade das fêmeas aos adenomas induzidos pela uretana. Em conclusão, a redução da expressão da Cx43 conferiu maior susceptibilidade ao desenvolvimento de adenomas pulmonares pela uretana. Este foi o primeiro estudo in vivo mostrando a influência da deleção de um único alelo da Cx43 na carcinogênese / Gap junctions are communicating protein channels formed between adjacent cells that allow the exchange of molecules and ions smaller than 1kDa; connexins are proteins that form these junctions. Studies in the literature have been showing the lower level of cell communication capacity and alterations in the expression and/or localization of connexins in neoplasia. This study was performed to verify the influence of the deletion of one allele of Cx43 on lung carcinogenesis. Genetically manipulated heterozygous (Cx43Gap junctions are communicating protein channels formed between adjacent cells that allow the exchange of molecules and ions smaller than 1kDa; connexins are proteins that form these junctions. Studies in the literature have been showing the lower level of cell communication capacity and alterations in the expression and/or localization of connexins in neoplasia. This study was performed to verify the influence of the deletion of one allele of Cx43 on lung carcinogenesis. Genetically manipulated heterozygous (Cx43± or wild type mice (Cx43+/+) were injected with 3g/kg of at the age of 15 and 17 days and were euthanized after 25-weeks. Macroscopic and microscopic quantification of pulmonary lesions revealed that Cx43± mice presented higher multiplicity of pulmonary adenomas. These presented also a higher cell proliferation index, as evaluated by counting PCNA positive nuclei. Cxs 26, 32, 43 and 46 expressions in the pulmonary epithelium were investigated by Real-Time PCR and by immunohistochemistry. Cx43 expression was about 50% lower in Cx43± mice, in comparison to Cx43+/+ mice, as expected. In vitro studies showed that the APTII cells extracted from Cx43± mice presented a reduced communication capacity. When treated with urethane, the expression of Cx43 was increased by 100%. Other Cxs were down-regulated after the treatment with urethane, and were not observed lung areas devoid of adenomas after the treatment with urethane. Cx43 and Cx32 were not detected in Cx43± mouse adenomas. However, Cx26 and Cx46 were correlated with papillary lesions. Cx32 was cumulated in the cytoplasm of the lung epithelial cells and its expression, together Cx43, were associated with the sex, maybe contributing to the lower susceptibility of the female mice to urethane. In conclusion, the reduced expression of Cx43 determines a higher susceptibility to the development of pulmonary adenomas by urethane. This study was the first in vivo showing the influence of the deletion of one allele of Cx43± or wild type mice (Cx43+/+) were injected with 3g/kg of at the age of 15 and 17 days and were euthanized after 25-weeks. Macroscopic and microscopic quantification of pulmonary lesions revealed that Cx43± mice presented higher multiplicity of pulmonary adenomas. These presented also a higher cell proliferation index, as evaluated by counting PCNA positive nuclei. Cxs 26, 32, 43 and 46 expressions in the pulmonary epithelium were investigated by Real-Time PCR and by immunohistochemistry. Cx43 expression was about 50% lower in Cx43± mice, in comparison to Cx43+/+ mice, as expected. In vitro studies showed that the APTII cells extracted from Cx43± mice presented a reduced communication capacity. When treated with urethane, the expression of Cx43 was increased by 100%. Other Cxs were down-regulated after the treatment with urethane, and were not observed lung areas devoid of adenomas after the treatment with urethane. Cx43 and Cx32 were not detected in Cx43± mouse adenomas. However, Cx26 and Cx46 were correlated with papillary lesions. Cx32 was cumulated in the cytoplasm of the lung epithelial cells and its expression, together Cx43, were associated with the sex, maybe contributing to the lower susceptibility of the female mice to urethane. In conclusion, the reduced expression of Cx43 determines a higher susceptibility to the development of pulmonary adenomas by urethane. This study was the first in vivo showing the influence of the deletion of one allele of Cx43 in carcinogenesis

Conexinas são proteínas que compõem as junções comunicantes do tipo gap, e a diminuição na sua expressão tem sido relacionada com diversas alterações fisiológicas, entre elas algumas síndromes, malformações genéticas, o aumento da proliferação celular e a carcinogênese. Dentre as isoformas das conexinas presentes nos tecidos animais, a Cx43 é a mais abundante e a mais estudada, tendo a sua importância relatada in vivo em camundongos que tiveram um dos alelos de Cx43 deletado (Cx43+/-), devido a morte desses animais logo após o nascimento devido a malformações cardíacas. Considerando o fato de esse gene ser expresso em dezenas de tipos celulares, tivemos como objetivo avaliar os outros tecidos em busca de anomalias ocorridas durante o desenvolvimento, e a possível interferência do background gentético. Para isso acompanhamos dia-a-dia o último terço gestacional de camundongos de background C57BL/6 e CD1, avaliando histologica e morfologicamente os fetos em busca de anomalias nos animais Cx43+/- e Cx43-/- em relação aos animais Cx43+/+. Exceto pelo tecido ósseo, não encontramos alterações nos órgãos que expressam esse gene, bem como alterações causadas pelo refluxo de sangue causado pela malformação da válvula tricúspide. Durante a osteogênese, por meio da avaliação das costelas e tíbia, percebemos um retardo no desenvolvimento, que se agrava conforme a deficiência do gene Cx43. Percebemos nitidamente que o processo de diferenciação celular ocorre de maneira menos eficiente, atrasando processos como deposição de colágeno e de matriz óssea. Conclui-se que a Cx43 é importante para o desenvolvimento ósseo na fase fetal em camundongos. / Connexins are proteins that compose the gap junctions, and the reduction in its expression has been related with diverse physiological alterations, like some syndromes, malformations, the increase of the cellular proliferation and carcinogenesis. Among isoforms of the connexins in animal cells, the Cx43 is the most abundant and studied, having its importance been shown up in alive mice that had one allele of Cx43 (Cx43+/-) deleted. REAUME et al. related that Cx43-/- mice presented cardiac malformation and died immediately after birth. Considering the fact that this gene is expressed in many cell types, we evaluate the possibility of other tissues also to present alterations during the fetal development. Due to this, we studied the mouse development initiating in 12.5 to 19.5 DE (embryologic day) and evaluated the histology of C57BL/6 and CD1 mice searching for anomalies of Cx43+/- and Cx43-/- mice in relation to the Cx43+/+ animals. We did not find alterations in the main organs that express Cx43, nor alterations due to blood out flow related to cardiac malformations. We only found significant difference was the bones; through the evaluation of the ribs and tibia. It has been observed a delay in the development, that was more important in Cx43 knockout mice. We observed clearly that the process of cellular differentiation occurs in less efficient way, delaying processes as deposition of collagen and bone matrix. In conclusion, this study showed that Cx43 is important for bone development in mice.

Duzentos e quarenta e cinco neoplasias de glândula perianal de cães dos arquivos do Departamento de Patologia da FMVZ/ USP, de 1984 à 2004, foram revisadas histologicamente. A grande maioria dos casos (34%) foi classificada como adenoma moderadamente diferenciado, grupo II, em machos com mais de oito anos de idade o que reflete a dependência androgênica dessas neoplasias. A análise quantitativa da proliferação celular e apoptose nos diferentes tipos histológicos de neoplasias, hiperplasia e tecido normal dessas glândulas determinou um padrão paralelo de aumento de ambas as quantificações. Com os resultados do índice de crescimento ajustado obtivemos que, embora os carcinomas tenham um nível de proliferação celular muito maior que os adenomas (grupo I), esses têm um potencial de crescimento maior, levando-se em conta a apoptose. Investigamos, também, a expressão de Cx43, 26 e 32 nessas glândulas perianais com métodos imunoistoquímicos. A Cx 43 expressava-se em glândulas perianais normais, hiperplásicas, adenomas (grupo I) e adenomas moderadamente diferenciados. Nos adenomas pouco diferenciados (grupo II), a expressão estava reduzida e não se expressava nos carcinomas (grupo III). A Cx 26 acumulava-se no citoplasma nas glândulas normais, hiperplásicas, adenomas (grupo I) e adenomas moderadamente diferenciados (grupo II). Nos adenomas pouco diferenciados (grupo II), a expressão estava reduzida e, ausente, nos carcinomas. A Cx 32 não foi identificada em nenhum dos grupos (I, II e III) ou glândulas normais e hiperplásicas. Concluindo, Cx 43 e Cx26 são importantes para a homeostasia de glândula perianal normal, podendo estar associadas aos receptores de andrógenos presentes em suas células. Este foi o primeiro estudo mostrando a apoptose e sua influência na fase promocional da carcinogênese e, também, o primeiro estudo mostrando a expressão de Cx43, Cx 26 citoplasmática e ausência de expressão da Cx 32 em glândulas perianais normais, hiperplásicas e neoplásicas em cães / Two hundred and forty five neoplasms of the perianal glands of dogs from the archives of the Department of Pathology of the FMVZ/USP, 1984 to 2004, have been reviewed hystologically. Most of the cases (34%) were classified as moderately differentiated adenomas, group II, in males over 8 years of age, which showed an androgenic dependence of these neoplasms. The quantitative analysis of the cellular proliferation and apoptosis, in the different hystologic types of neoplasia, hyperplasia and normal tissue of these glands, determined a parallel pattern of increase in both quantifications. The values of the net growth index showed that although carcinomas have a much higher level of proliferation than adenomas (group I), these latter have a much higher potential of growth, taking into account the effect of the apoptosis. The occurrence of Cx 43, 26 and 32 in these perianal glands was also investigated by immunohystochemical methods. Cx 43 expression was present in normal, hyperplasic, adenomas (group I) and moderately differentiated adenomas perianal glands. In poorly differentiated adenomas (group II), the expression was reduced and was absent in carcinomas. Cx 26 was accumulated in the cytoplasm in normal, hyperplasic, adenomas (group I) and moderately differentiated adenomas glands. In poorly differentiated adenomas (group II), the expression was reduced and was absent in carcinomas. Cx 32 was not found in all groups (I, II, III), normal and hyperplasic glands. In conclusion, Cx 43 and Cx 26 are important for homeostasis of normal canine perianal glands, being able to be associated with the androgen receptors present in their cells. This was the first study showing the apoptosis and its influence on the promotional phase of carcinogenesis and, also, the first study showing the occurrence of Cx 43, cytoplasmatic Cx 26 and no expression of Cx 32 in normal, hyperplasic and neoplastic canine perianal glands

Atualmente, as pesquisas sobre a as células epiteliais biliares expressam uma grande quantidade de conexina 43. O presente estudo avaliou influência do genótipo de camundongos Cx 43 +/- ou Cx 43 +/+, sobre a hiperplasia de ductos biliares, após a ligação do ducto biliar comum (colédoco). Neste estudo, foram utilizados seis grupos de camundongos BALB/c fêmeas de ambos os genótipos, que foram submetidos à ligação do ducto biliar com fio de seda por meio de procedimento cirúrgico, sendo eutanasiados nos tempos de 48 horas, 7 e 14 dias. Aproveitando-se o campo microscópico, cinco estruturas hepáticas: hepatócitos, arteríolas, vênulas, ductos biliares e veia centrolobular foram quantificados para verificar a existência de possível diferença entre os genótipos. Observou-se na análise quantitativa, diferença significante somente quanto ao número de vasos sanguíneos entre os dois grupos, sendo que as outras estruturas hepáticas quantificadas não apresentaram diferenças significativas. A proliferação celular foi avaliada pela técnica western blot para a proteína PCNA (Proliferation Cell Nuclear Antigen), somente dos grupos de 48 horas pós-cirúrgico, quando ocorre o pico de proliferação. Quanto à análise quantitativa do western blot, não houve diferenças significativas quanto ao genótipo. Já na das bandas protéicas, observou-se maior concentração nos camundongos controle, indicando maior número de células em divisão. / The goal in this study was evidence, after duct biliar common ligation (coledoco), using mice geneticly modified with connection in one of the alels to connexin 43 (Cx 43+/-) and comparing them to the group control (Cx 43+/+), in order to find out the participation of this protein, even though the epithelials biliary cells express a big quantity of cx 43. The experimental outline were made up of three mice groups BALB/c females of different genotypes which were subdued to surgery procedure using a wire of silk 3.0 and euthanased in each 48 hours during 7 to 14 days. Also using the microscopic view the fives hepatics structures such as, hepatocites, arterioles, veins, bile ductules and centrelobular vein were quantified, trying to obtain possible differences among the genetypes and to do the qualification of protein concentration was used the western blot to PCNA in order to compare the cells divided in 48 hours. It has observed in the quantitative analysis, strong difference only in the blood vein number among the two groups, however the orders quantitative hepatics structures did not present strong differences. About the quantitative analysis related to western blot did not have strong differences in the genotype, and finally in the proteic staff it has observed bigger concentration in the mice control showing bigger number of cells in division.

Duzentos e quarenta e cinco neoplasias de glândula perianal de cães dos arquivos do Departamento de Patologia da FMVZ/ USP, de 1984 à 2004, foram revisadas histologicamente. A grande maioria dos casos (34%) foi classificada como adenoma moderadamente diferenciado, grupo II, em machos com mais de oito anos de idade o que reflete a dependência androgênica dessas neoplasias. A análise quantitativa da proliferação celular e apoptose nos diferentes tipos histológicos de neoplasias, hiperplasia e tecido normal dessas glândulas determinou um padrão paralelo de aumento de ambas as quantificações. Com os resultados do índice de crescimento ajustado obtivemos que, embora os carcinomas tenham um nível de proliferação celular muito maior que os adenomas (grupo I), esses têm um potencial de crescimento maior, levando-se em conta a apoptose. Investigamos, também, a expressão de Cx43, 26 e 32 nessas glândulas perianais com métodos imunoistoquímicos. A Cx 43 expressava-se em glândulas perianais normais, hiperplásicas, adenomas (grupo I) e adenomas moderadamente diferenciados. Nos adenomas pouco diferenciados (grupo II), a expressão estava reduzida e não se expressava nos carcinomas (grupo III). A Cx 26 acumulava-se no citoplasma nas glândulas normais, hiperplásicas, adenomas (grupo I) e adenomas moderadamente diferenciados (grupo II). Nos adenomas pouco diferenciados (grupo II), a expressão estava reduzida e, ausente, nos carcinomas. A Cx 32 não foi identificada em nenhum dos grupos (I, II e III) ou glândulas normais e hiperplásicas. Concluindo, Cx 43 e Cx26 são importantes para a homeostasia de glândula perianal normal, podendo estar associadas aos receptores de andrógenos presentes em suas células. Este foi o primeiro estudo mostrando a apoptose e sua influência na fase promocional da carcinogênese e, também, o primeiro estudo mostrando a expressão de Cx43, Cx 26 citoplasmática e ausência de expressão da Cx 32 em glândulas perianais normais, hiperplásicas e neoplásicas em cães / Two hundred and forty five neoplasms of the perianal glands of dogs from the archives of the Department of Pathology of the FMVZ/USP, 1984 to 2004, have been reviewed hystologically. Most of the cases (34%) were classified as moderately differentiated adenomas, group II, in males over 8 years of age, which showed an androgenic dependence of these neoplasms. The quantitative analysis of the cellular proliferation and apoptosis, in the different hystologic types of neoplasia, hyperplasia and normal tissue of these glands, determined a parallel pattern of increase in both quantifications. The values of the net growth index showed that although carcinomas have a much higher level of proliferation than adenomas (group I), these latter have a much higher potential of growth, taking into account the effect of the apoptosis. The occurrence of Cx 43, 26 and 32 in these perianal glands was also investigated by immunohystochemical methods. Cx 43 expression was present in normal, hyperplasic, adenomas (group I) and moderately differentiated adenomas perianal glands. In poorly differentiated adenomas (group II), the expression was reduced and was absent in carcinomas. Cx 26 was accumulated in the cytoplasm in normal, hyperplasic, adenomas (group I) and moderately differentiated adenomas glands. In poorly differentiated adenomas (group II), the expression was reduced and was absent in carcinomas. Cx 32 was not found in all groups (I, II, III), normal and hyperplasic glands. In conclusion, Cx 43 and Cx 26 are important for homeostasis of normal canine perianal glands, being able to be associated with the androgen receptors present in their cells. This was the first study showing the apoptosis and its influence on the promotional phase of carcinogenesis and, also, the first study showing the occurrence of Cx 43, cytoplasmatic Cx 26 and no expression of Cx 32 in normal, hyperplasic and neoplastic canine perianal glands

Ca2+, a signal for death and life, is closely involved in the regulation of numerous important cellular events. Ca2+ carries out its function through its binding to Ca2+-receptors or Ca2+-binding proteins. The EF-hand protein, with a helix-loop-helix Ca2+-binding motif, constitutes one of the largest protein families. To facilitate our understanding of the role of Ca2+ in biological systems (denoted as calciomics) using genomic information, an improved pattern search method (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm) for the identification of EF-hand and EF-like Ca2+-binding proteins was developed. This fast and robust method allows us to analyze putative EF-hand proteins at the genome-wide level and further visualize the evolutionary scenario of the EF-hand protein family. This prediction method further enables us to locate a putative viral EF-hand Ca2+-binding motif within the rubella virus nonstructural protease that cleaves the nonstructural protein precursor into two active replicase components. A novel grafting approach has been used to probe the metal-binding properties of this motif by engineering the predicted 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2 domain 1. Structural and conformational studies were further performed on a purified, bacterially-expressed NS protease minimal metal-binding domain spanning the Zn2+- and EF-hand Ca2+-binding motif. It was revealed that Ca2+ binding induced local conformational changes and increased thermal stability. Furthermore, functional studies were carried out using RUB infectious cDNA clone and replicon constructs. Our studies have shown that the Ca2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. In addition, we have predicted and characterized a calmodulin-binding domain in the gap junction proteins connexin43 and connexin44. Peptides encompassing the CaM binding motifs were synthesized and their ability to bind CaM was determined using various biophysical approaches. Transient expression in HeLa cells of two mutant Cx43-EYFP constructs without the putative CaM-binding site eliminated the Ca2+-dependent inhibition of gap junction permeability. These results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 and Cx44 in a Ca2+-dependent manner, providing a molecular basis for the well-characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

No description available.