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Blood coagulation is a vital process for humans and other species. Following an injury to a blood vessel, a cascade of molecular signals is transmitted, inhibiting and activating more than a dozen coagulation factors and resulting in the formation of a fibrin clot that ceases the bleeding. In this process, the Coagulation factor V (FV) is a master regulator, coordinating critical steps of this process. Mutations to this factor result in spontaneous bleeding episodes and prolonged hemorrhage after trauma or surgery. Although the role of FV is well characterized, it is unclear how single-point mutations affect its structure. In this study, to understand the effect of mutations, we created a detailed network map of this protein, where each node is a residue, and two residues are connected if they are in close proximity in the three-dimensional structure. Overall, we analyzed 63 point-mutations from patients and identified common patterns underlying FV deficient phenotypes. We used structural and evolutionary patterns as input to machine learning algorithms to anticipate the effects of mutations and anticipated FV-deficiency with fair accuracy. Together, our results demonstrate how clinical features, genetic data and in silico analysis are converging to enhance treatment and diagnosis of coagulation disorders.
Assuntos
Fator V , Mutação Puntual , Humanos , Mutação , Algoritmos , Evolução BiológicaRESUMO
Introduction: Blood coagulation is an essential process to cease bleeding in humans and other species. This mechanism is characterized by a molecular cascade of more than a dozen components activated after an injury to a blood vessel. In this process, the coagulation factor VIII (FVIII) is a master regulator, enhancing the activity of other components by thousands of times. In this sense, it is unsurprising that even single amino acid substitutions result in hemophilia A (HA)-a disease marked by uncontrolled bleeding and that leaves patients at permanent risk of hemorrhagic complications. Methods: Despite recent advances in the diagnosis and treatment of HA, the precise role of each residue of the FVIII protein remains unclear. In this study, we developed a graph-based machine learning framework that explores in detail the network formed by the residues of the FVIII protein, where each residue is a node, and two nodes are connected if they are in close proximity on the FVIII 3D structure. Results: Using this system, we identified the properties that lead to severe and mild forms of the disease. Finally, in an effort to advance the development of novel recombinant therapeutic FVIII proteins, we adapted our framework to predict the activity and expression of more than 300 in vitro alanine mutations, once more observing a close agreement between the in silico and the in vitro results. Discussion: Together, the results derived from this study demonstrate how graph-based classifiers can leverage the diagnostic and treatment of a rare disease.
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COVID-19 has widely spread around the world, impacting the health systems of several countries in addition to the collateral damage that societies will face in the next years. Although the comparison between countries is essential for controlling this disease, the main challenge is the fact of countries are not simultaneously affected by the virus. Therefore, from the COVID-19 dataset by the Johns Hopkins University Center for Systems Science and Engineering, we present a temporal analysis on the number of new cases and deaths among countries using artificial intelligence. Our approach incrementally models the cases using a hierarchical clustering that emphasizes country transitions between infection groups over time. Then, one can compare the current situation of a country against others that have already faced previous waves. By using our approach, we designed a transition index to estimate the most probable countries' movements between infectious groups to predict next wave trends. We draw two important conclusions: (1) we show the historical infection path taken by specific countries and emphasize changing points that occur when countries move between clusters with small, medium, or large number of cases; (2) we estimate new waves for specific countries using the transition index.
Assuntos
Inteligência Artificial , COVID-19/epidemiologia , Previsões/métodos , Análise por Conglomerados , Bases de Dados Factuais , Humanos , PandemiasRESUMO
OBJECTIVE: The aim of the present study was to evaluate the effects of myoblast inoculation in combination with photobiomodulation therapy (PBMT) on skeletal muscle tissue following injury. MATERIALS AND METHODS: Sixty-five Wistar rats were divided into five groups: Control-animals not submitted to any procedure; Injury-cryoinjury of the tibialis anterior muscle; HBSS-animals submitted to cryoinjury and intramuscular Hank's Balanced Salt Solution; Injury + Cells-animals submitted to cryoinjury, followed by myogenic precursor cells (C2C12) transplantation; Injury + Cells + LLLT-animals submitted to cryoinjury, followed by myogenic precursor cells (C2C12) transplantation and PBMT (780 nm, 40 mW, 3.2 J in 8 points). The periods analyzed were 1, 3, and 7 days. The tibialis anterior muscle was harvest for histological analysis, collagen analysis, and immunolabeling of macrophages. RESULTS: No differences were found between the HBSS group and injury group. The Injury + Cells group exhibited an increase of inflammatory cells and immature fibers as well as a decrease in the number of macrophages on Day 1. The Injury + Cells + LLLT group exhibited a decrease in myonecrosis and inflammatory infiltrate at 7 days, but an increase in inflammatory infiltrate at 1 and 3 days as well as an increase in blood vessels at 3 and 7 days, an increase in macrophages at 3 days and better collagen organization at 7 days. CONCLUSION: Cell transplantation combined with PBMT led to an increase in the number of blood vessels, a reduction in myonecrosis and total inflammatory cells as well as better organization of collagen fibers during the skeletal muscle repair process. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.
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This study aimed to assess the effect of different breeds and crosses of broiler chickens and sex on growth, carcass characteristics, and tissue composition. The experimental design was a completely randomized design in a 6 × 2 factorial scheme, with six genotypes (New Hampshire NHS, Jersey Black Giant JBG, Rodhe Island Red RIR, Indigenous Giant IG, and the hybrids IG × NHS and IG × JBG) and two sexes (male and female). Each treatment was composed of five replications with three broiler chickens, totaling 180 broiler chickens. Zootechnical performance and growth were assessed through the Gompertz equation parameters. After slaughtering at 105 days, the characteristics of carcass and main cuts were determined by means of weight, yield, and proportion of tissue constituents (meat, bone, and skin). Regarding the Gompertz equation parameters, the genotype NHS presented a higher growth potential. Males presented higher live weight and carcass weights, except for IG. No difference was observed between genotypes and sexes (P > 0.05) for carcass yield. The genotype JBG presented the highest average for breast yield (24.4%). Males presented the highest average for leg yield (30.5%). The genotype IG presented the highest value of breast meat yield (71.2%), being similar to the genotypes from the crosses IG × NHS, and IG × JBG (68.9 and 68.3%, respectively). For meat to...(AU)
Objetivou-se avaliar o efeito de diferentes raças e cruzamentos de frangos e do sexo sobre as características de crescimento, carcaça e composição dos tecidos. O delineamento foi inteiramente casualizado disposto em esquema fatorial (6x2), sendo seis genótipos (New Hampshire - NHS; Gigante Negra de Jersey - GNJ; Rodhe Island Red - RIR; Índio Gigante - IG; cruzamento entre as raçasIG e NHS - IG x NHS e entre IG e GNJ - IG x GNJ) e dois sexos (macho e fêmea), com cinco repetições e três aves por repetição, totalizando 180 aves. Foi avaliado o desempenho zootécnico e o crescimento, através de parâmetros da equação de gompertz. Após o abate, que ocorreu aos 105 dias, foram determinadas as características de carcaça e dos principais cortes através do peso, do rendimento e da proporção dos constituentes teciduais (carne, osso e pele). Com relação aos parâmetros da equação de gompertz, as aves da raça NHS apresentaram maior potencial de crescimento. Os machos apresentaram médias superiores de peso vivo e de carcaça, exceto para o genótipo IG. Não houve diferença entre genótipos e sexos (P > 0,05) para o rendimento de carcaça. O genótipo GNJ apresentou a maior média de rendimento de peito (24,4 %). Para o rendimento de perna, os machos apresentaram a maior média (30,5 %). A raça IG apresentou maior valor de rendimento de carne do peito (71,2 %), sendo semelhante aos genótipos...(AU)
Assuntos
Animais , Galinhas/crescimento & desenvolvimento , Cruzamento , Hibridização Genética , Aves Domésticas/crescimento & desenvolvimento , Composição CorporalRESUMO
This study aimed to assess the effect of different breeds and crosses of broiler chickens and sex on growth, carcass characteristics, and tissue composition. The experimental design was a completely randomized design in a 6 × 2 factorial scheme, with six genotypes (New Hampshire NHS, Jersey Black Giant JBG, Rodhe Island Red RIR, Indigenous Giant IG, and the hybrids IG × NHS and IG × JBG) and two sexes (male and female). Each treatment was composed of five replications with three broiler chickens, totaling 180 broiler chickens. Zootechnical performance and growth were assessed through the Gompertz equation parameters. After slaughtering at 105 days, the characteristics of carcass and main cuts were determined by means of weight, yield, and proportion of tissue constituents (meat, bone, and skin). Regarding the Gompertz equation parameters, the genotype NHS presented a higher growth potential. Males presented higher live weight and carcass weights, except for IG. No difference was observed between genotypes and sexes (P > 0.05) for carcass yield. The genotype JBG presented the highest average for breast yield (24.4%). Males presented the highest average for leg yield (30.5%). The genotype IG presented the highest value of breast meat yield (71.2%), being similar to the genotypes from the crosses IG × NHS, and IG × JBG (68.9 and 68.3%, respectively). For meat to...
Objetivou-se avaliar o efeito de diferentes raças e cruzamentos de frangos e do sexo sobre as características de crescimento, carcaça e composição dos tecidos. O delineamento foi inteiramente casualizado disposto em esquema fatorial (6x2), sendo seis genótipos (New Hampshire - NHS; Gigante Negra de Jersey - GNJ; Rodhe Island Red - RIR; Índio Gigante - IG; cruzamento entre as raçasIG e NHS - IG x NHS e entre IG e GNJ - IG x GNJ) e dois sexos (macho e fêmea), com cinco repetições e três aves por repetição, totalizando 180 aves. Foi avaliado o desempenho zootécnico e o crescimento, através de parâmetros da equação de gompertz. Após o abate, que ocorreu aos 105 dias, foram determinadas as características de carcaça e dos principais cortes através do peso, do rendimento e da proporção dos constituentes teciduais (carne, osso e pele). Com relação aos parâmetros da equação de gompertz, as aves da raça NHS apresentaram maior potencial de crescimento. Os machos apresentaram médias superiores de peso vivo e de carcaça, exceto para o genótipo IG. Não houve diferença entre genótipos e sexos (P > 0,05) para o rendimento de carcaça. O genótipo GNJ apresentou a maior média de rendimento de peito (24,4 %). Para o rendimento de perna, os machos apresentaram a maior média (30,5 %). A raça IG apresentou maior valor de rendimento de carne do peito (71,2 %), sendo semelhante aos genótipos...
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Animais , Cruzamento , Galinhas/crescimento & desenvolvimento , Hibridização Genética , Aves Domésticas/crescimento & desenvolvimento , Composição CorporalRESUMO
Objective: To evaluate the effect of glide path creation on transportation promoted by NiTi and M-Wire instruments. Material and Methods: Sixty polyester resin blocks containing a simulated root canal were distributed into four groups (n=15), according to the protocols/systems used for root canal preparation: GPR group - glide path + Revo-S system; R group - no glide path + Revo-S system; GPPN group - glide path + ProTaper Next system and PN group - no glide path + ProTaper Next system. Root canals were photographed before and after preparation, and the images were superimposed to evaluate the transportation at the apical, middle and coronal thirds. The time spent to perform preparation was also measured (seconds). Data were submitted to the non-parametric Kruskal-Wallis test (p0.05). GPR and R groups were similar in the middle third (p>0.05). However, the transportation value in GPR group was statistically higher in comparison with GPPN and PN groups (p0.05). Only R group presented significant difference in comparison with GPPN and PN groups (p0.05). Conclusion: None of the systems were capable of maintaining the original trajectory of the simulated root canal, and the glide path had no effect on the transportation promoted by instruments. (AU)
Objetivo: Avaliar o efeito do glide path no transporte promovido por instrumentos fabricados em NiTi e M-Wire. Material e Métodos: Sessenta blocos de resina poliéster contendo um canal simulado foram separados em quatro grupos (n=15), de acordo com os protocolos/ sistemas utilizados para realização do preparo: Grupo GPR - glide path + sistema Revo-S; Grupo R - sem glide path + sistema Revo-S; Grupo GPPN - glide path + sistema ProTaper Next e Grupo PN - sem glide path + sistema ProTaper Next. Os canais foram fotografados antes e após o preparo, e as imagens sobrepostas para avaliação do transporte nos terços apical, médio e cervical. O tempo para realização do preparo também foi mensurado (segundos). Os dados foram submetidos ao teste não-paramétrico de Kruskal-Wallis (p0,05). Os grupos GPR e R foram semelhantes no terço médio (p>0,05), entretanto, o transporte no grupo GPR foi estatisticamente maior em comparação aos grupos GPPN e P (p0,05). Somente o grupo R apresentou diferença em comparação aos grupos GPPN e PN (p0,05). Conclusão: Nenhum sistema foi capaz de manter a trajetória original do canal simulado, e o glide path não teve efeito sobre o transporte promovido pelos instrumentos.(AU)
Assuntos
Instrumentos Odontológicos , Endodontia , Preparo de Canal RadicularRESUMO
O objetivo deste estudo foi verificar a contagem de mesófilos, coliformes totais, Staphylococcus aureus, Escherichia coli e presença de Salmonella sp. em cortes de traseiro de bovinos obtidos em um abatedouro frigorífico sob Inspeção Federal. Os resultados indicaram que todos os cortes mostraram-se ausentes para Salmonella sp. e a maior média de contagem para mesófilos foi no Filé de costela, seguido pela Picanha, Coxão mole, Fralda, Pera (recorte do Coxão Mole) e Maminha. Destes, a Pera e a Picanha apresentaram os maiores valores para Staphylococcus aureus. As maiores médias de contagens em UFC/g para coliformes totais foram nos cortes: Coxão mole, Alcatra, Pera e Filé-Mignon. De forma geral, os níveis de coliformes totais foram baixos, porém revelaram a presença de Escherichia coli principalmente no Coxão Mole. A Alcatra apresentou resultados elevados para coliformes totais, porém, sem a presença de E. coli. Os resultados dos cortes coletados mostraram que não houve desvio aparente nos processos quanto às operações do abate, a desossa e refile.
The aim of this study was to verify the mesophilic count, total coliforms, Staphylococcus aureus, Escherichia coli and the presence of Salmonella sp. in cattle rear cuts obtained in a slaughterhouse governed under Federal Inspection. The results indicated that all the cuts proved to be absent for Salmonella sp. and the highest average count for mesophilic was in rib steak, followed by Top Sirloin Cap, Topside, Flank Steak, Cut of Topside and Tenderloin. Of these, Cut of Topside and Top Sirloin Cap showed the highest values for S. aureus. The highest average scores in CFU/g for total coliforms were in cuts: Topside, Rump Steak, Cut of Topside and Tenderloin. The levels of total coliforms were low and showed the presence of Escherichia coli in the Topside. The results showed high thighs of coliforms, but without the presence of E. coli. The results of the collected cuttings showed no apparent shift in processes and operations during slaughter and boning and trimming.
Assuntos
Animais , Bovinos , Matadouros , Alimentos Resfriados , Carne/microbiologia , Salmonella , Vigilância Sanitária , Análise de Componente Principal , Escherichia coli , Microbiologia de AlimentosRESUMO
O objetivo deste estudo foi verificar a contagem de mesófilos, coliformes totais, Staphylococcus aureus, Escherichia coli e presença de Salmonella sp. em cortes de traseiro de bovinos obtidos em um abatedouro frigorífico sob Inspeção Federal. Os resultados indicaram que todos os cortes mostraram-se ausentes para Salmonella sp. e a maior média de contagem para mesófilos foi no Filé de costela, seguido pela Picanha, Coxão mole, Fralda, Pera (recorte do Coxão Mole) e Maminha. Destes, a Pera e a Picanha apresentaram os maiores valores para Staphylococcus aureus. As maiores médias de contagens em UFC/g para coliformes totais foram nos cortes: Coxão mole, Alcatra, Pera e Filé-Mignon. De forma geral, os níveis de coliformes totais foram baixos, porém revelaram a presença de Escherichia coli principalmente no Coxão Mole. A Alcatra apresentou resultados elevados para coliformes totais, porém, sem a presença de E. coli. Os resultados dos cortes coletados mostraram que não houve desvio aparente nos processos quanto às operações do abate, a desossa e refile.(AU)
The aim of this study was to verify the mesophilic count, total coliforms, Staphylococcus aureus, Escherichia coli and the presence of Salmonella sp. in cattle rear cuts obtained in a slaughterhouse governed under Federal Inspection. The results indicated that all the cuts proved to be absent for Salmonella sp. and the highest average count for mesophilic was in rib steak, followed by Top Sirloin Cap, Topside, Flank Steak, Cut of Topside and Tenderloin. Of these, Cut of Topside and Top Sirloin Cap showed the highest values for S. aureus. The highest average scores in CFU/g for total coliforms were in cuts: Topside, Rump Steak, Cut of Topside and Tenderloin. The levels of total coliforms were low and showed the presence of Escherichia coli in the Topside. The results showed high thighs of coliforms, but without the presence of E. coli. The results of the collected cuttings showed no apparent shift in processes and operations during slaughter and boning and trimming.(AU)
Assuntos
Carne/microbiologia , Alimentos Resfriados , Escherichia coli , Salmonella , Padrões de Referência , Bovinos , Colimetria , Inocuidade dos AlimentosRESUMO
The transition from gestation to lactation is characterized by a robust adaptation of maternal pancreatic ß-cells. Consistent with the loss of ß-cell mass, glucose-induced insulin secretion is down-regulated in the islets of early lactating dams. Extensive experimental evidence has demonstrated that the surge of prolactin is responsible for the morphofunctional remodeling of the maternal endocrine pancreas during pregnancy, but the precise molecular mechanisms by which this phenotype is rapidly reversed after delivery are not completely understood. This study investigated whether glucocorticoid-regulated expression of Rasd1/Dexras, a small inhibitory G protein, is involved in this physiological plasticity. Immunofluorescent staining demonstrated that Rasd1 is localized within pancreatic ß-cells. Rasd1 expression in insulin-secreting cells was increased by dexamethasone and decreased by prolactin. In vivo data confirmed that Rasd1 expression is decreased in islets from pregnant rats and increased in islets from lactating mothers. Knockdown of Rasd1 abolished the inhibitory effects of dexamethasone on insulin secretion and the protein kinase A, protein kinase C, and ERK1/2 pathways. Chromatin immunoprecipitation experiments revealed that glucocorticoid receptor (GR) and signal transducer and activator of transcription 5b (STAT5b) cooperatively mediate glucocorticoid-induced Rasd1 expression in islets. Prolactin inhibited the stimulatory effect of GR/STAT5b complex on Rasd1 transcription. Overall, our data indicate that the stimulation of Rasd1 expression by glucocorticoid at the end of pregnancy reverses the increased insulin secretion that occurs during pregnancy. Prolactin negatively regulates this pathway by inhibiting GR/STAT5b transcriptional activity on the Rasd1 gene.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Período Periparto/metabolismo , Prolactina/farmacologia , Proteínas ras/metabolismo , Animais , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Corticosterona/metabolismo , Dexametasona/farmacologia , Feminino , Imunoprecipitação , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcortina/genética , Transcortina/metabolismo , Proteínas ras/genéticaRESUMO
This study aimed to analyze the economic viability of the third milking in milk production systems using hand milking, aiming to provide information to technicians and farmers to help in the decision-making process. Specifically, we intended a) to estimate the cost of a milking; (b) to estimate, by means of simulation, the cost of the third milking using hired or family labor; (c) to develop a mathematical equation that allows estimating the minimum amount of milk produced in two milkings, from which it will be economically feasible to carry out the third milking. The data were collected in three milk production systems from November 2010 to March 2011, in a routine of two milkings per day, with three collections of data in each one, totaling nine. Considering the average data, it would be feasible to hold the third milking if the productivity of daily average lactating cows would be equal or greater than 157,75 and 9,29kg of milk, with hired or family labor, respectively. The mathematical equation developed may help the technician and the farmer to estimate quickly and precisely the minimum quantity of milk produced by a cow in two milkings, from which it will be economically feasible to keep the third milking. KEYWORDS: cost of production; dairy cattle; economy; family labor; frequency of milking.
Objetivou-se analisar a viabilidade econômica da terceira ordenha em sistemas de produção de leite utilizando ordenha manual, visando fornecer aos técnicos e pecuaristas informações que os auxiliem no processo de tomada de decisões. Especificamente, pretendeu-se, ainda: a) estimar o custo de uma ordenha; b) estimar o custo da terceira ordenha utilizando mão de obra contratada e familiar; c) desenvolver uma equação matemática que permita estimar a quantidade mínima de leite produzida em duas ordenhas, a partir da qual será economicamente viável a realização da terceira ordenha. Os dados foram coletados em três sistemas de produção de leite no período de novembro de 2010 a março de 2011, em uma rotina de duas ordenhas diárias, sendo realizadas três coletas de dados em cada um, perfazendo um total de nove. Considerando-se os dados médios, seria viável a realização da terceira ordenha se a produtividade média diária por vaca em lactação em duas ordenhas fosse igual ou superior a 157,75 e 9,20kg de leite, se a mão de obra fosse contratada e familiar, respectivamente. A equação matemática desenvolvida pode auxiliar o técnico e o pecuarista estimar a quantidade mínima de leite produzida por uma vaca em duas ordenhas, a partir da qual será viável economicamente a realização da terceira ordenha, com precisão e considerável rapidez.PALAVRAS-CHAVE: bovinocultura leiteira; custo de produçã
RESUMO
This study aimed to analyze the economic viability of the third milking in milk production systems using hand milking, aiming to provide information to technicians and farmers to help in the decision-making process. Specifically, we intended a) to estimate the cost of a milking; (b) to estimate, by means of simulation, the cost of the third milking using hired or family labor; (c) to develop a mathematical equation that allows estimating the minimum amount of milk produced in two milkings, from which it will be economically feasible to carry out the third milking. The data were collected in three milk production systems from November 2010 to March 2011, in a routine of two milkings per day, with three collections of data in each one, totaling nine. Considering the average data, it would be feasible to hold the third milking if the productivity of daily average lactating cows would be equal or greater than 157,75 and 9,29kg of milk, with hired or family labor, respectively. The mathematical equation developed may help the technician and the farmer to estimate quickly and precisely the minimum quantity of milk produced by a cow in two milkings, from which it will be economically feasible to keep the third milking. KEYWORDS: cost of production; dairy cattle; economy; family labor; frequency of milking.
Objetivou-se analisar a viabilidade econômica da terceira ordenha em sistemas de produção de leite utilizando ordenha manual, visando fornecer aos técnicos e pecuaristas informações que os auxiliem no processo de tomada de decisões. Especificamente, pretendeu-se, ainda: a) estimar o custo de uma ordenha; b) estimar o custo da terceira ordenha utilizando mão de obra contratada e familiar; c) desenvolver uma equação matemática que permita estimar a quantidade mínima de leite produzida em duas ordenhas, a partir da qual será economicamente viável a realização da terceira ordenha. Os dados foram coletados em três sistemas de produção de leite no período de novembro de 2010 a março de 2011, em uma rotina de duas ordenhas diárias, sendo realizadas três coletas de dados em cada um, perfazendo um total de nove. Considerando-se os dados médios, seria viável a realização da terceira ordenha se a produtividade média diária por vaca em lactação em duas ordenhas fosse igual ou superior a 157,75 e 9,20kg de leite, se a mão de obra fosse contratada e familiar, respectivamente. A equação matemática desenvolvida pode auxiliar o técnico e o pecuarista estimar a quantidade mínima de leite produzida por uma vaca em duas ordenhas, a partir da qual será viável economicamente a realização da terceira ordenha, com precisão e considerável rapidez.PALAVRAS-CHAVE: bovinocultura leiteira; custo de produçã
RESUMO
PURPOSE: Retinal melatonin synthesis occurs in the photoreceptor layer in a circadian manner, controlling several physiologic rhythmic phenomena, besides being the most powerful natural free radical scavenger. The purpose of the present work was to evaluate the diurnal profile of retinal melatonin content and the regulation of its synthesis in the retina of streptozotocin-induced diabetic rats. METHODS: Diabetes was induced in male Wistar rats (12 hour-12 hour light/dark cycle) with streptozotocin. Control, diabetic, and insulin-treated diabetic animals were killed every 3 hours throughout the light-dark cycle. Retinal melatonin content was measured by high-performance liquid chromatography, arylalkylamine N-acetyltransferase (AANAT) activity was analyzed by radiometric assay, Bmal1 gene expression was determined by qPCR, and cyclic adenosine monophosphate (cAMP) content was assessed by ELISA. RESULTS: Control animals showed a clear retinal melatonin and AANAT activity daily rhythm, with high levels in the dark. Diabetic rats had both parameters reduced, and the impairment was prevented by immediate insulin treatment. In addition, the Bmal1 expression profile was lost in the diabetic group, and the retinal cAMP level was reduced 6 hours after lights on and 3 hours after lights off. CONCLUSIONS: The present work shows a melatonin synthesis reduction in diabetic rats retinas associated with a reduction in AANAT activity that was prevented by insulin treatment. The Bmal1-flattened gene expression and the cAMP reduction seem to be responsible for the AANAT activity decrease in diabetic animals. The melatonin synthesis reduction observed in the pineal gland of diabetic rats is also observed in a local melatonin tissue synthesizer, the retina.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Melatonina/biossíntese , Retina/metabolismo , Fatores de Transcrição ARNTL/genética , Animais , Arilalquilamina N-Acetiltransferase/metabolismo , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Ritmo Circadiano/fisiologia , AMP Cíclico/metabolismo , Fragmentação do DNA , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Masculino , Glândula Pineal/cirurgia , Reação em Cadeia da Polimerase , Radiometria , Ratos , Ratos WistarRESUMO
Retinal melatonin synthesis occurs in the photoreceptor layer in a circadian manner, controlling several physiologic rhythmic phenomena, besides being the most powerful natural free radical scavenger. The purpose of the present work was to evaluate the diurnal profile of retinal melatonin content and the regulation of its synthesis in the retina of streptozotocin-induced diabetic rats.Diabetes was induced in male Wistar rats (12 hour-12 hour light/dark cycle) with streptozotocin. Control, diabetic, and insulin-treated diabetic animals were killed every 3 hours throughout the light-dark cycle. Retinal melatonin content was measured by high-performance liquid chromatography, arylalkylamine N-acetyltransferase (AANAT) activity was analyzed by radiometric assay, Bmal1 gene expression was determined by qPCR, and cyclic adenosine monophosphate (cAMP) content was assessed by ELISA.Control animals showed a clear retinal melatonin and AANAT activity daily rhythm, with high levels in the dark. Diabetic rats had both parameters reduced, and the impairment was prevented by immediate insulin treatment. In addition, the Bmal1 expression profile was lost in the diabetic group, and the retinal cAMP level was reduced 6 hours after lights on and 3 hours after lights off.The present work shows a melatonin synthesis reduction in diabetic rats retinas associated with a reduction in AANAT activity that was prevented by insulin treatment. The Bmal1-flattened gene expression and the cAMP reduction seem to be responsible for the AANAT activity decrease in diabetic animals. The melatonin synthesis reduction observed in the pineal gland of diabetic rats is also observed in a local melatonin tissue synthesizer, the retina.
Assuntos
Ratos , Células Fotorreceptoras , Estreptozocina/metabolismo , Melatonina/análise , Receptores de Melatonina/administração & dosagem , Diabetes Mellitus Experimental/induzido quimicamente , Insulina/uso terapêuticoRESUMO
It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-α serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats.
Assuntos
Gluconeogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Melatonina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Adiposidade/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Ritmo Circadiano , Ingestão de Alimentos/efeitos dos fármacos , Immunoblotting , Resistência à Insulina/fisiologia , Proteína Oncogênica v-akt/metabolismo , Fenilbutiratos/farmacologia , Fosforilação/efeitos dos fármacos , Glândula Pineal/fisiologia , Glândula Pineal/cirurgia , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Endocrine pancreas from pregnant rats undergoes several adaptations that comprise increase in ß-cell number, mass and insulin secretion, and reduction of apoptosis. Lactogens are the main hormones that account for these changes. Maternal pancreas, however, returns to a nonpregnant state just after the delivery. The precise mechanism by which this reversal occurs is not settled but, in spite of high lactogen levels, a transient increase in apoptosis was already reported as early as the 3rd day of lactation (L3). Our results revealed that maternal islets displayed a transient increase in DNA fragmentation at L3, in parallel with decreased RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation (pAKT), a known prosurvival kinase. Wortmannin completely abolished the prosurvival action of prolactin (PRL) in cultured islets. Decreased pAKT in L3-islets correlated with increased Tribble 3 (TRB3) expression, a pseudokinase inhibitor of AKT. PERK and eIF2α phosphorylation transiently increased in islets from rats at the first day after delivery, followed by an increase in immunoglobulin heavy chain-binding protein (BiP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islets from L3 rats. Chromatin immunoprecipitation (ChIP) and Re-ChIP experiments further confirmed increased binding of the heterodimer ATF4/CHOP to the TRB3 promoter in L3 islets. Treatment with PBA, a chemical chaperone that inhibits UPR, restored pAKT levels and inhibited the increase in apoptosis found in L3. Moreover, PBA reduced CHOP and TRB3 levels in ß-cell from L3 rats. Altogether, our study collects compelling evidence that UPR underlies the physiological and transient increase in ß-cell apoptosis after delivery. The UPR is likely to counteract prosurvival actions of PRL by reducing pAKT through ATF4/CHOP-induced TRB3 expression.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Apoptose/fisiologia , Ilhotas Pancreáticas/metabolismo , Lactação/fisiologia , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Células Cultivadas , Feminino , Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Modelos Animais , Fosforilação/fisiologia , Prolactina/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais , Fator de Transcrição CHOP/metabolismoRESUMO
Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phospho-ERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression.
Assuntos
Proliferação de Células/efeitos dos fármacos , Dexametasona/farmacologia , Fosfatase 1 de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Lactação/metabolismo , Fosforilação/efeitos dos fármacos , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Feminino , Glucocorticoides/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Gravidez , RatosRESUMO
Unfolded protein response (UPR)-mediated pancreatic beta-cell death has been described as a common mechanism by which palmitate (PA) and pro-inflammatory cytokines contribute to the development of diabetes. There are evidences that interleukin 6 (IL6) has a protective action against beta-cell death induced by pro-inflammatory cytokines; the effects of IL6 on PA-induced apoptosis have not been investigated yet. In the present study, we have demonstrated that PA selectively disrupts IL6-induced RAC-alpha serine/threonine-protein kinase (AKT) activation without interfering with signal transducer and activator of transcription 3 phosphorylation in RINm5F cells. The inability of IL6 to activate AKT in the presence of PA correlated with an inefficient protection against PA-induced apoptosis. In contrast to PA, IL6 efficiently reduced apoptosis induced by pro-inflammatory cytokines. In addition, we have demonstrated that IL6 is unable to overcome PA-stimulated UPR, as assessed by activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, X-box binding protein-1 gene mRNA splicing, and pancreatic eukaryotic initiation factor-2 alpha kinase phosphorylation, whereas no significant induction of UPR by pro-inflammatory cytokines was detected. This unconditional stimulation of UPR and apoptosis by PA was accompanied by the stimulation of CHOP and tribble3 (TRIB3) expression, irrespective of the presence of IL6. These findings suggest that IL6 is unable to protect pancreatic beta-cells from PA-induced apoptosis because it does not repress UPR activation. In this way, CHOP and ATF4 might mediate PA-induced TRIB3 expression and, by extension, the suppression of IL6 activation of pro-survival kinase AKT.
Assuntos
Apoptose , Insulinoma/metabolismo , Interleucina-6/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resposta a Proteínas não Dobradas , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , História do Século XVI , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Palmitatos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismoRESUMO
OBJECTIVE: To investigate the action of palmitate on insulin receptor (IR) signaling pathway in rat pancreatic islets. The following proteins were studied: IR substrate-1 and -2 (IRS1 and IRS2), phosphatidylinositol 3-kinase, extracellular signal-regulated protein kinase-1 and -2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3). METHODS: Immunoblotting and immunoprecipitation assays were used to evaluate the phosphorylation states of IRS1 and IRS2 (tyrosine [Tyr]), ERK1/2 (threonine 202 [Thr202]/Tyr204), and STAT3 (serine [Ser727]). RESULTS: The exposure of rat pancreatic islets to 0.1-mmol/L palmitate for up to 30 minutes produced a significant increase of Tyr phosphorylation in IRS2 but not in IRS1. The association of phosphatidylinositol 3-kinase with IRS2 was also upregulated by palmitate. Exposure to 5.6-mmol/L glucose caused a gradual decrease in ERK1/2 (Thr202/Tyr204) and STAT3 (serine [Ser727]) phosphorylations after 30-minute incubation. The addition of palmitate (0.1 mmol/L), associated with 5.6-mmol/L glucose, abolished these latter effects of glucose after 15-minute incubation. CONCLUSIONS: Palmitate at physiological concentration associated with 5.6-mmol/L glucose activates IR signaling pathway in pancreatic beta cells.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Palmitatos/farmacologia , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Caspase 3/metabolismo , Linhagem Celular , Feminino , Glucose/farmacologia , Immunoblotting , Imunoprecipitação , Técnicas In Vitro , Proteínas Substratos do Receptor de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Fator de Transcrição STAT3/metabolismo , Fatores de TempoRESUMO
OBJECTIVES: The effect of glucose and palmitate on the phosphorylation of proteins associated with cell growth and survival (extracellular signal-regulated kinase 1/2 [ERK1/2] and stress-activated protein kinase/c-Jun NH2-terminal kinase [SAPK/JNK]) and on the expression of immediate early genes was investigated. METHODS: Groups of freshly isolated rat pancreatic islets were incubated in 10-mmol/L glucose with palmitate, LY294002, or fumonisin B1 for the measurement of the phosphorylation and the content of ERK1/2, JNK/SAPK, and v-akt murine thymoma viral oncongene (AKT) (serine 473) by immunoblotting. The expressions of the immediate early genes, c-fos and c-jun, were evaluated by reverse transcription-polymerase chain reaction. RESULTS: Glucose at 10 mmol/L induced ERK1/2 and AKT phosphorylations and decreased SAPK/JNK phosphorylation. Palmitate (0.1 mmol/L) abolished the glucose effect on ERK1/2, AKT, and SAPK/JNK phosphorylations. LY294002 caused a similar effect. The inhibitory effect of palmitate on glucose-induced ERK1/2 and AKT phosphorylation changes was not observed in the presence of fumonisin B1. Glucose increased c-fos and decreased c-jun expressions. Palmitate and LY294002 abolished these latter glucose effects. The presence of fumonisin B1 abolished the effect induced by palmitate on c-jun expression. CONCLUSIONS: Our results suggest that short-term changes of mitogen-activated protein kinase and AKT signaling pathways and c-fos and c-jun expressions caused by glucose are abolished by palmitate through phosphatidylinositol 3-kinase inhibition via ceramide synthesis.