Expressão gênica de protaminas e proteínas nucleares de transição em testículos bovinos

Protamines (PRM) are the major DNA-binding proteins in the sperm nucleus and can pack the DNA into less than 5% of the volume of a somatic cell nucleus. It is already known that bulls only have the PRM1 protein on mature spermatozoa while most mammals also have the PRM2. Transition nuclear proteins (Tnps) and PRMs are fundamental to DNA integrity. It has already been reported the influence of PRM on chromatin structures, generating low fertility. However, molecular mechanisms underlying these effects are not known. The relative expression of PRM1, PRM2, PRM3, Tnp1 and Tnp2 was determined by real time RT-PCR, using bovine specific primers and β-actin as endogenous control. Quantification of mRNA relative expression showed a higher expression of PRM1 compared to the other genes. The PRM3 mRNA had the lowest relative expression. A significant (p < 0.05) and positive correlation was found between PRM1 and PRM2 (r = 0.518), PRM2 and Tnp1 (r = 0.750), PRM2 and Tnp2 (r = 0.706), PRM3 and Tnp1 (r = 0.542), PRM3 and Tnp2 (r = 0.731) and between Tnp1 and Tnp2 (r = 0.820). Since most of the knowledge about protamine 2 in bovine is based on a work from 1990 and according to new studies we know that PRM1 and PRM2 are important to bull fertility, more research is needed to elucidate the real function of protamines on bovines.Protaminas (PRM) são as principais proteínas ligantes do DNA espermático e podem compactar o núcleo do espermatozoide em menos de 5% do volume de uma célula somática. Já se sabe que o touro produz apenas a PRM1 em espermatozoide maduro, enquanto a maioria dos mamíferos também produz a PRM2. As proteínas nucleares de transição (Tnps) e as PRMs são fundamentais para a integridade do DNA. Já foi descrita a influência das protaminas na estrutura da cromatina e a associação destas com a fertilidade. Entretanto, os mecanismos moleculares que geram mudanças na cromatina espermática são desconhecidos. A expressão relativa da PRM1, PRM2, PRM3, Tnp1 e Tnp2 foi determinada para dez testículos de touros oriundos de matadouros comerciais, utilizando a técnica de RT-PCR em tempo real, com primers específicos para bovinos e a β-actina como controle endógeno. Ao quantificar a expressão relativa do RNAm, detectou-se alta expressão relativa da PRM1, em comparação aos outros genes. A expressão relativa da PRM3 foi a menor de todos os genes. Foram encontradas correlações positivas e significantes (p < 0,05) entre PRM1 e PRM2 (r = 0,518), PRM2 e Tnp1 (r = 0,750), PRM2 e Tnp2 (r = 0,706), PRM3 e Tnp1 (r = 0,542), PRM3 e Tnp2 (r = 0,731) e entre Tnp1 e Tnp2 (r = 0,820). Visto que a maioria dos conhecimentos sobre a PRM2 estão baseados em um trabalho de 1990 e, de acordo com recentes estudos se sabe que a PRM1 e a PRM2 são importantes para a fertilidade do touro, mais estudos são necessários para determinar a real função das protaminas em touros

In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein

The expression of milk proteins in vitro is essential to exploit the mammary gland cells as a biological model. Enzymatic tissue disaggregation has been widely used to establish mammary cell culture, but its effect in long-term ovine mammary cell culture is not completely elucidated. This study aimed at comparing mechanical/enzymatic and mechanical dissociation methods to establish ovine mammary cell culture. We compared cellular differentiation induced by lactating ewe sérum or fetal bovine serum based on the gene expression levels of milk proteins (beta-lactoglobulin, alpha s1-casein, and betacasein). Mechanically dissociated cells were positive immunostaining for cytokeratin 8.13, such as mammary epithelial cells. These cells are responsible for milk protein expression and they are low immunostaining for vimentin, mesenchymal marker. Mechanical/enzymatic dissociation cells were positive for vimentin. The fastest cell growth (cell/hour) was observed in the mechanical dissociation group cultured with 10% fetal bovine serum medium. Mechanically and mechanically/enzymatically derived cells were able to express beta-casein and beta-lactoglobulin, but not alpha s1-casein. The relative expression of beta-lactoglobulin was not affected by the tissue dissociation method or culture media, beta-casein relative expression was down regulated in mechanically dissociated cells cultured in the presence of lactating ewe serum, (P = 0.019). Beta-casein relative expression was also down regulated in mechanically/enzymatically dissociated cells cultured with fetal bovine sérum (P = 0.021). In the present conditions, we conclude that mechanical dissociation followed by culture with 10% of fetal bovine serum was the most efficient method to induce milk proteins’ mRNA expression by ovine mammary epithelial cells in vitro.A expressão in vitro de proteínas do leite e essencial para explorar as células da glândula mamaria como um modelo biológico. A desagregação tecidual via enzimática e amplamente utilizada para o estabelecimento cultivo de células mamarias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamaria ovina ainda não e bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamaria ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseina e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamarias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseina. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que o método de dissociação mecânica seguido pelo cultivo em meio com 10% de soro fetal bovino foi o método mais eficiente para induzir a expressão mRNA de proteínas do leite por células epiteliais mamarias ovinas in vitro

Comparison of different methods for exogenous DNA uptake by bovine spermatozoa

Apesar da manipulação genética de animais domésticos ser de grande interesse para a produção animal e para a indústria farmacêutica, a sua eficiência ainda é insatisfatória. A injeção pronuclear, a técnica mais utilizada para tal proposito, principalmente em camundongos, ainda apresenta limitações para esta espécie. Algumas alternativas têm sido desenvolvidas como o uso de espermatozoides como vetores para transferência genica, na qual a célula espermática tem habilidade espontânea de se ligar a molécula de DNA e internaliza-la. Dado o potencial da transferência genica mediada por espermatozoide para animais domésticos transgênicos, o objetivo do presente trabalho foi a avaliação de quatro métodos de incorporação de DNA para a transferência genica mediada por espermatozoides na espécie bovina: incubação com DNA, alteração da membrana plasmática induzida por cálcio ionóforo seguida por incubação com o DNA exógeno, eletroporação e lipofecção. Espermatozoides não expostos ao DNA exógeno foram usados como grupo controle. Os índices de clivagem, blastocisto e eclosão foram avaliados, respectivamente, as 72 horas após a inseminação dos oócitos, bem como, aos 9 e 12 dias de cultivo embrionário. Os embriões positivos para o DNA exógeno foram avaliados por PCR. Nenhum efeito de tratamento foi observado nos índices de clivagem, blastocisto e eclosão. Além disso, a porcentagem de blastocistos positivos para o DNA exógeno não diferiu entre os grupos experimentais. Apesar do baixo número de embriões positivos para DNA exógeno, os resultados obtidos mostram que todos os tratamentos apresentaram eficiências similares. A conclusão obtida foi que, apesar de os índices de desenvolvimento embrionário terem sido similares e constante em todos os grupos experimentais, outros fatores como a sequência, o tamanho e a concentração do DNA exógeno devem ser avaliados para melhorar a transferência genica mediada por espermatozoides.Although genetic manipulation of farm animals is of great interest for animal production and the pharmaceutical industry, its efficiency remains far from satisfactory. Pronuclear injection, which is the most widely used technique for such modification, mainly in mice, remains limited for this species. Some alternatives have been developed such as sperm mediated gene transfer, in which the spermatozoa are used as vectors for DNA delivery during in vitro fertilization. Mature sperm cells are able to spontaneously bind exogenous DNA molecules which may be internalized into sperm nuclei. Given the potential of sperm mediated gene transfer for livestock animals transgenesis, the aim of this study was to evaluate four methods of DNA uptake for sperm mediated gene transfer in bovine: incubation with DNA, plasma membrane alteration induced by calcium ionophore followed by incubation with DNA, electroporation and lipofection. Spermatozoa not exposed to exogenous DNA were used as control group. Cleavage, blastocyst and hatching rates were recorded at 72 hours post insemination (hpi), days 9 and 12 of embryo culture, respectively. Exogenous DNA-positive embryos were evaluated by PCR. No effect of treatment was observed on cleavage, blastocyst and hatching rates. In addition, percentage of DNA positive blastocysts did not differ among experimental groups. In spite of the low number of positive embryos, our results show that all treatments presented similar efficiencies for DNA delivery during in vitro fertilization. In conclusion, although the development rates were similar and constant in all groups, other factors such as exogenous DNA sequence, size and concentration should be considered to improve sperm mediated gene transfer

Serological survey of leptospirosis in horses with historical displacement through different geographic regions in Brazil

Leptospirosis has worldwide occurrence and shows higher incidence in tropical areas. Equines can present several serovars according to geographic region and it is important analyze de possible contribution of animal displacement on the Leptospira spp. strains occurrence. This research aimed to conduct a serological investigation in an equine regiment belonging to the Brazilian Army Guard Cavalry, resident in an urban area of Rio de Janeiro municipality, but with history of displacement between different regions in Brazil. A microscopic agglutination test was performed in a serum bank of 197 horses using 29 Leptospira spp. strains. The test showed 94.92% positivity, with 90.36% of the animals presenting a positive result for two or more serovars. Higher titers (?1:400) could be detected in 54.31% of horses for at least one serovar, whereas seropositivity for more than one bacterial type occurred mostly at lower titers (<1:400). All animals were clinically healthy. Occurrence of serogroup Icterohaemorraghiae in urban areas was confirmed; however, equine serovars Bratislava and Australis presented low frequencies. The serovars Hardjo, Wolffi, and Canicola were detected, and indicated the presence of ruminants and dogs as reservoirs in proximity in some of the places frequented by the animals. It is suggested that travel history has contributed to the high frequency found and a possible greater contact with different types of Leptospira spp. Therefore, veterinarians should consider the history of geographic displacement and other animal species coexistence with horses when conducting their clinical investigation.Leptospirosis has a worldwide occurrence and shows higher incidence in tropical areas. Equines can present several serovars according to geographic region and it is important to analyze the possible contribution of animal displacement on the Leptospira spp. strains occurrence. This research aimed to conduct a serological investigation in an equine regiment belonging to the Brazilian Army Guard Cavalry, resident in an urban area of Rio de Janeiro municipality, but with a history of displacement between different regions in Brazil. A microscopic agglutination test was performed in a serum bank of 197 horses using 29 Leptospira spp. strains. The test showed 94.92% positivity, with 89.84% of the animals presenting a positive result for two or more serovars. Higher titers (?1:400) could be detected in 57.22% of horses for at least one serovar, whereas seropositivity for more than one bacterial type occurred mostly at lower titers (<1:400). All animals were clinically healthy. The occurrence of serogroup Icterohaemorraghiae in urban areas was confirmed; however, equine serovars Bratislava and Australis presented low frequencies. The serovars Hardjo, Wolffi, and Canicola were detected and indicated the presence of ruminants and dogs as typical reservoirs in proximity to the equines. It is suggested that travel history has contributed to the high frequency found and possible greater contact with different types of Leptospira spp. Therefore, veterinarians should consider the history of geographic displacement and other animal species’ coexistence with horses when conducting their clinical investigation

Serological survey of leptospirosis in horses with historical displacement through different geographic regions in Brazil

Leptospirosis has worldwide occurrence and shows higher incidence in tropical areas. Equines can present several serovars according to geographic region and it is important analyze de possible contribution of animal displacement on the Leptospira spp. strains occurrence. This research aimed to conduct a serological investigation in an equine regiment belonging to the Brazilian Army Guard Cavalry, resident in an urban area of Rio de Janeiro municipality, but with history of displacement between different regions in Brazil. A microscopic agglutination test was performed in a serum bank of 197 horses using 29 Leptospira spp. strains. The test showed 94.92% positivity, with 90.36% of the animals presenting a positive result for two or more serovars. Higher titers (?1:400) could be detected in 54.31% of horses for at least one serovar, whereas seropositivity for more than one bacterial type occurred mostly at lower titers (<1:400). All animals were clinically healthy. Occurrence of serogroup Icterohaemorraghiae in urban areas was confirmed; however, equine serovars Bratislava and Australis presented low frequencies. The serovars Hardjo, Wolffi, and Canicola were detected, and indicated the presence of ruminants and dogs as reservoirs in proximity in some of the places frequented by the animals. It is suggested that travel history has contributed to the high frequency found and a possible greater contact with different types of Leptospira spp. Therefore, veterinarians should consider the history of geographic displacement and other animal species coexistence with horses when conducting their clinical investigation

Metabolically Healthy Obesity and High Carotid Intima-Media Thickness in Children and Adolescents: International Childhood Vascular Structure Evaluation Consortium

OBJECTIVE It has been argued that metabolically healthy obesity (MHO) does not increase cardiovascular disease (CVD) risk. This study examines the association of MHO with carotid intima-media thickness (cIMT), a proxy of CVD risk, in children and adolescents. RESEARCH DESIGN AND METHODS Data were available for 3,497 children and adolescents aged 6–17 years from five population-based cross-sectional studies in Brazil, China, Greece, Italy, and Spain. Weight status categories (normal, overweight, and obese) were defined using BMI cutoffs from the International Obesity Task Force. Metabolic status (defined as "healthy" [no risk factors] or "unhealthy" [one or more risk factors]) was based on four CVD risk factors: elevated blood pressure, elevated triglyceride levels, reduced HDL cholesterol, and elevated fasting glucose. High cIMT was defined as cIMT ≥90th percentile for sex, age, and study population. Logistic regression model was used to examine the association of weight and metabolic status with high cIMT, with adjustment for sex, age, race/ethnicity, and study center. RESULTS In comparison with metabolically healthy normal weight, odds ratios (ORs) for high cIMT were 2.29 (95% CI 1.58–3.32) for metabolically healthy overweight and 3.91 (2.46–6.21) for MHO. ORs for high cIMT were 1.44 (1.03–2.02) for unhealthy normal weight, 3.49 (2.51–4.85) for unhealthy overweight, and 6.96 (5.05–9.61) for unhealthy obesity. CONCLUSIONS Among children and adolescents, cIMT was higher for both MHO and metabolically healthy overweight compared with metabolically healthy normal weight. Our findings reinforce the need for weight control in children and adolescents irrespective of their metabolic status

Ocorrência de anticorpos anti-Toxoplasma gondii associada a fatores de risco em gatos com esporotricose oriundos da região metropolitana do Rio de Janeiro

Made available in DSpace on 2015-10-16T12:52:52Z (GMT). No. of bitstreams: 2 renata_barros_ini_mest_2013.pdf: 649069 bytes, checksum: ccf984958e0979b9f8cb5d6df59ab3fd (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-06-10Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.A toxoplasmose é uma zoonose causada pelo protozoário Toxoplasma gondii que acomete várias espécies de vertebrados, inclusive o ser humano. Os gatos, assim como os outros felinos, têm papel de suma importância na epidemiologia da infecção, pois são os hospedeiros definitivos do T. gondii. Esse trabalho teve como objetivo determinar a frequência de anticorpos anti-T.gondii associados a fatores de risco e co-infecções em 213 gatos com esporotricose oriundos da região metropolitana do Rio de Janeiro e assistidos no LAPCLIN-DERMZOO, no período de novembro de 2007 a fevereiro de 2011. Esses animais foram acompanhados mensalmente devido ao tratamento para esporotricose, até os seus desfechos clínicos. Foram realizadas sorologias seriadas para toxoplasmose por meio da hemaglutinação indireta (HAI) e pela reação da imunofluorescência indireta (RIFI) e diagnóstico para o feline imunnodeficiency virus (FIV) e o feline leukemia vírus (FeLV) através de um imunoensaio rápido. Dos 213 gatos, 14 (6,6%) apresentaram anticorpos anti-T. gondii na RIFI (IgG) e na HAI. Houve um caso único de soroconversão, no quarto acompanhamento Houve variação de pelo menos dois títulos na IgG-RIFI nos acompanhamentos de dois animais. Apenas um animal (7,1%) apresentou co-infecção de toxoplasmose com o FIV e três animais (21,4%) com o FeLV. Não foi detectada associação entre as variáveis e co-infecções estudadas e a presença de anticorpos anti-T. gondii, porém 78,6% dos gatos com infecção toxoplásmica apresentaram falência terapêutica no tratamento para esporotricose, sendo quatro deles (27,3%) FIV ou FeLV positivos. A frequência da infecção toxoplásmica nos gatos estudados foi baixa, houve uma maior frequência de animais soropositivos para T. gondii entre aqueles que tinham livre acesso a rua, conviviam com outros gatos e possuíam mais de três anos de idade e foi observada 100% de concordância no teste diagnóstico para T. gondii entre a RIFI e a HAIToxoplasmosis is a zoonotic disease caused by the Toxoplasma gondii protozoan that affects several species of vertebrates, incl uding human s . Cats, as well as other felines, a re important in the epidemiology of the infection because they are the definite hosts of T . gondii . This study aim ed to determine the frequency of anti - T . gondii antibodies associated with risk factors and coinfections in 213 cats infected with sporotrichosis in the metropolitan Rio de Janeiro and assisted in the LAPCLIN - DERMZOO , in the period of november 2007 to february 2011. These animals were monthly evaluate d due to sporotrichosis treatment until their sporotrichosis treatment outcomes. Serologic series for toxoplasmosis were performed through indirect he magglutination assay (IHA) and indirect fluorescence antibody test (I F A T) , and feline immunodeficiency vi rus (FIV) and feline leukemi a vi rus ( FeLV) diagnose s were made by fast immunoassay. Am ong the 213 studied cats, 14 (6. 6%) showed anti - T. gondii antibodies in the I F A T and in the IHA. There was only one occurrence of seroconversion in the fourth clinical evaluation . There was a variation of at least two titles in the I F A T - IgG in the clinical follow up of two animals. Just one animal (7. 1%) showed coinfection of toxoplasmosis with FIV a nd three animals (21. 4%) with FeLV. The association between variables a nd studied coinfections with the presence of T. gondii antibodies has not been detected, nonetheless 78.6% of the infected cats showed therapeutical failure in the sporotrichosis treatment , and four of them (27. 3%) were FIV or FeLV positive. The frequency of toxoplasmosis infection in the cats was low ; cats that had free access to the street , coexisted with other cats and were older than three years showed a higher rate of T. gondii positivity and a 100% concordance in the diagnostic test for T. gondii between IFAT and IHA was also observed