AVALIAÇÃO ULTRAESTRUTURAL DE ESPERMATOZOIDE CAPRINO CRIOPRESERVADO, ATRAVÉS DA MICROSCOPIA ELETRÔNICA DE TRANSMISSÃO

The ultrastructure evaluation allows the analysis of the sperm cell in a subcellular proportion that is not observed in optical microscopy. Transmission electron microscopy (MET) is a tool used to determine the size and shape of inorganic and biological structures based on the interaction of electrons incident on matter. In this sense, with the use of MET, the objective was to evaluate the ultrastructural changes after the semen manipulation, mainly in the freeze-thaw process, which were not observed in tests using optical microscopy, that is, the morphological integrity of the membranes and goat sperm organelles. The evaluation took place at the Institute of Biosciences of the University of Brasília (UnB). The semen straws were thawed and washed in PBS. Then, they were centrifuged and fixed, contrasted in bloc and subjected to dehydration. The samples were placed for polymerization and ultrathin cuts were made and stored until the time of MET evaluation. In the ultrastructural analyzes by MET in the present work, no harmful actions occurred in either the control or experimental groups. The head regions (plasma membrane and acrosome) remained preserved, with no change in DNA. In conclusion, MET is especially useful tool for cell evaluation.A avaliação da ultraestrutura permite a análise da célula espermática em uma proporção subcelular que não é observada na microscopia óptica. A microscopia eletrônica de transmissão (MET) é uma ferramenta utilizada para determinar o tamanho e a forma de estruturas inorgânicas e biológicas com base na interação de elétrons incidentes na matéria. Nesse sentido, com o uso do MET, objetivou-se avaliar as alterações ultraestruturais após a manipulação do sêmen, principalmente no processo de congelamento-descongelamento, que não foram observadas em testes com microscopia óptica, ou seja, a integridade morfológica das membranas e organelas de esperma de cabra. A avaliação aconteceu no Instituto de Biociências da Universidade de Brasília (UnB). As palhetas de sêmen foram descongeladas e lavadas em PBS. Em seguida, foram centrifugados e fixados, contrastados em bloco e submetidos à desidratação. As amostras foram colocadas para polimerização e cortes ultrafinos foram feitos e armazenados até o momento da avaliação do MET. Nas análises ultraestruturais por MET no presente trabalho, nenhuma ação prejudicial ocorreu nos grupos controle ou experimental. As regiões da cabeça (membrana plasmática e acrossoma) permaneceram preservadas, sem alterações no DNA. Em conclusão, MET é uma ferramenta especialmente útil para avaliação celular

PRODUÇÃO IN VIVO DE EMBRIÕES CAPRINOS E OVINOS SUBMETIDOS AO MEIO DE MANUTENÇÃO ACP®

The use of ACP® as a means of maintenance in the stages of multiple ovulation and embryo transfer, has not yet been reported in the literature, although ACP® is a plant fluid rich in nutrients. Thus, the objective was to compare the efficiency of ACP® as a substitute for embryo maintenance medium during MOTE biotechnology in goats and sheep. For this, three donor goats, Anglo-Nubian breed and three donor sheep, Dorper breed were used. Fifteen recipient females of each species were used. The donors were submitted to the superovulation protocol and inseminated, and later the embryos were collected. After harvesting, the embryos were submitted to the control maintenance medium, TQC Holding® or ACP® maintenance medium. The recipients were synchronized simultaneously with the donors, and after 30 days the pregnancy diagnosis was made. It was obtained 10% of pregnant in goats and 75% of pregnant in sheep, whose embryos were submitted to the ACP® maintenance medium before the innovation. It is concluded that the means of maintenance of ACP® embryos did not negatively influence the embryonic quality and the development of pregnancy in small ruminants.A utilização do ACP® como meio de manutenção nas fases de ovulação múltipla e transferência embrionária, ainda não foi relatada na literatura, embora o ACP® seja um fluido vegetal rico em nutrientes. Assim, o objetivo foi comparar a eficiência do ACP® como substituto do meio de manutenção de embriões durante a biotecnologia MOTE em caprinos e ovinos. Para isso, foram utilizadas três cabras doadoras, raça Anglo-Nubiana e três ovelhas doadoras, raça Dorper. Quinze fêmeas receptoras de cada espécie foram utilizadas. As doadoras foram submetidas ao protocolo de superovulação e inseminadas, e posteriormente os embriões foram coletados. Após a colheita, os embriões foram submetidos ao meio de manutenção controle, meio de manutenção TQC Holding® ou ACP®. As receptoras foram sincronizadas simultaneamente com as doadoras, e após 30 dias foi feito o diagnóstico de gestação. Obteve-se 10% de prenhes em cabras e 75% de prenhes em ovelhas, cujos embriões foram submetidos ao meio de manutenção ACP® antes da inovação. Conclui-se que o meio de manutenção dos embriões ACP® não influenciou negativamente na qualidade embrionária e no desenvolvimento da prenhez em pequenos ruminantes

Overexpression of the plg1 gene encoding pectin lyase in Penicillium griseoroseum

The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml−1 respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data

Pectin lyase production by recombinant Penicillium griseoroseum strain 105

Recombinant Penicillium griseoroseum strain 105 overproduces an extracellular pectin lyase (PL) under the transcriptional control of the strong gpdA promoter of Aspergillus nidulans. Our aim was to evaluate PL production by recombinant P. griseoroseum strain 105 in submerged fermentation system bioreactors BioFloIII and BioFloIV using 2 or 10 L working volumes under different growth conditions and to analyze the production of cellulase, polygalacturonase, pectin methylesterase, and protease. PL overproduction by recombinant P. griseoroseum strain 105 was 112 times higher than that of P. griseoroseum PG63 grown in sugarcane juice. Cellulases and proteases were not detected in the culture filtrate, and evaluation for extracellular proteins in the culture medium by SDS–PAGE showed the presence of a 36 kDa predominant band, similar to the molecular mass estimated from the nucleotide sequence of plg1 gene for PL of P. griseoroseum strain 105. This recombinant strain provides the advantage of PL production, which predominates over other extracellular proteins usually present in most commercial pectinase preparations, using sugarcane juice as a substrate of low cost

Genome organization and assessment of high copy number and increased expression of pectinolytic genes from Penicillium griseoroseum: a potential heterologous system for protein production

The fungus Penicillium griseoroseum has the potential for application on an industrial scale as a host for the production of homologous and heterologous proteins, mainly because it does not produce some mycotoxins or secrete proteases under the growth conditions for pectinase production. However, for the fungus to be used effectively as an expression heterologous system, an understanding of the organization of its genome, as well as the mechanisms of gene expression and protein production, is required. In the present study, the size of the P. griseoroseum genome was estimated to be 29.8–31.5 Mb, distributed among four chromosomes. An analysis of plg1 and pgg2 pectinolytic genes expression and copy number in recombinant multi-copy strains of P. griseoroseum demonstrated that an increase in the number of gene copies could increase enzyme production, but the transcription could be affected by the gene integration position. Placing a copy of the plg1 gene under the control of the gpd promoter of Aspergillus nidulans yielded a 200-fold increase in transcription levels compared to the endogenous gene, and two copies of the pgg2 gene produced an 1100-fold increase compared with the endogenous gene. These results demonstrated that transcription, translation, and protein secretion in the fungus P. griseoroseum respond to an increased number of gene copies in the genome. The processing capacity and efficiency of protein secretion in P. griseoroseum are consistent with our premise that this fungus can be used for the industrial-scale production of several enzymes

Overproduction of polygalacturonase by Penicillium griseoroseum recombinant strains and functional analysis by targeted disruption of the pgg2 Gene

Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production

Autocuidado da gestante adolescente na prevenção dos fatores de risco da Síndrome Hipertensiva Específica Da Gravidez (SHEG) - doi:10.5020/18061230.2007.p173

Adolescent pregnancy is considered a problem for public health, due to the high mortality related to hypertensive disorders in pregnancy – HDP. This was a descriptive study with the aim of analyzing the pregnant adolescent self-care in the prevention of HDP risk factors. It was carried out in the Nucleus of Integrated Medical Attention – NAMI, in Fortaleza – Ceará, with twenty women from the Dendê Community, taken care of in the prenatal ambulatory of that institution, during the months of September and October, 2005. The data were collected by means of interview. The pregnant adolescents informed precarious socio-economic conditions, low schooling and other risk factors for HDP, beyond the age: black color, familiar history, arterial hypertension (AH), diabetes mellitus, renal illness and emotional conflicts. The knowledge on the prevention of risk factors was restricted to five (25%) pregnant adolescents; however it was reduced to fragmented information, and the preventive behaviors related to feeding habit, smoke and alcoholism cessation, and physical exercise. Therefore, it was evidenced among the adolescents the unsatisfactory exercise of self-care activities, aiming at preventing HDP risk factors; that beyond the age, they presented other predisposing factors to this disorders; that they were susceptible to preventive behaviors and/or control, as in the case of chronic-degenerative illnesses – AH and diabetes mellitus. Probably, this behavior was associated to the elementary and fragmented knowledge, and the absence or the deficiency of family participation in health promotion actions, mainly in those inherent to the prenatal follow-up.A gestação na adolescência é considerada um problema para a saúde pública, em decorrência alta mortalidade relacionada à Síndrome Hipertensiva Específica da Gestação – SHEG. Estudo descritivo com o objetivo de analisar o autocuidado de gestantes adolescentes na prevenção dos fatores de risco da SHEG. Foi realizado no Núcleo de Atenção Médica Integrada – NAMI, em Fortaleza – Ceará, com vinte mulheres procedentes da Comunidade do Dendê, atendidas no ambulatório de pré-natal daquela instituição, durante os meses de setembro e outubro de 2005. Os dados foram coletados através de entrevista. As gestantes informaram precárias condições sócio-econômicas, baixa escolaridade e outros fatores de risco para SHEG, além da idade: cor negra, história familiar, hipertensão arterial (HA), diabetes mellitus, doença renal e conflitos emocionais. O conhecimento sobre a prevenção dos fatores de riscos era restrito a cinco (25%) gestantes, porém resumia-se a informações fragmentadas; e as condutas preventivas relacionavam-se com o hábito alimentar, a abolição de tabagismo e do alcoolismo, e exercício físico. Portanto, constatou-se entre as adolescentes que havia o exercício insatisfatório das atividades de autocuidado, com vista à prevenção dos fatores de risco da SHEG; que além da faixa etária, essas apresentavam outros fatores predisponentes a esse agravo e que eram passíveis de condutas preventivas e/ou de controle, como é o caso das doenças crônicodegenerativas – HA e diabetes mellitus. Provavelmente, esse comportamento estava associado ao saber elementar e fragmentado, e ausência ou deficiência na participação da família nas ações de promoção da saúde, principalmente àquelas inerentes ao acompanhamento no prénatal

The minimal regulatory region necessary for the expression of the Penicillium griseoroseum plg1 gene

The expression of the Penicillium griseoroseum plg1 gene is induced by citric pectin and repressed by glucose. In this work, the minimal region of the plg1 gene promoter essential for expression in pectin and sucrose plus yeast extract was identified by using constructs containing the gfp ORF under control of the plg1 gene promoter. The fragment A (283 bp) is essential for plg1 expression in sucrose plus yeast extract. Fragment B (309 bp plus 184; core promoter) was critical for expression in pectin and abolished the catabolic repression by glucose. Therefore, the fragment of 776 bp (fragment A and B) is essential for the expression of the plg1 gene in natural inducing conditions (pectin as carbon source) and in sucrose plus yeast extract. The fragment B is a promising minimal promoter usable for heterologous expression in filamentous fungi, since genes that contain it could be activated by the presence of peel from citric fruits (which contains citric pectin) and are not affected by glucose in these agricultural by-products

Barreiras para a prática de atividade física em pessoas com deficiência visual

Resumo Este estudo teve como objetivo avaliar as barreiras para a prática de atividade física em pessoas com deficiência visual. Foi aplicado um questionário específico por telefone a 114 pessoas (moradores do Distrito Federal-Brasil) com deficiência visual pouco ativas. Destacam-se várias barreiras, como problemas com as calçadas, falta de instalações/espaços apropriados, falta de políticas de apoio das entidades públicas, necessidade de guia, falta de oferta de atividades por instituições especializadas e falta de condições de segurança das instalações físicas para evitar acidentes. Devem ser implantadas medidas apropriadas que ajudem a remover ou diminuir o impacto das principais barreiras apontadas pelos participantes, de modo a promover a saúde e o bem-estar dessa população