Bovine sperm cells viability during incubation with or without exogenous DNA

The aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 10(6)/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.99% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Coupling Virus-Induced Gene Silencing to Exogenous Green Fluorescence Protein Expression Provides a Highly Efficient System for Functional Genomics in Arabidopsis and across All Stages of Tomato Fruit Development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of ""sectoring"" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Propagation, growth, and carbohydrates of Dendrobium Second Love (Orchidaceae) in vitro as affected by sucrose, light, and dark

In general, plant material grown in vitro has low photosynthetic ability to achieve positive carbon balances. Therefore, a continuous supply of carbohydrates from the culture medium is required, and sucrose has been the most commonly used carbon source. In this paper, we investigate the effects of different sucrose concentrations and the presence and absence of light on the endogenous levels of soluble carbohydrates and starch as well as on the proliferation and growth of Dendrobium Second Love (Orchidaceae) in vitro. The possibility of using etiolated stem segments as a means for micropropagating this hybrid was also verified. The results obtained indicated that the presence and absence of light and the sucrose concentrations used influenced the amounts of soluble carbohydrates and starch and the proliferation of D. Second Love shoots and roots. An increase in sucrose concentration caused a progressive increase in the amounts of total carbohydrates and starch. Under both light conditions, sucrose was the main sugar found in the shoots followed by glucose and fructose. The addition of sucrose to the culture medium up to 2% and 4% was advantageous to the number of shoots produced per explant and the root longitudinal growth in the presence and absence of light, respectively. Shoot and root dry matter and the number of roots formed per explant increased as sucrose concentration was raised up to 6% in both light treatments. The use of dark-grown shoot segments proved to be a useful and reliable alternative for the micropropagation of this hybrid.

The role of carbohydrates in seed germination and seedling establishment of Himatanthus sucuuba, an Amazonian tree with populations adapted to flooded and non-flooded conditions

Background and Aims In the Amazonian floodplains plants withstand annual periods of flooding which can last 7 months. Under these conditions seedlings remain submerged in the dark for long periods since light penetration in the water is limited. Himatanthus sucuuba is a tree species found in the `varzea` (VZ) floodplains and adjacent non-flooded `terra-firme` (TF) forests. Biochemical traits which enhance flood tolerance and colonization success of H. sucuuba in periodically flooded environments were investigated. Methods Storage carbohydrates of seeds of VZ and TF populations were extracted and analysed by HPAEC/PAD. Starch was analysed by enzyme (glucoamylase) degradation followed by quantification of glucose oxidase. Carbohydrate composition of roots of VZ and TF seedlings was studied after experimental exposure to a 15-d period of submersion in light versus darkness. Key Results The endosperm contains a large proportion of the seed reserves, raffinose being the main nonstructural carbohydrate. Around 93% of the cell wall storage polysaccharides (percentage dry weight basis) in the endosperm of VZ seeds was composed of mannose, while soluble sugars accounted for 2.5%. In contrast, 74% of the endosperm in TF seeds was composed of galactomannans, while 22% of the endosperm was soluble sugars. This suggested a larger carbohydrate allocation to germination in TF populations whereas VZ populations allocate comparatively more to carbohydrates mobilized during seedling development. The concentration of root non-structural carbohydrates in non-flooded seedlings strongly decreased after a 15-d period of darkness, whereas flooded seedlings were less affected. These effects were more pronounced in TF seedlings, which showed significantly lower root non-structural carbohydrate concentrations. Conclusions There seem to be metabolic adjustments in VZ but not TF seedlings that lead to adaptation to the combined stresses of darkness and flooding. This seems to be important for the survival of the species in these contrasting environments, leading these populations to different directions during evolution.

Changes in soluble carbohydrates and starch amounts during somatic and zygotic embryogenesis of Acca sellowiana (Myrtaceae)

Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myo-inositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.

Sub-tropical urban environment affecting content and composition of non-structural carbohydrates of Lolium multiflorum ssp italicum cv. Lema

This study analyzed the relationship between environmental factors, especially air pollution and climatic conditions, and non-structural carbohydrates (NSC) in plants of Lolium multiflorum exposed during 10 consecutive periods of 28 days at a polluted site (Congonhas) and at a reference site in Sao Paulo city (Brazil). After exposure, NSC composition and leaf concentrations of Al, Fe. Cu, Zn, Pb and Cd were measured. The seasonal pattern of NSC accumulation was quite similar in both sites, but plants at Congonhas showed higher concentrations of these compounds, especially fructans of low and medium degree of polymerization. Regression analysis showed that NSC in plants growing at the polluted site were explained by variations on temperature and leaf concentration of Fe (positive effect), as well as relative humidity and particulate material (negative effect). NSC in the standardized grass culture, in addition to heavy metal accumulation, may indicate stressing conditions in a sub-tropical polluted environment. (C) 2008 Elsevier Ltd. All rights reserved.

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat

Background and purpose: Recent findings suggest that the noxious gas H(2)S is produced endogenously, and that physiological concentrations of H(2)S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H(2)S to modulate carrageenan-induced synovitis in the rat knee. Experimental approach: Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H(2)S formation (dl-propargylglycine) or an H(2)S donor [Lawesson`s reagent (LR)]. Key results: Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters. Conclusions and implications: Whereas exogenous H(2)S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H(2)S exerts little immunomodulatory effect. These data further support the development and use of H(2)S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K`s regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3 beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis. (C) 2009 Published by Elsevier Inc.

Nickel hydroxide electrodes as amperometric detectors for carbohydrates in flow injection analysis and liquid chromatography

The present paper describes the utilization of nickel hydroxide modified electrodes toward the catalytic oxidation of carbohydrates (glucose, fructose, lactose and sucrose) and their utilization as electrochemical sensor. The modified electrodes were employed as a detector in flow injection analysis for individual carbohydrate detection, and to an ionic column chromatography system for multi-analyte samples aiming a prior separation step. Kinetic studies were performed on a rotating disk electrode (RDE) in order to determine both the heterogeneous rate constant and number of electrons transferred for each carbohydrate. Many advantages were found for the proposed system including fast and easy handling of the electrode modification, low cost procedure, a wide range of linearity (0.5-50 ppm), low detection limits (ppb level) and high sensitivities. (C) 2009 Elsevier B.V. All rights reserved.

O extrato de maracujá sobre a morfometria de hepatócitos da tilápia do Nilo

Avaliaram-se os efeitos do extrato de maracujá veiculado na dieta (0, 50, 100 e 200mg kg-1) sobre o consumo de alimento, o ganho em peso e os níveis de glicose e cortisol plasmático de juvenis de tilápias do Nilo (87,0±6,6g). Ao final do experimento (28 dias), os peixes foram eutanasiados para remoção do fígado, visando à avaliação da área citoplasmática, contagem de células e verificação dos estoques de glicogênio hepático. Os dados foram submetidos à ANOVA unidirecional, comparando-se as médias pelo Teste de Tukey (P<0,05), com posterior estudo de regressão, buscando estabelecer as curvas das áreas citoplasmáticas, em função das diferentes doses do extrato. A inclusão do extrato na dieta não afetou o consumo de alimento e o crescimento e todos os peixes apresentaram aumento da glicose e redução do cortisol plasmático, porém sem diferenças entre os tratamentos. As curvas de regressão indicaram aumento quadrático da área citoplasmática com a elevação da doses do extrato, principalmente para 100mg kg-1, resultando em uma curva dose-resposta em forma de "U" invertido. O aumento da área do citoplasma decorreu de um acúmulo de glicogênio hepático, conforme comprovado pela prova da amilase salivar. Concluiu-se que o extrato de maracujá pode ser fornecido na dieta de juvenis de tilápia, sem prejudicar o consumo alimentar e o crescimento dos animais e que o produto altera a morfometria dos hepatócitos, sugerindo a atividade de flavonóides sobre o metabolismo de carboidratos.

Características de fermentação da silagem de seis variedades de milho indicadas para a região semiárida Brasileira

Avaliaram-se as características fermentativas e a qualidade das silagens de seis variedades de milho, de ciclos precoce e superprecoce - BRS Caatingueiro, BRS Assum Preto, BR 5033 Asa Branca, BR 5028 São Francisco, Gurutuba e BRS 4103 - indicadas para a região semiárida brasileira. Foram utilizados silos experimentais, em delineamento inteiramente ao acaso, com seis tratamentos (variedades) e quatro repetições. Avaliaram-se: matéria seca (MS), matéria orgânica (MO), proteína bruta (PB), fibra em detergente neutro (FDN), fibra em detergente ácido (FDA), extrato etéreo (EE), carboidratos totais (CHO), carboidratos não fibrosos (CNF), pH, nitrogênio amoniacal como parte do nitrogênio total (N-NH3/NT), ácidos orgânicos e digestibilidade in vitro da matéria seca (DIVMS) das silagens. Os valores médios encontrados para a silagem foram: MS= 28,7%; MO= 94,9%; PB= 8,3%; FDN= 49,9%; FDA= 27,5%; EE= 3,8%; CHO= 82,7%; CNF= 32,8%; pH= 3,8; N-NH3/NT= 2,9%/NT; ácido láctico = 7,6%; ácido acético = 0,6%; ácido butírico = 0,3% e DIVMS= 57,9%. As variedades BR 5028 - São Francisco e Gurutuba destacaram-se das demais em relação ao teor de matéria seca. A variedade BRS Caatingueiro apresentou maior teor de carboidratos não fibrosos em relação às demais. As silagens de todas as variedades foram classificadas como de excelente qualidade, por apresentarem potencial para ensilagem no semiárido brasileiro

Desidratação e recomendações para a reposição hídrica em crianças fisicamente ativas

OBJETIVO: A prática de exercícios físicos, devido à produção inerente de calor, pode conduzir à desidratação. A maioria dos estudos que abordam os riscos da desidratação e fornecem recomendações de reposição hídrica é direcionada a indivíduos adultos residentes em regiões de clima temperado, porém, em regiões tropicais, pouco é conhecido sobre as necessidades de reposição hídrica em crianças fisicamente ativas. Esta revisão discute as recomendações para esta população e estabelece os riscos da prática esportiva em ambiente de clima tropical. FONTES DE DADOS: Análise sistemática com levantamento da literatura nacional (SciELO) e internacional (Medline) de artigos publicados entre 1972 e 2009, com os seguintes descritores isolados ou em combinação: hidratação, crianças, desidratação e reposição hídrica. Foram selecionados artigos publicados nas línguas portuguesa e inglesa. SÍNTESES DE DADOS: Observou-se que há riscos de desidratação e possível desenvolvimento de um quadro de hipertermia principalmente se as crianças são submetidas a condições climáticas desfavoráveis sem reposição hídrica adequada. O principal fator desencadeante da hipertermia é a menor adaptação das crianças aos extremos de temperatura, em comparação aos adultos, por possuírem área maior de superfície corporal e capacidade menor de termorregulação por evaporação. CONCLUSÕES: Conhecidos os fatores intervenientes da desidratação, a melhor recomendação, perante uma condição climática sabidamente desfavorável, é estabelecer um plano impositivo de hidratação com bebida com sabor e acréscimo de carboidratos e sódio, evitando-se uma perda hídrica significativa, diminuição da performance e, principalmente, com o objetivo de reduzir os riscos à saúde impostos pela hipertermia e desidratação a crianças fisicamente ativas.

Nutritional reserves of Vochysiaceae seeds: chemical diversity and potential economic uses

Contents of proteins, carbohydrates and oil of seeds of 57 individuals of Vochysiaceae, involving one species of Callisthene, six of Qualea, one of Salvertia and eight of Vochysia were determined. The main nutritional reserves of Vochysiaceae seeds are proteins (20% in average) and oils (21. 6%). Mean of carbohydrate contents was 5. 8%. Callisthene showed the lowest protein content (16. 9%), while Q. cordata was the species with the highest content (30% in average). The contents of ethanol soluble carbohydrates were much higher than those of water soluble carbohydrates. Oil contents lay above 20% for most species (30. 4% in V. pygmaea and V. pyramidalis seeds). The predominant fatty acids are lauric (Q. grandiflora), oleic (Qualea and Salvertia) or acids with longer carbon chains (Salvertia and a group of Vochysia species). The distribution of Vochysiaceae fatty acids suggests for seeds of some species an exploitation as food sources (predominance of oleic acid), for other species an alternative to cocoa butter (high contents or predominance of stearic acid) or the production of lubricants, surfactants, detergents, cosmetics and plastic (predominance of acids with C20 or C22 chains) or biodiesel (predominance of monounsaturated acids). The possibility of exploitation of Vochysiaceae products in a cultivation regimen and in extractive reserves is discussed.

Morfo-anatomia foliar de Myrcia multiflora (Lam.) DC. - Myrtaceae

Folhas de Myrcia multiflora (Lam.) DC. são usadas na medicina popular como hipoglicemiantes. O objetivo deste trabalho foi caracterizar morfológica e anatomicamente as folhas desta planta, de modo que os dados obtidos possam ser utilizados como referência em exames de controle de qualidade de amostras de fármacos, com vistas a verificar a autenticidade. Folhas inteiras foram diafanizadas e coradas para o estudo da nervação. Secções transversais do pecíolo e transversais e paradérmicas da lâmina foliar foram analisadas em microscópio óptico (MO) e a superfície do limbo foi observada, também, em microscopia eletrônica de varredura (MEV). Foram aplicados testes histoquímicos em material fresco, para identificação e localização de glicídios, amido, taninos, lignina, cristais e sílica. Morfologicamente, a folha é simples, oval-elíptica, com margem inteira, base aguda, ápice acuminado e textura cartácea. A venação é do tipo camptódromo-broquidódromo. Anatomicamente, a folha é hipostomática, com mesofilo compacto e dorsiventral, com três estratos de parênquima paliçádico. A epiderme é uniestratificada, silicificada em algumas regiões e as células exibem paredes anticlinais retas. Em posição subepidérmica ocorrem numerosas cavidades secretoras de óleos essenciais. Os feixes vasculares são colaterais e acompanhados por séries cristalíferas. Os dados obtidos são comparados com os de outras espécies de Myrtaceae e conclui-se que as características morfológicas e anatômicas de M. multiflora contribuem para a diagnose.

Induced spawning of the endangered Neotropical species Steindachneridion parahybae (Siluriformes: Pimelodidae)

The "surubim do Paraíba" (Steindachneridion parahybae) is a freshwater catfish endemic to the Paraíba do Sul River basin, Brazil. This species has been seriously threatened by environmental disturbances in the last several decades. Wild Steindachneridion parahybae males and females were collected in 2003 and taken to the hatchery of a power plant of the Companhia Energética de São Paulo (CESP). Steindachneridion parahybae broodstocks were artificially induced to reproduce in December 2003 using a combination of carp pituitary extract (CPE) and human chorionic gonadotropin (hCG). Oocytes and milt were stripped; the fertilized eggs were transferred to 60-liter conical incubators and hatched larvae distributed in nine horizontal trays. Exogenous feed was started just after yolk sac absorption. A high rate of cannibalism and photophobia were observed during the larval period, resulting in a 26% survival rate from larvae to fingerlings.