Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Phenotypic modulation of cultured vascular smooth muscle cells: a functional analysis focusing on MLC and ERK1/2 phosphorylation

Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction-relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction-relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.

Moderate exercise improves leucocyte function and decreases inflammation in diabetes

P>The genesis and progression of diabetes occur due in part to an uncontrolled inflammation profile with insulin resistance, increased serum levels of free fatty acids (FFA), proinflammatory cytokines and leucocyte dysfunction. In this study, an investigation was made of the effect of a 3-week moderate exercise regimen on a treadmill (60% of VO(2max), 30 min/day, 6 days a week) on inflammatory markers and leucocyte functions in diabetic rats. The exercise decreased serum levels of tumour necrosis factor (TNF)-alpha (6%), cytokine-induced neutrophil chemotactic factor 2 alpha/beta (CINC-2 alpha/beta) (9%), interleukin (IL)-1 beta (34%), IL-6 (86%), C-reactive protein (CRP) (41%) and FFA (40%) in diabetic rats when compared with sedentary diabetic animals. Exercise also attenuated the increased responsiveness of leucocytes from diabetics when compared to controls, diminishing the reactive oxygen species (ROS) release by neutrophils (21%) and macrophages (28%). Exercise did not change neutrophil migration and the proportion of neutrophils and macrophages in necrosis (loss of plasma membrane integrity) and apoptosis (DNA fragmentation). Serum activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were not modified in the conditions studied. Therefore, physical training did not alter the integrity of muscle cells. We conclude that moderate physical exercise has marked anti-inflammatory effects on diabetic rats. This may be an efficient strategy to protect diabetics against microorganism infection, insulin resistance and vascular complications.

Endotoxin levels correlate positively with a sedentary lifestyle and negatively with highly trained subjects

Introduction: A sedentary lifestyle increases the risk of developing cardiovascular disease, obesity, and diabetes. This phenomenon is supported by recent studies suggesting a chronic, low-grade inflammation status. Endotoxin derived from gut flora may be key to the development of inflammation by stimulating the secretion of inflammatory factors. This study aimed to examine plasma inflammatory markers and endotoxin levels in individuals with a sedentary lifestyle and/or in highly trained subjects at rest. Methods: Fourteen male subjects (sedentary lifestyle n = 7; highly trained subjects n = 7) were recruited. Blood samples were collected after an overnight fast (similar to 12 h). The plasmatic endotoxin, plasminogen activator inhibitor type-1 (PAI-1), monocyte chemotactic protein-1 (MCP1), ICAM/CD54, VCAM/CD106 and lipid profile levels were determined. Results: Endotoxinemia was lower in the highly trained subject group relative to the sedentary subjects (p < 0.002). In addition, we observed a positive correlation between endotoxin and PAI-1 (r = 0.85, p < 0.0001), endotoxin and total cholesterol (r = 0.65; p < 0.01), endotoxin and LDL-c (r = 0.55; p < 0.049) and endotoxin and TG levels (r = 0.90; p < 0.0001). The plasma levels of MCP-1, ICAM/CD54 and VCAM/CD106 did not differ. Conclusion: These results indicate that a lifestyle associated with high-intensity and high-volume exercise induces favorable changes in chronic low-grade inflammation markers and may reduce the risk for diseases such as obesity, diabetes and cardiovascular diseases.

Expression of Pancreatic Endocrine Markers by Prolactin-Treated Rat Bone Marrow Mesenchymal Stem Cells

Background. Mesenchymal stem cells (MSCs) are an attractive source for generation of cells with beta-cell properties. Previous studies have demonstrated the ability of prolactin to induce an increase in beta-cell mass and maturation, which suggests beneficial effects of its use in MSC differentiation protocols. Objective. To evaluate the expression of endocrine differentiation markers in rat MSCs treated in vitro with prolactin. Methods. Mesenchymal stem cells from bone marrow of Wistar rats were isolated, expanded, and characterized. Differentiation of MSCs was induced in medium containing 23 mmol/L of glucose, and nicotinamide, 2-mercaptoethanol, and exendin-4, in the presence or absence of 500 ng/mL of rat recombinant prolactin. Expression of endocrine markers and prolactin receptor genes was evaluated using real-time polymerase chain reaction, and compared between culture stages and presence vs absence of prolactin in the culture medium. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was also evaluated at immunofluorescence microscopy. Results. Isolated cells were mostly MSCs, as confirmed at fluorescent-activated cell sorting and cytochemistry. Pax6, Ngn-3, Isl1, NeuroD1, Nkx2.2, and Nkx6.1 exhibited varied expression during culture stages. The long form of the prolactin receptor messenger RNA was induced in prolactin-treated cultures (P < .05). The somatostatin gene was induced in early stages of differentiation (P < .05), and its expression was induced by prolactin, as confirmed using immunofluorescence. Conclusion. Culture of rat bone marrow MSCs in differentiation medium induces expression of pancreatic endocrine-specific genes, and somatostatin and prolactin receptor expression was also induced by prolactin.

Analytical Assessment of a Home Made Capillary Electrophoresis Equipment with Linear Charge Coupled Device for Visible Light Absorption Detection in the Determination of Food Dyes

The performance of modular home made capillary electrophoresis equipment with spectrophotometric detection, at a visible region by means of a miniaturized linear charge coupled device, was evaluated for the determination of four food dyes. This system presents a simple but efficient home made cell detection scheme. A computer program that converts the spectral data after each run into the electropherograms was developed to evaluate the analytical parameters. The dyes selected for analytical evaluation of the system were Brilliant Blue FCF, Fast Green FCF, Sunset Yellow FCF, and Amaranth. Separation was carried out in a 29cm length and 75 mu m I.D fused silica capillary, using 10mmolL-1 borate buffer at pH 9, with separation voltage of 7.5kV. The detection limits for the dyes were between 0.3 and 1.5mgL-1 and the method presented adequate linearity over the ranges studied, with correlation coefficients greater than 0.99. The method was applied for determination and quantification of these dyes in fruit juices and candies.

Hesitations and repair in German

The occurrence of pauses and hesitations in spontaneous speech has been shown to occur systematically, for example, "between sentences, after discourse markers and conjunctions and before accented content words." (Hansson [15]) This is certainly plausible in English, where pauses and hesitations can and often do occur before content words such as nominals, for example, "uh, there's a … man." (Chafe [8]) However, if hesitations are, in fact, evidence of "deciding what to talk about next," (Chafe [8]) then the complex grammatical system of German should render this pausing position precarious, since pre-modifiers must account for the gender of the nominals they modify.In this paper, I present data to test the hypothesis that pre-nominal hesitation patterns in German are dissimilar to those in English. Hesitations in German will be shown, in fact, to occur within noun phrase units. Nevertheless, native speakers most often succeed in supplying a nominal which conforms to the gender indicated by the determiner or pre-modifier. Corrections, or repairs, of infelicitous pre-modifiers indicate that the speaker was unable to supply a nominal of the same gender which the choice of pre-modifier had committed him/her to. The frequency of such repairs is shown to vary according to task, with fewest repairs occurring in elicited speech which allows for linguistic freedom and therefore is most like spontaneous speech. The data sets indicate that among German native speakers, hesitations occurring before noun phrase units (pre-NPU hesitations) indicate deliberation of what to say, while hesitations within or before the head of the noun phrase (pre-NPH hesitations) indicate deliberation of how to say what has already been decided (cf. Chafe [8]).

Acidentes de trânsito fatais e sua associação com indicadores sociais e adolescência

Introdução - Os acidentes de trânsito são um grave problema de saúde pública universal, em países desenvolvidos e subdesenvolvidos, estando entre as primeiras causas de morte em quase todos os países do mundo (DEL CIAMPO & RICCO, 1996). No Brasil, assu-mem grande relevância, especialmente pela alta morbidade e mortalidade, predominância em populações jovens e/ou economicamente ativas, maior perda de anos de vida produtiva e ele-vado custo direto e indireto para a sociedade. Objetivo - Os objetivos deste trabalho foram descrever a magnitude da mortali-dade por acidentes de trânsito, avaliar sua correlação com indicadores sociais e proporção de jovens na população e testar a sua associação com adolescência, sexo masculino e consumo de álcool. Material e Métodos - Foi realizado, inicialmente, um estudo ecológico envolven-do todas as capitais das unidades da federação e Distrito Federal (exceto o município do Rio de Janeiro), com coleta de dados sobre acidentes de trânsito com vítimas no Departamento Nacional de Trânsito. Foram descritos os índices de acidentes de trânsito com vítimas p/ 1.000 veículos (IAT-V) e de feridos p/ 1.000 veículos (IF-V) referentes aos anos de 1995, 1997 e 1998 e o índice de mortos p/ 10.000 veículos (IM-V) referente ao período de 1995 a 1998. Em seguida, avaliou-se a existência de correlação entre o IM-V e taxa de mortalidade infantil (TMI), índice municipal de desenvolvimento humano (IDH-M), índice de condições de vida (ICV), proporção de condutores adolescentes envolvidos em acidentes de trânsito com vítimas (PCJ-ATV) e proporção de residentes jovens (PRJ) nas diferentes capitais. Em um segundo momento, realizou-se um estudo de caso controle, onde foram estudados 863 condu-tores envolvidos em acidentes de trânsito com vítimas atendidos no Departamento Médico Legal de Porto Alegre, no período de 1998 a 1999. Os condutores foram divididos em dois grupos: condutores envolvidos em acidentes de trânsito com vítima fatal (casos) e com vítima não fatal (controles). Os grupos foram comparados com relação a adolescência, sexo mascu-lino e consumo de álcool, através da razão de chances e seu intervalo de confiança, com signi-ficância determinada pelo teste de qui-quadrado. Resultados - No estudo ecológico, observou-se, no Brasil, uma tendência decres-cente quanto aos indicadores de eventos relacionados ao trânsito no período de 1995 a 1998. Nas capitais das unidades da federação e Distrito Federal, apesar da ampla variação apresenta-da, a maioria manteve a mesma tendência decrescente observada para o país como um todo. Na análise das correlações entre o IM-V e os indicadores sociais, observou-se forte correlação positiva com a TMI (r = 0,57; P = 0,002), ou seja, quanto maior a TMI, maior a mortalidade no trânsito, além de correlação negativa com o IDH-M (r = - 0,41; P = 0,038) e com o ICV (r = - 0,58; P = 0,02). Quando se avaliaram o IDH-M e o ICV separados em suas dimensões, a dimensão renda de ambos indicadores foi a única que não demonstrou correlação com o IM- -V. As demais dimensões do IDH-M e ICV demonstraram correlação negativa, sendo que a dimensão infância (r = - 0,62; P = 0,001) apresentou a maior correlação. A análise da asso-ciação entre o IM-V e a PCJ-ATV não demonstrou correlação, mas, quando avaliada a asso-ciação com a PRJ nas capitais, houve forte correlação positiva (r = 0,59; P = 0,002). No estudo de caso controle, quando avaliada a relação entre condutores envolvidos em acidentes com vítima fatal e adolescência, sexo masculino e consumo de álcool, não foi observada asso-ciação importante em nenhum dos fatores em estudo. Conclusões - Apesar de os indicadores de eventos relacionados ao trânsito (IAT- -V, IF-V e IM-V) terem apresentado uma tendência decrescente durante o período de estudo, acidentes de trânsito continuam sendo um grave problema de saúde pública. O estudo ecológico evidenciou a existência de relação entre o IM-V e os indicadores sociais (TMI, IDH-M e ICV), sendo que a dimensão renda não demonstrou correlação e a dimensão infância apresen-tou a correlação negativa de maior valor. Quanto à PCJ-ATV, não foi encontrada associação relevante entre este indicador e o IM-V. Entretanto, observou-se forte associação entre a PRJ e o IM-V. O estudo de caso controle não evidenciou associação entre adolescência e os de-mais fatores estudados e maior risco para acidente de trânsito fatal.

A low cost one-camera optical tracking system for indoor wide-area augmented and virtual reality environments

In the last years the number of industrial applications for Augmented Reality (AR) and Virtual Reality (VR) environments has significantly increased. Optical tracking systems are an important component of AR/VR environments. In this work, a low cost optical tracking system with adequate attributes for professional use is proposed. The system works in infrared spectral region to reduce optical noise. A highspeed camera, equipped with daylight blocking filter and infrared flash strobes, transfers uncompressed grayscale images to a regular PC, where image pre-processing software and the PTrack tracking algorithm recognize a set of retro-reflective markers and extract its 3D position and orientation. Included in this work is a comprehensive research on image pre-processing and tracking algorithms. A testbed was built to perform accuracy and precision tests. Results show that the system reaches accuracy and precision levels slightly worse than but still comparable to professional systems. Due to its modularity, the system can be expanded by using several one-camera tracking modules linked by a sensor fusion algorithm, in order to obtain a larger working range. A setup with two modules was built and tested, resulting in performance similar to the stand-alone configuration.

Use of direct and iterative solvers for estimation of SNP effects in genome-wide selection

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Non invasive methods for genetic analysis applied to ecological and behavioral studies in Latino-America

Documenting the presence and abundance of the neotropical mammals is the first step for understanding their population ecology, behavior and genetic dynamics in designing conservation plans. The combination of field research with molecular genetics techniques are new tools that provide valuable biological information avoiding the disturbance in the ecosystems, trying to minimize the human impact in the process to gather biological information. The objective of this paper is to review the available non invasive sampling techniques that have been used in Neotropical mammal studies to apply to determine the presence and abundance, population structure, sex ratio, taxonomic diagnostic using mitochondrial markers, and assessing genetic variability using nuclear markers. There are a wide range of non invasive sampling techniques used to determine the species identification that inhabit an area such as searching for tracks, feces, and carcasses. Other useful equipment is the camera traps that can generate an image bank that can be valuable to assess species presence and abundance by morphology. With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in feces and amplify it to analyze the species diversity in an area, and the genetic variability at intraspecific level. This is particularly helpful in cases of sympatric and cryptic species in which morphology failed to diagnose the taxonomic status of several species of brocket deer of the genus Mazama.

Prevalence and virulence properties of Vibrio cholerae non-O1, Aeromonas spp. and Plesiomonas shigelloides isolated from Cambe Stream (State of Parana, Brazil)

The incidence of Vibrio cholerae, Aeromonas spp, and Plesiomonas shigelloides was determined in Rater samples from Cambe Stream. The samples were collected from seven different sites. The serogroups, virulence markers and drug resistance profiles were also evaluated. Twelve. Aer. hydrophila, 12 Aer. caviae, eight Aer. sobria, seven Ple. shigelloides and two V. cholerae non-O1 were isolated. They belonged to different serogroups and all produced haemolysis in different assays. Five of the Aeromonas strains and one of V, cholerae non-O1 were positive for enterotoxin activity. Haemagglutination and its inhibition, using erythrocytes of different origins, was variable for Aeromonas spp and V. cholerae, while none of the Plt. shigelloides haemagglutinated in association with any type of erythrocyte. All isolates exhibited multiple drug resistance. These results indicate that the occurrence of V. cholerae non-O1, Aeromonas spp, and Ple. shigelloides, in water used for vegetable irrigation, human recreation and animal consumption, among others, represents a potential risk for humans.

Predictors of health status do not change over three-year periods and exacerbation makes difference in chronic obstructive pulmonary disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hazards in non-pasteurized milk on retail sale in Brazil: prevalence of Salmonella spp, Listeria monocytogenes and chemical residues

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Independent clonal origin of multiple uterine leiomyomas that was determined by X chromosome inactivation and microsatellite analysis

Objective: In an attempt to clarify the clonality and genetic relationships that are involved in the tumorigenesis of uterine leiomyomas, we used a total of 43 multiple leiomyomas from 14 patients and analyzed the allelic status with 15 microsatellite markers and X chromosome inactivation analysis.Study design: We have used a set of 15 microsatellite polymorphism markers mapped on 3q, 7p, 11, and 15q by automated analysis. The X chromosome inactivation was evaluated by the methylation status of the X-linked androgen receptor gene.Results: Loss of heterozygosity analysis showed a different pattern in 7 of the 8 cases with allelic loss for at least 1 of 15 microsatellite markers that were analyzed. A similar loss of heterozygosity findings at 7p22-15 was detected in 3 samples from the same patient. X chromosome inactivation analysis demonstrated the same inactivated allele in all tumors of the 9 of 12 informative patients;. different inactivation patterns were observed in 3 cases.Conclusion: Our data support the concept that uterine leiomyomas are derived from a single cell but are generated independently in the uterus. Loss of heterozygosity findings at 7p22-15 are consistent with previous data that suggested the relevance of chromosomal aberrations at 7p that were involved in individual uterine leiomyomas. (C) 2005 Mosby, Inc. All rights reserved.