Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NES1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G2/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G2 checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G2/M checkpoint.

The receptor tyrosine kinase regulator sprouty1 is a target of the tumor suppressor WT1 and important for kidney development

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.

Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity

Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(2428)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization.

Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10

Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. ReIB activation is required for myeloid DC differentiation. Here, we show that antigen-exposed DCs in which ReIB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. DCs generated from CD40-deficient mice similarly confer suppression. Regulatory CD4(+) T cells induced by the DCs transfer antigen-specific Infectious tolerance to primed recipients in an interleukin10-dependent fashion. Thus CD40, regulated by ReIB activity, determines the consequences of antigen presentation by myeloid DCs. These observations have significance for autoimmune immunotherapy and suggest a mechanism by which peripheral tolerance might be constitutively maintained by RelB(-) CD40(-) DCs.

Alterations of neocortical pyramidal cell phenotype in the Ts65Dn mouse model of Down syndrome: Effects of environmental enrichment

Mental retardation in individuals with Down syndrome (DS) is thought to result from anomalous development and function of the brain; however, the underlying neuropathological processes have yet to be determined. Early implementation of special care programs result in limited, and temporary, cognitive improvements in DS individuals. In the present study, we investigated the possible neural correlates of these limited improvements. More specifically, we studied cortical pyramidal cells in the frontal cortex of Ts65Dn mice, a partial trisomy of murine chromosome 16 (MMU16) model characterized by cognitive deficits, hyperactivity, behavioral disruption and reduced attention levels similar to those observed in DS, and their control littermates. Animals were raised either in a standard or in an enriched environment. Environmental enrichment had a marked effect on pyramidal cell structure in control animals. Pyramidal cells in environmentally enriched control animals were significantly more branched and more spinous than non-enriched controls. However, environmental enrichment had little effect on pyramidal cell structure in Ts65Dn mice. As each dendritic spine receives at least one excitatory input, differences in the number of spines found in the dendritic arbors of pyramidal cells in the two groups reflect differences in the number of excitatory inputs they receive and, consequently, complexity in cortical circuitry. The present results suggest that behavioral deficits demonstrated in the Ts65Dn model could be attributed to abnormal circuit development.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyltransferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARgamma/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

Expression of hNP22 is altered in the frontal cortex and hippocampus of the alcoholic human brain

Background: Human neuronal protein (hNP22) is a gene with elevated messenger RNA expression in the prefrontal cortex of the human alcoholic brain. hNP22 has high homology with a rat protein (rNP22). These proteins also share homology with a number of cytoskeleton-interacting proteins. Methods: A rabbit polyclonal antibody to an 18-amino acid epitope was produced for use in Western and immunohistochemical analysis. Samples from the human frontal and motor cortices were used for Western blots (n = 10), whereas a different group of frontal cortex and hippocampal samples were obtained for immunohistochemistry (n = 12). Results: The hNP22 antibody detected a single protein in both rat and human brain. Western blots revealed a significant increase in hNP22 protein levels in the frontal cortex but not the motor cortex of alcoholic cases. Immunohistochemical studies confirmed the increased hNP22 protein expression in all cortical layers. This is consistent with results previously obtained using Northern analysis. Immunohistochemical analysis also revealed a significant increase of hNP22 immunoreactivity in the CA3 and CA4 but not other regions of the hippocampus. Conclusions: It is possible that this protein may play a role in the morphological or plastic changes observed after chronic alcohol exposure and withdrawal, either as a cytoskeleton-interacting protein or as a signaling molecule.

Práticas pedagógicas inclusivas no cotidiano da educação infantil : considerações sobre a infância e a criança com deficiência e transtornos globais do desenvolvimento

Esta dissertação tem como objetivo investigar as práticas pedagógicas inclusivas instituídas no cotidiano da educação infantil a partir de um olhar para a infância e para a criança com deficiência e transtornos globais do desenvolvimento. Apontamos como objetivos específicos: definir o que está sendo reconhecido como práticas educacionais inclusivas a partir de indicadores estabelecidos para identificá- las no contexto de uma escola de educação infantil; investigar como a escola reflete, dialoga sobre as questões da inclusão das crianças na primeira infância na unidade de educação infantil e como se configuram as propostas de formação dos professores neste espaço, a fim de constituir práticas pedagógicas inclusivas na unidade escolar; escutar as crianças com deficiência e transtornos globais do desenvolvimento e as demais crianças sobre como estão compreendendo o acontecimento das práticas pedagógicas em geral e as práticas pedagógicas inclusivas da unidade de ensino de educação infantil. Para tanto, desenvolvemos um estudo de natureza qualitativa, tendo como base a metodologia do estudo de caso etnográfico numa perspectiva colaborativa, no qual realizamos análise documental, entrevistas semiestruturadas, observações participantes, ciclos de formação com os professores e roda de conversa com as crianças, que foram registrados por meio de fotografias, áudio e videogravações. O estudo foi desenvolvido no contexto de uma escola pública de educação infantil do município de Cachoeiro de Itapemirim – ES. Os participantes foram oito crianças público-alvo da educação especial, com idade entre dois a seis anos de idade, dez professores, três pedagogas, uma diretora, uma coordenadora e duas auxiliares de turma que se envolveram direta ou indiretamente com o estudo. O estudo foi realizado durante quatorze meses, no período de 28 de outubro de 2011 a 10 de dezembro de 2012, em uma Escola Municipal de Educação Básica do município de Cachoeiro do Itapemirim/ES, que atende exclusivamente aos alunos da educação infantil, em duas turmas de creche e três de pré-escola. Os aportes teóricos fundamentam-se na abordagem histórico-cultural e nos estudos de Phelippe Meirieu. Os dados foram organizados em temáticas e “episódios interativos” e analisados por meio da abordagem microgenética e das análises das narrativas. A análise dos resultados evidenciou a importância do investimento na formação dos professores, a constituição de relações de colaboração entre professores regentes e de Educação Especial e a assunção de que toda criança tem capacidade de aprender, pois esses elementos influenciam as práticas pedagógicas constituídas nos espaços-tempos da Educação Infantil mediante o desafio de inclusão escolar de crianças com deficiência e com transtornos globais do desenvolvimento.

Economic analysis of occupational risk prevention: a case study in a textile company

Work accidents affect business and society as a whole. Fewer accidents mean fewer sick leaves, which results in lower costs and less disruption in the production process, with clear advantages for the employer. But workers and their households bear also a significant burden following a work accident, only partially compen-sated by insurance systems. Furthermore, the consequences of work accidents to the State and Society need also to be considered. When an organization performs an integrated risk analysis in evaluating its Occupational Health and Safety Management System, several steps are suggested to address the identified risk situations. Namely, to avoid risks, a series of preventive measures are identified. The organization should make a detailed analysis of the monetary impact (positive or negative) for the organization of each of the measures considered. Particularly, it is also important to consider the impact of each measure on society, involving an adequate eco-nomic cost-benefit analysis. In the present paper, a case study in a textile finishing company is presented. The study concentrates on the dyeing and printing sections. For each of the potential risks, several preventive measures have been identified and the corresponding costs and benefits have been estimated. Subsequently, the Benefit/Cost ratio (B/C) of these measures has been calculated, both in financial terms (from the organisa-tion’s perspective) and in economic terms (including the benefits for the worker and for the Society). Results show that, while the financial analysis in terms of the company does not justify the preventive measures, when the externalities are taken into account, the B/C ratio increases significantly and investments are fully justified.

Targeting lactate transport suppresses in vivo breast tumour growth

Background: Most cancers, including breast cancer, have high rates of glucose consumption, associated with lactate production, a process referred as “Warburg effect”. Acidification of the tumour microenvironment by lactate extrusion, performed by lactate transporters (MCTs), is associated with higher cell proliferation, migration, invasion, angiogenesis and increased cell survival. Previously, we have described MCT1 up-regulation in breast carcinoma samples and demonstrated the importance of in vitro MCT inhibition. In this study, we performed siRNA knockdown of MCT1 and MCT4 in basal-like breast cancer cells in both normoxia and hypoxia conditions to validate the potential of lactate transport inhibition in breast cancer treatment. Results: The effect of MCT knockdown was evaluated on lactate efflux, proliferation, cell biomass, migration and invasion and induction of tumour xenografts in nude mice. MCT knockdown led to a decrease in in vitro tumour cell aggressiveness, with decreased lactate transport, cell proliferation, migration and invasion and, importantly, to an inhibition of in vivo tumour formation and growth. Conclusions: This work supports MCTs as promising targets in cancer therapy, demonstrates the contribution of MCTs to cancer cell aggressiveness and, more importantly, shows, for the first time, the disruption of in vivo breast tumour growth by targeting lactate transport.

Working hours, work-life conflict and health in precarious and "permanent" employment

OBJECTIVE: The expansion of precarious employment in OECD countries has been widely associated with negative health and safety effects. Although many shiftworkers are precariously employed, shiftwork research has concentrated on full-time workers in continuing employment. This paper examines the impact of precarious employment on working hours, work-life conflict and health by comparing casual employees to full-time, "permanent" employees working in the same occupations and workplaces. METHODS: Thirty-nine convergent interviews were conducted in two five-star hotels. The participants included 26 full-time and 13 casual (temporary) employees. They ranged in age from 19 to 61 years and included 17 females and 22 males. Working hours ranged from zero to 73 hours per week. RESULTS: Marked differences emerged between the reports of casual and full-time employees about working hours, work-life conflict and health. Casuals were more likely to work highly irregular hours over which they had little control. Their daily and weekly working hours ranged from very long to very short according to organisational requirements. Long working hours, combined with low predictability and control, produced greater disruption to family and social lives and poorer work-life balance for casuals. Uncoordinated hours across multiple jobs exacerbated these problems in some cases. Health-related issues reported to arise from work-life conflict included sleep disturbance, fatigue and disrupted exercise and dietary regimes. CONCLUSIONS:This study identified significant disadvantages of casual employment. In the same hotels, and doing largely the same jobs, casual employees had less desirable and predictable work schedules, greater work-life conflict and more associated health complaints than "permanent" workers.

Ciclo testicular e espermatogênese de Devario aequipinnatus (cypriniformes, cyprinidae) submetido a diferentes temperaturas

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Citotoxicidade do ácido peracético: avaliação metabólica, estrutural e de morte em fibroblastos L929

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

Conservation of limpet populations: a heavily exploited resource in Azores, NE-Atlantic

10th International Temperate Reefs Symposium, The University of Western Australia, 12-17 de janeiro.

Functional validation of LexADBDGim chimeras in saccharomyces cerevisiae

GimC/Prefoldin is a hetero-oligomeric complex involved in cytoskeleton biogenesis. In order to identify by two-hybrid system targets that directly interact with Gims and support the stress phenotypes, this work aimed the functional validation of all Gims in saccharomyces cerevisiae.