Elucidating the molecular mechanisms that underlie the target control of motoneuron death

Approximately half of the motoneurons generated during normal embryonic development undergo programmed cell death. Most of this death occurs during the time when synaptic connections are being formed between motoneurons and their target, skeletal muscle. Subsequent muscle activity stemming from this connection helps determine the final number of surviving motoneurons. These observations have given rise to the idea that motoneuron survival is dependent upon access to muscle derived trophic factors, presumably through intact neuromuscular synapses. However, it is not yet understood how the muscle regulates the supply of such trophic factors, or if there are additional mechanisms operating to control the fate of the innervating motoneuron. Recent observations have highlighted target independent mechanisms that also operate to support the survival of motoneurons, such as early trophic-independent periods of motoneuron death, trophic factors derived from Schwann cells and selection of motoneurons during pathfinding. Here we review recent investigations into motoneuron cell death when the molecular signalling between motoneurons and muscle has been genetically disrupted. From these studies, we suggest that in addition to trophic factors from muscle and/or Schwann cells, specific adhesive interactions between motoneurons and muscle are needed to regulate motoneuron survival. Such interactions, along with intact synaptic basal lamina, may help to regulate the supply and presentation of trophic factors to motoneurons.

Diverse synaptic mechanisms generate direction selectivity in the rabbit retina

The synaptic conductance of the On-Off direction-selective ganglion cells was measured during visual stimulation to determine whether the direction selectivity is a property of the circuitry presynaptic to the ganglion cells or is generated by postsynaptic interaction of excitatory and inhibitory inputs. Three synaptic asymmetries were identified that contribute to the generation of direction-selective responses: (1) a presynaptic mechanism producing stronger excitation in the preferred direction, (2) a presynaptic mechanism producing stronger inhibition in the opposite direction, and (3) postsynaptic interaction of excitation with spatially offset inhibition. Although the on- and off-responses showed the same directional tuning, the off-response was generated by all three mechanisms, whereas the on- response was generated primarily by the two presynaptic mechanisms. The results indicate that, within a single neuron, different strategies are used within distinct dendritic arbors to accomplish the same neural computation.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Lipid rafts and caveolae as portals for endocytosis: New insights and common mechanisms

Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane of mammalian cells. While our understanding of the machinery regulating and driving clathrin-coated pit-mediated endocytosis has progressed dramatically, including the elucidation of the structure of individual components and partial in vitro reconstitution, the role of caveolae as alternative endocytic carriers still remains elusive 50 years after their discovery. However, recent work has started to provide new insights into endocytosis by caveolae and into apparently related pathways involving lipid raft domains. These pathways, distinguished by their exquisite sensitivity to cholesterol-sequestering agents, can involve caveolae but also exist in cells devoid of caveolins and caveolae. This review examines the current evidence for the involvement of rafts and caveolae in endocytosis and the molecular players involved in their regulation.

Early pregnancy factor treatment suppresses the inflammatory response and adhesion molecule expression in the spinal cord of SJL/J mice with experimental autoimmune encephalomyelitis and the delayed-type hypersensitivity reaction to trinitrochlorobenzene in normal BALB/c mice

Early pregnancy factor (EPF) is a secreted protein, present in serum during early pregnancy and essential for maintaining viability of the embryo. It is a homologue of chaperonin 10 (Cpn10) but, unlike Cpn10, it has an extracellular role. EPF has immunosuppressive and growth regulatory properties. Previously we have reported the preparation of recombinant EPF (rEPF) and shown that treatment with rEPF will suppress clinical signs of MBP-EAE in Lewis rats and PLP-EAE in SJL/J mice. In the present study, these findings have been extended to investigate possible mechanisms involved in the action of EPF. Following treatment of mice with rEPF from the day of inoculation, there were fewer infiltrating CD3+ and CD4+ cells in the parenchyma of the spinal cord during the onset of disease and after the initial episode, compared with mice treated with vehicle. Expression of the integrins LFA-1, VLA-4 and Mac-1 and of members of the immunoglobulin superfamily of adhesion molecules ICAM-1 and VCAM-1 was suppressed in the central nervous system (CNS) following rEPF treatment. The expression of PECAM-1 was not affected. To determine if rEPF suppressed T cell activation in the periphery, the delayed-type hypersensitivity (DTH) reaction of normal BALB/c mice to trinitrochlorobenzene (TNCB) following treatment with rEPF was studied. The results showed that treatment with rEPF suppressed the DTH reaction, demonstrating the ability of EPF to downregulate the cell-mediated immune response. These results indicate that suppression of immunological mechanisms by rEPF plays a major role in the reduction of clinical signs of disease in experimental autoimmune encephalomyelitis (EAE). (C) 2003 Elsevier Science B.V. All rights reserved.

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.

Mechanisms of exercise training in patients with heart failure

Background The reduction of exercise capacity because of fatigue and dyspnea in patients with heart failure can be improved with exercise training. We sought to examine the mechanisms of exercise training, as an adjunctive treatment strategy for patients with heart failure. Methods a reviewed the published data on the possible mechanisms of effect of exercise training in heart failure. Results Symptoms of heart failure may be explained on the basis of abnormal skeletal muscle perfusion and structure and endothelial function. Exercise training has been shown to engender changes in muscle structure and biochemistry and vascular function, although effects on cardiac function have not been detected uniformly and may require longer training periods. Conclusions A suitable, long-term program of exercise training may reverse unfavorable interactions among the heart, vessels, and skeletal muscles. These improvements may be preserved with an ongoing maintenance program.

Ventricular dysfunction in early diabetic heart disease: detection, mechanisms and significance

The detection of preclinical heart disease is a new direction in diabetes care. This comment describes the study by Vinereanu and co-workers in this issue of Clinical Science in which tissue Doppler echocardiography has been employed to demonstrate subtle systolic and diastolic dysfunction in Type 11 diabetic patients who had normal global systolic function and were free of coronary artery disease. The aetiology of early ventricular dysfunction in diabetes relates to complex intramyocardial and extramyocardial mechanisms. The initiating event may be due to insulin resistance, and involves abnormal myocardial substrate utilization and uncoupling of mitochondrial oxidative phosphorylation. Dysglycaemia plays an important role via the effects of oxidative stress, protein kinase C activation and advanced glycosylation end-products on inflammatory signalling, collagen metabolism and fibrosis. Extramyocardial mechanisms involve peripheral endothelial dysfunction, arterial stiffening and autonomic neuropathy. The clinical significance of the ventricular abnormalities described is unknown. Confirmation of their prognostic importance for cardiac disease in diabetes would justify routine screening for presymptomatic ventricular dysfunction, as well as clinical trials of novel agents for correcting causal mechanisms. These considerations could also have implications for patients with obesity and the metabolic syndrome.

Carreiras p??blicas em uma ordem democr??tica: entre os modelos burocr??tico e gerencial

Refletindo sobre modelo de carreira mais adequado para a Administra????o P??blica nesta era de economia globalizada e de maiores exig??ncias de efici??ncia e flexibilidade na gest??o dos aparatos governamentais, questiona-se aqui a id??ia de que o modelo burocr??tico deva ser substitu??do pelo modelo gerencial. Considerando essa id??ia apenas parcialmente verdadeira, nossa argumenta????o vai na seguinte dire????o: como a burocracia ?? a forma espec??fica de organiza????o do aparato administrativo do Estado de Direito, este modelo n??o pode ser rejeitado, sob pena de comprometimento desse Estado. Mas ele precisa ser flexibilizado, superando e reduzindo o formalismo excessivo e a rigidez que sua realiza????o hist??rica possa ter gerado nos diferentes pa??ses. Igualmente, deve ser aperfei??oado em seus mecanismos de controle, at?? como condi????o para sua manuten????o eficaz nos governos democr??ticos contempor??neos.

Mecanismos de sustentabilidade das pol??ticas de vigil??ncia sanit??ria: um olhar sobre a Anvisa

Este artigo pretende ser uma colabora????o para o debate na ??rea de gest??o estrat??gica da sa??de, com foco na vigil??ncia sanit??ria. Nele buscou-se compreender as configura????es organizacionais que viabilizam (maior) ades??o ?? formula????o de estrat??gias em uma ag??ncia reguladora, a Ag??ncia Nacional de Vigil??ncia Sanit??ria (Anvisa). Foram consultados documentos da Anvisa dispon??veis ao p??blico na Internet, al??m de documentos internos nos arquivos da ??rea de planejamento da Ag??ncia. Foram analisados dois momentos de planejamento estrat??gico da Ag??ncia, ?? luz dos aspectos relacionados aos conceitos de poder e cultura, da configura????o organizacional, al??m das formas de controle sobre as ag??ncias reguladoras. Conclui-se que mecanismos que refor??am a transpar??ncia institucional levam ao sucesso de uma formula????o estrat??gica e ?? maior sustentabilidade das pol??ticas.

Fundamentos para uma pol??tica de carreiras no contexto da reforma gerencial

Com o advento da administra????o gerencial no ??mbito do aparelho do Estado, em complementa????o ?? administra????o burocr??tica, verifica-se certa turbul??ncia na gest??o de pessoal na administra????o p??blica, decorrente da s??bita aus??ncia de fundamentos conceituais s??lidos que permitam pensar a administra????o do capital humano do Estado em um contexto de transforma????o. Este artigo procura estabelecer as bases para a recupera????o destes fundamentos, propondo tr??s crit??rios de design de carreiras: a estrutura de incentivos, os mecanismos de governan??a e a gest??o do conhecimento. Em seguida, exemplifica-se a utiliza????o destes crit??rios pela an??lise de seis temas pr??prios ?? discuss??o sobre carreiras no servi??o p??blico: remunera????o, sele????o, recrutamento e desenvolvimento, mobilidade, promo????o, carreiras para ag??ncias executivas e controle social da burocracia.

A lei geral de comunica????o eletr??nica de massa e a qualidade da programa????o televisiva

O presente artigo pretende analisar a quest??o da qualidade da programa????o na televis??o brasileira a partir da proposta de um novo marco regulat??rio para o setor de comunica????o social eletr??nica. Essa nova lei, entre outras disposi????es, ir?? regulamentar o artigo 221 da Constitui????o Federal, que trata dos princ??pios pelos quais o conte??do televisivo deve pautar-se. Com isso, define-se qualidade levando-se em considera????o dois aspectos: diversidade e ressalvas ?? liberdade de express??o, ambos previstos na Constitui????o Federal. A partir dessa conceitua????o, prop??e-se a instrumentaliza????o do controle social sobre o conte??do televisivo e a garantia de meios para a diversidade da programa????o. Com rela????o ao primeiro aspecto, recomenda-se a atua????o transparente de uma futura ag??ncia reguladora e a implementa????o de mecanismo de controle individual da programa????o. No que tange ?? diversidade, ressalta-se a import??ncia do fortalecimento das televis??es p??blicas e medidas governamentais no sentido de estimular a multiprograma????o propiciada pelo advento da tecnologia digital.

Insulamento burocr??tico, accountability e transpar??ncia: dez anos de regula????o da Ag??ncia Nacional de Vigil??ncia Sanit??ria

Este artigo estuda os instrumentos e mecanismos de transpar??ncia e accountability das ag??ncias reguladoras brasileiras. Por meio da caracteriza????o dos processos de controle, participa????o e acesso a informa????es da Ag??ncia Nacional de Vigil??ncia Sanit??ria (Anvisa), analisa-se como as ag??ncias t??m utilizado tais instrumentos e mecanismos para acolher e processar diversos interesses do processo regulat??rio, promover a estabilidade das regras do jogo e refor??ar a sua legitimidade no ambiente pol??tico e social em que elas est??o inseridas. Foram utilizados dados relativos aos diversos instrumentos de transpar??ncia e accountability, bem como ??s inst??ncias e mecanismos de participa????o da sociedade no processo regulat??rio da Anvisa. Conclui-se que a accountability das ag??ncias ?? um contrafluxo ?? tend??ncia de insulamento, ao mesmo tempo em que se pode configurar um esfor??o de reconhecimento pela sociedade da chegada de um novo aparato institucional no Estado brasileiro: as ag??ncias reguladoras.