Thiopalmitoylation of myelin proteolipid protein epitopes enhances immunogenicity and encephalitogenicity

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP104-117 and PLP139-151) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8(+) cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4(+) T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.

Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension

To test the hypothesis that Vegf-B contributes to the pulmonary vascular remodelling, and the associated pulmonary hypertension, induced by exposure of mice to chronic hypoxia. Methods: Right ventricular systolic pressure, the ratio of right ventricle/[left ventricle+septum] (RV/[LV+S]) and the thickness of the media (relative to vessel diameter) of intralobar pulmonary arteries (o.d. 50-150 and 151-420 mum) were determined in Vegfb knockout mice (Vegfb(-/-); n=17) and corresponding wild-type mice (Vegfb(+/+); n=17) exposed to chronic hypoxia (10% oxygen) or housed in room air (normoxia) for 4 weeks. Results: In Vegfb(+/+) mice hypoxia caused (i) pulmonary hypertension (a 70% increase in right ventricular systolic pressure compared with normoxic Vegfb(+/+) mice; P

Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy

The fundamental role of dendritic cells (DC in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6,0 AutoPBPC) provides an operator-independent reliable source of motionuclear cells (MNC for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17-179) BDC per 10-1 procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 151. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c(+)CD123(low) and CD11c(-)CD123(high)) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure. (C) 2002 Published by Elsevier Science B.V.

Effects of dietary level of protein, lysine and methionine and strain of bird on production and egg yolk cholesterol

The effects of dietary level of protein (151, 181 g/kg), lysine (nil, 10g L-lysine hydrochloride/kg) and methionine (nil, 5g DL-methionine/kg) on the production performance and egg yolk cholesterol of two strains of birds were studied for 12 weeks. Birds fed on the high protein diet had higher body weight gain, feed conversion ratio (FCR), rate of lay, egg weight and mass and yolk weight and mass. A high lysine diet decreased feed intake and improved FCR. High dietary level of methionine increased egg yolk cholesterol. There were differences between strains of laying bird in feed intake, rate of lay, egg and yolk weights and egg cholesterol content. It is concluded that strain of bird and dietary level of protein and lysine influenced the production performance of birds. Whilst, egg yolk cholesterol was not reduced by any of the factors studied.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines. drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway. in the case of certain drugs and carcinogens. it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP 11 library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenot (K-m and V-max values of 0.15 muM and 897.5 nmol/min/mg protein. respectively) and dopamine (K-m and V-max values of 175.3 muM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and recturm. (C) 2002 Elsevier Science Ltd. All rights reserved.

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.

Negocia????o entre Estado e sindicatos: instrumentos de negocia????o coletiva de trabalho no governo do Distrito Federal

O presente trabalho tem por objetivo analisar, ?? luz da conven????o da Organiza????o Internacional do Trabalho (OIT) 151, os instrumentos de negocia????o coletiva de trabalho entre o Governo do Distrito Federal e os sindicatos de servidores p??blicos. Para tanto, o referencial te??rico foi organizado em 4 t??picos: o primeiro ?? o conceito de inputs e outputs para se compreender melhor como ingressam as demandas - inputs de pol??ticas p??blicas ??? outputs ao Estado. Em seguida, ?? explicitado o conceito de agente principal, para esclarecer como os sindicatos de servidores p??blicos constituem-se num grupo de press??o corporativa relevante para a gest??o p??blica. No segundo cap??tulo ?? apresentado um breve hist??rico da consolida????o do modelo sindical brasileiro, qual a sua pauta e de que maneira ela orienta a a????o sindical na busca por influenciar as tomadas de decis??o do Estado. O terceiro cap??tulo aborda a import??ncia estrat??gica do Estado em dialogar com as press??es constitu??das pelo movimento sindical no setor p??blico, bem como a relev??ncia dos servidores p??blicos, por meio de gest??o participativa, com o instrumento da negocia????o coletiva para construir a governabilidade. No quarto cap??tulo s??o abordados os dispositivos legais para a constitui????o de espa??o de negocia????o coletiva entre sindicato e governo, considerando que o reconhecimento do di??logo com os sindicatos no setor p??blico depende do Estado se apropriar e aprofundar os mecanismos legais de negocia????o. Para tanto, s??o apresentados os instrumentos legais internacionais e nacionais, bem como os infra legais do Distrito Federal. Esta pesquisa foi realizada procurando apresentar a teoria e o que ?? praticado no GDF, no per??odo de 2011-2013, no governo Agnelo Queiroz, respons??vel por instituir o Dialoga-DF, que acolhe as demandas dos servidores p??blicos, por meio de um sistema de negocia????o permanente, que procura praticar o que postula a conven????o OIT 151. Para tanto, a vivencia do pesquisador foi importante para acesso ?? din??mica do processo negocial no GDF. Para complementar tal experi??ncia, foram feitas entrevistas semiestruturadas com as representa????es sindicais que participam de diversas mesas de negocia????o. Por fim, s??o apresentados alguns limites e avan??os na negocia????o coletiva no GDF, perante a cultura organizacional corporativa tradicional nos servi??os p??blicos, mas que vem mudando ?? medida que o Estado aperfei??oa a gest??o p??blica em busca da efici??ncia e de melhores servi??os p??blicos oferecidos ?? popula????o

Projeto RH NEW: equipe multiprofissional dedicada ao equacionamento das quest??es ligadas a recursos

Este projeto refere-se ao programa de acompanhamento sistematizado dos servidores t??cnico-administrativos da Universidade Federal de Santa Catarina que apresentem ind??cios de uso indevido de drogas, alcoolismo, problemas de relacionamento e outros que influenciam diretamente no comportamento funcional. Para o desenvolvimento deste programa, uma equipe multiprofissional (composta de pedagogo, t??cnico em assuntos educacionais e assistente social) da pr??pria universidade se envolveu com o trabalho. Os resultados s??o significativos principalmente no que se refere ?? inser????o do servidor no ambiente de trabalho, de forma mais produtiva

Revista do Servi??o P??blico de 1937 a 2007

Edi????o especial comemorativa dos 70 anos de exist??ncia da primeira publica????o brasileira especializada em gest??o e administra????o p??blicas. Ao longo de sete d??cadas, a revista publicou textos de servidores e intelectuais, que se tornaram refer??ncia no pa??s. Neste n??mero, a ENAP busca situar a revista dentro das transforma????es hist??ricas, sociais, pol??ticas e culturais, no per??odo 1937-2007, e delinear cen??rios para o futuro

Escolas de governo e profisionaliza????o do funcionalismo

Este estudo ?? parte integrante de um dos programas gerais de a????o definidos para esta Escola em 1994, referente ?? Forma????o de Quadros para a Administra????o P??blica, notadamente ?? profissionaliza????o de quadros superiores da burocracia federal, denominado Escolas de Governo e Profissionaliza????o do Funcionalismo. Faz-se uma an??lise das experi??ncias de forma????o levadas a cabo pela Escola Nacional de Administra????o P??blica - ENAP, bem como um mapeamento das experi??ncias das escolas de governo brasileiras e estrangeiras, que em certa medida possam ser comparadas, com a finalidade de oferecer subs??dios para a formula????o das pol??ticas governamentais dirigidas ?? forma????o e capacita????o de recursos humanos no setor p??blico brasileiro

Disciplina 3.6: conv??nios e termos de parceria

A disciplina teve como principais conte??dos: os conv??nios e os contratos de repasse de que trata o Decreto n?? 6.170/2007 e a Portaria Interministerial/CGU/MF/MP n?? 127/2008. A celebra????o, a execu????o e a presta????o de contas com o portal www.convenios.gov.br. Os dispositivos da LDO anual sobre as transfer??ncias volunt??rias. Termos de Parceria com Organiza????es da Sociedade Civil de Interesse P??blico ??? OSCIP (Lei n?? 9.790/99 e Decreto n?? 3.100). Julgados do Tribunal de Contas da Uni??o sobre o tema

Criando valor p??blico por meio de parcerias p??blico- privadas

O prop??sito deste trabalho ?? avan??ar em dire????o ao desenvolvimento de um marco anal??tico que ajude a avaliar as parcerias p??blico-privadas, tanto ?? luz do conceito geral, como de propostas espec??ficas concretas. Para isso, inicia-se o artigo com um breve caso para ilustrar o problema de modo geral e, em seguida, desenvolve-se um marco anal??tico para ajudar o setor p??blico a aprender como realizar suas responsabilidades de due diligence (checagem) de forma mais eficiente.

Conselhos municipais de pol??ticas p??blicas: uma an??lise explorat??ria

Os Conselhos Gestores s??o f??runs h??bridos onde se viabiliza a participa????o popular no desenho das pol??ticas p??blicas, nas negocia????es de interesses da coletividade, na elabora????o de programas e projetos sociais e na fiscaliza????o das a????es governamentais. O significado e a dimens??o dos Conselhos para a democracia participativa s??o enormes, justificando a import??ncia da realiza????o deste estudo, que se baseou na an??lise explorat??ria de 12 Conselhos locais do munic??pio mineiro de Vi??osa, com o objetivo de avaliar o desempenho dos mesmos. A avalia????o do desempenho levou em considera????o os mesmos par??metros dimensionais, tanto para a coleta de dados prim??rios quanto para a coleta secund??ria. A coleta de dados prim??rios foi realizada a partir da aplica????o de question??rios semi-estruturados aos presidentes dos Conselhos em an??lise. Utilizou-se o programa SPSS, vers??o 15.0, para an??lise estat??stica das quest??es objetivas dos question??rios e a t??cnica de an??lise de conte??do para exame das quest??es abertas. Ap??s essa etapa, realizou-se um workshop com os presidentes dos Conselhos, visando ?? obten????o de novos dados e informa????es. Os Conselhos municipais analisados apresentaram resultados pouco discrepantes entre a teoria aplicada ao tema e a sua efetiva pr??tica no campo p??blico. Contudo, a pesquisa revelou que o grau de desenvolvimento institucional dos Conselhos n??o ?? homog??neo, ou seja, h?? alguns melhores gestores que outros. A escolha independente e democr??tica dos membros n??o-governamentais dos Conselhos em Vi??osa revelou o seu grau de independ??ncia do Poder Executivo. Por??m, a capacita????o de conselheiros precisa ser implementada para evitar uma desfigura????o dos Conselhos. No que tange ?? governan??a, destacouse em Vi??osa o seu grau e formas de aplicabilidade observadas na gest??o de v??rias pol??ticas. Da mesma forma, foi observado um n??vel de governabilidade que confere legitimidade ??s pol??ticas implementadas. Por fim, verificou-se que a efetividade da accountability societal tem sido instrumentalizada pelos Conselhos no ??mbito das diversas pol??ticas setoriais. J?? os dados secund??rios da an??lise foram obtidos por meio das s??nteses dos relat??rios de fiscaliza????o de munic??pios do Estado de Minas Gerais, realizadas pela Controladoria Geral da Uni??o. Foram identificados todos os registros pertinentes aos Conselhos Gestores Municipais do Estado de Minas Gerais, no per??odo de 2003 a 2006. Verificou-se que os maiores problemas se referem aos Conselhos Municipais de Sa??de e Assist??ncia Social. Entre as in??meras falhas apontadas, destacam-se problemas graves na constitui????o e operacionaliza????o dos Conselhos, resultando na inoper??ncia desses mecanismos nos munic??pios fiscalizados. A avalia????o do desempenho dos Conselhos locais de Vi??osa possibilitou concluir que esses mecanismos apresentaram um desempenho bem satisfat??rio na gest??o e controle das pol??ticas p??blicas no munic??pio.

Gest??o p??blica e pesquisa operacional: avalia????o de desempenho em ag??ncias da Previd??ncia Social

O presente trabalho utiliza a metodologia DEA (Data Envelopment Analysis ), para avaliar a efici??ncia das 22 Ag??ncias da Previd??ncia Social ??? Ger??ncia Executiva Fortaleza (APS-GEXFOR). DEA utiliza programa????o linear cujo n??cleo das estruturas anal??ticas ?? formado a partir do modelo original CCR (Charnes, Cooper, e Rhodes). Aplicada ??s DMU (Decision Making Units), define uma Fronteira de Efici??ncia identificando Unidades Eficientes e Ineficientes. Foi utilizado o modelo DEA-CCR implementado pelo software DEA Solver??. A Previd??ncia Social (INSS) disp??e de indicadores de desempenho. Algumas vari??veis utilizadas no modelo implementado derivam desses indicadores, outras informa????es foram disponibilizadas pelos sistemas de informa????o da institui????o. A avalia????o de efici??ncia DEA das APS-GEXFOR permitiu identificar as melhores pr??ticas, mensurar a participa????o de cada vari??vel envolvida na avalia????o da unidade e projetar as unidades ineficientes na fronteira de efici??ncia, identificando metas a serem atingidas para torn??-las eficientes no conjunto observado.