Protection of mice from group A streptococcal infection by intranasal immunisation with a peptide vaccine that contains a conserved M protein B cell epitope and lacks a T cell autoepitope

Infection with group A streptococci (GAS) can lead to rheumatic fever (RF) and rheumatic heart disease (RHD) which are a major health concern particularly in indigenous populations worldwide, and especially in Australian Aboriginals. A primary route of GAS infection is via the upper respiratory tract, and therefore, a major goal of research is the development of a mucosal-based GAS vaccine, The majority of the research to date has focused on the GAS M protein since immunity to GAS is mediated by M protein type-specific opsonic antibodies. There are two major impediments to the development of a vaccine-the variability in M proteins and the potential for the induction of an autoimmune response. To develop a safe and broad-based vaccine, we have therefore focused on the GAS M protein conserved C-region, and have identified peptides, J8 and the closely related J8 peptide (J14), which may be important in protective immunity to GAS infection. Using a mucosal animal model system, our data have shown a high degree of throat GAS colonisation in B10.BR mice 24 h following intranasal immunisation with the mucosal adjuvant, cholera toxin B subunit (CTB), and/or diptheria toxoid (dT) carrier, or PBS alone, and challenge with the M1 GAS strain. However, GAS colonisation of the throat was significantly reduced following intranasal immunisation of mice with the vaccine candidate J8 conjugated to dT or J14-dT when administered with CTB. Moreover, J8-dT/CTB and J14-dT/CTB-immunised mice had a significantly higher survival when compared to CTB and PBS-immunised control mice. These data indicate that immunity to GAS infection can be evoked by intranasal immunisation with a GAS M protein C-region peptide vaccine that contains a protective B cell epitope and lacks a T cell autoepitope. (C) 2002 Published by Elsevier Science Ltd.

Conserved modularity and potential for alternate splicing in mouse and human Slit genes

The vertebrate Slit gene family currently consists of three members;Slit1,Slit2 and Slit3. Each gene encodes a protein containing multiple epidermal growth factor and leucine rich repeat motifs, which are likely to have importance in cell-cell interactions. In this study, we sought to fully define and characterise the vertebrate Slit gene family. Using long distance PCR coupled with in silico mapping, we determined the genomic structure of all three Slit genes in mouse and man. Analysis of EST and genomic databases revealed no evidence of further Slit family members in either organism. All three Slit genes were encoded by 36 (Slit3) or 37 (Slit1 and Slit2) exons covering at least 143 kb or 183 kb of mouse or human genomic DNA respectively. Two additional potential leucine-rich repeat encoding exons were identified within intron 12 of Slit2. These could be inserted in frame, suggesting that alternate splicing may occur in Slit2 A search for STS sequences within human Slit3 anchored this gene to D5S2075 at the 5' end (exon 4) and SGC32449 within the 3' UTR, suggesting that Slit3 may cover greater than 693 kb. The genomic structure of all Slit genes demonstrated considerable modularity in the placement of exon-intron boundaries such that individual leucine-rich repeat motifs were encoded by individual 72 by exons. This further implies the potential generation of multiple Slit protein isoforms varying in their number of repeat units. cDNA library screening and EST database searching verified that such alternate splicing does occur.

Functional significance of a highly conserved glutamate residue of the human noradrenaline transportere

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of IIE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in V-max values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.

The role of conserved CXXXRXG motif in the expression and function of the human norepinephrine transporter

Highly conserved motifs in the monoamine transporters, e.g. the human norepinephrine transporter (hNET) GXXXRXG motif which was the focus of the present study, are likely to be important structural features in determining function. This motif was investigated by mutating the glycines to glutamate (causing loss of function) and alanine, and the arginine to glycine. The effects of hG117A, hR121G and hG123A mutations on function were examined in COS-7 cells and compared to hNET. Substrate K-m values were decreased for hG117A and hG123A, and their K values for inhibition of [3 H]nisoxetine binding were decreased 3-4-fold and 4-6-fold, respectively. Transporter turnover was reduced to 65% of hNET for hG117A and hR121G and to 28% for hG123A, suggesting that substrate translocation is impaired. K values of nisoxetine and desipramine for inhibition of [H-3]norepinephrine uptake were increased by 5-fold for hG117A, with no change for cocaine. The K-i value of cocaine was increased by 3-fold for hG123A, with no change for nisoxetine and desipramine. However, there were no effects of the mutations on the K-d of [H-3]nisoxetine binding or K-i values of desipramine or cocaine for inhibition of [H-3]nisoxetine binding. Hence, glycine residues of the GXXXRXG motif are important determinants of NET expression and function, while the arginine residue does not have a major role. This study also showed that antidepressants and psychostimulants have different NET binding sites and provided the first evidence that different sites on the NET are involved in the binding of inhibitors and their competitive inhibition of substrate uptake. (C) 2002 Elsevier Science B.V. All rights reserved.

Characterization of highly conserved microsatellite loci in Araucaria cunninghamii and related species

Ten microsatellite loci are described in Araucaria cunninghamii, the first reported in the Araucariaceae. Eight were tested in sections Eutacta and Bunya, which diverged more than 200 MYA, and to the sister genus Agathis. Specific amplification products within the expected size range were obtained for six to eight loci in section Eutacta (depending on species), five loci in section Bunya and three. loci in Agathis. Two of the loci (CRCAc1 and CRCAc2, both GA repeats) produced specific amplification products in all taxa, with orthology confirmed by sequence analysis. The repeats were perfect in all taxa. The flanking sequences were extremely conserved, with sequence divergence of 0% to 2.0% within Araucaria species and 2.9% to 7.5% between Araucaria and Agathis. These microsatellites represent some of the most conserved microsatellite loci reported in plants. This may be due to a low evolutionary rate in Araucariaceae genome or the loci may be closely associated with highly conserved, unreported genes.

Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.

Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development

Blepharophimosis ptosis epicanthus inversus syndrome (BPES) is a human disorder caused by mutations in the forkhead transcription factor gene FOXL2 and is characterized by facial dysmorphology combined in some cases with ovarian failure. To better understand the role of FOXL2 in the etiology of ovarian failure in BPES, we examined its expression in embryonic ovaries of mice, chickens, and red-eared slider turtles, representatives of three phylogenetically distant vertebrate groups that have different mechanisms of sex determination. Expression of Foxl2 was detected in early ovaries of all three species around the time of sex determination and was associated with both somatic and germ cell populations in mice. Expression was sexually dimorphic in all cases. Sequence analysis of turtle and chicken FoxL2 orthologues indicated an unusually high degree of structural conservation during evolution. FoxL2 was found to be autosomal in chickens, and therefore unlikely to represent the dominant ovarian-determining gene that has been postulated to exist as a possible explanation for female heterogamety in birds. Our observations suggest that BPES may result from early abnormalities in regulating the development of the fetal ovary, rather than premature degeneration of the postnatal or adult ovary. Further, our results suggest that FOXL2 is a highly conserved early regulator of vertebrate ovarian development.

The value of idiosyncratic markers and changes to conserved tRNA sequences from the mitochondrial genome of hard ticks (Acari: Ixodida: Ixodidae) for phylogenetic inference

Idiosyncratic markers are features of genes and genomes that are so unusual that it is unlikely that they evolved more than once in a lineage of organisms. Here we explore further the potential of idiosyncratic markers and changes to typically conserved tRNA sequences for phylogenetic inference. Hard ticks were chosen as the model group because their phylogeny has been studied extensively. Fifty-eight candidate markers from hard ticks ( family Ixodidae) and 22 markers from the subfamily Rhipicephalinae sensu lato were mapped onto phylogenies of these groups. Two of the most interesting markers, features of the secondary structure of two different tRNAs, gave strong support to the hypothesis that species of the Prostriata ( Ixodes spp.) are monophyletic. Previous analyses of genes and morphology did not strongly support this relationship, instead suggesting that the Prostriata is paraphyletic with respect to the Metastriata ( the rest of the hard ticks). Parallel or convergent evolution was not found in the arrangements of mitochondrial genes in ticks nor were there any reversals to the ancestral arthropod character state. Many of the markers identified were phylogenetically informative, whereas others should be informative with study of additional taxa. Idiosyncratic markers and changes to typically conserved nucleotides in tRNAs that are phylogenetically informative were common in this data set, and thus these types of markers might be found in other organisms.

Educação infantil em jornada de tempo integral : dilemas e perspectivas

Vania Carvalho de Araújo (Org.)

Estudantes público-alvo da educação especial na educação em tempo integral : um estudo em Vitória-ES

Este estudo tem como tema “Estudantes Público-Alvo da Educação Especial na Educação em Tempo Integral no Município de Vitoria-ES”. Seu objetivo geral foi investigar os modos de atendimento aos estudantes público-alvo da Educação Especial do Programa Educação em Tempo Integral – Educação Ampliada, oferecido pelo ensino público municipal nas Escolas de Ensino Fundamental (EMEFs) do município de Vitória, pela fala dos gestores e executores do programa. Seus objetivos específicos foram compreender a configuração da proposta de Educação em Tempo Integral – Educação Ampliada no município de Vitória-ES; caracterizar o conjunto de estudantes público-alvo da Educação Especial participantes do Programa Educação em Tempo Integral – Educação Ampliada no município; compreender os sentidos e significados da inclusão de estudantes público-alvo da Educação Especial no Programa Educação em Tempo Integral – Educação Ampliada no município de Vitória-ES. Trata-se de pesquisa qualitativa, cuja metodologia envolveu revisão de literatura e estudo de caso. O instrumento de pesquisa foi uma entrevista semiestruturada, por permitir explorar mais amplamente as abordagens e proporcionar aos entrevistados maior liberdade para emitir as suas opiniões. Os sujeitos da pesquisa foram os gestores responsáveis pelo desenvolvimento do Programa Educação em Tempo Integral – Educação Ampliada da Secretaria Municipal de Educação do município de Vitória (Seme) e a equipe de uma das EMEFs que compôs o grupo focal, profissionais diretamente envolvidos no exercício das práticas pedagógicas com os estudantes matriculados no Programa da Unidade de Ensino. Os resultados das análises da pesquisa foi que a Educação em Tempo Integral – Educação Ampliada é uma realidade no município de Vitória-ES, desde a sua implantação em 1989. O programa atende hoje a 3.203 estudantes. Desse total, apenas 35, ou seja, 1,9%, compõem o público-alvo da Educação Especial atendido pelo programa em foco. A análise das informações documentais, os relatos dos gestores que se revezaram desde a implantação do Peti e as entrevistas com os sujeitos desta pesquisa revelaram que a educação em tempo integral se processa no município de Vitória de forma lenta e descontínua, especialmente por ocasião do revezamento entre uma administração e outra, quando muda a gestão política.

La adopción del cuadro de mando integral en organizaciones portuguesas: variables contingentes e institucionales

Este artículo tiene como objetivo identificar y analizar un conjunto de variables contingentes e institucionales que pueden potencialmente influir en el conocimiento e implementación del Cuadro de Mando Integral (CMI) en las organizaciones portuguesas pertenecientes al sector público y privado.

Determinantes de la adopción del cuadro de mando integral en organizaciones portuguesas del setor público y privado

Esta comunicación tiene como objetivo identificar y analizar un conjunto de variables que pueden potencialmente influir en la adopción del Cuadro de Mando Integral (CMI) en organizaciones públicas y privadas portuguesas. Definimos un conjunto de hipótesis de investigación y para comprobar empíricamente las hipótesis formuladas hemos recurrido a los datos obtenidos mediante la aplicación de un cuestionario postal a 591 organizaciones públicas (ayuntamientos, hospitales, empresas municipales y empresas intermunicipales) y 549 organizaciones privadas (grandes empresas y pequeñas y medianas empresas) portuguesas, con una tasa de respuesta del 31,3%. Los resultados obtenidos indican que el compromiso de la alta dirección, el desarrollo de mapas estratégicos y el establecimiento de relaciones causa-efecto son factores que están asociados con la implementación del CMI.

La adopción del cuadro de mando integral en organizaciones portuguesas: variables contingentes e institucionales

Esta comunicación tiene como objetivo identificar y analizar un conjunto de variables contingentes e institucionales que pueden potencialmente influir en el conocimiento e implementación del Cuadro de Mando Integral (CMI) en las organizaciones portuguesas pertenecientes al sector público y privado. Para comprobar empíricamente las hipótesis de investigación formuladas hemos recurrido a los datos obtenidos mediante la aplicación de un cuestionario postal a 591 organizaciones públicas y 549 organizaciones privadas portuguesas, con una tasa de respuesta total del 31,3%. Los resultados obtenidos indican la asociación entre el conocimiento CMI y el grado de formación de los empleados, así como entre la implementación del CMI y la realización de cursos de formación. Por otro lado, no hemos obtenido evidencia empírica respecto a la posible relación entre la implementación del CMI y el grado de comunicación interna, el recurso a consultores externos, cuestiones de “modas de gestión” y la búsqueda de legitimidad.

El cuadro de mando integral, el activity based costing y la activity based management: un estudio empírico de su integración

La presente comunicación tiene como objetivo analizar la complementariedad y la posibilidad de integración entre las herramientas de gestión Cuadro de Mando Integral (CMI), Activity Based Costing (ABC) y Activity Based management (ABM). Para la realización del estudio empírico hemos recurrido a los datos obtenidos mediante la aplicación de un cuestionario postal a 591 organizaciones públicas (ayuntamientos, hospitales, empresas municipales y empresas intermunicipales) y 549 organizaciones privadas (grandes empresas y pequeñas y medianas empresas) portuguesas, con una tasa de respuesta total del 31,3%. Los resultados obtenidos indican que, contrariamente al registrado en las organizaciones públicas, observamos que un número significativo de organizaciones privadas ya utilizaba los sistemas ABC/ABM antes del CMI y que los han integrado (total o parcialmente) o esperan integrar en el CMI.