Divergência genética em café conilon

O objetivo deste trabalho foi avaliar a divergência genética entre 32 clones de café conilon (Coffea canephora Pierre ex Frohener) componentes de três variedades clonais melhoradas, com vistas à identificação dos mais dissimilares, para o estabelecimento de programas de cruzamentos dirigidos. A divergência genética foi avaliada por procedimentos multivariados: distância generalizada de Mahalanobis, método de agrupamento de otimização de Tocher e técnica de variáveis canônicas. Sete caracteres foram avaliados em experimento conduzido em Marilândia, ES. Os genótipos ES 92, ES 25 e ES 22 são os mais divergentes, sendo os dois últimos os mais indicados para cruzamento com os demais, tendo em vista aliarem divergência genética a um bom desempenho produtivo.

Development of stable cell lines for production or regulated expression using matrix attachment regions.

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Glucocorticoid-induced MIF expression by human CEM T cells.

Macrophage migration inhibitory factor (MIF) is an upstream activator of the immune response that counter-regulates the immunosuppressive effects of glucocorticoids. While MIF is released by cells in response to diverse microbial and invasive stimuli, evidence that glucocorticoids in low concentrations also induce MIF secretion suggests an additional regulatory relationship between these mediators. We investigated the expression of MIF from the human CEM T cell line, which exists in two well-characterized, glucocorticoid-sensitive (CEM-C7) and glucocorticoid-resistant (CEM-C1) variant clones. Dexamethasone in low concentrations induced MIF secretion from CEM-C7 but not CEM-C1 T cells by a bell-shaped dose response that was similar to that reported previously for the release of MIF by monocytes/macrophages. Glucocorticoid stimulation of CEM-C7 T cells was accompanied by an MIF transcriptional response, which by promoter analysis was found to involve the GRE and ATF/CRE transcription factor binding sites. These data support a glucocorticoid-mediated MIF secretion response by T cells that may contribute to the regulation of the adaptive immune response.

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.

Role of lymph node fibroblasts in T cell priming

SummarySecondary lymphoid organs, such as lymph nodes or spleen, are the only places in our body where primary adaptive immune responses are efficiently elicited. These organs have distinct Β and Τ cell rich zones and Τ lymphocytes constantly migrate from the bloodstream into Τ zones to scan dendritic cells (DCs) for antigens they present. Specialized fibroblasts, the Τ zone reticular cells (HR.Cs), span the Τ zone in the form a three-dimensional network. lK.Cs guide incoming Τ cells in their migration, both chemically, by the secretion of the chemokines CCL19 and CCL21, and physically, by construction of a road system to which also DCs adhere. In this way TRCs are thought to facilitate encounters of Τ cells with antigen-bearing DCs and thereby accelerate the selection of rare antigen-specific Τ cells. The resulting Τ cell activation, proliferation and differentiation all take place within the TRC network. However, the influence of TRCs on Τ cell activation has so fer not been elucidated with the possible reasons being that TRCs represent a relative rare cell population and that mice devoid of TRCs have not been described.To circumvent these technical limitations, we established TRC clones and lines to have an abundant source to functionally characterize TRCs. Both the clones and lines show a fibroblastic phenotype, express a surface marker profile comparable to ex vivo TRCs and produce extracellular matrix molecules. However, expression of Ccl19, Ccl21 and ZL-7 is lost and could not be restored by cytokine stimulation. When these TRC clones or lines were cultured in a three-dimensional cell culture system, their morphology changed and resembled that of in vivo TRCs as they formed networks. By adding Τ cells and antigen-loaded DCs to these cultures we successfully reconstructed lymphoid Τ zones that allowed antigen-specific Τ cell activation.To characterize the role of TRCs in Τ cell priming, TRCs were co-cultured with antigen-specific Τ cells in the presence antigen-loaded DCs. Surprisingly, the presence of TRC lines and ex vivo TRCs inhibited rather than enhanced CD8+ Τ cell activation, proliferation and effector cell differentiation. TRCs shared this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. TRCs were identified as a strong source of nitric oxide (NO) thereby directly dampening Τ cell expansion as well as reducing the Τ cell priming capacity of DCs. The expression of inducible NO synthase (iNOS) was up- regulated in a subset of TRCs by both DC-signals as well as interferon-γ produced by primed CD8+ Τ cells. Importantly, iNOS expression was induced during viral infection in vivo in both lymph node TRCs and DCs. Consistent with a role for NO as a negative regulator, the primary Τ cell response was exaggerated in iNOS-/- mice. Our findings highlight that in addition to their established positive roles in Τ cell responses TRCs and DCs cooperate in a negative feedback loop to attenuate Τ cell expansion during acute inflammation.RésuméLes organes lymphoïdes secondaires, comme les ganglions lymphoïdes ou la rate, sont les seuls sites dans notre corps où la réponse primaire des lymphocytes Β et Τ est initiée efficacement. Ces organes ont des zones différentes, riches en cellules Β ou T. Des lymphocytes Τ circulent constamment du sang vers les zones T, où ils échantillonent la surface des cellules dendritiques (DCs) pour identifier les antigènes qu'ils présentent. Des fibroblastes spécialisés - nommés Τ zone reticular cells (TRCs)' forment un réseau tridimensionnel dans la zone T. Les TRCs guident la migration des cellules Τ par deux moyens: chimiquement, par la sécrétion des chimiokines CCL19 et CCL21 et physiquement, par la construction d'un réseau routier en trois dimensions, auquel adhèrent aussi des DCs. Dans ce? cas, on pense que la présence des TRCs facilite les rencontres entre les cellules Τ et les DCs chargées de l'antigène et accélère la sélection des rares cellules Τ spécifiques. Ensuite, l'activation de cellules T, ainsi que la prolifération et la différenciation se produisent toutes à l'intérieur du réseau des TRCs. L'influence des TRCs sur l'activation des cellules T n'est que très peu caractérisée, en partie parce que les TRCs représentent une population rare et que les souris déficientes dans les TRCs n'ont pas encore été découvertes.Pour contourner ces limitations techniques, nous avons établi des clones et des lignées cellulaires de TRC pour obtenir une source indéfinie de ces cellules permettant leur caractérisation fonctionnelle. Les clones et lignées établis ont un phénotype de fibroblaste, ils expriment des molécules de surface similaires aux TRCs ex vivo et produisent de la matrice extracellulaire. Mais l'expression de Ccl19, Ccl21 et 11-7 est perdue et ne peut pas être rétablie par stimulation avec différentes cytokines. Les clones TRC ou les lignées cultivées en un système tridimensionnel de culture cellulaire, montrent une morphologie changée, qui ressemble à celle de TRC ex vivo inclus la construction de réseaux tridimensionnels.Pour caractériser le rôle des TRC dans l'activation des cellules T, nous avons cultivé des TRCs avec des cellules T spécifiques et des DCs chargées avec l'antigène. Etonnamment, la présence des TRC (lignées et ex vivo) inhibait plutôt qu'elle améliorait l'activation, la prolifération et la différenciation des lymphocytes T CDS+. Les TRCs partageaient cette fonction avec des fibr-oblastes des organes non lymphoïdes et des cellules souches du type mésenchymateux. Dans ces conditions, les TRCs sont une source importante d'oxyde nitrique (NO) et par ce fait limitent directement l'expansion des cellules T et réduisent aussi la capacité des DCs à activer les cellules T. L'expression de l'enzyme NO synthase inductible (ïNOS) est régulée à la hausse par des signaux dérivés des DCs et par l'interféron-γ produit par des cellules T de type CD8+ activées. Plus important, l'expression d'iNOS est induite pendant une infection virale in vivo, dans les TRCs et dans les DCs. Par conséquent, la réponse primaire de cellules T est exagérée dans des souris iNOS-/-. Nos résultats mettent en évidence qu'en plus de leur rôle positif bien établi dans la réponse immunitaire, les TRCs et les DCs coopèrent dans une boucle de rétroaction négative pour atténuer l'expansion des cellules T pendant l'inflammation aigiie pour protéger l'intégrité et la fonctionnalité des organes lymphoïdes secondaires.

Th2 activities induced during virgin T cell priming in the absence of IL-4, IL-13, and B cells.

Virgin T cells being primed to Th2-inducing or Th1-inducing Ags, respectively, start to synthesize IL-4 or IFN-gamma as they begin to proliferate. Parallel respective induction of B cells to produce gamma1 or gamma2a switch transcripts provides additional evidence of early divergent Th activity. This report concerns the roles of IL-4, IL-13, and B cells in these early events in vivo. Th2 responses were induced in lymph nodes against hapten-protein given s.c. with killed Bordetella pertussis adjuvant. In T cell proliferation in wild-type mice, IL-4 message up-regulation and gamma1 and epsilon switch transcript production were underway 48-72 h after immunization. The absence of IL-4, IL-13, or B cells did not alter the early T cell proliferative response. The gamma1 and epsilon switch transcript production was still induced in the absence of IL-4, IL-13, or both, but at a reduced level, while the dominance of switching to IgG1 in the extrafollicular hapten-specific plasma cell response was retained. The up-regulation of IL-4 message was not reduced or delayed in the absence of B cells and was only marginally reduced by the absence of IL-13. It is concluded that signals delivered by dendritic cells, which are not dependent on the presence of IL-4, IL-13, or B cells, can prime virgin T cells and induce the early Th2 activities studied. These early events that direct virgin T cells toward Th2 differentiation contrast with the critical later role of Th2 cytokines in selectively expanding Th2 clones and driving further IL-4 synthesis.

Distribuição do sistema radicular de porta-enxertos de umezeiro enxertados com o pessegueiro 'Aurora-1'

O objetivo deste trabalho foi estudar a distribuição do sistema radicular de três porta-enxertos de umezeiro (Prunus mume Siebold et Zucc.), Clone 05, Clone 15 e a cultivar Rigitano, propagados por estacas herbáceas, em condições de campo. As plantas, enxertadas com o pessegueiro 'Aurora-1' [Prunus persica (L.) Batsch], foram conduzidas no espaçamento de 6x1 m em Argissolo Vermelho-Amarelo eutrófico de textura arenosa média. Aos 34 meses após o transplantio, foram avaliadas duas plantas de cada porta-enxerto, tendo-se demarcado 36 monólitos (0,5x0,5x0,4 m) ao redor de cada planta, com barras de ferro (0,6 m) e fitas de plástico. O solo foi removido com jatos de água até a profundidade de 0,4 m. Não houve diferença entre os porta-enxertos, na massa de matéria fresca e seca de raízes, e na distribuição das raízes finas e grossas ao redor da planta. Mesmo sem a formação de uma raiz pivotante típica, as raízes grossas apresentaram crescimento vertical, além dos 0,4 m avaliados, e concentraram-se a 0,5 m ao redor do tronco da planta. As raízes finas apresentaram crescimento horizontal, além da projeção da copa, e também além dos 1,5 m avaliados, no sentido transversal à linha de plantio. Os Clones 05, 15 e a cultivar Rigitano de umezeiro, usados como porta-enxerto de pessegueiro, apresentam ancoragem satisfatória de plantas.

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

Caracterização morfoagronômica e coeficientes de trilha de caracteres componentes da produção em mandioca

Os objetivos deste trabalho foram caracterizar agronomicamente cem clones de mandioca (Manihot esculenta Crantz) e calcular os coeficientes de trilha entre a produção de raízes tuberosas e cinco componentes da produção, de modo a auxiliar na seleção de clones superiores. O experimento foi conduzido em área experimental da Universidade Federal de Lavras, em 2005 e 2006. Cem clones de mandioca foram avaliados em delineamento látice quadrado 10x10. A unidade experimental foi constituída por quatro plantas espaçadas de 1,0x0,6 m. As análises estatísticas foram realizadas considerando-se as oito características individualmente, utilizando-se o teste de Scott-Knott para agrupamento das médias. Os clones 87 e 88 mostraram-se promissores tanto para serem utilizados em cruzamentos quanto para fixação como novas cultivares em virtude do excelente desempenho nas características comprimento, número e produção de raízes tuberosas por planta. A análise de trilha mostrou que o número de raízes por planta e o peso total da parte aérea podem ser utilizados como critérios na seleção indireta para produção de raízes tuberosas em mandioca.

Vitellogenin genes A1 and B1 are linked in the Xenopus laevis genome.

Genomic clones containing the Xenopus laevis vitellogenin gene B1 have been isolated from DNA libraries and characterized by heteroduplex mapping in the electron microscope, restriction endonuclease analysis, and in vitro transcription in a HeLa whole-cell extract. Sequences from the 3'-flanking region of the previously isolated A1 vitellogenin gene were found in the 5'-flanking region of this B1 gene. Thus, the two genes are linked, with 15.5 kilobase pairs of DNA between them. Their length is about 22 kilobase pairs (A1 gene) and 16.5 kilobase pairs (B1 gene) and they have the following arrangement: 5'-A1 gene-spacer-B1 gene-3'. The analysis of heteroduplexes formed between the two genes revealed several regions of homology. Both genes are in the same orientation and, therefore, are transcribed from the same DNA strand. The possible events by which the vitellogenin gene family arose in Xenopus laevis are discussed.

Marcadores fAFLP na caracterização de três genótipos de umezeiro selecionados como porta-enxertos para pessegueiro

O objetivo deste trabalho foi caracterizar a diversidade genética existente em três genótipos de umezeiro (Clone 05, cv. Rigitano e Clone 15) e identificar marcadores moleculares fAFLP (fluorescent Amplified Fragment Lenght Polymorphism) passíveis de serem utilizados na discriminação dos três genótipos de umezeiro selecionados como porta-enxertos para pessegueiro. Foram utilizadas 24 diferentes combinações de primers seletivos fAFLP que geraram 648 marcas, das quais 272 foram diferenciadoras dos três genótipos entre si. As marcas diferenciadoras permitiram o agrupamento dos clones de umezeiro de acordo com sua similaridade através do Método da Distância e algorítmo Neighbour Joining. As mesmas marcas foram utilizadas para calcular a distância genética entre os clones. Com o uso de marcadores fAFLP foi possível discriminar os três genótipos de umezeiro entre si, destacando-se as combinações Fam ACT/CAT, Joe AGG/CTT e Ned AGC/CAA, que permitiram a diferenciação individual de cada um dos clones. A maior distância genética foi encontrada entre a cv. Rigitano e o Clone 15. Os marcadores fAFLP revelaram maior proximidade genética entre o Clone 05 e a cv. Rigitano.

Diversidade genética entre acessos de cacau de fazendas e de banco de germoplasma na Bahia

O objetivo deste trabalho foi avaliar a diversidade genética de acessos de cacau, selecionados previamente como produtivos e resistentes à vassoura-de-bruxa na Bahia, e estudar suas inter-relações com genótipos no banco de germoplasma. Amostras de DNA de folhas dos 120 acessos, coletados em 17 fazendas de sete municípios do Sul da Bahia, foram amplificadas pela técnica de RAPD ("random amplified polymorphic DNA"). Os coeficientes de dissimilaridade genética, calculados pelo método de Jaccard a partir das bandas RAPD, permitiram evidenciar, pela análise de agrupamento, que a maioria das seleções das fazendas (89,2%) agrupou-se com acessos do banco de germoplasma considerados representativos da diversidade de cacau (híbridos, trinitários, Scavinas, amazônicos e cacau-comum). As demais seleções distribuíram-se em outros sete grupos distintos. Há elevada diversidade genética entre as seleções das fazendas, e algumas delas devem ter-se originado de genitores não incluídos nesta análise. Esses materiais apresentam potencial para seleção de clones com maior diversidade para novos cruzamentos ou uso pelos agricultores.

Differential gene expression, induced by salicylic acid and Fusarium oxysporum f. sp. lycopersici infection, in tomato

The objective of this work was to determine the transcript profile of tomato plants (Lycopersicon esculentum Mill.), during Fusarium oxysporum f. sp. lycopersici infection and after foliar application of salicylic acid. The suppression subtractive hybridization (SSH) technique was used to generate a cDNA library enriched for transcripts differentially expressed. A total of 307 clones was identified in two subtractive libraries, which allowed the isolation of several defense-related genes that play roles in different mechanisms of plant resistance to phytopathogens. Genes with unknown roles were also isolated from the two libraries, which indicates the possibility of identifying new genes not yet reported in studies of stress/defense response. The SSH technique is effective for identification of resistance genes activated by salicylic acid and F. oxysporum f. sp. lycopersici infection. Not only the application of this technique enables a cost effective isolation of differentially expressed sequences, but also it allows the identification of novel sequences in tomato from a relative small number of sequences.

Closed soilless growing system for producing strawberry bare root transplants and runner tips

The objective of this work was to test a closed soilless growing system for producing bare root transplants and runner tips of two strawberry clones, using two categories of substrates. The system used corrugated roofing panels of fiber-cement, over which a substrate layer was used as a growing bed. The nutrient solution was pumped from a reservoir toward the upper end of the roofing panels and drained back to a reservoir. Plant growth and development were determined for two advanced strawberry clones, grown in sand or in Plantmax organic substrate. Growth of the stock plants and the number and dry mass of bare root transplants were similar in the substrates, but bare roots differed in their crown diameters by substrate. For number of runner tips, no significant differences were found in total, small, and medium categories in the substrates. A mean production of about 590 runner tips per square meter and 145 bare root transplants per square meter was obtained. For both clones, a large number of bare root transplants and runner tips of adequate size were produced in the closed soilless growing system using sand or organic substrate.

Estimativas de repetibilidade de caracteres de produção em laranjeiras-doces no Acre

O objetivo deste trabalho foi estimar o coeficiente de repetibilidade em laranjeiras-doces e o número mínimo de avaliações a serem feitas para a determinação do valor real dos indivíduos. De março a junho de 1999, foram coletadas borbulhas de laranjeiras pé-franco, em fase de produção, em nove municípios do Acre. As borbulhas foram enxertadas sobre porta-enxertos de limoeiro 'Cravo', o que resultou em 54 clones. Esses clones foram avaliados em conjunto com a cultivar Aquiri, recomendada para o Estado. O delineamento experimental foi o de blocos ao acaso, com 55 tratamentos (55 clones), três repetições, com uma planta por parcela. O número total de frutos por planta, a produção de frutos por planta e o peso médio de fruto foram avaliados em 2002, 2003, 2004, 2005, 2006 e 2008. Foram realizadas as análises de variância, de componentes principais e estrutural. A estimativa de repetibilidade para peso médio do fruto demonstrou regularidade na classificação dos clones, de um ciclo para outro, e foram necessários cinco ciclos de avaliação para a predição do valor real dos indivíduos, com acurácia de 90%, pelo método dos componentes principais (matriz de co-variância). Apesar de as características número de frutos total por planta e produção de frutos por planta serem influenciadas pelo ambiente, oito e nove medições, respectivamente, permitem obter coeficientes de determinação de 95%.