An allele-specific, stochastic gene expression process controls the expression of multiple Ly49 family genes and generates a diverse, MHC-specific NK cell receptor repertoire.

Mouse NK cells express MHC class I-specific inhibitory Ly49 receptors. Since these receptors display distinct ligand specificities and are clonally distributed, their expression generates a diverse NK cell receptor repertoire specific for MHC class I molecules. We have previously found that the Dd (or Dk)-specific Ly49A receptor is usually expressed from a single allele. However, a small fraction of short-term NK cell clones expressed both Ly49A alleles, suggesting that the two Ly49A alleles are independently and randomly expressed. Here we show that the genes for two additional Ly49 receptors (Ly49C and Ly49G2) are also expressed in a (predominantly) mono-allelic fashion. Since single NK cells can co-express multiple Ly49 receptors, we also investigated whether mono-allelic expression from within the tightly linked Ly49 gene cluster is coordinate or independent. Our clonal analysis suggests that the expression of alleles of distinct Ly49 genes is not coordinate. Thus Ly49 alleles are apparently independently and randomly chosen for stable expression, a process that directly restricts the number of Ly49 receptors expressed per single NK cell. We propose that the Ly49 receptor repertoire specific for MHC class I is generated by an allele-specific, stochastic gene expression process that acts on the entire Ly49 gene cluster.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus from 4 Cuban hospitals.

During a 1-year period, 87 methicillin-resistant Staphylococcus aureus isolates were collected from 4 major Cuban hospitals for epidemiological analysis. The majority (86%) were related to the community-associated USA300 clone, whereas the remaining belonged to a new clone ST72-V. Interestingly, no hospital-associated clone was found in these Cuban hospitals.

Vigor de clones de umezeiro e pessegueiro 'Okinawa' propagados por estacas herbáceas

O objetivo deste trabalho foi avaliar o vigor de três clones de umezeiro (Prunus mume Sieb. et Zucc.) e do pessegueiro 'Okinawa' [Prunus persica (L.) Batsch], propagados por estacas herbáceas, em condições de campo. O experimento foi conduzido em blocos ao acaso, com quatro tratamentos (genótipos) e cinco repetições. As plantas foram espaçadas 0,5 m entre si. O pessegueiro 'Okinawa' apresentou maior diâmetro do tronco, em relação aos clones de umezeiro. Na análise conjunta das variáveis, o Clone 10 revela-se o menos vigoroso, indicando a possibilidade de sucesso como porta-enxerto ananizante para pessegueiro.

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination.

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Postsynaptic density protein PSD-95 expression in Alzheimer's disease and okadaic acid induced neuritic retraction.

In order to understand how plasticity is related to neurodegeneration, we studied synaptic proteins with quantitative immunohistochemistry in the entorhinal cortex from Alzheimer patients and age-matched controls. We observed a significant decrease in presynaptic synaptophysin and an increase in postsynaptic density protein PSD-95, positively correlated with beta amyloid and phosphorylated Tau proteins in Alzheimer cases. Furthermore, Alzheimer-like neuritic retraction was generated in okadaic acid (OA) treated SH-SY5Y neuroblastoma cells with no decrease in PSD-95 expression. However, in a SH-SY5Y clone with decreased expression of transcription regulator LMO4 (as observed in Alzheimer's disease) and increased neuritic length, PSD-95 expression was enhanced but did not change with OA treatment. Therefore, increased PSD-95 immunoreactivity in the entorhinal cortex might result from compensatory mechanisms, as in the SH-SY5Y clone, whereas increased Alzheimer-like Tau phosphorylation is not related to PSD-95 expression, as suggested by the OA-treated cell models.

Viabilidade econômica de diferentes sistemas de sangria em clones de seringueira

O objetivo deste trabalho foi avaliar o desempenho produtivo e os aspectos econômicos de três clones de seringueira [Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell. Arg.], sob nove sistemas de sangria. O experimento foi instalado sob delineamento de blocos ao acaso com parcelas subdivididas no tempo. Os tratamentos principais foram os clones PR 255, RRIM 600 e GT 1, submetidos aos seguintes sistemas de sangria: ½S d/3 6d/7.11m/y.ET 2,5% Pa La 8/y; ½S d/3 6d/7.11m/y.ET 5,0% Pa La 8/y; ½S d/4 6d/7.11m/y.ET 2,5% Pa La 8/y; ½S d/4 6d/7.11m/y.ET 5,0% Pa La 8/y; ½S d/5 6d/7.11m/y.ET 2,5% Pa La 8/y; ½S d/5 6d/7.11m/y.ET 5,0% Pa La 8/y; ½S d/7 6d/7.11m/y.ET 2,5% Pa La 8/y; ½S d/7 6d/7.11m/y.ET 5,0% Pa La 8/y e ½S d/2 6d/7.11m/y (testemunha). As variáveis estudadas foram: perímetro do caule, produtividade de borracha seca e secamento do painel. Também foi avaliada a viabilidade econômica dos sistemas de sangria. Observaram-se maior produtividade e rentabilidade dos sistemas ½S d/3.ET 2,5% 8/y para os clones PR 255 e RRIM 600 e ½S d/7.ET 2,5% 8/y para o clone GT 1, comparados com a testemunha. A maior e a menor porcentagem de secamento do painel foram observadas nos sistemas ½S d/3 ET 5,0% 8/y e ½S d/7.ET 5,0% 8/y, respectivamente.

Desenvolvimento vegetativo e custo de produção de porta-enxertos de citros em recipientes para fins de subenxertia

O objetivo deste trabalho foi avaliar o desenvolvimento vegetativo e estimar o custo de produção de 11 porta-enxertos de citros para fins de subenxertia, em diferentes recipientes. Avaliaram-se limão 'Cravo' clone Limeira (Citrus limonia Osbeck); citrumelo 'Swingle' (Poncirus trifoliata (L.) Raf. x Citrus paradisi Macf.); tangerina 'Cleópatra' (Citrus reshni Hort. ex Tanaka); tangerina 'Sunki' (Citrus sunki Hort. ex Tanaka); limão 'Volkameriano' clone Catânia 2 (Citrus volkameriana Pasquale); laranja 'Caipira' clone DAC (Citrus sinensis L. Osbeck); limão 'Rugoso da África' clone Mazoe (Citrus jambhiri Lush.); Poncirus trifoliata 'Davis A'; tangerina 'Sun Shu Sha Kat' (Citrus sunki Hort. ex Tanaka); tangerina 'Sunki' clone 2506 ou Fruto Grande (Citrus sunki Hort. ex Tanaka) e Poncirus trifoliata 'Barnes'. Foram utilizados tubetes de 290 mL, sacolas de 1,7 L, e porta-enxertos transplantados de tubetes de 75 mL para sacolas de polietileno de 1,7 e 4,5 L. Porta-enxertos produzidos diretamente em sacolas de 1,7 L atingem ponto ideal de subenxertia em menor tempo, de 100 a 150 dias após a semeadura, e permitem a obtenção de plantas maiores e com sistema radicular adequado, porém com custo de produção superior ao sistema de produção em tubetes de 290 mL.

Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells.

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)

Sistemas de explotação de seringueira utilizados em clones asiáticos Prang Besar no Oeste paulista

O objetivo deste trabalho foi avaliar o desempenho produtivo e os aspectos fisiológicos e econômicos de quatro clones asiáticos Prang Besar de seringueira [Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg.], sob diferentes sistemas de sangria. O experimento foi instalado na Fazenda Santa Gilda, Município de Guararapes, SP, em delineamento de blocos ao acaso, com parcelas subdivididas no tempo. Os tratamentos principais foram os clones PB 260, PB 235, PB 330 e PB 217, submetidos a nove sistemas de sangria: ½S d/3.ET 2,5% 8/y; ½S d/3.ET 5% 8/y; ½S d/4.ET 2,5% 8/y; ½S d/4.ET 5% 8/y; ½S d/5.ET 2,5% 8/y; ½S d/5.ET 5% 8/y; ½S d/7.ET 2,5% 8/y; ½S d/7.ET 5% 8/y e ½S d/2 (testemunha). As variáveis avaliadas foram: perímetro do caule, produção de borracha seca, secamento do painel; foi avaliada, também, a viabilidade econômica. Os resultados mostram a superioridade econômica dos sistemas ½S d/5.ET 2,5% 8/y e ½S d/7.ET 5% 8/y, para o clone PB 260; ½S d/7.ET 2,5% 8/y para o clone PB 235; e ½S d/3.ET 2,5% 8/y e ½S d/3.ET 5% 8/y, para os clones PB 330 e PB 217, comparados com a testemunha. A menor incidência de secamento do painel foi observada no sistema ½S d/7.ET 5% 8/y, exceto para o clone PB 235.

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.

Microbial communities in a porphyry copper tailings impoundment and their impact on the geochemical dynamics of the mine waste

The distribution and diversity of acidophilic bacteria of a tailings impoundment at the La Andina copper mine, Chile, was examined. The tailings have low sulfide (1.7% pyrite equivalent) and carbonate (1.4% calcite equivalent) contents and are stratified into three distinct zones: a surface (0-70-80 cm) `oxidation zone' characterized by low-pH (2.5-4), a `neutralization zone' (70-80 to 300-400 cm) and an unaltered `primary zone' below 400 cm. A combined cultivation-dependent and biomolecular approach (terminal restriction enzyme fragment length polymorphism and 16S rRNA clone library analysis) was used to characterize the indigenous prokaryotic communities in the mine tailings. Total cell counts showed that the microbial biomass was greatest in the top 125 cm of the tailings. The largest numbers of bacteria (10(9) g(-1) dry weight of tailings) were found at the oxidation front (the junction between the oxidation and neutralization zones), where sulfide minerals and oxygen were both present. The dominant iron-/sulfur-oxidizing bacteria identified at the oxidation front included bacteria of the genus Leptospirillum (detected by molecular methods), and Gram-positive iron-oxidizing acidophiles related to Sulfobacillus (identified both by molecular and cultivation methods). Acidithiobacillus ferrooxidans was also detected, albeit in relatively small numbers. Heterotrophic acidophiles related to Acidobacterium capsulatum were found by molecular methods, while another Acidobacterium-like bacterium and an Acidiphilium sp. were isolated from oxidation zone samples. A conceptual model was developed, based on microbiological and geochemical data derived from the tailings, to account for the biogeochemical evolution of the Piuquenes tailings impoundment.

Inhibition and enhancement of in vitro helper activity of apocytochrome c-specific murine T cell populations and clones by anti-apocytochrome c-specific monoclonal antibodies.

A murine monoclonal antibody (mAb) specific for apocytochrome c was found to be able to either inhibit or enhance the helper activity of mouse apocytochrome c-specific T cell clones and populations in a hapten (trinitrophenyl)-carrier (apocytochrome c) system of T-B cell cooperation. This effect of the mAb was carrier specific, could not be ascribed simply to a shift in the kinetics of the antibody response and was observed using apocytochrome c T helper cells of different mouse haplotypes. In addition, the anti-apocytochrome c mAb was able to inhibit specific T helper cell activity even when the T cells were triggered with antigen-presenting cells pulsed with antigen. Taken together, these results suggested that the mAb was inhibiting helper activity due to its ability to modify the interaction between T cells and antigen-presenting cells.

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.

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.