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 herb��ceas

O objetivo deste trabalho foi avaliar o vigor de tr��s clones de umezeiro (Prunus mume Sieb. et Zucc.) e do pessegueiro 'Okinawa' [Prunus persica (L.) Batsch], propagados por estacas herb��ceas, em condi����es de campo. O experimento foi conduzido em blocos ao acaso, com quatro tratamentos (gen��tipos) e cinco repeti����es. As plantas foram espa��adas 0,5 m entre si. O pessegueiro 'Okinawa' apresentou maior di��metro do tronco, em rela����o aos clones de umezeiro. Na an��lise conjunta das vari��veis, 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 econ��mica de diferentes sistemas de sangria em clones de seringueira

O objetivo deste trabalho foi avaliar o desempenho produtivo e os aspectos econ��micos de tr��s 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 vari��veis estudadas foram: per��metro do caule, produtividade de borracha seca e secamento do painel. Tamb��m foi avaliada a viabilidade econ��mica 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 produ����o 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 produ����o de 11 porta-enxertos de citros para fins de subenxertia, em diferentes recipientes. Avaliaram-se lim��o 'Cravo' clone Limeira (Citrus limonia Osbeck); citrumelo 'Swingle' (Poncirus trifoliata (L.) Raf. x Citrus paradisi Macf.); tangerina 'Cle��patra' (Citrus reshni Hort. ex Tanaka); tangerina 'Sunki' (Citrus sunki Hort. ex Tanaka); lim��o 'Volkameriano' clone Cat��nia 2 (Citrus volkameriana Pasquale); laranja 'Caipira' clone DAC (Citrus sinensis L. Osbeck); lim��o 'Rugoso da ��frica' 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 ap��s a semeadura, e permitem a obten����o de plantas maiores e com sistema radicular adequado, por��m com custo de produ����o superior ao sistema de produ����o 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 explota����o de seringueira utilizados em clones asi��ticos Prang Besar no Oeste paulista

O objetivo deste trabalho foi avaliar o desempenho produtivo e os aspectos fisiol��gicos e econ��micos de quatro clones asi��ticos 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, Munic��pio 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 vari��veis avaliadas foram: per��metro do caule, produ����o de borracha seca, secamento do painel; foi avaliada, tamb��m, a viabilidade econ��mica. Os resultados mostram a superioridade econ��mica 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 incid��ncia de secamento do painel foi observada no sistema ½S d/7.ET 5% 8/y, exceto para o clone PB 235.

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

O objetivo deste trabalho foi estudar a distribui����o do sistema radicular de tr��s porta-enxertos de umezeiro (Prunus mume Siebold et Zucc.), Clone 05, Clone 15 e a cultivar Rigitano, propagados por estacas herb��ceas, em condi����es de campo. As plantas, enxertadas com o pessegueiro 'Aurora-1' [Prunus persica (L.) Batsch], foram conduzidas no espa��amento de 6x1 m em Argissolo Vermelho-Amarelo eutr��fico de textura arenosa m��dia. Aos 34 meses ap��s o transplantio, foram avaliadas duas plantas de cada porta-enxerto, tendo-se demarcado 36 mon��litos (0,5x0,5x0,4 m) ao redor de cada planta, com barras de ferro (0,6 m) e fitas de pl��stico. O solo foi removido com jatos de ��gua at�� a profundidade de 0,4 m. N��o houve diferen��a entre os porta-enxertos, na massa de mat��ria fresca e seca de ra��zes, e na distribui����o das ra��zes finas e grossas ao redor da planta. Mesmo sem a forma����o de uma raiz pivotante t��pica, as ra��zes grossas apresentaram crescimento vertical, al��m dos 0,4 m avaliados, e concentraram-se a 0,5 m ao redor do tronco da planta. As ra��zes finas apresentaram crescimento horizontal, al��m da proje����o da copa, e tamb��m al��m dos 1,5 m avaliados, no sentido transversal �� linha de plantio. Os Clones 05, 15 e a cultivar Rigitano de umezeiro, usados como porta-enxerto de pessegueiro, apresentam ancoragem satisfat��ria 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 caracteriza����o de tr��s gen��tipos de umezeiro selecionados como porta-enxertos para pessegueiro

O objetivo deste trabalho foi caracterizar a diversidade gen��tica existente em tr��s gen��tipos de umezeiro (Clone 05, cv. Rigitano e Clone 15) e identificar marcadores moleculares fAFLP (fluorescent Amplified Fragment Lenght Polymorphism) pass��veis de serem utilizados na discrimina����o dos tr��s gen��tipos de umezeiro selecionados como porta-enxertos para pessegueiro. Foram utilizadas 24 diferentes combina����es de primers seletivos fAFLP que geraram 648 marcas, das quais 272 foram diferenciadoras dos tr��s gen��tipos entre si. As marcas diferenciadoras permitiram o agrupamento dos clones de umezeiro de acordo com sua similaridade atrav��s do M��todo da Dist��ncia e algor��tmo Neighbour Joining. As mesmas marcas foram utilizadas para calcular a dist��ncia gen��tica entre os clones. Com o uso de marcadores fAFLP foi poss��vel discriminar os tr��s gen��tipos de umezeiro entre si, destacando-se as combina����es Fam ACT/CAT, Joe AGG/CTT e Ned AGC/CAA, que permitiram a diferencia����o individual de cada um dos clones. A maior dist��ncia gen��tica foi encontrada entre a cv. Rigitano e o Clone 15. Os marcadores fAFLP revelaram maior proximidade gen��tica 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.

Characterization of the neuronal microtubule regulator SCG10 in transfected cells and " in vivo "

SUMMARY The ability of neuronal processes to find their way along complex paths and to establish appropriate connections depends on continual rearrangements of the cytoskeletal components. The regulation of microtubules plays an important role for morphological changes underlying nevrite outgrowth, axonal elongation, and growth cone steering. SCG10 (superior cervical ganglion clone 10) is a neuronal growthassociated protein developmentally regulated and highly enriched in the neuronal growth cones. SCG10 presents a microtubule destabilizing activity that could participate to the regulation of microtubule dynamics and thus explain microtubule behaviors in the growth cone during axonal elongation and turning. It is here suggested that a tight control of the opposite effects on microtubules of SCG10 and the stabilizing microtubule-associated protein MAP1B allows a fine tuning of cytoskeletal rearrangement and may provide the required microtubule dynamic instability to promote axonal growth. Moreover, antibodyblockade of SCG10 function, that leads to growth cone pauses similar as those triggered by the guidance molecule EphB, and the modulation of SCG10 activity by the Rho GTPase Rnd1 suggest a potential role for SCG10 in the signal transduction pathways of extracellular guidance cues. The identification of the active zone protein Bassoon as a potential interaction partner for the SCG10-related protein NPC2, using atomic force microscopy as well as COS-7 and neuronal cell cultures, also gives new insights for a role of this protein family into the processes of synapse genesis or plasticity. Finally, SCG10 mutant mice generated by gene targeting and expressing a soluble form of the protein have been characterized during early postnatal development and in the adulthood. Due to the deletion of its membrane binding domain, SCG10 specific subcellular targeting to growth cones is compromised and results in impairments of motor and coordination development. Further histological analysis in the sciatic nerve reveal that these symptoms are associated with neurodegenerative signs. RESUME Une navigation correcte des prolongements cellulaires neuronaux leur permettant de former des connections appropri��es repose sur de continuels r��arrangements des constituants de leur cytosquelette. La r��gulation des microtubules joue notamment un r��le important dans les changements morphologiques qui accompagnent la croissance axonale et les r��orientations du c��ne de croissance. SCG10 (superior cervical ganglion clone 10) est une prot��ine ��troitement associ��e �� la croissance neuronale, hautement r��gul��e durant le d��veloppement et abondante au niveau du c��ne de croissance. SCG10 pr��sente une activit�� d��stabilisatrice sur les microtubules qui pourrait permettre une r��gulation des param��tres dynamiques propres aux microtubules et ainsi expliquer leur comportement durant la navigation du c��ne de croissance. Il est ici propos�� qu'un contr��le pr��cis des effets oppos��s de SCG10 et d'une autre prot��ine stabilisante associ��e aux microtubules (MAP1 B) permette un r��glage fin des r��arrangements du cytosquelette et puisse ainsi produire l'instabilit�� dynamique n��cessaire �� la croissance anale. Par ailleurs, le blocage de la fonction de SCG10 par un anticorps sp��cifique, conduisant �� des pauses du c��nes de croissance similaires �� celles provoqu��es par la mol��cule de guidage EphB, ainsi que la modulation de l'activit�� de SCG10 par la Rho GTPase Rnd1 sugg��rent une potentielle implication de SCG10 dans les voies de transduction des signaux provenant de mol��cules de guidage extracellulaires. L'identification d'une interaction de la prot��ine synaptique Bassoon avec la prot��ine NPC2 apparent��e �� SCG10, au moyen de la microscopie �� force atomique et dans des cultures de cellules neuronales et COS-7, ouvre des perspectives concernant ces prot��ines dans la formation et la plasticit�� synaptiques. Finalement, des souris mutantes pour SCG10 produites par ciblage de g��ne et exprimant une forme soluble de la prot��ine ont ��t�� caract��ris��es durant la phase pr��coce du d��veloppement et �� l'��ge adulte. La d��l��tion du domaine permettant l'ancrage de SCG10 aux membranes compromet sa sub-localisation au niveau du c��ne de croissance et r��sulte en l'apparition de troubles moteurs et de la coordination. Des analyses histologiques compl��mentaires au niveau du nerf sciatique montrent que ces sympt��mes sont associ��s avec des signes neurod��g��n��ratifs.