HIV rebounds from latently infected cells, rather than from continuing low-level replication.

Rapid rebound of plasma viremia in patients after interruption of long-term combination antiretroviral therapy (cART) suggests persistence of low-level replicating cells or rapid reactivation of latently infected cells. To further characterize rebounding virus, we performed extensive longitudinal clonal evolutionary studies of HIV env C2-V3-C3 regions and exploited the temporal relationships of rebounding plasma viruses with regard to pretreatment sequences in 20 chronically HIV-1-infected patients having undergone multiple 2-week structured treatment interruptions (STI). Rebounding virus during the short STI was homogeneous, suggesting mono- or oligoclonal origin during reactivation. No evidence for a temporal structure of rebounding virus in regard to pretreatment sequences was found. Furthermore, expansion of distinct lineages at different STI cycles emerged. Together, these findings imply stochastic reactivation of different clones from long-lived latently infected cells rather than expansion of viral populations replicating at low levels. After treatment was stopped, diversity increased steadily, but pretreatment diversity was, on average, achieved only >2.5 years after the start of STI when marked divergence from preexisting quasispecies also emerged. In summary, our results argue against persistence of ongoing low-level replication in patients on suppressive cART. Furthermore, a prolonged delay in restoration of pretreatment viral diversity after treatment interruption demonstrates a surprisingly sustained evolutionary bottleneck induced by punctuated antiretroviral therapy.

Influência da irrigação e do genótipo na produção de castanha em cajueiro-anão-precoce

Avaliou-se a influência da irrigação e do genótipo na produção de castanha em cajueiro-anão-precoce (Anacardium occidentale L.) durante três anos. Foram estudados três clones (CP 09, CP 76 e CP 1001) e quatro regimes hídricos (testemunha sem irrigação e intervalos de irrigação de um, três e cinco dias). O delineamento experimental foi em blocos ao acaso, em parcelas subsubdivididas, com quatro repetições, com os regimes hídricos nas parcelas, os clones nas subparcelas, cada uma com quatro plantas, e os anos de produção nas subsubparcelas. A quantidade de água aplicada nos três tratamentos irrigados baseou-se na evaporação do tanque classe A. Em relação à produção de castanha, os clones de cajueiro-anão-precoce não apresentaram comportamento diferencial em resposta à irrigação; os clones CP 09 e CP 76 mostraram-se superiores ao CP 1001 quanto à estabilidade de safra; independentemente do regime hídrico estudado, o clone CP 76 mostrou-se menos produtivo do que os clones CP 09 e CP 1001.

Correlações inter e intragerações e herdabilidade de cor de chips, matéria seca e produção em batata

Os objetivos deste trabalho foram determinar correlações inter e intragerações clonais, estimar herdabilidade quanto à cor de chips, teor de matéria seca e produção de batata, e suas implicações na seleção. Duzentos e cinqüenta clones de dez famílias foram escolhidos aleatoriamente de uma população de primeira geração clonal, destinada ao processamento de batatas chips, do programa de melhoramento genético de batata da Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado. Os clones foram avaliados em segunda (G2), terceira (G3) e quarta (G4) gerações, respectivamente, no outono e primavera de 1999, e outono de 2000, em Pelotas, RS. Os coeficientes de correlação entre gerações e as estimativas de herdabilidade dentro das gerações clonais foram baixas em relação à cor de chips, baixas a moderadas quanto à matéria seca e incrementais com as gerações nos componentes de produção. Os coeficientes de correlação entre as características de qualidade e os componentes de produção dentro de cada geração foram baixos e, na maioria, não-significativos. As estimativas de herdabilidade dos dados conjuntos da G3 e G4 foram moderada, moderadamente alta e alta, respectivamente, em relação à cor de chips, teor de matéria seca e produção.

Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC-peptide and antibody Fab' conjugates.

The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26-35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.

Repetibilidade de características agroindustriais em cana-de-açúcar

Os objetivos deste trabalho foram estabelecer estimativas de repetibilidade de características agroindustriais em 20 genótipos de cana-de-açúcar, determinar a previsibilidade de cada caráter e indicar a predição do valor verdadeiro de cada clone. O delineamento utilizado foi blocos casualizados, com cinco repetições. Utilizaram-se a análise de variância com dois fatores de variação (cortes e genótipos) e a análise dos componentes principais para estimar o coeficiente de repetibilidade der=0 width=19 height=21 id="_x0000_i1026" src="../../../../img/revistas/pab/v39n4/20437s1.gif" align=absmiddle>, a partir de três cortes. Foram obtidas estimativas de repetibilidade acima de 0,5 para fibra e toneladas de cana por hectare, em ambos os métodos, com confiabilidade maior que 84% pelo método dos componentes principais. As características que ficaram abaixo de 0,5, com previsibilidade inferior a 74%, necessitam de um maior número de avaliações. Os métodos dos componentes principais e análise de variância indicaram, em cinco cortes, uma previsibilidade maior que 80% para fibra, porcentagem de pol (sacarose) no caldo da cana, toneladas de cana por hectare e toneladas de pol no caldo da cana por hectare, embora o primeiro tenha sido mais eficiente. Considerando toneladas de cana por hectare e toneladas de pol no caldo da cana por hectare, os clones RB9371, RB9350 e RB9364 são os melhores.

Recognition of major histoincompatibilities after transplantation with marrow from HLA closely matched donors.

BACKGROUND: To determine the extent to which major histoincompatibilities are recognized after bone marrow transplantation, we characterized the specificity of the cytotoxic T lymphocytes isolated during graft-versus-host disease. We studied three patients transplanted with marrow from donors who were histoincompatible for different types of HLA antigens. METHODS: Patient 1 was mismatched for one "ABDR-antigen" (HLA-A2 versus A3). Two patients were mismatched for antigens that would usually not be taken into account by standard selection procedures: patient 2 was mismatched for an "HLA-A subtype" (A*0213 versus A*0201), whereas patient 3 was mismatched for HLA-C (HLA-C*0501 versus HLA-C*0701). All three HLA class I mismatches were detected by a pretransplant cytotoxic precursor test. RESULTS: Analysis of the specificity of the cytotoxic T lymphocyte clones isolated after transplantation showed that the incompatibilities detected by the pretransplant cytotoxic precursor assay were the targets recognized during graft-versus-host disease. CONCLUSIONS: Independent of whether the incompatibility consisted of a "full" mismatch, a "subtype" mismatch, or an HLA-C mismatch, all clones recognized the incompatible HLA molecule. In addition, some of these clones had undergone antigen selection and were clearly of higher specificity than the ones established before transplantation, indicating that they had been participating directly in the antihost immune response.

Análise da castanha do cajueiro por tomografia de ressonância magnética

O objetivo deste trabalho foi avaliar a técnica de tomografia de ressonância magnética na análise de castanhas de cajueiro, em relação ao método tradicional, visando sua aplicação na seleção de clones. Amostras de castanhas de 40 clones de cajueiro comum, colhidas na safra de 2002, foram analisadas por ambos os métodos. Pelo método tradicional, a maioria dos clones apresentou altos e médios valores dos indicadores industriais massa de castanha, massa de amêndoa e rendimento industrial e baixos índices de quebra das amêndoas. Pela tomografia, a maioria dos clones apresentou castanhas com espaços vazios entre a amêndoa e o endocarpo, que podem proteger a amêndoa durante a decorticação. Os resultados dos dois métodos foram complementares, e a tomografia, além de alternativa promissora na avaliação da qualidade de castanha, pode subsidiar outras áreas de pesquisa relacionadas ao estudo da castanha.

Cloning, functional expression, and chromosomal localization of the human pancreatic islet glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is a hormone secreted by the endocrine K-cells from the duodenum that stimulates glucose-induced insulin secretion. Here, we present the molecular characterization of the human pancreatic islet GIP receptor. cDNA clones for the GIP receptor were isolated from a human pancreatic islet cDNA library. They encoded two different forms of the receptor, which differed by a 27-amino acid insertion in the COOH-terminal cytoplasmic tail. The receptor protein sequence was 81% identical to that of the rat GIP receptor. When expressed in Chinese hamster lung fibroblasts, both forms of the receptor displayed high-affinity binding for GIP (180 and 600 pmol/l). GIP binding was displaced by < 20% by 1 mumol/l glucagon, glucagon-like peptide (GLP-I)(7-36) amide, vasoactive intestinal peptide, and secretin. However exendin-4 and exendin-(9-39) at 1 mumol/l displaced binding by approximately 70 and approximately 100% at 10 mumol/l. GIP binding to both forms of the receptor induced a dose-dependent increase in intracellular cAMP levels (EC50 values of 0.6-0.8 nmol/l) but no elevation of cytoplasmic calcium concentrations. Interestingly, both exendin-4 and exendin-(9-39) were antagonists of the receptor, inhibiting GIP-induced cAMP formation by up to 60% when present at a concentration of 10 mumol/l. Finally, the physical and genetic chromosomal localization of the receptor gene was determined to be on 19q13.3, close to the ApoC2 gene. These data will help study the physiology and pathophysiology of the human GIP receptor.

CleanEx: a database of heterogeneous gene expression data based on a consistent gene nomenclature.

The main goal of CleanEx is to provide access to public gene expression data via unique gene names. A second objective is to represent heterogeneous expression data produced by different technologies in a way that facilitates joint analysis and cross-data set comparisons. A consistent and up-to-date gene nomenclature is achieved by associating each single experiment with a permanent target identifier consisting of a physical description of the targeted RNA population or the hybridization reagent used. These targets are then mapped at regular intervals to the growing and evolving catalogues of human genes and genes from model organisms. The completely automatic mapping procedure relies partly on external genome information resources such as UniGene and RefSeq. The central part of CleanEx is a weekly built gene index containing cross-references to all public expression data already incorporated into the system. In addition, the expression target database of CleanEx provides gene mapping and quality control information for various types of experimental resource, such as cDNA clones or Affymetrix probe sets. The web-based query interfaces offer access to individual entries via text string searches or quantitative expression criteria. CleanEx is accessible at: http://www.cleanex.isb-sib.ch/.

Avaliação agronômica de híbridos interespecíficos entre capim-elefante e milheto

O objetivo deste trabalho foi avaliar o comportamento agronômico de híbridos entre capim-elefante (Pennisetum purpureum Schum.) e milheto (P. glaucum (E.) Leek), a fim de determinar seu potencial para o melhoramento da forragem e a seleção de híbridos para futuras avaliações. Foram utilizadas 12 cultivares de milheto e 11 clones de capim-elefante, cruzados em esquema de dialelo parcial. As 132 combinações híbridas, além de duas testemunhas, foram avaliadas em experimentos em blocos casualizados, com três repetições. Anotaram-se dados de produção de matéria seca, altura de plantas, porcentagem de matéria seca, relação entre folha e caule e de qualidade da forragem (porcentagem de proteína bruta, porcentagem de fibra em detergente neutro e ácido e digestibilidade in vitro da matéria orgânica). Foi verificada existência de variabilidade entre os híbridos interespecíficos de capim-elefante e milheto, na maioria das características. A superioridade de alguns híbridos, em relação às testemunhas, demonstra o potencial do cruzamento entre P. purpureum e P. glaucum para a obtenção de cultivares melhoradas. Considerando-se tanto características de produção como de qualidade da forragem, os melhores híbridos avaliados foram 108 (F91-2-5 x M-60), 53 (F93-4-2 x M-27), 35 (F94-28-3 x M-42), 36 (F94-28-3 x M-60) e 4 (F92-101-2 x M-35).

Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells.

Direct identification as well as isolation of antigen-specific T cells became possible since the development of "tetramers" based on avidin-fluorochrome conjugates associated with mono-biotinylated class I MHC-peptide monomeric complexes. In principle, a series of distinct class I MHC-peptide tetramers, each labelled with a different fluorochrome, would allow to simultaneously enumerate as many unique antigen-specific CD8(+) T cells. Practically, however, only phycoerythrin and allophycocyanin conjugated tetramers have been generally available, imposing serious constraints for multiple labeling. To overcome this limitation, we have developed dextramers which are multimers based on a dextran backbone bearing multiple fluorescein and streptavidin moieties. Here we demonstrate the functionality and optimization of these new probes on human CD8(+) T cell clones with four independent antigen specificities. Their applications to the analysis of relatively low frequency antigen-specific T cells in peripheral blood, as well as their use in fluorescence microscopy, are demonstrated. The data show that dextramers produce a stronger signal than their fluoresceinated tetramer counterparts. Thus, these could become the reagents of choice as the antigen-specific T cell labeling transitions from basic research to clinical application.

The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia.

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.

Molecular Analysis of the PTEN Gene in a Choroidal Schwannoma in the Context of a Hamartomatous Syndrome

Purpose: To investigate the molecular involvement of PTEN, a tumor suppressor gene, in a case of cellular pigmented choroidal Schwannoma in a patient with hamartomatous syndrome due to heterozygous PTEN germline mutation. Methods: Histopathological, immunohistochemical, and electron microscopy analyses were performed by standard procedures. Paraffin-embedded samples of normal and tumor eye tissues were collected and DNA was extracted. A 145 bp region flanking the heterozygous c.406T>C mutation in exon 5 of PTEN was amplified by PCR and sequenced. To evaluate the allelic status of PTEN in the tumor sample, we cloned different PCR products in E. coli using a TA cloning procedure. Results: Histopathology demonstrated a posterior choroidal mass measuring 1.3 x 1.6 x 1.4 cm. The tumor was composed by fascicles of spindle cells with wavy cytoplasm. No Verrocay bodies could be identified. Scattered histiocytes with clear cytoplasm were present. By immunohistochemistry, the cells were expressing S100 and focally Melan A proteins. Pericellular type IV collagen could be demonstrated. Interlacing cytoplasmic processes covered by thick basement membrane could be found by electron microscopy as well as few premelanosomes. Moderate PTEN expression by immunohistochemistry was identified in some cells. As expected, the germline mutation could be detected by DNA sequencing in both the paraffin-embedded normal and tumor eye tissues. Analysis of 33 E. coli colonies bearing clones from the tumor eye tissue DNA surprisingly revealed that most of them contained the PTEN wild-type allele (29 vs. 4, Fisher's test p-value = 0.002). Conclusions: This is the first reported case of choroidal cellular Schwannoma arising in the context of a PTEN hamartomatous syndrome. Allelic analysis of PTEN in the tumor suggests a statistically-significant partial loss of heterozygozity in favor of the wild-type allele. Our findings are in clear contrast with what is usually observed in cancer tissues, for which mutated alleles of tumor suppressor genes are usually brought to homozygosity. Similar results were previously reported in human non-Hodgkin's lymphomas, displaying an overexpression of the wild-type form of the tumor suppressor gene p53. We are in the process of investigating additional DNA derived from other fresh and paraffin-embedded tissues from the patient, in order to gain insights on the molecular bases of PTEN involvement in this rare choroidal Schwannoma.

Selection via simulated individual BLUP based on family genotypic effects in sugarcane

The objective of this work was to propose a new selection strategy for the initial stages of sugarcane improvement, based on the methodology 'simulated individual BLUP (BLUPIS)', which promotes a dynamic allocation of individuals selected in each full-sib family, using BLUP as a base for both the genotypic effects of the referred families and plot effects. The method proposed applies to single full-sib families or those obtained from unbalanced or balanced diallel crosses, half-sib families and self-pollinated families. BLUPIS indicates the number of individuals to be selected within each family, the total number of clones to be advanced, and the number of families to contribute with selected individuals. Correlation between BLUPIS and true BLUP was 0.96, by method validation. Additionally, BLUPIS allows the identification of which replication contains the best individuals of each family.

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.