Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.

The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide.

The trans-activation response element (TAR) found near the 5' end of the viral RNA of the human immunodeficiency virus contains a 3-nt bulge that is recognized by the virally encoded trans-activator protein (Tat), an important mediator of transcriptional activation. Insertion of the TAR bulge into double-stranded RNA is known to result in reduced electrophoretic mobility, suggestive of a bulge-induced bend. Furthermore, NMR studies indicate that Arg causes a change in the structure of the TAR bulge, possibly reducing the bulge angle. However, neither of these effects has been quantified, nor have they been compared with the effects of the TAR-Tat interaction. Recently, an approach for the quantification of bulge-induced bends has been described in which hydrodynamic measurements, employing the method of transient electric birefringence, have yielded precise estimates for the angles of a series of RNA bulges, with the angles ranging from 7 degrees to 93 degrees. In the current study, transient electric birefringence measurements indicate that the TAR bulge introduces a bend of 50 degrees +/- 5 degrees in the absence of Mg2+. Addition of Arg leads to essentially complete straightening of the helix (to < 10 degrees) with a transition midpoint in the 1 mM range. This transition demonstrates specificity for the TAR bulge: no comparable transition was observed for U3 or A3 (control) bulges with differing flanking sequences. An essentially identical structural transition is observed for the Tat-derived peptide, although the transition midpoint for the latter is near 1 microM. Finally, low concentrations of Mg2+ alone reduce the bend angle by approximately 50%, consistent with the effects of Mg2+ on other pyrimidine bulges. This last observation is important in view of the fact that most previous structural/binding studies were performed in the absence of Mg2+.

Reciprocal modulations between p53 and Tat of human immunodeficiency virus type 1.

Infection by human immunodeficiency virus type 1 (HIV-1) causes acquired immunodeficiency syndrome (AIDS) after a long clinical latency. This disease is associated with a spectrum of cancers. Here we report that wild-type p53 is a potent suppressor of Tat, a major transactivator of HIV-1. Reciprocally, Tat inhibits the transcription of p53. Downregulation of p53 by upregulated tat may be important for the establishment of productive viral infection in a cell and also may be involved in the development of AIDS-related malignancies.

Angiogenic properties of human immunodeficiency virus type 1 Tat protein.

Extracellular human immunodeficiency virus type 1 (HIV-1) Tat protein promotes growth of spindle cells derived from AIDS-associated Kaposi sarcoma (AIDS-KS), an angioproliferative disease very frequent in HIV-1-infected individuals. Normal vascular cells, progenitors of AIDS-KS cells, proliferate in response to Tat after exposure to inflammatory cytokines, whose levels are augmented in HIV-1-infected individuals and in KS lesions. Here we show that Tat also promotes AIDS-KS and normal vascular cells to migrate and to degrade the basement membrane and stimulates endothelial cell morphogenesis on a matrix substrate. These effects are obtained at picomolar concentrations of exogenous Tat and are promoted by the treatment of the cells with the same inflammatory cytokines stimulating expression of the receptors for Tat, the integrins alpha 5 beta 1 and alpha v beta 3. Thus, under specific circumstances, Tat has angiogenic properties. As Tat and its receptors are present in AIDS-KS lesions, these data may explain some of the mechanisms by which Tat can induce angiogenesis and cooperate in the development of AIDS-KS.

O Teste de Apercepção Temática (TAT) em adultos: dados normativos para o sistema morvaliano

No contexto da pesquisa e utilização prática das técnicas de autoexpressão, o Teste de Apercepção Temática (TAT) apresenta reconhecidas vantagens. Apesar de linhas de pesquisa bastante desenvolvidas internacionalmente, não existem estudos atuais sobre as propriedades deste instrumento no Brasil, levantando a necessidade de conhecer seus alcances e limites nesse contexto cultural. Desse modo, o desenvolvimento de normas que descrevam o desempenho típico de uma população não clínica se mostra um importante passo inicial rumo à retomada da pesquisa com o TAT e sua adoção por profissionais. Tendo em vista que a validação das técnicas de autoexpressão se refere aos sistemas de classificação e não aos instrumentos em si, o presente estudo adotou, dentre a miríade de sistemas de categorização das histórias do TAT descritos na literatura, o sistema proposto por Monique Morval, que foi desenvolvido para estudantes e profissionais com pouca experiência com o TAT, utilizando pressupostos da Personologia de Henry Murray, Psicologia Cognitiva e Psicologia do Ego, possibilitando leituras objetivas e qualitativas do material. Considerando o potencial de utilização desse sistema por parte dos psicólogos brasileiros - visto sua semelhança com o atual sistema aprovado para uso pelo Sistema de Avaliação dos Testes Psicológicos (SATEPSI) do Conselho Federal de Psicologia - foram feitos aprimoramentos no assim chamado sistema morvaliano, possibilitando sua adoção no estudo normativo proposto pelo presente projeto. Tais aprimoramentos consistiram na revisão e adição de categorias e subcategorias, a partir de trabalhos brasileiros e internacionais sobre o instrumento. Desta forma, o presente estudo teve por objetivo desenvolver normas para o TAT (Sistema Morvaliano) em adultos, a partir de uma amostra não clínica, aleatória e estratificada por critérios de idade, sexo, escolaridade e nível socioeconômico (NSE) da cidade de Ribeirão Preto (SP). Os participantes foram 100 adultos (25 a 44 anos de idade), selecionados através de visita a domicílios sorteados em bairros de diferentes NSE. Foram realizadas entrevistas de avaliação psicológica individuais, utilizando um roteiro de entrevista semiestruturada, o Teste de Inteligência Geral Não-Verbal (TIG-NV) e 20 cartões do TAT, pré-selecionados de acordo com as recomendações do manual original do instrumento. As histórias do TAT foram categorizadas no sistema morvaliano revisado e seus dados passaram por análises estatísticas e sínteses qualitativas em busca de diferenças de desempenho relacionadas ao sexo, escolaridade e NSE. Os resultados mostram poucas diferenças entre os grupos que justifiquem a elaboração de normas específicas; apesar disso, foi identificada uma tendência (ainda que pequena) de o nível de escolaridade e o NSE favorecerem um melhor desempenho nas variáveis relativas à organização formal das histórias. São discutidas as implicações dos dados obtidos em termos do perfil normativo de desempenho nas variáveis do sistema morvaliano, suas evidências preliminares de validade e futuras direções para estudos sobre o TAT nesse sistema.

Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system

The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general.

Talfīq al-akhbār wa-talqīḥ al-āthār fī waqāʻiʻ Qāzān wa-Bulghār wa-mulūk al-Tatār

athar M. M. al-Ramzī.

Hādhihi al-musammāh bi-Yāqūtat al-ʻilm / lil-Shaykh Abī Isḥāq al-Tūnisī : manuscript, [1629]

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