Avaliação de bentonita sódica purificada e bentonita sódica intercalada como promotores de dissolução de clorpropamida em comprimidos preparados por granulação úmida e compressão direta / Evaluation of purified sodium bentonite and intercalated sodium bentonite as dissolution enhancers for chlorpropamide in tablets produced by wet granulation and direct compression

O presente estudo avaliou sistemas baseados em bentonita sódica purificada e bentonita sódica purificada intercalada com colina como promotores de dissolução de clorpropamida. A intercalação da bentonita sódica foi avaliada por difração de raios X, análise termo gravimétrica, análise calorimétricade varredura e espectrometria no infravermelho. Prepararam-se misturas físicas, empastamentos (kneadings) e granulados e comprimidos, por granulação úmida e compressão direta; ambos os sistemas (físicos e comprimidos) com diversas relações fármaco: promotores de dissolução. Todos os sistemas físicos e os comprimidos foram avaliados quanto à dissolução do fármaco segundo metodologia descrita na Farmacopéia Norte-Americana (USP). Os comprimidos ainda foram avaliados quanto à dureza e friabilidade. O empastamento hidroalcoólico mostrou ser o melhor sistema físico para aumento da dissolução, entretanto é inviável para produção industrial de comprimidos. Os comprimidos de clorpropamida preparados por granulação úmida com proporção de fármaco e promotores de dissolução de 1:0,25 p/p apresentaram melhores resultados no teste de dissolução em relação aos comprimidos sem os promotores. Não houve diferença significativa entre a liberação de clorpropamida com a bentonita sódica e bentonita sódica intercalada. Os comprimidos preparados por compressão direta apresentaram, comparativamente, o melhor desempenho no teste de dissolução. Não foi evidenciado perda de cristalinidade do fármaconos sistemas estudados. O poder de desintegração da bentonita e a possível interação molecular entre o fármaco e a bentonita são as prováveis causas do aumento da dissolução da clorpropamida com tais sistemas baseados em bentonita.

A general method for purification of H1 histones that are active for repression of basal RNA polymerase II transcription.

H1 histones were purified from extracts of salt-treated nuclei as a co-product of RNA polymerase II transcription factors from both Drosophila embryos and HeLa cells by a simple and general method. This procedure was also used to purify H1 as co-product of the core histones from calf thymus. The key steps in this purification exploit the solubility of H1 in 2.26 M ammonium sulfate and the chromatographic properties of the highly charged H1 molecules on a phenyl-Sepharose resin. H1 that is prepared by this procedure is active for in vitro repression of basal RNA polymerase II transcription. This method provides a new means of purifying H1 by a mild procedure that is likely to be generally useful for studies of transcription and chromatin structure.

Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription.

To understand the principles of control and selectivity in gene expression, the biochemical mechanisms by which promoter- and enhancer-binding factors regulate transcription by RNA polymerase II were analyzed. A general observed repressor of transcription was purified and identified as histone H1. Since many aspects of H1 binding to naked DNA resemble its interaction with chromatin, purified H1 bound to naked DNA was used as a model for the repressed state of the DNA template. Three sequence-specific transcription factors, Sp1, GAL4-VP16, and GAGA factor, were shown to counteract H1-mediated repression (antirepression). In addition, Sp1 and GAL4-VP16, but not the GAGA factor, activated transcription in the absence of H1. Therefore, true activation and antirepression appear to be distinct activities of sequence-specific factors. Furthermore, transcription antirepression by GAL4-VP16 was sustained for several rounds of transcription. These findings, together with previous studies on H1, suggest that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.

Sequence-specific transcriptional antirepression of the Drosophila Krüppel gene by the GAGA factor.

We have analyzed the proximal promoter of the Drosophila Krüppel (Kr) gene. A 44-base pair fragment containing the RNA start sites contains significant promoter activity, and this minimal promoter is flanked both upstream and downstream by binding sites for the GAGA factor. The GAGA factor is the predominant sequence-specific DNA binding factor that interacts with the Kr promoter region, and the purified protein activates Kr transcription in vitro. However, strong transcriptional activation of Kr as well as of Ultrabithorax, another GAGA factor-responsive gene, requires the presence of a DNA binding transcriptional repressor. The GAGA factor is able to relieve this repression in a binding site-dependent manner, and, thus, these data suggest that the GAGA factor functions as an antirepressor, rather than an activator, of the Kr gene.

Sequence analysis of the EF-1 alpha gene family of Mucor racemosus.

Our previous studies have shown that Mucor racemosus possesses three genes (TEF-1, -2 and -3) for EF-1 alpha, and that all three genes are transcribed. However, the level of transcription varies markedly between the three genes, with TEF-1 mRNA levels being approximately two fold higher than TEF-3 and 6 fold higher than TEF-2. We have now completed the DNA sequence of both strands of all three genes and have found that these genes are highly homologous. TEF-2 and TEF-3 are more similar to each other than they are to TEF-1. The TEF-2 and the TEF-3 coding regions differ from TEF-1 at 30 and 37 positions respectively out of 1374 nucleotides. Twenty-six of these nucleotide substitutions were common to both TEF-2 and TEF-3, and the majority of the substitutions were clustered in the 5' region of the coding sequences. While the majority of these changes were silent, TEF-2 and TEF-3 differed from TEF-1 by having a lysine instead of a glutamate at amino acid position 41. In addition, TEF-2 and -3, but not TEF-1, each have an intron located near the 5' end of the coding region, although its size and sequence is not conserved between the two genes. All three genes have a conserved intron near the 3' end of the coding region. The sequence data have been analyzed with respect to the structure and function of EF-1 alpha in protein biosynthesis.

The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus.

We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.