Molecular models of NS3 protease variants of the Hepatitis C virus

Background: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.Results: the atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures.Conclusions: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

Caracterização de hemoglobina N-Baltimore em doador de sangue de São José do Rio Preto, SP

As alterações que envolvem as globinas devem-se a modificações em genes responsáveis pela seqüência e estrutura das cadeias polipeptídicas, bem como aos genes reguladores da síntese destas cadeias. Hemoglobinas variantes apresentam estrutura química diferente da hemoglobina normal correspondente, resultante de mutações em uma ou mais bases nitrogenadas, ocasionando a troca de aminoácidos nas globinas alfa, beta, delta ou gama. A hemoglobina N-Baltimore é uma variante de globina beta, com substituição da lisina, na posição 95, por ácido glutâmico, apresentando mobilidade eletroforética mais rápida que a hemoglobina A em pH alcalino. Nas análises eletroforéticas em pH alcalino realizadas em doadores de sangue do Hemocentro de São José do Rio Preto (SP) identificamos a presença de portador de hemoglobina rápida em heterozigose, posteriormente confirmada por focalização isoelétrica e cromatografia líquida de alta pressão (HPLC). Os estudos de hemoglobinas anormais em doadores de sangue permitem a identificação de variantes raras e possibilitam o aconselhamento genético adequado a cada caso com estudo familial.

Intermolecular interactions and characterization of the novel factor Xa exosite involved in macromolecular recognition and inhibition: Crystal structure of human Gla-domainless factor Xa complexed with the anticoagulant protein NAPc2 from the hematophagous nematode Ancylostoma caninum

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K-i = 8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty. (c) 2006 Elsevier Ltd. All rights reserved.

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Síntese, caracterização e estudos de interação de um análogo da antitoxina CcdA empregando fluorescência no estado estacionário

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Maturação, formas de secagem e qualidade fisiológica de sementes de mucuna-preta

O objetivo deste trabalho foi verificar o efeito das formas de secagem, durante a maturação, sobre o desenvolvimento e a qualidade fisiológica de sementes de mucuna-preta. No primeiro experimento, inflorescências foram etiquetadas no início de florescimento e as colheitas iniciaram-se duas semanas após; no segundo, as colheitas foram iniciadas 40 dias após 50% de florescimento das plantas; em ambos, as colheitas foram feitas a intervalos semanais até o estádio de vagens secas. As sementes de cada colheita do primeiro experimento foram secadas, em condições ambientais de laboratório, no interior de vagens fechadas ou abertas (sementes expostas) e determinados a massa de 100 sementes, o comprimento, a largura e a espessura das sementes, enquanto no segundo foram secadas no interior de vagens fechadas ou extraídas das vagens e submetidas ao teste de germinação. A secagem no interior das vagens promove o desenvolvimento de sementes imaturas, antecipa a formação de sementes duras e aumenta o número dessas nas imaturas comparativamente às sementes secadas em vagens abertas ou após a extração.

Intensidade de dormência durante a maturação de sementes de mucuna-preta

A mucuna-preta é uma leguminosa empregada para adubação verde e como forrageira, cujas sementes apresentam dormência causada pela impermeabilidade do tegumento à água. O objetivo deste trabalho foi estudar a intensidade de dormência das sementes de mucuna-preta em diferentes estádios de maturação quando submetidas à secagem no interior das vagens. Rácemos foram colhidos, semanalmente, a partir de 35 dias do início de florescimento (35 DAF) até o estádio de vagens secas (98 DAF). As sementes de cada colheita foram secadas no interior das vagens, em condições ambientais não controladas de laboratório; quando secas foram extraídas, avaliadas quando a massa de 100 sementes, espessura das sementes e submetidas ao teste de germinação. Foram analisadas as porcentagens de sementes duras, de germinação, de embebição e o índice de velocidade de embebição. Pode-se concluir que o estádio de maturação em que ocorre a secagem afeta a intensidade de dormência das sementes de mucuna-preta.

Recuperação de solos degradados na exploração mineral de cassiterita: biomassa microbiana e atividade da desidrogenase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detecção e conscientização de portadores de hemoglobinopatias nas regiões de São José do Rio Preto e Presidente Prudente, SP (Brasil)

Foram analisadas 7.657 amostras de sangue provenientes de 48 cidades das regiões de São José do Rio Preto e de Presidente Prudente, com o objetivo de detectar e conscientizar os portadores de hemoglobinas anormais. As análises efetuadas mostraram que 3,47% tinham hemoglobinas anormais, sendo 2,26% portadores de variantes moleculares (Hbs, AS, AC, SS, SC, AJ, AB2) e 1,21% de talasse-mias (alfa e beta). Os resultados obtidos evidenciaram que estudos semelhantes, além de propiciar o melhor conhecimento das causas genéticas, bioquímicas e hematológicas dessas alterações hereditárias, oferecem também a oportunidade de estimar a importância que essas patologias representam para a saúde pública do nosso país. A ação preventiva foi estabelecida por meio de reuniões de esclarecimentos médicos e biológicos aos portadores de hemoglobinas anormais.

Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungus Scytalidium thermophilum

An extracellular (conidial) and an intracellular (mycelial) alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum were purified by DEAE-cellulose and Concanavalin A-Sepharose chromatography. These enzymes showed allosteric behavior either in the presence or absence of MgCl2, BaCl2, CuCl2, and ZnCl2. All of these ions increased the maximal velocity of both enzymes. The molecular masses of the conidial and mycelial enzymes, estimated by gel filtration, were 162 and 132 kDa, respectively. Both proteins migrated on SDS-PAGE as a single polypeptide of 63 and 58.5 kDa, respectively, suggesting that these enzymes were dimers of identical subunits. The best substrate for the conidial and mycelial phosphatases was p-nitrophenylphosphate, but,beta -glycerophosphate and other phosphorylated compounds also served as substrates. The optimum pH for the conidial and mycelial alkaline phosphatases was 10.0 and 9.5 in the presence of AMPOL buffer, and their carbohydrate contents were about 54% and 63%, respectively. The optimum temperature was 70-75 degreesC for both activities. The enzymes were fully stable up to 1 h at 60 degreesC. These and other properties suggested that the alkaline phosphatases of S. thermophilum might be suitable for biotechnological applications.

AN ULTRASTRUCTURAL-STUDY ON THE ENDOCRINE PANCREAS OF CAIMAN-LATIROSTRIS, WITH SPECIAL REFERENCE TO PANCREATIC MOTILIN CELLS

Pancreatic endocrine cells of Caiman latirostris were investigated by electron microscopy using conventional and immunocytochemical methods. Ultrastructurally, four types of endocrine cells were classified according to the morphology of their secretory granules. Three types of endocrine cells were identified as either glucagon, insulin or somatostatin cells by the presence of such characteristic granules well established in mammals. The remaining endocrine cell type could not be classified by its ultrastructural features alone.Immunocytochemical observations confirmed the ultrastructural classification of glucagon, insulin and somatostatin cells. In addition, endocrine cells immunoreactive for either pancreatic polypeptide (PP) or motilin were identified. Morphometric analysis of PP- and motilin-immunoreactive granules demonstrated that they were the most polymorphous and smallest granules among the pancreatic endocrine cell granules. Although both PP and motilin granules closely resemble each other, motilin granules were smaller in size and more spherical in shape than PP granules.

A SYSTEMIC CONCEPT OF THE GENE

The univocal correspondence between one gene and one polypeptide has been challenged by many examples of ambiguities. A rapidly expanding list of one-to-many or many-to-one correspondences includes: genomic rearrangements, alternative processing of transcripts, overlapping translation frames, RNA editing, alternative translation modes, and polyprotein cleavage.The genomic message requires interpretation through decoding by a sophisticated information retrieval system which should also carry some kind of information. The full meaning of the whole cell, as a unit, is emphasized.The gene is a combination of (one or more) nucleic acid (DNA or RNA) sequences, defined by the system (the whole cell, interacting with the environment, or the environment alone, in subcellular or pre-cellular systems), that gives origin to a product (RNA or polypeptide).

Frustration and hydrophobicity interplay in protein folding and protein evolution

A lattice model is used to study mutations and compacting effects on protein folding rates and folding temperature. In the context of protein evolution, we address the question regarding the best scenario for a polypeptide chain to fold: either a fast nonspecific collapse followed by a slow rearrangement to form the native structure or a specific collapse from the unfolded state with the simultaneous formation of the native state. This question is investigated for optimized sequences, whose native state has no frustrated contacts between monomers, and also for mutated sequences, whose native state has some degree of frustration. It is found that the best scenario for folding may depend on the amount of frustration of the native structure. The implication of this result on protein evolution is discussed. (c) 2006 American Institute of Physics.

Sulfation of intrinsic glycoproteins of the rabbit vitreous

The experiments reported here were designed to characterize the intrinsic vitreous glycoproteins and to understand the process of their sulfation. Rabbits were injected intravitreally with S-35-sodium sulfate and killed at several time intervals after injection. In another series of experiments, rabbits were injected either with S-35-sodium sulfate, H-3-fucose or H-3-tyrosine, associated or not associated with tunicamycin administration. Vitreous from the control eyes was also digested with N-glycosidase.. Furthermore, ciliary bodies, the putative source of the intrinsic vitreous glycoproteins, were incubated with S-35-sodium sulfate in the presence or absence of the protein synthesis inhibitor cycloheximide, and the culture media recovered for analysis. These and the vitreous samples of the other experiments were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Except for serum albumin, practically all polypeptide bands of the vitreous and culture media were labeled with radioactive sulfate and were shown to undergo renewal. The experiments using tunicamycin or enzyme treatment suggest that radioactive sulfate was incorporated not only into the carbohydrate side chains of the glycoproteins but also into the amino acid tyrosine of the polypeptide backbone of these glycoproteins. (C) 1998 Academic Press.