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.

SPH simulations of clumps formation by dissipative collision of molecular clouds - I. Non magnetic case

Computer experiments of interstellar cloud collisions were performed with a new smoothed-particle-hydrodynamics (SPH) code. The SPH quantities were calculated by using spatially adaptive smoothing lengths and the SPH fluid equations of motion were solved by means of a hierarchical multiple time-scale leapfrog. Such a combination of methods allows the code to deal with a large range of hydrodynamic quantities. A careful treatment of gas cooling by H, H(2), CO and H II, as well as a heating mechanism by cosmic rays and by H(2) production on grains surface, were also included in the code. The gas model reproduces approximately the typical environment of dark molecular clouds. The experiments were performed by impinging two dynamically identical spherical clouds onto each other with a relative velocity of 10 km s(-1) but with a different impact parameter for each case. Each object has an initial density profile obeying an r(-1)-law with a cutoff radius of 10 pc and with an initial temperature of 20 K. As a main result, cloud-cloud collision triggers fragmentation but in expense of a large amount of energy dissipated, which occurred in the head-on case only. Off-center collision did not allow remnants to fragment along the considered time (similar to 6 Myr). However, it dissipated a considerable amount of orbital energy. Structures as small as 0.1 pc, with densities of similar to 10(4) cm(-3), were observed in the more energetic collision.

Surface pressure-area isotherms for Langmuir monolayers and a docking molecular orientation of perylene tetracarboxylic derivatives on a water surface

The surface pressure-molecular area (pi-A) isotherms for Langmuir monolayers of four perylenetetracarboxylic (PTCD) derivatives, measured with varying subphase temperatures and compression speeds, are reported. The behavior of these PTCD derivatives at the water-air interface is modeled using the rigid docking method. This approach is the first attempt to model the molecular orientation of PTCD on the water surface to be compared with experimental Langmuir isotherms. Through this methodology, it would be possible to anticipate aggregation and determine if favorable spatial orientations of perylenes are generated on the water surface. The pi-A isotherm experiments show that these molecules can support high surface pressures, indicating strong packing on the water surface and that the isotherms are compression speed independent but temperature dependent. The molecular orientation and stacking was further examined in Langmuir-Blodgett (LB) monolayers deposited onto glass and glass coated with Ag island films using UV-visible absorption and surface-enhanced fluorescence (SEF) measurements.

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger-fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane em,, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behaviour. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P 1-30 was estimated by measuring the permeability PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the PEGs of different pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St 11 conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation. (c) 2005 Wiley Periodicals, Inc.

Testing transposable elements as genetic drive mechanisms using Drosophila P element constructs as a model system

The use of transposable elements (TEs) as genetic drive mechanisms was explored using Drosophila melanogaster as a model system. Alternative strategies, employing autonomous and nonautonomous P element constructs were compared for their efficiency in driving the ry(+) allele into populations homozygous for a ry(-) allele at the genomic rosy locus. Transformed flies were introduced at 1%, 5%, and 10% starting frequencies to establish a series of populations that were monitored over the course of 40 generations, using both phenotypic and molecular assays. The transposon-borne ry(+) marker allele spread rapidly in almost all populations when introduced at 5% and 10% seed frequencies, but 1% introductions frequently failed to become established. A similar initial rapid increase in frequency of the ry(+) transposon occurred in several control populations lacking a source of transposase. Constructs carrying ry(+) markers also increased to moderate frequencies in the absence of selection on the marker. The results of Southern and in situ hybridization studies indicated a strong inverse relationship between the degree of conservation of construct integrity and transposition frequency. These finding have relevance to possible future applications of transposons as genetic drive mechanisms.

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17 alpha-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment. (C) 1999 Elsevier B.V. B.V. All rights reserved.

A Dielectric Model of Self-Assembled Monolayer Interfaces by Capacitive Spectroscopy

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

Optimal laser control of molecular photoassociation along with vibrational stabilization

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The monosodium glutamate (MSG) obese rat as a model for the study of exercise in obesity

Obesity is an increasing problem in several countries, leading to health problems. Physical exercise, in turn, can be used effectively by itself or in combination with dietary restriction to trigger weight loss. The present study was designed to evaluate the effects of aerobic exercise training on lipid profile of obese male Wistar rats in order to verify if this model may be of value for the study of exercise in obesity. Obesity was induced by MSG administration (4mg/g, each other day, from birth to 14 days old) After 14 from drug administration, the rats were separated into two groups: MSG-S (sedentary) and MSG-T (exercise trained). Exercise training consisted in 1h/day, 5 days/week, with an overload of 5% bw, for 10 weeks. Rats of the same age and strain, receiving saline at birth, were used as control (C), and subdivided into two groups: C-S and C-T. At the end of the experimental period, MSG-T and C-T rats showed similar blood lactate and muscle glycogen responses to exercise training and acute exercise. MSG-S rats showed significantly higher carcass fat, serum triacylglycerol, serum insulin and liver total fat than C-S rats. On the other hand, MSG-T rats had lower carcass fat, serum triacylglycerol and liver total fat than MSG-S rats. There were no statistical differences in food intake and serum free fatty acids among the groups studied. These data indicate that this model may be of value for the study of exercise effects on tissue and circulating lipid profile in obesity.

Mean-field model of jet formation in a collapsing Bose-Einstein condensate

We perform a systematic numerical study, based on the time-dependent Gross-Pitaevskii equation, of jet formation in collapsing and exploding Bose-Einstein condensates as in the experiment by Donley et al (2001 Nature 412 295). In the actual experiment, via a Feshbach resonance, the scattering length of atomic interaction was suddenly changed from positive to negative on a pre-formed condensate. Consequently, the condensate collapsed and ejected atoms via explosion. On disruption of the collapse by suddenly changing the scattering length to zero, a radial jet of atoms was formed in the experiment. We present a satisfactory account of jet formation under the experimental conditions and also make predictions beyond experimental conditions which can be verified in future experiments.

Acute and short-term insulin-induced molecular adaptations of GLUT2 gene expression in the renal cortex of diabetic rats

Increased GLUT2 gene expression in the renal proximal tubule of diabetic rats is an adaptive condition, which may be important in the diabetic nephropathy development. We investigated the effects of insulin treatment upon the renal GLUT2 overexpression of diabetic rats. Acute treatment, surprisingly, induced a rapid further increase in GLUT2 mRNA content. Twelve hours after insulin injection, GLUT2 mRNA was twice the value of saline-injected rats (P < 0.001), when GLUT2 protein remained unchanged. In response to short-term treatment, both GLUT2 mRNA and protein were increased in 1-day treated rats (P < 0.05 versus saline-injected), decreasing after that, and reaching, within 6 days, values close to those of non-diabetic rats. Concluding, insulin treatment induced: initially, an additional upregulation of GLUT2 gene expression, involving posttranscriptional modulation; thereafter, downregulation of GLUT2 expression, which returns to non-diabetic levels. The former may be related to increased insulin concentration, the latter may be due to glycemic control. © 2005 Elsevier B.V. All rights reserved.

The inhibition of 5-enolpyruvylshikimate-3-phosphate synthase as a model for development of novel antimicrobials

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of βαβαββ-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future. © 2007 Bentham Science Publishers Ltd.

Heteropicnotic chromatin and nucleolar activity in meiosis and spermiogenesis of Limnogonus aduncus (Heteroptera, Gerridae): a stained nucleolar organizing region that can serve as a model for studying chromosome behavior.

Males of Limnogonus aduncus were found to have the sex chromosome system X0 and chromosome number 2n = 23 (22A + X0). Testis cells were stained with lacto-acetic orcein and silver nitrate so that changes in the morphology and degree of staining of the heteropicnotic chromatin and the nucleolar material could be observed during meiosis and spermiogenesis. These structures share the same nuclear position and could be seen until almost the end of spermiogenesis. A chromosome region stained with silver nitrate was indicative of a nucleolar organizing region (NOR), which is rarely detected in Heteroptera with this technique. The NOR is located at one end of a single member of an autosome pair. The finding of this stained region enabled us to observe that the telomeric association of sister chromatids that characterizes the Heteroptera does not include the chromosome ends, where NORs are located; we also observed in anaphase that the chromosome end through which it is pulled to the pole is the one containing the NOR. Another observation was that the single nucleolar body present in the cells at anaphase never goes to the cell pole that does not receive the NOR. We conclude that L. aduncus is a good model for cytogenetic studies involving nucleolar activity and also may be useful for studying the mechanisms of activation and inactivation of kinetic activity at the chromosome ends. Although the chromosomes of Heteroptera are known to be holocentric, whether kinetic activity is restricted to one or involves both chromosome ends is still not well understood.

Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae)

Toadlets of the genus Brachycephalus are endemic to the Atlantic rainforests of southeastern and southern Brazil. The 14 species currently described have snout-vent lengths less than 18. mm and are thought to have evolved through miniaturization: an evolutionary process leading to an extremely small adult body size. Here, we present the first comprehensive phylogenetic analysis for Brachycephalus, using a multilocus approach based on two nuclear (Rag-1 and Tyr) and three mitochondrial (Cyt b, 12S, and 16S rRNA) gene regions. Phylogenetic relationships were inferred using a partitioned Bayesian analysis of concatenated sequences and the hierarchical Bayesian method (BEST) that estimates species trees based on the multispecies coalescent model. Individual gene trees showed conflict and also varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. The concatenated gene tree was completely resolved and is identical in topology and degree of statistical support to the individual mtDNA gene tree. On the other hand, the BEST species tree showed reduced significant node support relative to the concatenate tree and recovered a basal trichotomy, although some bipartitions were significantly supported at the tips of the species tree. Comparison of the log likelihoods for the concatenated and BEST trees suggests that the method implemented in BEST explains the multilocus data for Brachycephalus better than the Bayesian analysis of concatenated data. Landmark-based geometric morphometrics revealed marked variation in cranial shape between the species of Brachycephalus. In addition, a statistically significant association was demonstrated between variation in cranial shape and genetic distances estimated from the mtDNA and nuclear loci. Notably, B. ephippium and B. garbeana that are predicted to be sister-species in the individual and concatenated gene trees and the BEST species tree share an evolutionary novelty, the hyperossified dorsal plate. © 2011 Elsevier Inc.