Die Flora von Kremsier in Mähren

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Mechanism of flexibility control for ATP access of hepatitis C virus NS3 helicase

Hepatitis C virus (HCV) NS3 helicase couples adenosine triphosphate (ATP) binding and hydrolysis to polynucleotide unwinding. Understanding the regulation mechanism of ATP binding will facilitate targeting of the ATP-binding site for potential therapeutic development for hepatitis C. T324, an amino acid residue connecting domains 1 and 2 of NS3 helicase, has been suggested as part of a flexible hinge for opening of the ATP-binding cleft, although the detailed mechanism remains largely unclear. We used computational simulation to examine the mutational effect of T324 on the dynamics of the ATP-binding site. A mutant model, T324A, of the NS3 helicase apo structure was created and energy was minimized. Molecular dynamics simulation was conducted for both wild type and the T324A mutant apo structures to compare their differences. For the mutant structure, histogram analysis of pairwise distances between residues in domains 1 and 2 (E291-Q460, K210-R464 and R467-T212) showed that separation between the two domains was reduced by ～10% and the standard deviation by ～33%. Root mean square fluctuation (RMSF) analysis demonstrated that residues in close proximity to residue 324 have at least 30% RMSF value reductions in the mutant structure. Solvent RMSF analysis showed that more water molecules were trapped near D290 and H293 in domain 1 to form an extensive interaction network constraining cleft opening. We also demonstrated that the T324A mutation established a new atomic interaction with V331, revealing that an atomic interaction cascade from T324 to residues in domains 1 and 2 controls the flexibility of the ATP-binding cleft.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress

An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605–Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596–Ala1669 sequence (VWF74) and (2) VWF Asp1596–Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

A Fast Method that Uses Polygenic Scores to Estimate the Variance Explained by Genome-wide Marker Panels and the Proportion of Variants Affecting a Trait

Several methods have been proposed to estimate the variance in disease liability explained by large sets of genetic markers. However, current methods do not scale up well to large sample sizes. Linear mixed models require solving high-dimensional matrix equations, and methods that use polygenic scores are very computationally intensive. Here we propose a fast analytic method that uses polygenic scores, based on the formula for the non-centrality parameter of the association test of the score. We estimate model parameters from the results of multiple polygenic score tests based on markers with p values in different intervals. We estimate parameters by maximum likelihood and use profile likelihood to compute confidence intervals. We compare various options for constructing polygenic scores, based on nested or disjoint intervals of p values, weighted or unweighted effect sizes, and different numbers of intervals, in estimating the variance explained by a set of markers, the proportion of markers with effects, and the genetic covariance between a pair of traits. Our method provides nearly unbiased estimates and confidence intervals with good coverage, although estimation of the variance is less reliable when jointly estimated with the covariance. We find that disjoint p value intervals perform better than nested intervals, but the weighting did not affect our results. A particular advantage of our method is that it can be applied to summary statistics from single markers, and so can be quickly applied to large consortium datasets. Our method, named AVENGEME (Additive Variance Explained and Number of Genetic Effects Method of Estimation), is implemented in R software.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.