Study of the structure of streptokinase conjugates with a hydrophilic vinylpyrrolidone copolymer

The modification of streptokinase by a synthetic N-vinylpyrrolidone copolymer leads to formation of conjugates varying in structures according to the proportions of the components in the reaction medium. Based on data obtained from spectrophotometry, as well as sedimentation and diffusion analyses, it is shown that in the presence of excess protein in the reaction medium, formation of the main chain takes place via the copolymer associated with several protein globules. Under conditions of excess modifier copolymer, either single-site and/or multiple-site bonding is possible for the protein backbone, depending on the molecular weight of the copolymer. One of the models for the conjugates obtained in this manner has been corroborated by small-angle X-ray scattering data. CD spectral analyses has been performed in order to demonstrate that covalent modification does not alter the secondary structure of streptokinase in the conjugate whereas the tertiary structure undergoes local changes in conformation.     

The polysaccharide component of Pseudomonas pseudomallei slime

The polysaccharide component contained in the slime of the causative agent of melioidosis was obtained. This component was found to be a biopolymer, mainly of the carbohydrate nature, consisting of galactose, glucose, mannose, rhamnose and two unidentified carbohydrates. The slime polysaccharide component contained two thermostable and acid-resistant antigens. The action of alkali led to the loss of one of these antigens. Rabbit antisera to the preparations of the slime polysaccharide component with titers of 1:64 to 1:256, determined in the immunodiffusion test, were obtained. In the precipitation test the slime polysaccharide component reacted with antisera from sick experimental animals, thus confirming the suggestion of its secretion in the process of the development of melioidosis infection.     

Extracellular electrical signals in a neuron-surface junction: model of heterogeneous membrane conductivity

Signals recorded from neurons with extracellular planar sensors have a wide range of waveforms and amplitudes. This variety is a result of different physical conditions affecting the ion currents through a cellular membrane. The transmembrane currents are often considered by macroscopic membrane models as essentially a homogeneous process. However, this assumption is doubtful, since ions move through ion channels, which are scattered within the membrane. Accounting for this fact, the present work proposes a theoretical model of heterogeneous membrane conductivity. The model is based on the hypothesis that both potential and charge are distributed homogeneously on the membrane surface, concentrated near channel pores, as the direct consequence of the inhomogeneous transmembrane current. A system of continuity equations having non-stationary and quasi-stationary forms expresses this fact mathematically. The present work performs mathematical analysis of the proposed equations, following by the synthesis of the equivalent electric element of a heterogeneous membrane current. This element is further used to construct a model of the cell-surface electric junction in a form of the equivalent electrical circuit. After that a study of how the heterogeneous membrane conductivity affects parameters of the extracellular electrical signal is performed. As the result it was found that variation of the passive characteristics of the cell-surface junction like conductivity of the cleft and the cleft height could lead to different shapes of the extracellular signals.     

A Genome-Scale Metabolic Reconstruction of Mycoplasma genitalium,iPS189

With a genome size of ∼580 kb and approximately 480 protein coding regions, Mycoplasma genitalium is one of the smallest known self-replicating organisms and, additionally, has extremely fastidious nutrient requirements. The reduced genomic content of M. genitalium has led researchers to suggest that the molecular assembly contained in this organism may be a close approximation to the minimal set of genes required for bacterial growth. Here, we introduce a systematic approach for the construction and curation of a genome-scale in silico metabolic model for M. genitalium. Key challenges included estimation of biomass composition, handling of enzymes with broad specificities, and the lack of a defined medium. Computational tools were subsequently employed to identify and resolve connectivity gaps in the model as well as growth prediction inconsistencies with gene essentiality experimental data. The curated model, M. genitalium iPS189 (262 reactions, 274 metabolites), is 87% accurate in recapitulating in vivo gene essentiality results for M. genitalium. Approaches and tools described herein provide a roadmap for the automated construction of in silico metabolic models of other organisms.     

Calculations of atomic structures in the problems of interaction of heavy-ion beams with targets

The process of energy conversion in heavy-ion inertial confinement fusion is associated with the deceleration of heavy ions in a low-temperature plasma that is produced when the beam ionizes the target material. In order to calculate the deceleration of heavy ions in a target, it is necessary to determine the wave functions, energy levels, and oscillator strengths for atoms and for ions in different charge states. The models that have been developed thus far to calculate deceleration processes apply only to gas targets. In the present paper, a method is proposed that is based on the Hartree-Fock-Slater model and makes it possible to perform calculations for experiments with both low-density (gas) and high-density (solid) targets. The method applies to neutral atoms and also to ions in different charge states. Results are presented from calculations carried out for nitrogen, oxygen, aluminum, and silicon atoms and are compared with the results obtained by other authors and with the experimental data. It is shown that, for high-density targets, the method proposed provides better agreement with experiments than do the models developed earlier.     

Dissection of the structural and functional role of a conserved hydration site in RNase T1

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites (WAT1) were analyzed by protein engineering, X-ray crystallography, and (17)O and 2H nuclear magnetic relaxation dispersion (NMRD). The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93N epsilon2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side-chain atom, at the center of this intricate hydrogen bond network. Comparison of the (17)O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 degrees C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.     

The mapping and reconstitution of a conformational discontinuous B-cell epitope of HIV-1

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.     

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo pi-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.     

Structural organization of the human genome: distribution of nucleotides, Alu-repeats and exons in chromosomes 21 and 22]

Analysis of DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (Y waves phi) with a period of about 3 Mbp. Distribution of G + C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G + C than chromosome 22. Both exons and Alu repeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alu repeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alu repeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Alu distribution patterns along the chromosome, the concurrent distribution of Alu repeats in both orientations along the chromosome, and the equal copy numbers for Alu in direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alu repeats belong to parasitic (junk) DNA.