Activity and Stability of Native and Modified Subtilisins in Various Media

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.     

Detection of nitric oxide from pig trachea by a fluorescence method

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was used to detect nitric oxide (NO) from freshly excited tissues. This approach is based on the phenomenon of the intramolecular fluorescence quenching of the fluorophore fragment by the nitroxide. The pyrene-nitronyl (PN) reacts with NO to yield a pyrene-imino nitroxide radical (PI) and NO(2). Conversion of PN to PI is accompanied by changes in the electron paramagnetic resonance (EPR) spectrum from a five-line pattern (two equivalent N nuclei) into a seven-line pattern (two nonequivalent N nuclei). The transformation of the EPR signal is accompanied by an increase in the fluorescence intensity since the imino nitroxide radical is a weaker quencher than the nitronyl one. The results indicate that the fluorescence measurements enable detection of nanomolar concentrations of NO compared to a sensitivity threshold of only several micromolar for the EPR technique. The method was applied to the determination of NO and S-nitroso compounds in tissue from pig trachea epithelia. The measured basal flux of S-nitroso compounds obtained from the tissues was about 1.2 nmol/g x min, and NO-synthase stimulated by extracellular adenosine 5'-triphosphate produced NO flux of 0.9 nmol/g x min.     

Mobster: accurate detection of mobile element insertions in next generation sequencing data

Mobile elements are major drivers in changing genomic architecture and can cause disease. The detection of mobile elements is hindered due to the low mappability of their highly repetitive sequences. We have developed an algorithm, called Mobster, to detect non-reference mobile element insertions in next generation sequencing data from both whole genome and whole exome studies. Mobster uses discordant read pairs and clipped reads in combination with consensus sequences of known active mobile elements. Mobster has a low false discovery rate and high recall rate for both L1 and Alu elements. Mobster is available at http://sourceforge.net/projects/mobster.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0488-x) contains supplementary material, which is available to authorized users.     

Structural Features of Transiently Modified Beta-Lactoglobulin Relevant to the Stable Binding of Large Hydrophobic Molecules

Binding sites for hydrophobic molecules on bovine β-lactoglobulin, and their susceptibility to temperature, were studied by using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual probes was found to be required for binding to β-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible denaturation. Binding became extremely tight and stable upon cooling of the protein–probe mixture. Comparison among the various probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially—and reversibly—modified form of β-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes binding to β-lactoglobulin may be modulated by simple physical treatment of the protein.     

Structural and biological characterization of the tubulin interaction with dinitroanilines

Characteristics of the interaction of dinitroaniline compounds with tubulin molecules have an extremely high selectivity: these substances efficiently bind to the tubulins of both plant and protozoan origins and practically do not interact with any animal and fungal tubulins despite a very high similarity between the corresponding sequences. This work summarizes and comprehensively analyzes the specific structural features and mechanisms of these interactions, in particular, the patterns of the structure and arrangement of dinitroaniline binding sites on the surface of different tubulin subunits and tubulins of various origins. Dinitroaniline binding sites are localized to the surface of longitudinal contacts between tubulin subunits and contain diamine amino acid residues (lysine or arginine), which bind the nitrile group of dinitroanilines. The localizations of these sites on the surface of identical subunits of different origins (for example, α-tubulins of plants and protozoans) coincide; however, the location of these binding sites on the surfaces of tubulin α- and β-subunits is different. The characterized sites can also be potential binding sites for other antimicrotubule compounds, in particular, cyanoacrylates.     

Novel immobilized biocatalyst for microbiological synthesis of pharmaceutical steroids

Applied Biochemistry and Microbiology - The steroid-transforming activity of free and immobilized cells of Pimelobacter simplex VKPM Ac-1632 entrapped in an operationally stable macroporous...     

Replicated high-density genetic maps of two great tit populations reveal fine-scale genomic departures from sex-equal recombination rates

Linking variation in quantitative traits to variation in the genome is an important, but challenging task in the study of life-history evolution. Linkage maps provide a valuable tool for the unravelling of such trait−gene associations. Moreover, they give insight into recombination landscapes and between-species karyotype evolution. Here we used genotype data, generated from a 10k single-nucleotide polymorphism (SNP) chip, of over 2000 individuals to produce high-density linkage maps of the great tit (Parus major), a passerine bird that serves as a model species for ecological and evolutionary questions. We created independent maps from two distinct populations: a captive F2-cross from The Netherlands (NL) and a wild population from the United Kingdom (UK). The two maps contained 6554 SNPs in 32 linkage groups, spanning 2010 cM and 1917 cM for the NL and UK populations, respectively, and were similar in size and marker order. Subtle levels of heterochiasmy within and between chromosomes were remarkably consistent between the populations, suggesting that the local departures from sex-equal recombination rates have evolved. This key and surprising result would have been impossible to detect if only one population was mapped. A comparison with zebra finch Taeniopygia guttata, chicken Gallus gallus and the green anole lizard Anolis carolinensis genomes provided further insight into the evolution of avian karyotypes.     

Sorption of microorganisms by wide-porous agarose cryogels containing grafted aliphatic chains of different length

The possibility of fractionation of heterogeneous bacterial populations using wide-porous agarose cryogels containing grafted aliphatic groups with the chain lengths of 4, 7, and 12 carbon atoms was demonstrated for the first time. The maximum sorption of vegetative cells of gram-positive bacteria (60%) was shown for the polymeric carrier with the chain length of 4 carbon atoms, while the hypometabolic cells appearing in the population after prolonged (60-day) cultivation were trapped by wide-porous affinity sorbents with C₇- and C₁₂-aliphatic groups much better than vegetative cells.     

Swelling behavior of poly(vinyl alcohol) cryogels employed as matrices for cell immobilization

Poly(vinyl alcohol) cryogels are prepared from aqueous solutions of the polymer by freezing and thawing and are employed as matrices for cell immobilization. The swelling behavior of these macroporous gel carriers in pure water and in solutions of certain compounds (salts, amino acids, and glucose) was studied to elucidate the osmotic properties of the cryogels during long-term exposure to aqueous media. It was shown that after the initial sol fraction was washed out, the residual gel matrix possessed high stability even at extreme pH conditions (acid or alkali concentration up to 1.0 mol l⁻¹) or in the presence of strong chaotropic salts such as sodium rhodanide. Although the macroporous supermolecular structure of the carriers under consideration underwent certain changes as a result of aging processes during prolonged washing of the gel, the high porous morphology of the material was retained.