Influence of cultivation conditions on spatial structure and functional activity of OmpF-like porin from outer membrane of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

The influence of cultivation conditions of pseudotuberculosis bacteria on the spatial structure and the functional activity of nonspecific OmpF-like porin was studied by means of optical spectroscopy, scanning microcalorimetry, and bilayer lipid membrane technique. With this goal, porin samples isolated from microbial masses grown at different temperatures, nutrient medium densities, and growth phases were characterized. According to CD data, the porin samples under investigation represent beta-sheet proteins. It was found that the protein isolated from the colonial culture of pseudotuberculosis bacteria grown at low temperature has the most compact structure. Using intrinsic protein fluorescence, it was shown that different conditions of pseudotuberculosis bacteria cultivation (temperature, medium, growth phase) led to the changes in spectral properties of porin fluorescence due to the redistribution of the contributions of tyrosine and different classes of tryptophan residues to the total protein emission. Heat inactivation of porin samples was studied using CD spectroscopy, intrinsic protein fluorescence, and scanning microcalorimetry. Spatial features of the porin samples were found to affect their functional activities. Considering all these data, it is possible to correlate the spatial structure and functional activity of porin samples isolated under different cultivation conditions of bacteria and the composition of the outer membrane lipid matrix.     

Compatibility of cells of the nervous system with structured biodegradable chitosan-based hydrogel matrices

Hydrogel matrices for cell cultivation have been generated by two-photon laser polymerization of unsaturated chitosan derivatives and methacrylated hyaluronic acid. The adhesive and toxic properties of the matrices have been assessed, and the matrices have been shown to have a good compatibility with primary hippocampal cell cultures. The formation of morphologically normal neural networks by cells of the nervous system cultured on the surface of hydrogel matrices has been observed. The metabolic status of dissociated hippocampal cells cultured on the matrices was similar to that of the control cultures, as shown by the results of MTT reductase activity assay. Thus, matrices based on unsaturated polysaccharide derivatives crosslinked by laser irradiation showed good compatibility with differentiated cells of the nervous system and considerable potential for use in neurotransplantation.     

The selection and properties of Penicillium verruculosum mutants with enhanced production of cellulases and xylanases

The paper describes three Penicillium verruculosum 28K mutants with about threefold enhanced production of five industrially important carbohydrases. The two-stage fermentation process that we developed provided a further two- to threefold increase in the production of carbohydrases. Physiological and biochemical studies showed that the synthesis of all five carbohydrases is inducible. Carboxymethylcellulase, xylanase, and -glucanase are synthesized under a common regulatory control, as is evident from the concurrent increase in the synthesis of these enzymes in the presence of microcrystalline cellulose. The synthesis of avicelase and -glucosidase is evidently induced by other cellulose- and hemicellulose-containing compounds present in the fermentation medium and, hence, is regulated independently of the three aforementioned enzymes.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 172–178.Original Russian Text Copyright © 2005 by Soloveva, Okunev, Velkov, Koshelev, Bubnova, Kondrateva, Skomarovskii, Sinitsyn.     

The origin of the “dark” absorption band near 675 nm in the purple bacterial core light-harvesting complex LH1: two-photon measurements of LH1 and its subunit B820

Photosynthesis Research - A comparative two-photon excitation spectroscopic study of the exciton structure of the core antenna complex (LH1) and its subunit B820 was carried out. LH1 and its...     

Infection with metacercaria of the family diplostomidae and activity of digesting enzymes of the young of the Siberian dace <Emphasis Type=Italic>Leuciscus leuciscus baicalensis</Emphasis> (Dyb.) in the Kargat River of the basin of Lake Chany

In 2008 and 2009 on the Kargat River (basin of Lake Chany) we studied activity of the intestinal digestive enzymes (general activity of proteases, α-amilase, nonspecific esterases and pancreatic lipase) of the young of the Siberian dace Leuciscus leuciscus baicalensis (Dyb.) infected with metacercaria of the family Diplostomidae. The relation between the intensity of the invasion with metacercaria of the family Diplostomidae and activity of the intestinal digestive hydrolases was studied. The negative correlation of the activity of the digestive enzymes and the intensity of the invasion with metacercaria both in various segments and in the whole intestine was revealed.     

Morphoecological aspects of preimaginal stages of development of the parasitic wasp <Emphasis Type="Italic">Minotetrastichus frontalis</Emphasis> (Nees, 1834) (Insecta,Hymenoptera, Eulophidae)

This work is devoted to studying the morphology and biology of preimaginal stages of development of the parasitic wasp Minotetrastichus frontalis. The morphological characteristics of eggs and larvae, the features of interaction of the parasitic wasp with the host (Phyllonorycter issikii), and other biological characteristics were determined. For the species identification of the studied specimens of insects, we performed molecular genetic analysis using a nuclear 28S RNA gene fragment as a molecular marker. The result showed a strong genetic polymorphism of the populations of the studied species for the selected marker.     

Liposomal Drug with Carnosine and Lipoic Acid: Preparation,Antioxidant and Antiplatelet Properties

The conditions for producing phosphatidylcholine liposomes containing lipoic acid and carnosine together were determined. The obtained liposomes are 180–250-nm spherical particles with an efficiency of lipoic acid inclusion of 50–70% (for carnosine, 17–33%). Based on the model of the oxidation of phosphatidylcholine by hydrogen peroxide, an antioxidant effect of carnosine, lipoic acid or lipoic acid with carnosine together was demonstrated; it consisted in inhibition of lipid peroxidation process, which was manifested in a decrease in the formation of lipid peroxidation products that react with thiobarbituric acid. It was established that lipoic acid (5 mM) and carnosine (0.1–10 mM) in liposomes exhibit an antioxidant effect. At the same time, it was demonstrated that the content of the appropriate lipid peroxidation products in liposomes with antioxidants (lipoic acid + carnosine) was 15 times lower than in control liposomes (without antioxidants). The effect of the obtained liposomal drugs on the platelet aggregation induced by arachidonic acid was evaluated. It was found that the liposomal drug containing lipoic acid (1.5 mM) and carnosine (2.1 mM) inhibited platelet aggregation by 50–55% relative to the control (platelets and arachidonic acid), while liposomes without antioxidants and water-soluble forms of carnosine and lipoic acid had almost no effect on platelet aggregation caused by arachidonic acid.

     

Methods of molecular cytogenetics for studying interphase chromosomes in human brain cells

One of the main genetic factors determining the functional activity of the genome in somatic cells, including brain nerve cells, is the spatial organization of chromosomes in the interphase nucleus. For a long time, no studies of human brain cells were carried out until high-resolution methods of molecular cytogenetics were developed to analyze interphase chromosomes in nondividing somatic cells. The purpose of the present work was to assess the potential of high-resolution methods of interphase molecular cytogenetics for studying chromosomes and the nuclear organization in postmitotic brain cells. A high efficiency was shown by such methods as multiprobe and quantitative fluorescence in situ hybridization (Multiprobe FISH and QFISH), ImmunoMFISH (analysis of the chromosome organization in different types of brain cells), and interphase chromosome-specific multicolor banding (ICS-MCB). These approaches allowed studying the nuclear organization depending on the gene composition and types of repetitive DNA of specific chromosome regions in certain types of brain cells (in neurons and glial cells, in particular). The present work demonstrates a high potential of interphase molecular cytogenetics for studying the structural and functional organizations of the cell nucleus in highly differentiated nerve cells. Analysis of interphase chromosomes of brain cells in the normal and pathological states can be considered as a promising line of research in modern molecular cytogenetics and cell neurobiology, i. e., molecular neurocytogenetics.     

New derivatives of eremomycin containing 15N or F atoms for NMR study

New semisynthetic derivatives of eremomycin containing ¹⁵N or F atoms were obtained for studying the antibiotic-target interaction in intact cells of Gram-positive bacteria by REDOR NMR method. Interaction of the terminal carboxyl group of amino acid 7 (AA7) of eremomycin with amines in the presence of PyBOP and TBTU reagents resulted in the corresponding [¹⁵N]-amide, p-fluorobenzylamide, p-fluorophenylpiperazide, and 6-N-(p-fluorobenzyl)aminohexylamide. A selective method of [¹⁵N]-amidation of carboxyl group of amino acid 3 (AA3) of carboxyeremomycin was developed, and the amide of eremomycin containing [¹⁵N] in AA3 amide group near the antibiotic binding pocket was obtained. Carboxyeremomycin bisamides substituted at AA3 and AA7 and containing two atoms of [¹⁵N] or F were obtained from carboxyeremomycin and [¹⁵N]NH₄Cl or the corresponding p-fluorobenzylamine hydrochloride in the presence of PyBOP at pH ～8. The Edman degradation of eremomycin p-fluorobenzylamide gave de-(D-MeLeu)-eremomycin p-fluorobenzylamide, a hexapeptide derivative incapable of the antibiotic binding with-D-Ala-D-Ala fragment of growing cell wall peptidoglycan. Among the compounds studied, carboxyeremomycin bis-p-fluorobenzylamide showed the best activity against both the glycopeptides-sensitive and glycopeptides-resistant strains of staphylococci and enterococci.     

Chaperone Skp from <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> exhibits immunoglobulin G binding ability

A low-molecular-weight cationic protein that can bind human and rabbit immunoglobulins G has been isolated from Yersinia pseudotuberculosis cells. This immunoglobulin binding protein (IBP) interacts with IgG Fc-fragment, the association constant of the resulting complex being 3.1 μM^?1. MALDI-TOF mass spectrometry analysis of IBP revealed its molecular mass of 16.1 kDa, and capillary isoelectrofocusing analysis showed pI value of 9.2. N-Terminal sequence determination by Edman degradation revealed the sequence of the 15 terminal amino acid residues (ADKIAIVNVSSIFQ). Tryptic hydrolysate of IBP was subjected to MALDI-TOF mass spectrometry for proteolytic peptide profiling. Based on the peptide fingerprint, molecular mass, pI, and N-terminal sequence and using bioinformatic resources, IBP was identified as Y. pseudotuberculosis periplasmic chaperone Skp. Using the method of comparative modeling a spatial model of Skp has been built. This model was then used for modeling of Skp complexes with human IgG1 Fc-fragment by means of molecular docking.