A study of the effect of low alpha-tocopherol concentrations on the dynamical parameters of microsomal membranes by the method of spin probes

The effect of alpha-tocopherol (alpha-tp) prepared in solvents of different polarity in a wide range of concentrations (10(-4) M - 10(-25) M) on lipid phase structural characteristics of microsomal membranes isolated from mouse liver cells has been investigated in vitro. Structural changes in membranes were detected on a Bruker-200D ESR-spectrometer (Germany) by the method of spin probes. Changes in the rigidity of surface lipid bilayer regions (8 A) and microviscosity of deep membrane layers (20 A) were studied using the stable nitroxyl radicals 5- and 16-doxylstearic acids, correspondingly. As a result, nonlinear multimodal dose dependences were obtained. It was demonstrated that the physiological (10(-4) M - 10(-9) M) and ultralow doses of alpha-tocopherol up to "apparent" concentrations (10(-11) M - 10(-25) M) increased the rigidity of surface lipid bilayer regions and microviscosity in the depth of membrane. Additionally, these doses of alpha-tp induced an increase in the number of thermoinduced structural transitions in deep lipid bilayer regions. The effect at "apparent" concentrations (< 10(-18) M) has only been observed in polar alpha-tocopherol solutions. The results obtained are statistically reliable with a significance level of 95%.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Stimulated ionization scattering of a wave beam forming a discharge channel in a magnetic mirror trap

The stimulated scattering of a whistler wave beam forming an extended discharge channel in a magnetic mirror trap is discovered and investigated experimentally. It is shown that the beam is scattered by relaxaction oscillations of the lattice of plasma inhomogeneities excited by the beam field. The spectrum of the pump field in the RF discharge plasma is found to broaden considerably and to contain individual modulation peaks corresponding to lattice oscillations. The peaks are observed at working gas pressures at which the electron mean free path is close to the wavelength of the standing wave forming the discharge channel. A physical model describing the phenomena observed is developed.     

Organization of infectious disease prevention in town Verkhnyaya Pyshma

Data on organization of antiepidemic measures in town Verkhnyaya Pyshma are presented. Infectious diseases incidence for several years was analyzed, its dependence from quality of preventive measures was shown. Experience on ensuring the epidemiological welfare for vaccine-preventable diseases was described. Results of implementation of regional programs, which allowed to improve the system for infectious diseases surveillance, as well as interactions between territorial administration and services and agencies providing the epidemiologic safety of the area.     

Identification of natural killer cell receptor clusters in the platypus genome reveals an expansion of C-type lectin genes

Natural killer (NK) cell receptors belong to two unrelated, but functionally analogous gene families: the immunoglobulin superfamily, situated in the leukocyte receptor complex (LRC) and the C-type lectin superfamily, located in the natural killer complex (NKC). Here, we describe the largest NK receptor gene expansion seen to date. We identified 213 putative C-type lectin NK receptor homologs in the genome of the platypus. Many have arisen as the result of a lineage-specific expansion. Orthologs of OLR1, CD69, KLRE, CLEC12B, and CLEC16p genes were also identified. The NKC is split into at least two regions of the genome: 34 genes map to chromosome 7, two map to a small autosome, and the remainder are unanchored in the current genome assembly. No NK receptor genes from the LRC were identified. The massive C-type lectin expansion and lack of Ig-domain-containing NK receptors represents the most extreme polarization of NK receptors found to date. We have used this new data from platypus to trace the possible evolutionary history of the NK receptor clusters. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

GBF1, a Guanine Nucleotide Exchange Factor for Arf,Is Crucial for Coxsackievirus B3 RNA Replication

The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.Enteroviruses are small, nonenveloped, positive-stranded RNA viruses that include many important pathogens, such as poliovirus (PV), coxsackievirus, echovirus, and human rhinovirus. Following virus entry and uncoating, the 7.5-kb enteroviral RNA genome is directly translated into a large polyprotein. This polyprotein is proteolytically processed by the virus-encoded proteases 2A^pro, 3C^pro, and 3CD^pro into the structural P1 region proteins and the nonstructural P2 and P3 region proteins that are involved in viral RNA replication.All RNA viruses with a positive-stranded genome induce the remodeling of cellular membranes to create a scaffold for genomic RNA replication. The organelle origin and morphology of these membranous replication sites, however, appear to vary for different viruses. Enteroviruses replicate their RNA genomes in nucleoprotein complexes that are associated with small vesicular membrane structures (6). The enteroviral proteins 2B, 2C, and 3A have been implicated in vesicle formation (4, 6, 27), but the mechanism and pathway of membrane reorganization are poorly understood. There are strong indications that these vesicular membranous structures, which are referred to here as “vesicles,” are derived from the early secretory pathway. Vesicles produced in PV-infected cells may form at the endoplasmic reticulum (ER) by the cellular COP-II budding machinery and may therefore share components with the membranous vesicles mediating ER-to-Golgi network transport (26). Further support for the involvement of the secretory pathway stems from the observation that brefeldin A (BFA), a well-known inhibitor of ER-to-Golgi network transport, completely inhibits enteroviral RNA replication (17, 20). In addition, the autophagocytic pathway appears to contribute to the formation of the membrane vesicles, many of which exhibit a double-membrane morphology characteristic of autophagosomes (18, 27). The utilization of individual components or reactions from different membrane metabolic pathways, rather than subversion of an entire pathway in toto, may represent a common strategy for building viral replication machinery.BFA inhibits activation of the small monomeric GTPase ADP ribosylation factor 1 (Arf1), a major regulator of intracellular protein transport (2). Arf1 cycles between an inactive, GDP-bound, cytosolic state and an active, GTP-bound, membrane-associated state, and this cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (13). BFA blocks the activities of the large GEFs GBF1, BIG1, and BIG2 by stabilizing an intermediate, abortive complex with inactive Arf1 (23), thus efficiently preventing activation of Arf1 and eventually formation of transport intermediates.Not only the fact that BFA blocks enteroviral replication suggests a role for Arf1 and/or its large GEFs in this process; recently, it was shown that Arf1 accumulates on membranes during PV infection (3). Arf1 translocation to membranes can be induced independently by enterovirus protein 3A or 3CD in vitro (5), but the underlying mechanisms seem to differ; the 3A protein specifically triggers the recruitment of GBF1 to membranes, most likely through a direct interaction with this GEF (32, 33), whereas 3CD recruits BIG1 and BIG2 to membranes (3). Here, we report the involvement of Arf1 and its large BFA-sensitive GEFs in coxsackievirus B3 (CVB3) replication.     

Synthesis, structure and electron-mediator properties of the mono- and difunctionalized macrobicyclic iron(II) tris-dioximates with thiol terminated ribbed spacer substituents

Nucleophilic substitution of the reactive chlorine atoms of mono- and dichlorine clathrochelate FeBd₂(HGmCl)(BF)₂ and FeBd₂(Cl₂Gm)(BF)₂ precursors (where Bd²⁻, HGmCl²⁻ and Cl₂Gm²⁻ are α-benzyldioxime, chloroglyoxime and dichloroglyoxine dianions, respectively) with HSCH₂CH₂SH and (HSCH₂CH₂)₂S in the presence of triethylamine afforded the FeBd₂(HGmSCH₂CH₂SH)(BF)₂ (1) and FeBd₂(HGm(SCH₂CH₂)₂SH)(BF)₂ clathrochelates with a sole thiol terminated spacer substituent and the FeBd₂((HS(CH₂CH₂S)₂)₂Gm)(BF)₂ clathrochelate with two longchain thiol-sulfide ribbed groups. The crystal and molecular structures of these complexes were obtained by X-ray crystallography. The clathrochelate molecules possess a geometry intermediate between a trigonal prism (TP) and a trigonal antiprism (TAP). The X-ray data are in a good agreement with the ⁵⁷Fe Mössbauer parameters for complexes studied. These parameters characterize them as the low-spin iron(II) complexes (the isomeric shift values) with TP-TAP geometry (the quadrupole splitting values). Application of clathrochelate self-assembled monolayer (SAM) as the redox mediators for the determination of hydrogen peroxide is discussed. The calibration curve for hydrogen peroxide concentrations was found to be in the range from 2.0 · 10⁻⁶ to 1.6 · 10⁻⁵ mol · L⁻¹ with limit of detection equal to 1.0 · 10⁻⁶ mol · L⁻¹. The clathrochelate SAM on gold electrode provides precise and sensitive amperometric determination of hydrogen peroxide.     

Molecular genetic determinants of intraspecific polymorphism of the phytopathogenic fungus Cryphonectria parasitica

The review summarizes the current evidence on the phytopathogenic fungus Cryphonectria parasitica, which is a classic object for studying hypovirulence. Phenotypic manifestations of hypovirulence and themolecular mechanisms of action of the mycovirus Cryphonectria hypovirus (CHV) infecting the fungus are described in detail. Genetic determinants of vegetative incompatibility in C. parasitica (a phenomenon increasing polymorphism of the fungus and preventing CHV expansion) are considered. The data on C. parasitica polymorphism are correlated with the data on the distribution of different CHV species in the European, American, and Asian populations of the fungus.