Shift in oligosaccharide specificities of hemagglutinin and neuraminidase of influenza B viruses resistant to neuraminidase inhibitors

Influenza virus neuraminidase inhibitors (NAIs), currently used as anti-influenza drugs, can lead to the appearance of drug-resistant variants. Resistance to NAIs appears due to mutations in the active site of the neuraminidase (NA) molecule that decrease the NA enzymatic activity and sometimes in the hemagglutinin (HA) that decrease its affinity for cell receptors and, therefore, reduce the requirement for NA activity in releasing mature virions from infected cells. Using a set of sialo-oligosaccharides, we evaluated changes in the receptor-binding specificity of the HA and substrate specificity of the NA of influenza B viruses that had acquired resistance to NAIs. The oligosaccharide specificity of two pairs of field influenza B viruses, namely: i) B/Memphis/20/96 and its NAI-resistant variant, B/Memphis/20-152K/96, containing mutation R152K in the NA and 5 amino acid substitutions in the HA1, and ii) B/Hong Kong/45/2005 and its NAI-resistant variant B/Hong Kong/36/2005, containing a single R371K mutation in the NA, was evaluated. Wild type viruses bound strictly to a “human type” receptor, α2-6-sialo-oligosaccharide 6`SLN, but desialylated it is approximately 8 times less efficiently than the α2-3 sialosaccharides. Both drug-resistant viruses demonstrated the ability to bind to “avian type” receptors, α2-3 sialo-oligosaccharides (such as 3`SLN), whereas their affinity for 6`SLN was noticeably reduced in comparison with corresponding wild type viruses. Thus, the development of the NAI resistance in the studied influenza B viruses was accompanied by a readjustment of HA-NA oligosaccharide specificities.     

Plants and birds on the shore of the Sea of Okhotsk: Balance, crisis, adaptation

Mutual relationships between sea colonial birds and plants on the northern shore of the Sea of Okhotsk are considered. The birds form huge flocks on small islands thus transforming the island landscape and providing specific vegetation at nesting sites. The intensity of ornithogenic load determines different relations between the productivity of plant communities and their species richness. As a rule, flora is depleted. The plants thriving at rookeries react to the intensive action of birds by forming specific ecobiomorphs. On the islands of the northern part of the Sea of Okhotsk tussocks of pine purple grass existing on the majority of nesting colonies are of the highest significance.     

Fumarate reductase and succinate oxidase activity of Escherichia coli complex II homologs are perturbed differently by mutation of the flavin binding domain

The Escherichia coli complex II homologues succinate:ubiquinone oxidoreductase (SQR, SdhCDAB) and menaquinol:fumarate oxidoreductase (QFR, FrdABCD) have remarkable structural homology at their dicarboxylate binding sites. Although both SQR and QFR can catalyze the interconversion of fumarate and succinate, QFR is a much better fumarate reductase, and SQR is a better succinate oxidase. An exception to the conservation of amino acids near the dicarboxylate binding sites of the two enzymes is that there is a Glu (FrdA Glu-49) near the covalently bound FAD cofactor in most QFRs, which is replaced with a Gln (SdhA Gln-50) in SQRs. The role of the amino acid side chain in enzymes with Glu/Gln/Ala substitutions at FrdA Glu-49 and SdhA Gln-50 has been investigated in this study. The data demonstrate that the mutant enzymes with Ala substitutions in either QFR or SQR remain functionally similar to their wild type counterparts. There were, however, dramatic changes in the catalytic properties when Glu and Gln were exchanged for each other in QFR and SQR. The data show that QFR and SQR enzymes are more efficient succinate oxidases when Gln is in the target position and a better fumarate reductase when Glu is present. Overall, structural and catalytic analyses of the FrdA E49Q and SdhA Q50E mutants suggest that coulombic effects and the electronic state of the FAD are critical in dictating the preferred directionality of the succinate/fumarate interconversions catalyzed by the complex II superfamily.     

Mutations in Hemagglutinin and Polymerase Alter the Virulence of Pandemic A(H1N1) Influenza Virus

To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.     

Search for additional influenza virus to cell interactions

Sialyl oligosaccharides have long been considered to be the sole receptors for influenza virus. However, according to [1] some viruses are able to grow in sialic-free MDCK cells. Here we attempted to reveal a possible second, non-sialic receptor, hypothesizing the involvement of additional carbohydrate lectin recognition in influenza virus reception process, first of all in situations when a lectin of the host cell could recognize the viral carbohydrate ligand. We tested the presence of galactose- and sialic acid-binding lectins, as well as mannoside- and sulfo-N-acetyllactosamine-recognizing properties of MDCK and Vero cells using polyacrylamide neoglycoconjugates and antibodies. MDCK cells bind galactoside probes stronger than Vero cells, whereas Vero cells bind preferentially sialoside, mannoside and various sulfo-oligosaccharide probes. The probing of viruses with the neoglycoconjugates revealed specific 6′-HSO ₃ LacNAc (but not other sulfated oligosaccharides) binding property of A and B human strains. Affinity of 6′-HSO ₃ LacNAc probe was comparable with affinity of 6′-SiaLac probe but the binding was not inhibited by the sialooligosaccharide.     

Mathematical analysis of myelin structure of nerves of the brachial plexus in antenatal and early postnatal ontogenesis of man]

A gradual increase of the information entropy indices and a decrease of the excess percentage is observed in the course of antenatal and early postnatal ontogenesis of man. The indices of the information entropy reach their maximum and the excess percentage reaches its minimum in children younger than 3 years. At that time the myelinated fibres have the most various diameters. In the antenatal development of man the prevalence of fine (1--4 mkm) myelinated fibres makes the structure of the humeral plexus nerves more definite while in the postnatal ontogenesis their structure is less definite since the myelinated fibres of different diameter are met with almost the same probability.     

Differences in protonation of ubiquinone and menaquinone in fumarate reductase from Escherichia coli

Escherichia coli quinol-fumarate reductase operates with both natural quinones, ubiquinone (UQ) and menaquinone (MQ), at a single quinone binding site. We have utilized a combination of mutagenesis, kinetic, EPR, and Fourier transform infrared methods to study the role of two residues, Lys-B228 and Glu-C29, at the quinol-fumarate reductase quinone binding site in reactions with MQ and UQ. The data demonstrate that Lys-B228 provides a strong hydrogen bond to MQ and is essential for reactions with both quinone types. Substitution of Glu-C29 with Leu and Phe caused a dramatic decrease in enzymatic reactions with MQ in agreement with previous studies, however, the succinate-UQ reductase reaction remains unaffected. Elimination of a negative charge in Glu-C29 mutant enzymes resulted in significantly increased stabilization of both UQ-* and MQ-* semiquinones. The data presented here suggest similar hydrogen bonding of the C1 carbonyl of both MQ and UQ, whereas there is different hydrogen bonding for their C4 carbonyls. The differences are shown by a single point mutation of Glu-C29, which transforms the enzyme from one that is predominantly a menaquinol-fumarate reductase to one that is essentially only functional as a succinate-ubiquinone reductase. These findings represent an example of how enzymes that are designed to accommodate either UQ or MQ at a single Q binding site may nevertheless develop sufficient plasticity at the binding pocket to react differently with MQ and UQ.     

Localization of the 3' end of Escherichia coli 16 S RNA by electron microscopy of antibody-labelled subunits.

The 3′ end of 16 S RNA is localized on the 30 S subunit of Escherichia coli ribosomes by immune electron microscopy. It is located in the groove between the side “ledge” and the “head” of the subunit on the level of the ledge top. Thus, we have localized the 30 S subunit functional site which is believed to be responsible for binding of the specific messenger RNA sequence preceding the initiation codon. The localization of the 3′ end of 16 S RNA has been done by a new approach in immune electron microscopy. It is based on the covalent binding of low molecular weight ligands, containing the residue of phenyl-β-d-lactoside hapten, to certain points of RNA and the localization of the binding site of the antibody specific to this hapten by electron microscopy. The advantages of this approach in comparison with conventional methods of immune electron microscopy are discussed.     

New <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Strains as Promising Probiotics

The properties of new B. subtilis strains GM2 and GM5, isolated from potato rhizosphere and possessing high antimicrobial activity, were studied. The potential of the strains for their use as probiotics was characterized. The strains were resistant to bile and to a wide range of the ambient pH. B. subtilis strains GM2 and GM5 possessed proteolytic and phytate-hydrolyzing activity and proved to be safe for model animals. The strains were characterized by antagonistic properties against phytopathogenic micromycetes, as well as against pathogenic and opportunistic enterobacteria. B. subtilis GM2 and GM5 were concluded to be promising strains for use as probiotics.