Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses

Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers.     

Changes in distribution and molecular weight of the acrosomal protein acrin2 (MC41) during guinea pig spermiogenesis and epididymal maturation

In this study, we examined the localization and characteristics of an intra-acrosomal protein, acrin2 (MC41), during guinea pig spermiogenesis and post-testicular sperm maturation in the epididymis, using the monoclonal antibody MC41. Immunoelectron microscopy demonstrated not only a specific domain localization of acrin2 in the apical segment of the guinea pig sperm acrosome, but also its dynamic behavior according to the spermatid differentiation and passage through the epididymis, as follows: acrin2 was exclusively localized in the membrane of the endoplasmic reticulum of early-stage spermatids but was not detectable in the developing acrosome until spermatids reached the maturation phase. In the final stage of spermiogenesis, acrin2 became localized in the outer acrosomal membrane (OAM)/matrix-associated materials both in the small region posterior to the dorsal matrix and along the ventral margin of the acrosomal apical segment. The acrosomal location of acrin2 in caput epididymidal sperm was almost identical to that observed in the final step spermatids, but during maturation it became progressively more restricted in area until on distal cauda epididymidal sperm it remained only in the dorsal region. In Western blot analysis, the MC41 antibody recognized a 165-kDa protein in the mature sperm extract. Furthermore, it was demonstrated that molecular weight reduction of the protein occurred during sperm passage through the epididymis. These findings indicate that acrin2 changes progressively in both distribution and size during development and maturation of the acrosome.     

Cloning and Physical Mapping of the EcoRI Fragments of the Giant Linear Plasmid SCP1

A cosmid library was constructed for the 350-kb giant linear plasmid SCP1 and aligned on a successive linear map. Only a 0.8-kb gap has remained uncloned in the terminal inverted repeats close to both ends. Partial digestion of the aligned cosmids with EcoRI and hybridization with the flanking fragments of the vector enabled physical mapping of all of the EcoRI fragments. On this map, the methylenomycin biosynthetic gene cluster, the insertion sequence IS466, and the sapCDE genes coding for spore-associated proteins were localized.     

Possible involvement of volatile compounds in the after-ripening of cocklebur seeds

The mechanism of emergence from primary dormancy, the process of after-ripening, in cocklebur (Xanthium pennsylvanicum) seeds was examined in relation to the involvement of volatile compounds and to the relative humidity (RH) in which the seeds were stored. The after-ripening of these seeds proceeds only at water contents between 7 and 14% which are conditioned under RHs of 33% to 53% and are identified with water-binding region II. After-ripening of cocklebur seeds occurred even in water-binding region I. imposed by 12% RH. when exposed to HCN gas during the storage period. Exposure of dormant seeds to acetaldehyde (ethanal) retarded after-ripening. even in water-binding region II. thus decreasing germinability. This decrease of germinability by ethanal was found also in the after-ripened seeds, suggesting that ethanal accelerates seed deterioration rather than retarding the after-ripening. The contents of ethanal. ethanal and HCN were high only in the dormant seeds held at 12% RH. Regardless of RH. a possible conversion of ethanal to ethanol. perhaps via alcohol dehydrogenase. was far larger in dormant than in non-dormant seeds. In contrast, the reverse conversion of ethanol to ethanal was more profound in non-dormant seeds. Pre-exposure of both types of seeds to HCN reduced the contents of both ethanal and ethanol at 12% RH. The contents of various adenylales including ATP in seed tissues were higher in dormant seeds stored at 12% RH than in non-dormant seeds after-ripened at 44% RH. It is suggested that emergence of cocklebur seeds from primary dormancy by HCN treatment at 12% RH may result from the reduction in the contents of ethanal via an unknown mechanism incurring the consumption of ATP. This implies involvement of volatile compound metabolism at the water-binding region II in the after-ripening process of cocklebur seeds.     

Characterization of a Novel Plasmid,pMAH135, from Mycobacterium avium Subsp. hominissuis

Mycobacterium avium complex (MAC) causes mainly two types of disease. The first is disseminated disease in immunocompromised hosts, such as individuals infected by human immunodeficiency virus (HIV). The second is pulmonary disease in individuals without systemic immunosuppression, and the incidence of this type is increasing worldwide. M. avium subsp. hominissuis, a component of MAC, causes infection in pigs as well as in humans. Many aspects of the different modes of M. avium infection and its host specificity remain unclear. Here, we report the characteristics and complete sequence of a novel plasmid, designated pMAH135, derived from M. avium strain TH135 in an HIV-negative patient with pulmonary MAC disease. The pMAH135 plasmid consists of 194,711 nucleotides with an average G + C content of 66.5% and encodes 164 coding sequences (CDSs). This plasmid was unique in terms of its homology to other mycobacterial plasmids. Interestingly, it contains CDSs with sequence homology to mycobactin biosynthesis proteins and type VII secretion system-related proteins, which are involved in the pathogenicity of mycobacteria. It also contains putative conserved domains of the multidrug efflux transporter. Screening of isolates from humans and pigs for genes located on pMAH135 revealed that the detection rate of these genes was higher in clinical isolates from pulmonary MAC disease patients than in those from HIV-positive patients, whereas the genes were almost entirely absent in isolates from pigs. Moreover, variable number tandem repeats typing analysis showed that isolates carrying pMAH135 genes are grouped in a specific cluster. Collectively, the pMAH135 plasmid contains genes associated with M. avium’s pathogenicity and resistance to antimicrobial agents. The results of this study suggest that pMAH135 influence not only the pathological manifestations of MAC disease, but also the host specificity of MAC infection.     

Establishment of a New Quality Control and Vaccine Safety Test for Influenza Vaccines and Adjuvants Using Gene Expression Profiling

We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.     

Disruption of the Putative Cell Surface Polysaccharide Biosynthesis Gene SO3177 in Shewanella oneidensis MR-1 Enhances Adhesion to Electrodes and Current Generation in Microbial Fuel Cells

A microbial fuel cell (MFC) was inoculated with a random transposon insertion mutant library of Shewanella oneidensis MR-1 and operated with lactate as the sole fuel to select for mutants that preferentially grew in it. Agar plate cultivation of the resultant MFC enrichment culture detected an increased number of colonies exhibiting rough morphology. One such isolate, strain 4A, generated 50% more current in an MFC than wild-type MR-1. Determination of the transposon insertion site in strain 4A followed by deletion and complementation experiments revealed that the SO3177 gene, encoding a putative formyltransferase and situated in a cell surface polysaccharide biosynthesis gene cluster, was responsible for the increased current. Transmission electron microscopy showed that a layered structure at the cell surface, stainable with ruthenium red, was impaired in the SO3177 mutant (ΔSO3177), confirming that SO3177 is involved in the biosynthesis of cell surface polysaccharides. Compared to the wild type, ΔSO3177 cells preferentially attached to graphite felt anodes in MFCs, while physicochemical analyses revealed that the cell surface of ΔSO3177 was more hydrophobic. These results demonstrate that cell surface polysaccharides affect not only the cell adhesion to graphite anodes but also the current generation in MFCs.Dissimilatory metal-reducing bacteria (DMRB) conserve energy for growth by coupling the oxidation of organic compounds to the reduction of metal compounds (29). DMRB are of great interest not only for their importance in the biogeochemical cycling of metals (25) but also for their utility in biotechnological processes, such as microbial fuel cells (MFCs) (24, 40). In recent years, the ability of many DMRB, including members of the genera Shewanella (5, 12, 20, 31), Geobacter (2), Aeromonas (34), Desulfobulbus (19), and Phodoferax (9), to generate current in MFCs has been described.Among DMRB, Shewanella oneidensis MR-1 is one of the most extensively studied due to its metabolic versatility (28), annotated genome sequence (17), and genetic accessibility. In addition, since the first report in 1999 when this microorganism was shown to have the ability to transfer electrons to an anode without an exogenously added mediator (20), it has become a model organism for the study of microbial current generation in MFCs. Extensive studies have been performed to understand the mechanisms of extracellular electron transfer (EET) to solid materials, such as MFC anodes and metal oxides, in strain MR-1. Multiple mechanisms, including direct EET through the physical contact of bacterial cells via outer membrane (OM) cytochromes (42) and conductive nanowires (16) and mediated EET via self-produced electron shuttles such as quinones and flavins (27, 30, 39, 41), have been identified.Although OM cytochromes and electron shuttles have been identified to play central roles in EET, it is reasonable to speculate that this complex catabolic process is also influenced by other (extra)cellular components. To date, only limited studies have been done to investigate other cellular components involved in EET (7). A useful approach for identifying unknown cellular components (and genes) associated with a particular phenotype involves the construction and screening of a random mutant library for obtaining mutants with altered phenotypes. In the present study, we constructed a random transposon (Tn) insertion mutant library of S. oneidensis MR-1 and obtained mutants with altered colony morphologies (rough morphotypes) after the selection of mutants in an MFC. Analyses of one of such mutants suggest that cell surface capsular polysaccharides affect not only the adhesion of cells to graphite anodes but also the current generation in MFCs.