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A bovine IgG‐enriched whey fraction contains antibodies against various bacterial antigens. We investigated the protective effects of a bovine whey fraction preparation against infections with Enterohaemorrhagic Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, and Mycobacterium avium in mouse models. After infection with these pathogens, the IgG‐enriched fraction or skim milk was given ad libitum at a 5% solution instead of water. The mice given the IgG‐enriched fraction were significantly resistant to orally challenged EHEC O157:H7 (LD50: 4.0 × 105 CFU/mouse) infections compared with the mice given skim milk (LD50

Drug resistance in Mycobacterium tuberculosis (Mtb) is caused by mutations in restricted regions of the genome. Mutations in katG, the promoter region of the mabA-inhA operon, and inhA are those most frequently responsible for isoniazid (INH) resistance. Several INH-resistant (INH(r) ) Mtb clinical isolates without mutations in these regions have been described, however, indicating that there are as yet undetermined mechanisms of INH resistance. We identified the mabA(g609a) silent mutation in a significant number of INH(r) Mtb clinical isolates without known INH resistance mutations. A laboratory strain, H37Rv, constructed with mabA(g609a) , was resistant to INH. We show here that the mabA(g609a) mutation resulted in the upregulation of inhA, a gene encoding a target for INH, converting the region adjacent to the mutation into an alternative promoter for inhA. The mabA(g609a) silent mutation results in a novel mechanism of INH resistance, filling in a missing piece of INH resistance in Mtb.

Please cite this paper as: Miyoshi-Akiyama et al. (2012) Discrimination of influenza A subtype by antibodies recognizing host-specific amino acids in the viral nucleoprotein. Influenza and Other Respiratory Viruses 6(6), 434–441. Background Nucleoprotein (NP) of influenza viruses is utilized to differentiate between the A, B, and C viral serotypes. The availability of influenza genome sequence data has allowed us to identify specific amino acids at particular positions in viral proteins, including NP, known as “signature residues,” which can be used to discriminate human influenza A viruses from H5N1 highly pathogenic avian influenza in human cases (HPAI) and pandemic H1N1(2009) (H1N1/2009) viruses. Methods Screening and epitope mapping of monoclonal antibodies (mAb) against NP of influenza A, which reacted differently with NP from human influenza A virus from HPAI and H1N1/2009 A virus. To identify the epitope(s) responsible for the discrimination of viral NP by mAbs, we prepared mutant NP proteins in the 293 cell expression system because some of the mAbs reacted with non-linear epitopes. Results and Conclusions In the present study, we identified 3 mAbs. The results of epitope mapping showed that the epitopes were located at the signature residues. These results indicated that signature residues of NP could discriminate influenza A viruses from different origin.

Isoniazid (INH) is a key agent in the treatment of tuberculosis. In Mycobacterium tuberculosis, INH is converted to its active form by KatG, a catalase-peroxidase, and attacks InhA, which is essential for the synthesis of mycolic acids. We sequenced furA-katG and fabG1-inhA in 108 INH-resistant (INH(r) ) and 51 INH-susceptible (INH(s) ) isolates, and found three mutations in the furA-katG intergenic region (Int(g-7a) , Int(a-10c) and Int(g-12a) ) in four of 108 INH(r) isolates (4%), and the furA(c41t) mutation with an amino acid substitution in 18 INH(r) isolates (17%). These mutations were not found in any of 51 INH(s) isolates tested. We reconstructed these mutations in isogenic strains to determine whether they conferred INH resistance. We found that the Int(g-7a) , Int(a-10c) and Int(g-12a) single mutations in the furA-katG intergenic region decreased katG expression and conferred INH resistance. In contrast, the furA(c41t) mutation was not sufficient to confer INH resistance. These results suggested that downregulation of katG is a mechanism of INH resistance in M. tuberculosis and that mutations in the furA-katG intergenic region play a role in this resistance mechanism.

We analyzed genetic variations of angiotensin‐converting enzyme 2 (ACE2), considering that it might influence patients' susceptibility to severe acute respiratory syndrome‐associated coronavirus (SARS‐CoV) or development of SARS as a functional receptor. By cloning of the full‐length cDNA of the ACE2 gene in the lung, where replication occurs on SARS‐CoV, it was shown that there are different splicing sites. All exons including the new alternative exon, exon‐intron boundaries, and the corresponding 5′‐flanking region of the gene were investigated and 19 single nucleotide polymorphisms (SNPs) were found. Out of these, 13 SNPs including one non‐synonymous substitution and three 3′‐UTR polymorphisms were newly identified. A case control study involving 44 SARS cases, 16 anti‐SARS‐CoV antibody‐positive contacts, 87 antibody‐negative contacts, and 50 non‐contacts in Vietnam, failed to obtain any evidence that the ACE2 gene polymorphisms are involved in the disease process in the population. Nevertheless, identification of new 5′‐untranslated exon and new SNPs is considered helpful in investigating regulation of ACE2 gene expression in the future. © 2005 Wiley‐Liss, Inc.

We show that Proteus vulgaris O25 (PO25) lipopolysaccharide (LPS) induced an anaphylactoid reaction not only in wild-type and in lipid A non-responding mice but also in recombinase-activating gene-2-deficient (RAG-2(-/-)) and in mast cell-deficient (W/Wv) animals. Western blot analysis indicated that PO25 LPS bound to Ra-reactive factor (RaRF), the complex of mannan-binding lectins (MBL) and MBL-associated serine proteases. Binding of RaRF to PO25 LPS led to the activation of C4 component without participation of either C1 or Ig, via the lectin pathway. Relative concentration of RaRF and hemolytic activity in mouse serum decreased rapidly during the process of anaphylactoid reaction. A significant drop of MBL-A, but not MBL-C level was observed. Administrationwith antiserum to RaRF prevented animals from death as a consequence of the inhibition of interaction of RaRF with the carbohydrate target and complement activation. These results indicate that complement-lectin pathway activation is responsible for the anaphylactoid reaction induced with LPS in muramyldipeptide-primed mice. RaRF also activated fibrinogen in vitro suggesting the involvement of the coagulation system in the process investigated.

Bacteroides vulgatus has been shown to be involved in the aggravation of colitis. Previously, we separated two potent virulence factors, capsular polysaccharide (CPS) and lipopolysaccharide (LPS), from a clinical isolate of B. vulgatus and characterized the structure of CPS. In this study, we elucidated the structures of O-antigen polysaccharide (OPS) and lipid A in the LPS. LPS was subjected to weak acid hydrolysis to produce the lipid A fraction and polysaccharide fraction. Lipid A was isolated by preparative TLC, and its structure determined by MS and NMR to be similar to that of Bacteroides fragilis except for the number of fatty acids. The polysaccharide fraction was subjected to gel-filtration chromatography to give an OPS-rich fraction. The structure of OPS was determined by chemical analysis and NMR spectroscopy to be a polysaccharide composed of the following repeating unit: [-->4)alpha-L-Rhap(1-->3)beta-D-Manp(1-->].

The structure of a capsular polysaccharide (CPS) from a clinical isolate of Bacteroides vulgatus was elucidated. B. vulgatus IMCJ 1204 was isolated from feces of a patient with Crohn's disease. CPS was prepared by phenol/water extraction of the bacterial cells followed by hydrophobic interaction chromatography and then gel filtration chromatography of the extract. The structure of CPS was determined by chemical analysis and NMR spectroscopy including DQF-COSY, TOCSY, ROESY, HSQC-TOCSY, HMQC and HMBC to be a polysaccharide composed of the following repeating unit: -->3)beta-D-Glcp(1-->6)[alpha-D-GalpNAc(1-->2)beta-D-Galp(1-->4)]beta-D-GlcpNAc(1-->3)alpha-D-Galp(1-->4)beta-D-Manp(1-->.

Escherichia coli O25 strains that produce heat-stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST-producing and non-producing strains of E. coli O25 was investigated. All eight ST-producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder-banding of LPS in SDS-polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed-field gel electrophoresis (PFGE). In contrast, ST-non-producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.

The surface antigen CD14 is known to play a central role in the recognition of lipopolysaccharide by macrophages. We characterized a mutant cell line, J7.DEF.3, derived from a murine macrophage-like cell line, J774.1, to be defective in the ability to express the membrane-associated form of CD14 (mCD14) but not in the ability to release the soluble form of CD14 (sCD14), and used these parent and mutant cells to investigate the role of CD14 in lipopolysaccharide signaling. In response to lipopolysaccharide stimulation, mutant cells produced slightly less tumor necrosis factor than parent cells, and produced much less (negligible level) nitric oxide than parent cells. Production of both tumor necrosis factor and nitric oxide by parent cells upon lipopolysaccharide stimulation was suppressed by anti-CD14 serum. Expression of interferon-β mRNA by stimulation with lipopolysaccharide, detected in parent cells, was barely detectable in mutant cells and in enzymatically mCD14-eliminated parent cells. Lipopolysaccharide-induced nitric oxide production in parent cells was suppressed by anti-(murine interferon-β), and its production in the mutant cells appeared and increased dose dependently on exogenously supplied murine interferon-β in the presence of lipopolysaccharide. These results provide new insight into the lipopolysaccharide signaling pathway, indicating that the lipopolysaccharide signal for interferon-β production is transduced through a mCD14-dependent pathway and that the endogenously generated interferon-β is an essential cofactor leading to nitric oxide production. Nuclear translocation of a transcription factor, nuclear factor κB, was observed in both parent and mutant cells following stimulation with a low dose of lipopolysaccharide, and mitogen-activated protein kinases were also activated in both types of cell, although a higher dose of lipopolysaccharide was required by the mutant cells than by the parent cells. These results indicate that these signaling factors may participate in the mCD14-independent lipopolysaccharide signaling pathway rather than in the mCD14-dependent interferon-β-producing pathway.

The role of CD14 in the phagocytosis and killing of microorganisms was investigated using macrophage-like cell lines, CD14-positive J774.1 cells and CD14-negative mutant J7.DEF.3 cells derived from J744.1 cells. The cells were infected with Salmonella typhimurium organisms of the smooth (S)-form LT2, mutant rough (R)-form TV148 or Staphylococcus aureus 248betaH. At 30 or 180 min incubation, the cells were washed and disrupted. Colony-forming units (CFUs) liberated from the disrupted cells were determined by quantitative cultivation, and the phagocytic index and killing rate were calculated. Both the phagocytic index and killing rate of J774.1 cells against LT2 organisms were greater than those of J7.DEF.3 cells. However, the index and rate of J774.1 cells against TV148 and 248betaH organisms were similar to those of the J7.DEF.3 cells. The phagocytosis of LT2 organisms by J774.1 cells was partially inhibited by S-form LPS (S-LPS) and anti-CD14 antibody, but not by R-chemotype LPS (R-LPS). These results suggest that CD14 participates in the phagocytosis of S-form Salmonella.

A new sulfoalkylresorcinol (1) was isolated from the marine-derived fungus, Zygosporium sp. KNC52. The structure of 1 was elucidated by spectroscopic methods including MS and NMR, and the absolute stereochemistry was determined by the modified Mosher's method. Compound 1 inhibited FtsZ polymerization in vitro and exhibited weak antimicrobial activity against multi-drug-resistant bacteria.

Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase-sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS-binding protein on the murine erythrocyte membrane, a unique method to detect LPS-binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate-buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non-background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1% to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti-Re LPS antibodies (IgG) and then to alkaline phosphatase-conjugated anti-IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96 kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS-binding protein on the erythrocyte membrane. There was no difference between LPS-responder and LPS-nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS-binding protein.

An attempt was made to establish long-term cultures of marrow cells from genetically anaemic W/Wv mice. Two batches of horse sera were used. One batch of horse serum (HS-lot A) supported long-term maintenance (up to 20 weeks) of granulopoiesis in vitro. The number of suspension cells in W/Wv marrow culture was maintained at the same level as that in the control +/+ culture, but the number of granulocyte-macrophage progenitor cells (GM-CFC) and the ratio of immature to mature granulocytes were at a lower level than those in +/+ culture. These data suggest that haemopoietic progenitors in W/Wv cultures maintain a higher level of differentiation, and hence an increased self-renewal than those in +/+ cultures. Another batch of horse serum (HS-lot B) was less effective in the maintenance of the cultures, and the cultures deteriorated within 10 weeks. Addition of bacterial lipopolysaccharide (LPS) induced increased granulopoiesis in +/+ cultures, whereas such treatment resulted in the depletion of suspension cells in W/Wv cultures. The results suggest that haemopoietic cells of W/Wv mouse cannot cope with the strong stimulus for differentiation that occurs after the administration of LPS, although the cells can continue a moderately increased self-renewal and differentiation, as indicated by the results in the culture with HS-lot A.