Isolation and molecular characterization of multidrug-resistant Gram-negative bacteria from imported flamingos in Japan

Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, β-lactamase-encoding genes, bla _TEM-1 and bla _CTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another.     

Studies on Mold Dextranases

Nine strains capable of producing dextranase were isolated from soil. Among them, a strain belonging to the genus Aspergillus was chosen as the best producer of the enzyme. The mold produced greater amounts of dextranase than those found in some strains in the genus Penicillium, when grown aerobically at 28°C for 5 to 6 days in medium containing 1% dextran, 1% NaNO₃ or polypeptone, 0.2% yeast extracts, 0.4% K₂HPO₄ and small amounts of inorganic salts, pH 8.5. From the comparative taxonomic experiments, the mold used here was identified to be a strain belonging to Aspergillus carneus.     

A novel guanine-guanine base pairing: crystal structure of a complex between 7-methylguanosine and its iodide.

7-Methylguanosine, one of the biologically important minor nucleosides, could be crystallized as a complex of its zwitterionic form and its iodide, and the crystal structure was determined by the X-ray diffraction method. The crystals belong to the triclinic space group P1 with the unit cell dimensions: a = 7.678(1), b = 18.094(3), and c = 5.711(1) A, alpha = 79.32(1), beta = 80.14(1) and gamma = 76.90(1) degrees. The structure was solved by the heavy atom method and refined by the least-squares method to give a final R index of 0.075. The novel reverse Watson-Crick type base pairing observed between a positively charged molecule and a deprotonated one indicates that the deprotonation at the N(1) position promoted by the alkylation at the N(7) position may interrupt the formation of the normal Watson-Crick type GC base pair. The conformations about the glycosidic bond and the sugar puckering are quite different between the two molecules: the former has anti and C(4')-exo,C(3')-endo and the latter syn and C(1')-exo-C(2')-endo.     

Synergistic action of gastrin and ghrelin on gastric acid secretion in rats

Gastrin and ghrelin are secreted from G cells and X/A-like cells in the stomach, respectively, and respective hormones stimulate gastric acid secretion by acting through histamine and the vagus nerve. In this study, we examined the relationship between gastrin, ghrelin and gastric acid secretion in rats. Intravenous (iv) administration of 3 and 10 nmol of gastrin induced transient increases of ghrelin levels within 10 min in a dose-dependent manner. Double immunostaining for ghrelin and gastrin receptor revealed that a proportion of ghrelin cells possess gastrin receptors. Although (iv) administration of gastrin or ghrelin induced significant gastric acid secretion, simultaneous treatment with both hormones resulted in a synergistic, rather than additive, increase of gastric acid secretion. This synergistic increase was not observed in vagotomized rats.These results suggest that gastrin may directly stimulate ghrelin release from the stomach, and that both hormones may increase gastric acid secretion synergistically.     

Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

Studies on Bacteriolytic Enzymes

About 100 soil samples were subjected to screening for microorganisms which were capable of producing lytic enzyme toward Staphylococcus aureus. A strain belonging to Streptomyces was isolated and found to produce lytic enzyme(s) noninduciblly, when grown aerobically at 37°C for 25 hr in a medium containing 7.5% soybean cake extract, 2% dextrin, 0.6% K₂HPO₄, 0.02% each of MgSO₄·7H₂O and KCl, pH 7.0. The crude enzyme preparation was active at pH values of 8.5 and 5.8 toward S. aureus, B. subtilis, L. bulgaricus and Str. faecalis but was completely inert against M. lysodeikticus, indicating the enzyme(s) to be distinguished from other bacteriolytic enzymes of Streptomyces so far reported.     

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.     

UDP-<Emphasis Type="SmallCaps">d</Emphasis>-galactose synthesis by UDP-glucose 4-epimerase 4 is required for organization of the <Emphasis Type="Italic">trans</Emphasis>-Golgi network/early endosome in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> root epidermal cells

Endomembrane organization is essential for cell physiology. We previously identified an Arabidopsis thaliana mutant in which a plasma membrane (PM) marker GFP-NIP5;1 and trans-Golgi network/early endosome (TGN/EE) markers were accumulated in intracellular aggregates in epidermal cells of the root elongation zone. The mutant was identified as an allele of UDP-glucose epimerase 4 (UGE4)/root hair defective 1/root epidermal bulgar 1, which was previously described as a mutant with swollen root epidermal cells and has an altered sugar composition in cell wall polysaccharides. Importantly, these defects including aggregate formation were restored by supplementation of d-galactose in the medium. These results suggested that UDP-d-galactose synthesis by UGE4 is important for endomembrane organization in addition to cell wall structure. Here, we further investigated the nature of the aggregates using various markers of endomembrane compartments and BOR1-GFP, which traffics from PM to vacuole in response to high-B supply. The markers of multi-vesicular bodies/late endosomes (MVB/LEs) and BOR1-GFP were strongly accumulated in the intracellular aggregates, while those of the endoplasmic reticulum (ER), the vacuolar membrane, and the Golgi were only slightly affected in the uge4 mutant. The abnormal localizations of these markers in the uge4 mutant differed from the effects of inhibitors of actin and microtubule polymerization, although they also affected endomembrane organization. Furthermore, electron microscopy analysis revealed accumulation of abnormal high-electron-density vesicles in elongating epidermal cells. The abnormal vesicles were often associated or interconnected with TGN/EEs and contained ADP-ribosylation factor 1, which is usually localized to the Golgi and the TGN/EEs. On the other hand, structures of the ER, Golgi apparatus, and MVB/LEs were apparently normal in uge4 cells. Together, our data indicate the importance of UDP-d-galactose synthesis by UGE4 for the organization and function of endomembranes, especially TGN/EEs, which are a sorting station of the secretory and vacuolar pathways.