RESUMEN
A facultatively psychrophilic bacterium, previously described as Pseudomonas sp. strain E-3, has been reassigned by phenotypic characterization, chemotaxonomic analysis, DNA-DNA hybridization, and 16S rRNA gene phylogenetic analysis. The organism was a gram-negative, aerobic. straight rod with polar flagella. It was catalase positive and oxidase positive, able to grow at -1 degree C but not at 40 degree C, and produced acid from D-glucose under aerobic conditions. The major isoprenoid quinone was ubiquinone-9, and the DNA G + C content was 57.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacterium is a member of the genus Pseudomonas and was closest to Pseudomonas fragi. Determination of the DNA-DNA relatedness between strain E-3 and P. fragi revealed too low a level of homology (47.9%-51.3%) to identify them as the same species. On the basis of phenotypic characteristics, phylogenetic analysis, and DNA-DNA relatedness data, it is concluded that strain E-3 represents an individual species. Accordingly, the name Pseudomonas psychrophila is proposed. The type strain is E-3T (= JCM 10889).
Asunto(s)
Pseudomonas/clasificación , Pseudomonas/genética , Composición de Base , Frío , ADN Bacteriano/química , ADN Bacteriano/genética , Microscopía Electrónica , Fenotipo , Filogenia , Pseudomonas/química , Pseudomonas/ultraestructura , Quinonas/análisis , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
The expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) on lymphatic and blood vessels of the human tongue was examined with fluorescence and transmission electron microscopy (TEM). The study used anti-desmoplakins antiserum for light microscopic identification of the lymphatic vessels, plus a pre-embedding immunogold electron microscopic technique for TEM observations. Before making TEM observations, cryostat serial sections were immunostained with anti-desmoplakins or anti-PECAM-1 and then embedded. Semithin sections from each cryostat section were photographed under a light microscope and compared in order to identify the lymphatic vessels expressing PECAM-1. In fluorescence microscopy, PECAM-1 expression on lymphatic vessels was weaker than that on blood vessels. TEM observations showed that PECAM-1 expression on the blood vessels was observed only on the luminal surface of the endothelium. In lymphatic vessels, PECAM-1 expression was found both on the luminal and abluminal surfaces of the endothelium. The density of the PECAM-1 reaction products was lower in lymphatic vessels than in blood vessels. The density of PECAM-1 reaction products on the luminal surface of lymphatic vessels was higher than on the abluminal surfaces. The results suggest that blood vessels are more active than lymphatic vessels in leukocyte migration. The expression of PECAM-1 on the abluminal surface of lymphatic endothelium may allow leukocytes to adhere to the endothelium and interact in their migration from tissue into lymphatic vessels.
Asunto(s)
Endotelio Linfático/química , Sistema Linfático/química , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Lengua/química , Lengua/ultraestructura , Endotelio Linfático/ultraestructura , Humanos , Sistema Linfático/ultraestructura , Microscopía Electrónica , Microscopía InmunoelectrónicaRESUMEN
Facultatively psychrophilic alkaliphilic strains were isolated from seawater obtained off the coast of Rumoi, Hokkaido, Japan. They were Gram-negative, aerobic straight rods with polar flagella. The isolates were catalase- and oxidase-positive and able to grow at 4 degrees C, but not at 40 degrees C. They produced acid from D-glucose under aerobic conditions. The isolates reduced nitrate to nitrite and hydrolysed casein and gelatin, but not starch or DNA. NaCl was required for growth at pH 10 but was not required at neutral pH. The major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 62.3-63.2 mol%. The whole-cell fatty acids mainly consisted of C16:0, C16:1(9c) and C18:1(9c), with 3-OH C10:0 and 3-OH C12:0 as the hydroxyl fatty acids. A larger amount of trans-unsaturated fatty acid, C16:1(9t) was observed when the cells were grown at pH 7 compared to when cells were grown at pH 10. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacteria are members of the genus Pseudomonas. Analysis of DNA-DNA relatedness data with several close phylogenetic neighbours revealed a low level of hybridization (less than 61%). On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, it is concluded that these isolates represent a separate new species. Accordingly, the name Pseudomonas alcaliphila is proposed. The type strain is AL15-21T (= JCM 10630T = IAM 14884T).
Asunto(s)
Pseudomonas/clasificación , Microbiología del Agua , Álcalis , Técnicas de Tipificación Bacteriana , Carbohidratos , ADN Ribosómico , Ácidos Grasos/análisis , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Pseudomonas/citología , Pseudomonas/genética , Pseudomonas/aislamiento & purificación , ARN Ribosómico 16S , Agua de Mar , Terminología como AsuntoRESUMEN
The usefulness of immunostaining with anti-desmoplakin antibody for light microscopic identification of lymphatic vessels was examined in cryostat sections of the human tongue. The results were compared with laminin, 5'-nucleotidase (5'-Nase), and factor VIII staining. Immunoelectron microscopic observation was also performed to confirm that the vessels reacting with anti-desmoplakin were lymphatic vessels. Under the immunoelectron microscopic, the vessels reacting with anti-desmoplakin showed ultrastructural features characteristic of lymphatic vessels: thin endothelial walls, no or incomplete basal lamina, open junctions, and overlapping endothelium. In general, lymphatic vessels identified by anti-desmoplakin reacted strongly with 5'-Nase, but showed weak or no reactivity with anti-laminin and anti-factor VIII. Blood vessels showed no reactivity with anti-desmoplakin, but reacted strongly with anti-laminin and anti-factor VIII. However, some blood and lymphatic vessels showed intermediate reactivity with anti-laminin, anti-factor VIII, and 5'-Nase. It was difficult to identify these as blood or lymphatic vessels only by the reactivity differences. The results indicate that anti-desmoplakin antibody specifically distinguishes lymphatic vessels and is useful for studying the fine distribution of lymphatic vessels under light microscopy.
Asunto(s)
Proteínas del Citoesqueleto , Sistema Linfático , Biomarcadores , Desmoplaquinas , Humanos , InmunohistoquímicaRESUMEN
Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA-DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment.
Asunto(s)
Bacillus/química , Bacillus/ultraestructura , Pared Celular/química , Ácidos Grasos/análisis , Álcalis , Bacillus/clasificación , Bacillus/crecimiento & desarrollo , Pared Celular/ultraestructura , Clasificación , Medios de Cultivo/química , ADN Bacteriano/genética , Ácidos Grasos/química , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ARNRESUMEN
Strains 61AT (T = type strain) and 61B2, the first bacteriochlorophyll (BChl) a-containing obligate aerobes to be classified in the beta-subclass of the Proteobacteria, were isolated from river water. The strains were originally isolated as degraders of poly(hexamethylene carbonate) (PHC). The organisms can utilize PHC and some other biodegradable plastics. The strains grow only under aerobic conditions. Good production of BChl a and caroterioid pigments is achieved on PHC agar plates and an equivalent production is observed under oligotrophic conditions on agar medium. Spectrometric results suggest that BChl a is present in light-harvesting complex I and the photochemical reaction centre. The main carotenoids are spirilloxanthin and its precursors. Analysis of the 16S rRNA gene sequence indicated that the phylogenetic positions of the two strains are similar to each other and that their closest relatives are the genera Rubrivivax, ideonella and Leptothrix with similarities of 96.3, 96.2 and 96.1%, respectively. The cells are motile, straight rods and contain poly-beta-hydroxybutyrate granules. Ubiquinone-8 is the predominant quinone. Vitamins are not required for growth. The G + C content of genomic DNA is 66.2-66.3 mol%. Genetic and phenotypic features suggest that the strains represent a new genus in the beta-subclass which is evenly distant from known genera. Consequently, the name Roseateles depolymerans gen. nov., sp. nov. is proposed for the strains; the type strain of Roseateles depolymerans is strain 61AT (= DSM 11813T).
Asunto(s)
Bacterias Aerobias/clasificación , Bacterioclorofilas/metabolismo , Bacterias Aerobias/fisiología , Bacterias Aerobias/ultraestructura , Carotenoides/metabolismo , ADN Ribosómico/genética , Genes de ARNr , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
We investigated activity of bone sialoprotein (BSP) to mediate attachment of cells to hydroxyapatite using a model peptide, Glu7-Pro-Arg-Gly-Asp-Thr, which contains a putative hydroxyapatite-binding site (poly-Glu) and a cell-attachment site. The peptide has affinity to hydroxyapatite with a dissociation constant of 13.5 microM. The peptide affected in vitro mineralization in a gel system, indicating interaction between this peptide and calcium phosphate. The osteoblastic cell line MC3T3-E1 was incubated with hydroxyapatite powder coated with the peptide or proteins. Attachment of the cells was observed on the powder coated with BSP, but not on the powder coated with serum albumin. The cells were attached to the powder coated with the peptide. The cells were flattened on the powder, and pseudopods developed. The attachment of the cells was inhibited by an excessive amount of Gly-Arg-Gly-Asp-Ser peptide. In conclusion, BSP mediated attachment of osteoblastic cells to hydroxyapatite, and this activity could be accomplished only by the poly-Glu sequence and the Arg-Gly-Asp sequence.
Asunto(s)
Durapatita/química , Oligopéptidos/química , Osteoblastos/química , Sialoglicoproteínas/química , Secuencia de Aminoácidos , Unión Competitiva , Adhesión Celular , Cristalización , Sialoproteína de Unión a Integrina , Datos de Secuencia MolecularRESUMEN
Non-collagenous proteins of dentin and bone have important effects on mineralization which have been studied by various in vitro systems. We developed an in vitro mineralization system using electrophoretic gels as diffusion media of calcium and phosphate ions. Calcium and phosphate ions were diffused naturally or propelled by electric potential. Calcium phosphate was precipitated in the gel, and the precipitation was affected by proteins in the gel which had been separated by electrophoresis. We applied this system to analysis of non-collagenous proteins of dentin. Among the proteins, phosphophoryns promoted calcium phosphate precipitation in the natural-diffusion system. A non-collagenous protein having a molecular mass of 60 kDa inhibited precipitation. The results were different, however, in the electric-diffusion system, in which phosphophoryns had a negative effect. The present system enabled us to compare the effects of plural proteins rapidly, even using unpurified material.
Asunto(s)
Dentina/metabolismo , Fosfoproteínas/metabolismo , Calcificación de Dientes , Animales , Fosfatos de Calcio/antagonistas & inhibidores , Fosfatos de Calcio/química , Fosfatos de Calcio/metabolismo , Bovinos , Precipitación Química , Dentina/química , Difusión , Electroforesis en Gel de Poliacrilamida , Proteínas de la Matriz Extracelular/metabolismo , Geles , Fosforilación , RatasRESUMEN
The inner ear of mutant bustling mice, BUS/Idr, was examined histopathologically. LM examinations revealed an age-dependent degeneration of the auditory organ of Corti in BUS homozygotes, but not heterozygotes. Cochlear base-to-apex gradient in severity of the degeneration was noted. First signs of degeneration were found in the outer hair cells of the cochlear basal turn at about 3 weeks of age, followed by degeneration of the spiral ganglion cells which occurred slowly. As examined by SEM, stereociliary derangements of both the inner and outer hair cells were apparent in homozygotes as early as after 10 days. No normal arrays of stereocilia were found in homozygotes examined at 10 days through 6 months. The results of immunohistochemical examinations suggest that the sensory cells of the Corti's organ of homozygotes are structurally once normally innervated. No significant difference was found in the expression of protooncogene c-mos in the CNS between BUS homozygotes and control mice. We propose that BUS mice be categorized as a member of the so-called "waltzer-shaker" mutants group.
Asunto(s)
Órgano Espiral/ultraestructura , Factores de Edad , Envejecimiento , Animales , Conducta Animal , Femenino , Heterocigoto , Homocigoto , Inmunohistoquímica , Ratones , Ratones Mutantes , Degeneración Nerviosa , Proteínas Proto-Oncogénicas c-mos/fisiología , Reflejo de SobresaltoRESUMEN
Osteonectin, an acidic noncollagenous protein of bone and dentin, has affinity to hydroxyapatite crystals. Binding sites to hydroxyapatite of this protein were determined by a proteolytic experiment and an in vitro binding experiment using synthetic peptide analogues. Osteonectin was adsorbed on hydroxyapatite crystals and digested with trypsin. A peptide was left adsorbed on the crystal even after the digestion. The peptide was identified as an amino terminal peptide containing glutamic acid-rich sequences, which have been assumed to be possible hydroxyapatite-binding sites. Poly glutamic acid sequences were synthesized as models of the binding sites. Glu6 peptide was bound to the hydroxyapatite with a dissociation constant of 2.4 microM. Peptides containing fewer glutamic acids had lower affinity to the crystal. Effects of these peptides on in vitro mineralization were examined by a gel system in microtiter plates. The Glu6 peptide had a positive effect on the mineralization in this system, whereas Asp6 peptide had a negative effect. These effects indicate the presence of an interaction between these peptides and mineral crystals.
Asunto(s)
Durapatita/metabolismo , Osteonectina/química , Adsorción , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Sitios de Unión , Huesos/química , Fosfatos de Calcio/química , Bovinos , Precipitación Química , Cromatografía Líquida de Alta Presión , Cristalización , Microscopía Electrónica , Datos de Secuencia Molecular , Peso Molecular , Osteonectina/metabolismo , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Fosfoproteínas , Ácido Poliglutámico/química , Ácido Poliglutámico/metabolismo , Unión Proteica , Tripsina/metabolismoRESUMEN
Bone sialoprotein (BSP) has an affinity to collagen fibrils [25]. A role of carbohydrate chains in the affinity was examined by removing sialic acids of BSP. Neuraminidase treatment of the BSP increased the binding to collagen. Binding sites of BSP on collagen were examined by biochemical and electron-microscopic methods. Purified bovine BSP was labeled with biotin. Collagen alpha chains or CNBr peptides were separated by electrophoresis and transfered to nitrocellulose membranes. The membranes were incubated with the biotin-labeled BSP, and the bound BSP was visualized with avidin conjugated with alkaline phosphatase. The labeled BSP was preferentially bound to the alpha 2 chain, and peptides derived from alpha 2 chain. In another experiment, the labeled BSP was incubated with reconstituted native collagen fibrils. The mixture was put on a copper grid, reacted with avidin conjugated with gold particles, and observed with an electron microscope. The gold particles were seen mainly within hole zones of the fibrils. BSP bound to the alpha 2 chain within the hole zones may regulate the onset of calcification at hole zones and the cell binding to collagen fibrils.
Asunto(s)
Colágeno/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Sitios de Unión , Bovinos , Colágeno/química , Colágeno/ultraestructura , Técnicas In Vitro , Sialoproteína de Unión a Integrina , Microscopía Electrónica , Neuraminidasa , Unión Proteica , Ácidos Siálicos/química , Sialoglicoproteínas/químicaRESUMEN
Inhibition of hemagglutinin (HA) activity in a membrane fraction of Bacteroides gingivalis was examined using various compounds. Leupeptin and anti-pain inhibited the HA activity at nM order. This potency was lost when the aldehyde group of leupeptin was converted to an alcohol moiety. Irreversible protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK), p-chloromercuribenzoate (PCMB), and N-ethylmaleimide (NEM) were also inhibitory. From the inhibition experiments, we speculate that the HA possesses protease activity and that the same site of the molecule participates in the erythrocyte binding and the substrate binding.
Asunto(s)
Bacteroides/enzimología , Hemaglutininas/metabolismo , Péptido Hidrolasas/metabolismo , Bacteroides/ultraestructura , Benzoilarginina-Nitroanilida/metabolismo , Pruebas de Inhibición de Hemaglutinación , Leupeptinas/metabolismo , Inhibidores de Proteasas/farmacologíaRESUMEN
Fimbriae and their constituent protein (fimbrilin) were purified to homogeneity from the bacterial wash fluid and cell lysate fraction, respectively, of Bacteroides gingivalis 381. Fimbriae, observed by negative staining, were curly, single-stranded filaments with a diameter of ca. 5 nm. The apparent molecular weight of the fimbrilin was 43,000. Fimbriae were resistant to sodium dodecyl sulfate denaturation at 70 degrees C. Heating at 100 degrees C in sodium dodecyl sulfate was needed to completely dissociate them to monomers of fimbrilin. Different sets of antigenic determinants seemed to be exposed on the surfaces of fimbriae and sodium dodecyl sulfate-denatured fimbrilin. Purified fimbriae did not show either hemagglutinating activity or hemagglutination inhibitory activity, although it has been inferred on the basis of circumstantial evidence that fimbriae are correlated to hemagglutinating activity of the organism. Hemagglutinin activity, however, was detected in culture supernatant, and this observation suggests that fimbriae of a different type or a lectin-like protein may be acting as hemagglutinin in B. gingivalis.