Fibronectin-Binding Proteins of a Human Oral Spirochete Treponema denticola

Major polypeptides from a human oral spirochete Treponema denticola ATCC 33520 were examined to demonstrate their ability to bind to human plasma fibronectin by immunoblot analysis. Of three main polypeptides separated on sodium dodecyl sulfate polyacrylamide gels 53,000-daltons (53-kDa) and 72-kDa surface antigenic proteins and a 38-kDa axial flagellar protein showed the ability to bind to fibronectin, suggesting that fibronectin on host cells can mediate cytoadherence of T. denticola by its binding to the surface proteins or the exposed 38-kDa axial flageller protein.     

Occurrence of 2-keto-3-deoxyoctonate (KDO) and KDO phosphate in lipopolysaccharides ofBacteriodes species

Occurrence of 2-keto-3-deoxyoctonate (KDO) in lipopolysaccharides (LPS) of genusBacteroides (some strains have recently been reclassified asPorphyromonas orPrevotella) was examined. Strong-acid treatment of LPS isolated fromBacteroides fragilis, Bacteroides (Porphyromonas) gingivalis andBacteroides intermedius, (Prevotella intermedia) released periodate/thiobarbituric acid reaction-positive substances that were not detectable under conventional hydrolysis conditions. These substances were demonstrated to be KDO phosphate by high voltage paper electrophoresis before and after alkaline phosphatase treatment. KDO phosphate was also identified in these LPS by gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. KDO was identified as well in both mild and strong-acid hydrolysates of LPS isolated fromBacteriodes melaninogenicus (Prevotella melaninogenica). Neither KDO nor KDO phosphate was detectable in LPS ofBacteriodes asaccharolyticus (Porphyromonas asaccharolytica) even after the strong-acid treatment of LPS. These findings indicate that there are possible structural variations in the inner core region ofBacteroides LPS.     

Isolation,partial purification and preliminary characterization of a bacteriocin from Streptococcus mutans Rm-10

An extracellular bactericidal substance was isolated from the supernatant of Streptococcus mutans Rm-10 culture fluid and partially purified with 60% ammonium sulfate precipitation, differential centrifugation, and gel filtration on Sephadex G-200. There was a good correlation of the sensitivity profiles of indicator strains whether assayed on solid medium or with purified material from cell-free culture fluid, indicating that the same inhibitory substance is produced on solid medium and in broth. Vapor from organic solvents such as chloroform, acetone, ethanol, and ether as well as heat treatment at 100 degrees C for 30 min had little effect on the bactericidal factor. It was sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme, and phospholipase C. The inhibitor was not infective, and electron microscopic studies failed to reveal phage or phage-like particles in concentrated solutions of the bactericidal material. The results indicate that the extracellular bactericidal substance is indeed a bacteriocin. Activity in broth cultures reached a maximum only after exponential growth had ceased. It was active against other streptococcal strains as well as strains of Actinomyces naeslundii, A. viscosus, Bacillus subtilis and Staphylococcus aureus, but not against strains of Fusobacterium nucleatum and Escherichia coli.     

Lanthionine as an essential constituent of cell wall peptidoglycan ofFusobacterium nucleatum

Lanthionine, a sulfur-containing diamino acid which had not previously been reported as one of the main amino acids of any bacterial cell wall peptidoglycan, was demonstrated inFusobacterium nucleatum peptidoglycan isolated by sodium dodecyl sulfate extraction and protease digestion. Lysine, diaminopimelic acid, and ornithine were absent. Lanthionine seems to be an essential dibasic amino acid, involved in cross-linkages betwen stem peptide subunits inF. nucleatum.     

The effects of habitat fragmentation on persistence of source-sink metapopulations in systems with predators and prey or apparent competitors.

We consider systems with one predator and one prey, or a common predator and two prey species (apparent competitors) in source and sink habitats. In both models, the predator species is vulnerable to extinction, if productivity in the source is insufficient to rescue demographically deficient sink populations. Conversely, in the model with two prey species, if the source is too rich, one of the prey species may be driven extinct by apparent competition, since the predator can maintain a large population because of the alternative prey. Increasing the rate of predator movement from the source population has opposite effects on prey and predator persistence. High emigration rate exposes the predator population to danger of extinction, reducing the number of individuals that breed and produce offspring in the source habitat. This may promote coexistence of prey by relaxing predation pressure and apparent competition between the two prey species. The number of sinks and spatial arrangement of patches, or connectivity between patches, also influence persistence of the species. More sinks favor the prey and fewer sinks are advantageous to the predator. A linear pattern with the source at one end is profitable for the predator, and a centrifugal pattern in which the source is surrounded by sinks is advantageous to the prey. When the dispersal rate is low, effects of the spatial structure may exceed those of the number of sinks. In brief, productivity in patches and patterns of connectivity between patches differentially influence persistence of populations in different trophic levels.     

Electron Microscopy of the Spherical Body of Oral Spirochetes In Vitro: Further Studies

The surface and inner structure of the spherical bodies (SB) produced by the human oral treponeme strain G7201, similar to Treponema macrodentium, were studied by electron microscopy. Ultrathin sectioning and scanning techniques demonstrated that in the presence of a high concentration of sucrose, the outer envelope of one or both terminal ends of this oral spirochete changed into a swollen structure, the SB. Spirochetal cells adhered firmly to the surface of the resultant body. The membrane of the SB, i.e. the outer envelope, enclosed the coiled protoplasmic cylinder and five axial fibrils which were located between the envelope and the cylinder. Large expanded protoplasmic cylinders were observed, surrounded by a partially disrupted double membrane in some SBs. A number of frizzly fibrous structures, which differed from axial fibrils in number and shape, were also observed within these SBs. Except for abnormal or partially broken cylinders, the protoplasmic cylinders tended to be located close to the inner surface of the SB membrane, resulting in a central vacant space with occasional axial fibrils. These findings suggest that the oral spirochete produces an SB by terminal expansion of the outer envelope in the presence of high concentrations of sucrose. The outer envelope of the SB, which consists of two electron-dense layers, has the property of binding spirochetal cells to its outer layer and the protoplasmic cylinder and axial fibrils to the inner layer. Some protoplasmic cylinders were also observed to be swollen in the presence of high sucrose concentrations.     

Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development,Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight genes—fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a—had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a – ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.     

Crystal structures and inhibitor binding properties of plant class V chitinases: the cycad enzyme exhibits unique structural and functional features

A class V (glycoside hydrolase family 18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/β)₈ barrel fold with an (α + β) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face‐to‐face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between ?9.5 and ?9.8 kcal mol^?1. However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.     

Relation of Cheddar Cheese Ripening to Bacterial Lipolysis

This investigation was undertaken to find the relationship between fat hydrolysis and lipolytic activities of lactic acid bacteria participated in Cheddar cheese ripening. Increases in titratable acidities due to lactic fermentation were completed at early stage of ripening. Ripening indices (ratio of water-soluble nitrogen to total nitrogen) increased rapidly until 90 days and thereafter gradually up to 150 days. Considerable amounts of free fatty acids were released from cheese fat throughout the ripening period. Cheese bacteria were enumerated on the media of tomato-glucose-agar and acetate-agar. About 70% of bacteria isolated from cheese at age of 150 days were classified into Lactobacillus casei and L. plantarum. Lipolytic activities of lactobacilli isolated were detected definitely on double-layered agar plates containing Victoria blue-stained olive oil. Lipase activities were determined in cheese extracts during ripening.