Application of rpoB and zinc protease gene for use in molecular discrimination of Fusobacterium nucleatum subspecies.

Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase beta-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.

Stereoselectivity of fructose-1,6-bisphosphate aldolase in Thermus caldophilus.

It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.

Phenol/water extract of Treponema socranskii subsp. socranskii as an antagonist of Toll-like receptor 4 signalling.

Treponema socranskii is one of the most frequently found oral spirochaetes in periodontitis and endodontic infections. LPS or glycolipids from bacteria are potent stimulators of innate immune and inflammatory systems. In this study the bioactivity of a phenol/water extract from T. socranskii subsp. socranskii (TSS-P) was analysed. TSS-P showed minimal endotoxicity and no inducing potential for proinflammatory cytokines (TNF-alpha and IL-8) or for intercellular adhesion molecule-1 (ICAM-1) in human monocyte cell line THP-1 cells and primary cultured human gingival fibroblasts. Rather, it inhibited ICAM-1 expression and IL-8 secretion from cells stimulated by the LPS of Escherichia coli and Actinobacillus actinomycetemcomitans, which are known to be Toll-like receptor 4 (TLR4) agonists. However, this antagonistic activity was not shown in cells stimulated by peptidoglycan or IL-1beta. As its antagonistic mechanism, TSS-P blocked the binding of E. coli LPS to LPS-binding protein (LBP) and CD14, which are molecules involved in the recruitment of LPS to the cell membrane receptor complex TLR4-MD-2 for the intracellular signalling of LPS. TSS-P itself did not bind to MD-2 or THP-1 cells, but inhibited the binding of E. coli LPS to MD-2 or to the cells in the presence of serum (which could be replaced by recombinant human LBP and recombinant human CD14). The results suggest that TSS-P acts as an antagonist of TLR4 signalling by interfering with the functioning of LBP/CD14.

Identification of novel allele on the locus 47z (DXYS5) in the Korean population.

Two homogenous sequences of 47z (DXYS5) are located on the X (DXYS5X) and Y (DXYS5Y) chromosomes, and these are known to be useful polymorphic markers for tracing male-specific gene flow such as the migration routes of human populations. Using the 47z/StuI PCR-RFLP system, we found a novel allele which showed two bands, in contrast to the previous two allele types, one band (Y1) and three bands (Y2). This means that copies of PCR products derived from both the DXYS5X and DXYS5Y loci were clearly cut by the StuI enzyme, implying that the DXYS5X locus of the X chromosome is polymorphic. Allelic frequencies examined in 267 male Korean individuals showed that 95.8% had Y1, 3.4% Y2, and 0.8% had the novel allele. Our findings should contribute to a better understanding of genetic polymorphism on X and Y chromosomes, the molecular evolution mechanism of sex chromosomes, and how the migration route of Koreans is related to those of other East Asian populations.

Blocking tumor cell migration and invasion with biphenyl isoxazole derivative KRIBB3, a synthetic molecule that inhibits Hsp27 phosphorylation.

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nM, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.

A hexokinase with broad sugar specificity from a thermophilic bacterium.

A recombinant thermophilic Thermus caldophilus GK24 hexokinase, one of the ROK-type (repressor protein, open reading frames, and sugar kinase) proteins, exists uniquely as a 120 kDa molecule with four subunits (31 kDa), in contrast to eukaryotic and bacterial sugar kinases which are monomers or dimers. The optimal temperature and pH for the enzyme reaction are 70-80 degrees C and 7.5, respectively. This enzyme shows broad specificity toward glucose, mannose, glucosamine, allose, 2-deoxyglucose, and fructose. To understand the sugar specificity at a structural level, the enzyme-ATP/Mg2+-sugar binding complex models have been constructed. It has been shown that the sugar specificity is probably dependent on the interaction energy occurred by the positional proximity of sugars bound in the active site of the enzyme, which exhibits a tolerance to modification at C2 or C3 of glucose.

Mechanistic study of the intramolecular conversion of maltose to trehalose by Thermus caldophilus GK24 trehalose synthase.

This paper questions what types of molecular transformation are involved in the conversion of maltose to trehalose by trehalose synthase from Thermus caldophilus GK24. The reverse reaction pathway has been examined with the aid of alpha,alpha-(2,4,6,6',2',4',6",6"'-(2)H(8))trehalose (1). The mass data of the isolated reaction products clearly indicate that deuterated glucose is confined only to substrate molecules, and thus the reversible enzymatic conversion of trehalose into maltose proceeds through an intramolecular pathway.