The partial 60 kDa heat-shock protein (HSP60) genes of 36 Bifidobacterium strains representing 30 different Bifidobacterium species and subspecies and of the type strain of Gardnerella vaginalis were cloned and sequenced using a pair of universal degenerate HSP60 PCR primers. The HSP60 DNA sequence similarities were determined for the taxa at various ranks as follows : 994-100 % within the same species, 96 % at the subspecies level, and 73-96 % (mean 85 %) at the interspecies level (and 98 % in the case of two groups of closely related species, Bifidobacterium animalis and Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium suis, whose 16S rRNA sequence similarities are all above 99 %). The HSP60 DNA sequence similarities between different Bifidobacterium species and G. vaginalis, a closely related bacterium according to 16S rRNA analysis, ranged from 71 to 79 % (mean 75 %). Although the topology of the phylogenetic tree constructed using the HSP60 sequences determined was basically similar to that for 16S rRNA, it seemed to be more clear-cut for species delineation, and the clustering was better correlated with the DNA base composition (mol % GMC) than that of the 16S rRNA tree. In the HSP60 phylogenetic tree, all of the high-GMC (55-67 mol %) bifidobacteria were grouped into one cluster, whereas the low-GMC species Bifidobacterium inopinatum (45 mol %) formed a separate cluster with G. vaginalis (42 mol %) and Bifidobacterium denticolens (55 mol %) ; a Bifidobacterium species of intermediate GMC content formed another cluster between the two. This study demonstrates that the highly conserved and ubiquitous HSP60 gene is an accurate and convenient tool for phylogenetic analysis of the genus Bifidobacterium.

Hydrophilic interaction chromatography (HILIC) provides a complementary separation mode to RPLC and thus has been gaining increased utilization in LC-MS/MS based quantitative bioanalysis. It has proven to be a powerful tool for separation of polar compounds and has afforded increased selectivity, higher sensitivity, and improved efficiency for quantitation of drug and their metabolites in complex biological matrices. Practical knowledge has been gained for some of the challenges with HILIC applications and effective remedies have been adopted to overcome these challenges. Due to its orthogonality to RPLC, HILIC has been coupled with RP sample preparation or separation techniques, either off-line or on-line to achieve better assay selectivity. The low back-pressure with HILIC enables fast separations under higher flow rates. Small particle columns can be operated under HILIC conditions with regular system back-pressure, eliminating the requirement for ultra-high-pressure liquid chromatographic systems. Matrix effects under HILIC have also been systematically investigated. Effective approaches to reduce or eliminate matrix effects have included optimizing sample preparation, modifying chromatographic conditions, and/or using valve-switching techniques. Finally, the utilization of HILIC is not limited to quantitation of polar drugs and their metabolites, but extended to quantitation of relatively non-polar compounds, peptides and biomarkers.

Background-The proinflammatory and vascular actions of cysteinyl leukotrienes (CysLTs) are mediated by 2 receptors: cysteinyl leukotriene 1 receptor (CysLT 1 R) and cysteinyl leukotriene 2 receptor (CysLT 2 R). However, the distinct contribution of CysLT 2 R to the vascular actions of CysLTs has not been addressed. Methods and Results-We generated an endothelial cell-specific human CysLT 2 R (EC-hCysLT 2 R) transgenic (TG) mouse model using the Tie2 promoter/enhancer. Strong expression of hCysLT 2 R in TG lung and endothelial cells, detected by real-time polymerase chain reaction, markedly enhanced CysLT-stimulated intracellular calcium mobilization compared with endogenous expression in cells from nontransgenic mice. The permeability response to exogenous LTC 4 and to endogenous CysLTs evoked by passive cutaneous anaphylaxis was augmented in TG mice. The rapid, systemic pressor response to intravenous LTC 4 was also diminished in TG mice coincidentally with augmented production of nitric oxide. Conclusions-The development of EC-hCysLT 2 R mice has permitted detection of distinct vascular effects of CysLTs, which can be mediated via the CysLT 2 R in vivo.

A new phenotypic group of Bifidobacterium strains, isolated from an anaerobic digester for the treatment of waste water from a bean-curd farm, was described previously. In this study, the DNA-DNA relatedness between strain 36 (type strain, AS 1.2282 T ) of this new group and the type strains of other described Bifidobacterium species was analysed. The low level of DNA homology (0-589 %) as well as comparison of the 16S rDNA sequences confirmed the distinct phylogenetic position of strain 36. In addition, the new species differed from other Bifidobacterium species in its phenotypic characteristics, such as its growth at moderately thermophilic conditions (495 SC) and at relatively low pH (40), as well as its sugar-fermentation pattern. On the basis of phenotypic, genetic and phylogenetic studies, a new Bifidobacterium species, Bifidobacterium thermacidophilum sp. nov., was designated.