Three new thermotolerant methylotrophic yeasts, Candida krabiensis sp. nov., Candida sithepensis sp. nov., and Pichia siamensis sp. nov., isolated in Thailand

Five strains of thermotolerant methylotrophic yeasts isolated in Thailand were found to represent three new species in the genera Pichia and Candida, based on phylogenetic analysis of D1/D2 domain of 26S rDNA, in addition to the morphological, physiological, biochemical and chemotaxonomic characterization. Three strains, FS96 and FS101 from flowers and M02 from tree flux, were characterized by ubiquinone Q7, multilateral budding, and the formation of hat-shaped ascospores that are liberated at maturation. These strains showed identical nucleotide sequences in the D1/D2 domain and formed a cluster with Candida thermophila, "Pichia salicis" and Pichia angusta. They differed by 1.9% of nucleotide substitutions from Candida thermophila, the nearest species. They were considered to represent a single new species and are described as Pichia siamensis sp. nov. Two strains, N051 and S023, isolated from soil did not produce ascospores, proliferated by multilateral budding, did not demonstrate urease or DBB color reaction, and lacked sexual stages. These characteristics correspond to the genus Candida. Strains N051 and S023 differed by 2.8% and 1.9% of nucleotide substitutions in the D1/D2 domain from the nearest species, Candida nemodendra and Candida ovalis, respectively, and are considered to represent respective new species. N051 and S023 are described as Candida krabiensis sp. nov. and Candida sithepensis sp. nov., respectively.     

Structure of Thermus thermophilus 2-Keto-3-deoxygluconate kinase: evidence for recognition of an open chain substrate

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate (KDGP). The genome sequence of Thermus thermophilus HB8 contains an open reading frame that has a 30% identity to Escherichia coli KDGK. The KDGK activity of T.thermophilus protein (TtKDGK) has been confirmed, and its crystal structure has been determined by the molecular replacement method and refined with two crystal forms to 2.3 angstroms and 3.2 angstroms, respectively. The enzyme is a hexamer organized as a trimer of dimers. Each subunit is composed of two domains, a larger alpha/beta domain and a smaller beta-sheet domain, similar to that of ribokinase and adenosine kinase, members of the PfkB family of carbohydrate kinases. Furthermore, the TtKDGK structure with its KDG and ATP analogue was determined and refined at 2.1 angstroms. The bound KDG was observed predominantly as an open chain structure. The positioning of ligands and the conservation of important catalytic residues suggest that the reaction mechanism is likely to be similar to that of other members of the PfkB family, including ribokinase. In particular, the Asp251 is postulated to have a role in transferring the gamma-phosphate of ATP to the 5'-hydroxyl group of KDG.     

Characterization of human CD4 helper T cell responses against Aurora kinase A

Aurora kinase A (Aurora-A) is a cell cycle-associated serine–threonine kinase that is overexpressed by various types of cancer and is highly associated with poor prognosis. Since the expression of Aurora-A in normal tissues has been shown to be significantly lower as compared to tumor cells, this protein is being considered as a potential tumor-associated antigen for developing immunotherapies. The goal in the present study was to identify CD4 helper T lymphocyte (HTL) epitopes for Aurora-A for the design of T cell-based immunotherapies against Aurora-A-expressing tumors. Synthetic peptides corresponding to potential HTL epitopes were identified from Aurora-A and used to stimulate CD4 T lymphocytes in vitro to generate antigen-specific HTL clones that were evaluated for antigen specificity, MHC restriction and for their ability to interact with Aurora-A-expressing tumor cells. The results show that two peptides (Aurora-A_161–175 and Aurora-A_233–247) were effective in generating HTL responses that were restricted by more than one MHC class II allele (i.e., promiscuous responses). The CD4 HTL clones were able to directly recognize Aurora-A-expressing tumor cells in an antigen-specific and MHC class II-restricted manner and some of the clones displayed cytolytic activity toward Aurora-A + tumor cells. Both of these peptides were capable of stimulating in vitro T cell responses in patients with bladder cancer.     

3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective EP3 receptor antagonists. Part 1: Discovery and exploration of the carboxyamide side chain

A series of 3-(2-aminocarbonyl-4-phenoxymethylphenyl)propanoic acid analogs were synthesized and evaluated for their EP3 antagonist activity in the presence of additive serum albumin. Several compounds were biologically evaluated for their in vivo efficacy with respect to the PGE₂-induced uterine contraction in pregnant rats as well as their pharmacokinetics. The discovery process of these potent and selective EP3 antagonists and their structure activity relationship are also presented.     

Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum

In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions—the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100β. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish.     

Analysis of urinary 8-nitroguanine, a marker of nitrative nucleic acid damage, by high-performance liquid chromatography-electrochemical detection coupled with immunoaffinity purification: association with cigarette smoking

We have developed an analytical method to quantitate urinary 8-nitroguanine, a product of nitrative nucleic acid damage caused by reactive nitrogen species such as peroxynitrite and nitrogen dioxide. 8-Nitroguanine was purified from human urine using immunoaffinity columns with an anti-8-nitroguanine antibody, followed by quantitation by high-performance liquid chromatography-electrochemical detection. Four sequential electrodes were used to (a) oxidize interfering compounds (+250 mV), (b) reduce nitrated bases (two online electrodes at -1000 mV), and (c) quantitate reduced derivatives (+150 mV). Using this system 8-nitroxanthine can also be detected, with the detection limits being 25 and 50 fmol/injection for 8-nitroguanine and 8-nitroxanthine, respectively. The method was used to analyze both adducts in the urine of smokers (n=12) and nonsmokers (n=17). We found that smokers excrete more 8-nitroguanine [median, 6.1 fmol/mg creatinine; interquartile range (IQR), 23.8] than nonsmokers (0; IQR, 0.90) (p=0.018), and although 8-nitroxanthine was detected in human urine, its level was not related to smoking status. This is the first report to show that 8-nitroguanine is present in human urine and the methodology developed can be used to study the pathogenic roles of this adduct in the etiology of cancers associated with cigarette smoking and inflammation.     

Combined effect of salinomycin and feeding on whole body glucose kinetics in sheep fed a high-concentrate diet

The aim of this study was to investigate the effects of salinomycin (SL) and feeding on whole body glucose kinetics in sheep fed a high-concentrate diet (25% orchardgrass hay and 75% commercial concentrate). Four adult sheep were fed the diet with or without 20 mg x kg(-1) diet of SL once daily for each 3 wk. The rates of glucose entry and utilization were determined before and during 3 h after feeding using a [ (13)C(6)] glucose dilution approach. Ruminal characteristics and concentrations of blood volatile fatty acids (VFA) and plasma glucose and insulin were also measured. Metabolizable energy intake was unaffected (P = 0.22) with SL. Salinomycin decreased (P = 0.06) the ratio of acetate to propionate in rumen fluid. Salinomycin increased (P = 0.01) both rates of entry and utilization of glucose, but did not affect (P > 0.10) concentrations of blood VFA or plasma glucose or insulin. Feeding caused gradual increases in concentrations of blood acetate (P < 0.01) and propionate (P = 0.01), a transient increase in plasma insulin concentration (P = 0.05), a transient decrease in plasma glucose concentration (P < 0.01), and persistent increases in both rates of glucose entry (P < 0.01) and utilization (P < 0.01). No SL x feeding interaction was observed (P > 0.10) on any measurements. We conclude that SL and feeding would have an additive effect on both rates of glucose entry and utilization without modifications with SL to feeding responses of peripheral concentrations of blood VFA, plasma glucose and insulin.     

Capacity of dental pulp differentiation after tooth transplantation

Under pathological conditions, dental pulp elaborates both bone and dentin matrix in which the contribution of periodontal tissue cannot be excluded. This study has aimed to clarify the capability of dental pulp to deposit bone matrix in an auto-graft experiment by using (1) immunohistochemistry for 5-bromo-2′-deoxyuridine (BrdU) and nestin and (2) histochemistry for tartrate-resistant acid phosphatase (TRAP). Following the extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately transplanted into the sublingual region. On Days 5–7, tubular dentin formation commenced next to the pre-existing dentin at the pulp horn in which nestin-positive odontoblast-like cells were arranged. Up until Day 14, bone-like tissue formation occurred in the pulp chamber in which intense TRAP-positive cells appeared. These results suggest that odontoblast- and osteoblast-lineage cells reside in the dental pulp. Overall, specific dental pulp regeneration should provide fundamental knowledge for the realization of human tooth regeneration in the near future.This work was supported in part by a grant from MEXT to promote the 2001-multidisciplinary research project (in 2001–2005), KAKENHI (B) (no. 16390523 to H.O.) from MEXT, and the Japan-Korea Joint Research Project from JSPS and KOSEF (no. F01-2005-000-10212-0).     

TRIM50 Protein Regulates Vesicular Trafficking for Acid Secretion in Gastric Parietal Cells

Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.