A conventional and easy method to establish a murine oral candidiasis model, which has not only a stable yeast population in the oral cavity but also symptoms characteristic of oral thrush, was developed by using a sedative agent. Mice were immunosuppressed with prednisolone and were given tetracycline hydrochloride. They were orally infected with 106 viable cells of Candida albicans by means of a cotton swab and enough chlorpromazine chloride had been injected to keep them in a sedative state about for 3 hr after inoculation. From day 3 to day 7 post inoculation, 105–106 colony forming units of Candida were recovered from the oral cavity of each mouse and whitish, curd‐like patches were observed on most parts of tongue. Microscopically, germ tubes had appeared on the tongue surface. This model would be a useful experimental oral candidiasis for investigating the pathogenesis of C. albicans oral infection and the efficacy of various antifungal agents microbiologically and symptomatically.

Oligodeoxyribonucleotides were synthesized using H-phosphonate monomers without amino protection. The H-phosphonate monomers of deoxyadenosine, deoxycytidine, and deoxyguanosine bearing the free amino groups were synthesized in good yields by O-selective phosphonylation of the parent 5‘-O-(dimethoxytrityl)deoxyribonucleosides. It was found that the amino groups of the nucleosides were not modified during internucleotidic bond formation where (benzotriazol-1-yloxy)carbonium and -phosphonium compounds were employed as condensing reagents. The most effective condensing reagent for rapid internucleotidic bond formation was found to be 2-(benzotriazol-1-yloxy)-1,1-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium hexafluorophosphate (BOMP). In the present H-phosphonate method, 2-(phenylsulfonyl)-3-(3-nitrophenyl)oxaziridine (PNO) was employed as a new oxidizing reagent for the oxidation of internucleotidic H-phosphonate linkages under anhydrous conditions in the presence of N,O-bis(trimethylsilyl)acetamide. The reaction mechanism for the O-selective condensation was investigated in detail by means of 31P NMR spectroscopy. Unprecedented oxidation of the H-phosphonate monomers was observed during activation of the monomers with (benzotriazol-1-yloxy)phosphonium and -carbonium condensing reagents in the absence of the 5‘-hydroxyl components. A mechanism for the O-selective condensation was proposed on the basis of ab initio molecular orbital calculations for the model compounds at the HF/6-31G* level.

N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to ϳ1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of 125 I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the upregulation of the endocytosis of EGFR.

Genetic diagnoses, such as single nucleotide polymorphism (SNP) typing, allow elucidation of gene-based physiological differences, such as susceptibility to diseases and response to drugs, among individuals. Many detection technologies, including allele-specific hybridization, allele-specific primer extension and oligonucleotide ligation, are being used to discriminate SNP alleles. These methods still have many unsolved practical issues. In general they require adequate and specific hybridizations of primer or probe DNAs with target DNAs. This frequently needs optimization of the probe/primer structures and operating conditions. In nature, highly homology-sensitive hybridization is assisted by a nucleic acid chaperone that reduces the energy barrier associated with breakage and reassociation of nucleic base pairs. Here we report a simple, quick, precise but enzyme-free method for SNP analysis. The method uses cationic comb-type copolymers (CCCs) producing high nucleic acid chaperone activities. A single-base mismatch in 20-mer DNA can be detected within a few minutes at ambient temperatures (25-37 degrees C). Even without careful optimization processes, the method has the sensitivity to detect the mismatches causing subtle changes (Delta T(m) equals approximately 1 degree C) in duplex thermal stability. CCCs may have various bioanalytical applications where precise hybridization of nucleic acids is needed.

Water-soluble neutral cosolutes can be used to quantify biomolecular properties in the particular molecular environment occurring in a cell. We studied the conformation and the thermal stability of DNA and RNA structures in the presence of PEG [poly(ethylene glycol)] and smaller cosolutes of glycerol, ethylene glycol, 1,3-propanediol, 2-methoxyethanol, and 1,2-dimethoxyethane. Although the neutral cosolutes destabilized the oligonucleotide duplex and the hairpin structures, the left-handed Z-form duplex was more energetically favored in the cosolute-containing solutions. These observations were due to the contribution of water molecule on the nucleotide structure formations because the cosolutes act as an osmolyte to reduce the water activity of a solution. Moreover, the sodium ion condensation for the duplex and the hairpin formations was reduced in the presence of PEG, while that for the transition from the B-form to the Z-form was unaltered. The CD (circular dichroism) and EPR (electron paramagnetic resonance) spectra demonstrated that the cosolutes changed the helical conformation of the unstructured oligonucleotides, but not those of the ordered structures. The results of the favorable formation of the noncanonical nucleotide structures, and minimized conformational and thermal perturbations of the ordered nucleotide structures in the cosolute-containing solutions implicate the significance of the intracellular environment on DNA and RNA structures in a cell.

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Four anthocyanin components of black currant, delphinidin 3-O-beta-rutinoside (D3R), cyanidin 3-O-beta-rutinoside (C3R), delphinidin 3-O-beta-glucoside (D3G), and cyanidin 3-O-beta-glucoside (C3G), were successfully isolated as crystalline forms on a preparative scale. In this process, selective hydrolysis of the glucosides (D3G and C3G) and rhamnosides (D3R and C3R) was achieved by treatment with beta-glucosidase and hesperidinase (alpha-rhamnosidase), respectively, to improve resolution of anthocyanin components. Especially, selective conversion of the rutinosides into glucosides made the amounts of D3G and C3G increase about 4- and 7-fold, respectively. D3R, C3R, D3G, and C3G were isolated from enzymatic hydrolysates of black currant anthocyanins through Amberlite XAD-7HP absorption followed by preparative HPLC separation, and their crystals were obtained as the flavylium chloride.

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