Development of 16S rRNA-Gene-Targeted Group-Specific Primers for the Detection and Identification of Predominant Bacteria in Human Feces

For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.     

Cryopreservation of hairy root cultures of Vinca minor (L.) by encapsulation-dehydration

Hairy root cultures of Vinca minor and Ajuga reptans var. atropurpurea could be cryopreserved when the roots were precultured and encapsulated in 2% (w/v) alginate beads with 0.3 M sucrose and 0.5 M glycerol and dehydrated until the bead weight reached 25% of the initial weight before cooling in liquid nitrogen. Preculture and encapsulation of the roots with abscisic acid was effective in increasing the survival rates. For V. minor root tips moreover a sufficiently high survival rate of more than 70% was attained by eliminating glycerol from the preculture medium and dehydration of beads until 23% of the initial weight was reached instead of 25%.     

Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay.

A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4-benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio (B/B(o)%) of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N(omega)-nitro-L-arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35.21 +/- 4.87 ng/mL (135.4 +/- 18.7 nM). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41.40 +/- 20.96 ng/mL (159.02 +/- 80.6 nM). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources.     

Facile and stereoselective synthesis of 2'-5'-oligothioadenylate by UO2(2+) ion catalyst.

The synthesis and characterization of 2'-5'-oligothioadenylate by UO2(2+) ion catalyst are described. The polymerization of imidazole-activated thioadenylate or thioinosylate yielded oligomers containing mainly 2'-5'-internucleotidyl blond and Rp configuration at phosphorous atom.     

Application of high-performance liquid chromatography with a chemiluminescence detection system to determine catecholamines in urine

Chemiluminescence generated with the reaction of bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide was applied to a detection system for high-performance liquid chromatography to determine fluorescamine-labeled catecholamines. The sensitivity of the chemiluminescence detection system with 25 fmol of detection limit was approximately 20 times higher than that of a conventional fluorescence detection system. Norepinephrine and dopamine in human urine were determined by the use of the new high-performance liquid chromatography detection system with the coefficient of variation of less than 4.0%. Good correlations (r = 0.998 for norepinephrine and r = 0.999 for dopamine) were obtained between the values by the present method and the conventional method.     

Chemical analysis of late stages of pheomelanogenesis: conversion of dihydrobenzothiazine to a benzothiazole structure

Pheomelanogenesis is a complex pathway that starts with the oxidation of tyrosine (or DOPA, 3,4‐dihydroxyphenylalanine) by tyrosinase in the presence of cysteine, which results in the production of 5‐S‐cysteinyldopa and its isomers. Beyond that step, relatively little has been clarified except for a possible intermediate produced, dihydro‐1,4‐benzothiazine‐3‐carboxylic acid (DHBTCA). We therefore carried out a detailed study on the course of pheomelanogenesis using DOPA and cysteine and the physiological enzyme tyrosinase. To elucidate the later stages of pheomelanogenesis, chemical degradative methods of reductive hydrolysis with hydroiodic acid and alkaline peroxide oxidation were applied. The results show that: (1) DHBTCA accumulates after the disappearance of the cysteinyldopa isomers, (2) DHBTCA is then oxidized by a redox exchange with dopaquinone to form ortho‐quinonimine, which leads to the production of pheomelanin with a benzothiazine moiety, and (3) the benzothiazine moiety gradually degrades to form a benzothiazole moiety. This latter process is consistent with the much higher ratio of benzothiazole‐derived units in human red hair than in mouse yellow hair. These findings may be relevant to the (photo)toxic effects of pheomelanin.     

Ions released from a S-PRG filler induces oxidative stress in <Emphasis Type="Italic">Candida albicans</Emphasis> inhibiting its growth and pathogenicity

Candida albicans causes opportunistic fungal infections usually hidden among more dominant bacteria and does not exhibit high pathogenicity in vivo. Among the elderly, due to reduced host resistance to pathogens attributable to immunoscenesence, oral candidiasis is more likely to develop often leading to systemic candidiasis. Surface pre-reacted glass ionomer filler (S-PRG filler) is an ion-releasing functional bioactive glass that can release and recharge six ions which in turn strengthens tooth structure, inhibits demineralization arising from dental caries, and suppresses dental plaque accumulation. However, its effects on C. albicans have never been elucidated. Here, we evaluated the effects of ion released from S-PRG filler on C. albicans. Results show that extraction liquids containing released ions (ELIS) decreased the amount of hydrogen peroxide and catalase activity in C. albicans. Moreover, ELIS presence was found to affect C. albicans: (1) suppression of fungal growth and biofilm formation, (2) prevent adherence to denture base resin, (3) inhibit dimorphism conversion, and (4) hinder the capability to produce secreted aspartyl proteinase. Taken together, our findings suggest that ELIS induces oxidative stress in C. albicans and suppresses its growth and pathogenicity. In this regard, we propose that ELIS has the potential to be clinically used to help prevent the onset and inhibition of oral candidiasis among the elderly population.     

Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.