Human tyrosinase is able to oxidize both enantiomers of rhododendrol

Racemic RS‐4‐(4‐hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a topical skin‐whitening agent until it was recently reported to induce leukoderma. We then showed that oxidation of RD with mushroom tyrosinase rapidly produces RD‐quinone, which is quickly converted to RD‐cyclic quinone and RD‐hydroxy‐p‐quinone. In this study, we examined whether either or both of the enantiomers of RD can be oxidized by human tyrosinase. Using a chiral HPLC column, racemic RD was resolved optically to R(?)‐RD and S(+)‐RD enantiomers. In the presence of a catalytic amount of l ‐dopa, human tyrosinase, which can oxidize l ‐tyrosine but not d ‐tyrosine, was found to oxidize both R(?)‐ and S(+)‐RD to give RD‐catechol and its oxidation products. S(+)‐RD was more effectively oxidized than l ‐tyrosine, while R(?)‐RD was less effective. These results support the notion that the melanocyte toxicity of RD depends on its tyrosinase‐catalyzed conversion to toxic quinones and the concomitant production of reactive oxygen species.     

Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.     

Development of nuclear microsatellite markers for the threatened wetland plant Geranium soboliferum var. kiusianum (Geraniaceae)

Nuclear microsatellite markers were developed for the threatened plant Geranium soboliferum var. kiusianum, which has decreased its population size as a result of loss of its wetland habitat in Kyushu, Japan. Utilizing RNA‐seq data obtained by next‐generation sequencing techniques, 10 polymorphic microsatellite markers with 3–16 alleles in a nuclear genome were developed and characterized. Two to 15 alleles were observed in G. soboliferum. These markers will be used to investigate the genetic circumstance of remnant populations of G. soboliferum var. kiusianum and their phylogenetic relationship with G. soboliferum.     

Single gene deletions of mrpA to mrpG and mrpE point mutations affect activity of the Mrp Na+/H+ antiporter of alkaliphilic Bacillus and formation of hetero-oligomeric Mrp complexes

Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.     

CO binding studies of nitric oxide synthase: effects of the substrate, inhibitors and tetrahydrobiopterin

The dissociation constant (K_d) for CO from neuronal nitric oxide synthase heme in the absence of the substrate and cofactor was less than 10⁻³ μM. In the presence of

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-Arg, it dramatically increased up to 1 μM. In the presence of inhibitors such as N^G-nitro-

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-arginine methyl ester and 7-nitroindazole (NI), the K_d value further increased up to more than 100 μM. Addition of the cofactor, 5,6,7,8-tetrahydrobiopterin (H₄B), increased the K_d value by 10-fold in the presence of

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-Arg, whereas it decreased the value to less than one 250th in the presence of NI. Addition of H₄B increased the recombination rate constant (k_on) for CO by more than two-fold in the presence of

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-Arg or N⁶-(1-iminoethyl)-

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-lysine, whereas it decreased the k_on value by three-fold in the presence of

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-thiocitrulline. Thus, the binding fashion of some of inhibitors, such as NI, may be different from that of

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-Arg with respect to the H₄B effect.     

PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA

Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2α(PGF2α)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA (CHO-PGF2α·R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756, 1994). In the present study, we investigated PGF2α-induced PLD activation in CHO-PGF2α·R cells. PLD activation was examined by measuring the production of [³H]phosphatidylbutanol ([³H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2α-induced [³H]PBut formation was concentration-dependent with the maximal level obtained at 1 μM PGF2α. The maximal [³H]PBut formation was observed at 2 min after addition of PGF2α. Depletion of extracellular Ca²⁺ with EGTA suppressed PGF2α-induced PLD activation by 50%. PKC inhibitors Ro31–8425 and calphostin C inhibited PGF2α-induced [³H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2α-induced PLD activation. A combination of maximal effective concentrations of PGF2α (1 μM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKCα and decreased PGF2α-induced PLD activation. These results suggest that PLD activation by PGF2α is mediated by both PKC-dependent and -independent pathways and that PKCα is involved in the former pathway.     

Superoxide Scavenging Activity of Spin-Labeled Nitrosourea and Triazene Derivatives

Superoxide scavenging activities (SSA) of newly synthesized spin-labeled nitrosourea and triazene derivatives, and their precursor nitroxides were investigated by the ESR/spin-trapping method using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and hypoxanthine/xanthine oxidase as the superoxide-generating system. The spin-labeled nitrosoureas, triazenes and their precursor nitroxides exhibited excellent SSA, whereas clinically used nitrosourea and triazene, which do not contain the nitroxide moiety, did not show any SSA. Furthermore, it was deduced that these nitroxides scavenge superoxide by redox cycling between nitroxide and corresponding hydroxylamine.     

Occurrence of Yersinia enterocolitica in wild-living birds and Japanese serows.

Yersinia spp. were isolated from 34 of 500 birds representing nine species. The highest isolation rate, 5 of 21 (23.8%), was found in blue magpies (Cyanopia cyanus), followed by pheasants (Phasianus colchicus tohkaidi), 5 of 33 (15.2%); gray starlings (Sturnus cineraceus), 6 of 57 (10.5%); tree sparrows (Passer montanus), 1 of 14 (7.1%); bulbuls (Hypsipetes amaurotis), 4 of 57 (7.0%); crows (Corvus levailantii or Corvus corone), 7 of 117 (6.0%); eastern turtledoves (Streptopelia orientalis), 4 of 118 (3.4%); Chinese bamboo pheasants (Bumbusicola thoracica thoracica), 1 of 36 (2.8%); and domestic pigeons (Columba livia domestica), 1 of 47 (2.1%). The isolates were identified as Yersinia enterocolitica O:3, O:4, O:4,32, O:5A, O:6,30, O:7,8, and O:14, Yersinia frederiksenii, Yersinia intermedia, and Yersinia kristensenii. Yersinia spp. were isolated from 35 of 157 wild-living Japanese serows (Capricornis cripus). The isolates were identified as Y. enterocolitica O:4, O:4,32, O:5A, O:7, O:7,8, O:9, O:14, O:18, and O:34, Y. frederiksenii, Y. intermedia, and Y. kristensenii.     

Mutagenicities of quinoline and its derivatives.

Quinoline, recently reported to be carcinogenic in rats [12], was mutagenic to Salmonella typhimurium tester strains TA100 and TA98 in the presence of the metabolic activation system S-9 mix. 2-Chloroquinoline, a non-carcinogen [12], was non-mutagenic with or without S-9 mix. 8-Hydroxyquinoline, which is t known to be carcinogenic, was mutagenic with S-9 mix to both bacterial strains. The mutagenicities of 17 other quinoline derivatives that are not known to be carcinogenic were tested, and 12 of these compounds were mutagenic.