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.     

Peroxisomal membrane protein Pmp47 is essential in the metabolism of middle-chain fatty acid in yeast peroxisomes and Is associated with peroxisome proliferation

Pmp47 of the methylotrophic yeast Candida boidinii belongs to a mitochondrial family of solute transporters and is localized in peroxisomal membranes. Its human homolog, Pmp34, is also known. In this study, we characterized the role of Pmp47 in fatty acid metabolism and peroxisome proliferation using the PMP47-deleted strain of C. boidinii (strain pmp47Delta). The wild-type strain grew well on a middle-chain fatty acid, laureate, as the single carbon source, and mild peroxisome proliferation was observed during its growth. The pmp47Delta strain could not grow on laureate but could grow on long-chain fatty acids including palmitate, myristate, and oleate. The levels of laureate oxidation activity in intact cells and in semi-permeabilized cells of strain pmp47Delta were lower than the respective level in the wild-type strain, although the level of laureate oxidation activity in the cell lysate and the level of lauroyl-CoA oxidation in semi-permeabilized cells of strain pmp47Delta were indistinguishable from the respective level in the wild-type strain. When lauroyl-CoA was provided in the cytosol of strain pmp47Delta through expression of Saccharomyces cerevisiae Faa2p (lauroyl-CoA synthetase) in which its peroxisome targeting signal was deleted, the growth of strain pmp47Delta on laureate was recovered to the level of growth of the wild-type strain. Laureate is converted to its CoA form in peroxisomes by the action of lauroyl-CoA synthetase. These results suggested that Pmp47 is involved in the transport of a small molecule (possibly ATP) required in the conversion of laureate to its CoA form in peroxisomes and that the absence of Pmp47 causes impairment of laureate metabolism, which results in the inability of pmp47Delta cells to grow on laureate. In addition, Pmp47 may be involved in peroxisome proliferation, because the pmp47Delta strain contained a reduced number of peroxisomes, as judged from the fluorescence analysis of cells expressing green fluorescent protein tagged with the peroxisome targeting signal 1 (GFP-AKL).     

Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica

Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation.     

Extracellular production of cycloisomaltooligosaccharide glucanotransferase and cyclodextran by a protease-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> host–vector system

A cycloisomaltooligosaccharide (CI; cyclodextran) production system was developed using a Bacillus subtilis expression system for the cycloisomaltooligosaccharide glucanotransferase (CITase) gene. The CITase gene of Bacillus circulans T-3040, along with the α-amylase promoter (PamyQ) and amyQ signal sequence of Bacillus amyloliquefaciens, was cloned into the Bacillus expression vector pUB110 and subsequently expressed in B. subtilis strain 168 and its alkaline (aprE) and neutral (nprE) protease-deficient strains. The recombinant CITase produced by the protease-deficient strains reached 1 U/mL in the culture supernatant within 48 h of cultivation, which was approximately 7.5 times more than that produced by the industrial CITase-producing strain B. circulans G22-10 derived from B. circulans T-3040. When aprE- and nprE-deficient B. subtilis 168 harboring the CITase gene was cultured with 10% dextran 40 for 48 h, 17% of the dextran in the culture was converted to CIs (CI-7 to CI-12), which was approximately three times more than that converted by B. circulans G22-10 under the same dextran concentration. The B. subtilis host–vector system enabled us to produce CIs by direct fermentation of dextran along with high CITase production, which was not possible in B. circulans G22-10 due to growth inhibition by dextran at high concentrations and limited production of CITase.     

Discovery and SAR of a novel series of Natriuretic Peptide Receptor-A (NPR-A) agonists

Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring to a quinazoline ring, introduction of the basic functional group and optimization of the substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with an EC₅₀ value of 0.073 μM, indicating 350-fold potency compared to the hit compound 3.     

Effect of N-terminal residues on the structural stability of recombinant horse L-chain apoferritin in an acidic environment

The denaturation of recombinant horse L-chain apoferritin (rLF), which is composed of 24 L-chain subunits, in acidic solution was studied. Using two rLF mutants, lacking four (Fer4) or eight (Fer8) N-terminal amino acid residues, the effect of N-terminal residues on the protein's stability was investigated. Of the two mutants and wild-type rLF, the tertiary and secondary structures of Fer8 were found to be most sensitive to an acidic environment. The Fer8 protein dissociated easily into subunit dimers at or below pH 2.0. Comparing the crystal structures of the mutant proteins, deletion of the N-terminal residues was found to result in fewer inter- and intra-subunit hydrogen bonds. The loss of these bonds is assumed to be responsible for lower endurance against acidic denaturation in N-terminus-deleted mutants. These results indicated that the inter- and intra-subunit hydrogen bonds of N-terminal residues affect the denaturation, especially oligomer formation of apoferritin subunits and will be of use in designing ferritin-based nanodevices.     

Variation in the activity of some enzymes of photorespiratory metabolism in C4 grasses

BACKGROUND AND AIMS: Photorespiration occurs in C4 plants, although rates are small compared with C3 plants. The amount of glycine decarboxylase in the bundle sheath (BS) varies among C4 grasses and is positively correlated with the granal index (ratio of the length of appressed thylakoid membranes to the total length of all thylakoid membranes) of the BS chloroplasts: C4 grasses with high granal index contained more glycine decarboxylase per unit leaf area than those with low granal index, probably reflecting the differences in O2 production from photosystem II and the potential photorespiratory capacity. Thus, it is hypothesized that the activities of peroxisomal enzymes involved in photorespiration are also correlated with the granal development. METHODS: The granal development in BS chloroplasts was investigated and activities of the photorespiratory enzymes assayed in 28 C4 grasses and seven C3 grasses. KEY RESULTS: The NADP-malic enzyme grasses were divided into two groups: one with low granal index and the other with relatively high granal index in the BS chloroplasts. Both the NAD-malic enzyme and phosphoenolpyruvate carboxykinase grasses had high granal index in the BS chloroplasts. No statistically significant differences were found in activity of hydroxypyruvate reductase between the C3 and C4 grasses, or between the C4 subtypes. The activity of glycolate oxidase and catalase were smaller in the C4 grasses than in the C3 grasses. Among the C4 subtypes, glycolate oxidase activities were significantly smaller in the NADP-malic enzyme grasses with low granal index in the BS chloroplasts, compared with in the C4 grasses with substantial grana in the BS chloroplasts. CONCLUSIONS: There is interspecies variation in glycolate oxidase activity associated with the granal development in the BS chloroplasts and the O2 production from photosystem II, which suggests different potential photorespiration capacities among C4 grasses.     

Production of human erythropoietin by chimeric chickens

The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells.