The pyrogallol related compounds reduce UV-induced mutations in Escherichia coli B/r WP2

Plant components with bio-antimutagenic activity were screened on UVC (254 nm)-induced mutagenesis using E. coli B/r WP2. The components with a pyrogallol moiety including gallic acid, (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) reduced the mutation induction, but other components such as caffeic acid, chlorogenic acid and quercetin did not. The above compounds with a pyrogallol moiety were also effective on UVAB (295-400 nm)-induced mutagenesis, while they showed little effect on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis. As this bio-antimutagenic effect was not seen in the DNA excision-repair-deficient strains WP2s and ZA159, the activity by the above plant components might be based on the promotion of the excision-repair system in E. coli B/r WP2.     

Methyl cinnamate derivatives enhance UV-induced mutagenesis due to the inhibition of DNA excision repair in Escherichia coli B/r

UV-induced mutagenesis in Escherichia coli B/r WP2 was enhanced by certain derivatives of methyl cinnamate which themselves were not mutagenic. Methyl ferulate, methyl isoferulate and methyl sinapate showed this effect markedly. Such an enhancement effect was absent with the derivatives of cinnamic acid and ethyl cinnamate and was not observed in Escherichia coli WP2s uvrA. Methyl sinapate also enhanced 4NQO-induced mutation and suppressed liquid-holding recovery in the above repair-proficient strain. The presence of methyl sinapate in plating agar medium decreased the survival of UV-irradiated cells of a recombination-repair-deficient strain, CM571 recA. However, the effect was not observed with those of WP2s uvrA. In an in vitro experiment in which the removal rate of thymine dimers was measured, methyl sinapate clearly inhibited this repair event. From these results, we conclude that methyl sinapate inhibits DNA excision repair, thus enhancing UV mutagenicity.     

Characterization of a Xenopus laevis skin peptidylglycine alpha-hydroxylating monooxygenase expressed in insect-cell culture.

The C-terminal amide structure of peptide hormones and neurotransmitters is synthesized via a two-step reaction catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase. A Xenopus laevis PHM expressed in insect-cell culture by the baculovirus-expression-vector system was purified to homogeneity and characterized. Using a newly established assay system for PHM, the kinetic features of this enzyme were investigated. As expected, the enzyme required copper ions, L-ascorbate and molecular oxygen for turnover. Salts like KI and KCl, and catalase stabilized the enzyme in the presence of L-ascorbate. The optimum pH value for the enzyme reaction was around six when Mes buffer was used and around seven when phosphate buffer was used under the same assay condition. Below pH 6, acetate, iodide and chloride ions activated the reaction. The kinetic analysis is consistent with a ping-pong mechanism with respect to peptide and L-ascorbate, and the peptide showed substrate inhibition. The substrate specificity of the enzyme at the penultimate position was examined by competitive assay using tripeptides with glycine at the C-termini and the inhibitory potency of these peptides in descending order was methionine > aromatic > non-polar amino acids.     

Development and characterization of ten novel microsatellite loci for the emu (Dromaius novaehollandiae) and genetic diversity of Japanese farm populations

Molecular Biology Reports - The emu (Dromaius novaehollandiae) is a useful poultry animal farmed for fat, meat, and eggs. Genetic structure and relationships among farmed emu populations in Japan...     

Leucine Dehydrogenase of a Thermophilic Anaerobe,Clostridium thermoaceticum: Gene Cloning,Purification and Characterization

The leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of 46%. The enzyme of E. coli- pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 15 min; at 55°C and various pH’s between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD⁺ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin- amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].     

The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases

We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal beta-glucosidases classified in subfamily B. We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant beta-glucosidase in the periplasm fraction of the recombinant yeast. A. kawachii can produce two extracellular beta-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound beta-glucosidase. A. kawachii in which the bglA gene was disrupted produced none of the three beta-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound beta-glucosidases in A. kawachii.     

A temperature-sensitive dcw1 mutant of Saccharomyces cerevisiae is cell cycle arrested with small buds which have aberrant cell walls

Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Deltadcw1 Deltadfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Deltadfg5). When DC61 cells were incubated at 37 degrees C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37 degrees C. At 37 degrees C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37 degrees C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37 degrees C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Deltadcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.     

Functional properties of wasabi and horseradish

Wasabi (Wasabi japonica) and horseradish (Cholearia arnoracia) are used as spices of daily foodstuffs. Allylisothiocyanate (AIT) is a potent component in both plants and occurs by grating them. It is well known that AIT shows inhibitory effect on the growth of food poisoning bacteria and fungi. In this work, several functional properties of roots and leaves from wasabi and horseradish were examined in vitro. Each sample showed peroxidase activity. They also exhibited antioxidative and superoxide scavenging potency. Antimutagenic activity was observed toward 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline [MeIQx], a well-known mutagen/carcinogen in broiled fish and meat. They also decreased His+ revertant colonies of 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone (MX) in the Ames test, a strong mutagen and carcinogen in chlorine disinfected tap water. Isolation of antimutagenic components in wasabi root was done. Three components including (-)-(R)-7-methylsulfinylheptyl isothiocyanate were identified. These data show that wasabi and horseradish might be potent functional foods for keeping human health.     

Molecular breeding of yeast with higher metal-adsorption capacity by expression of histidine-repeat insertion in the protein anchored to the cell wall

A fusion protein of hexa-histidine repeat (His) and glycosylphosphatidylinositol (GPI)-anchor region of Saccharomyces cerevisiae Cwp1 with Aspergillus oryzae Taka-amylase A (TAA) was expressed on the yeast cell surface. The expressed fusion protein (TAA-His-Cwp1) was localized on the cell wall and demonstrated amylolytic activity. In comparison with the TAA-Cwp1 expressing strain, these cells exhibited 1.6- to 2.8-fold higher adsorbing capacity for Cu(2+), Ni(2+), and Zn(2+).