Protein phosphatase-type 2B is involved in the regulation of the acrosome reaction but not in the temperature-dependent flagellar movement of fowl spermatozoa

The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenized inner perivitelline layers (IPVL) prepared from laid fowl eggs, was almost negligible at 40 degrees C. However, motility became vigorous even at 40 degrees C when 2 mmol CaCl2/l was added, and the acrosome reaction was also stimulated in the presence, but not in the absence, of IPVL. The presence of deltamethrin or fenvalerate, specific inhibitors of protein phosphatase-type 2B (PP2B), did not permit the restoration of motility at 40 degrees C but, in the presence of IPVL, these compounds stimulated the acrosome reaction in a dose-dependent manner in the range of 1-1000 nmol/l. These results suggest that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca2+ plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of the acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e. protein dephosphorylation by PP2B in the former but not in the latter case.     

Production of two types of ice crystal-controlling proteins in Antarctic bacterium

Pseudomonas fluorescens KUAF-68, which was isolated from Antarctica, had both ice-nucleating protein and antifreeze protein activities in the culture broth. We found that both proteins were separately produced based on the results of column chromatography, SDS-PAGE analysis and Southern hybridization. The activity of the ice-nucleating protein was stimulated by the addition of glycine (0.020 N%), whereas the activity of the antifreeze protein was stimulated by the addition of L-asparagine (0.025 N%). This is the first report on the production of two types of ice crystal-controlling proteins in one bacterial strain.     

Characterization and molecular cloning of cellobiose dehydrogenase from the brown-rot fungus Coniophora puteana

Cellobiose dehydrogenase (CDH) was purified from the brown-rot fungus Coniophora puteana grown in culture containing crystalline cellulose as a carbon source. The purified enzyme gave a single band at 115 kDa on SDS-PAGE and showed a typical flavocytochrome absorption spectrum. The enzyme oxidized both cellobiose and cellooligosaccharides, but not their monomer, glucose, suggesting typical kinetic features of CDH. A cDNA encoding CDH was cloned by RT-PCR using primers designed from the consensus sequences of known CDHs from white-rot fungi. The cDNA consists of 2448 bp, including an open reading frame encoding the 18 amino acids of the putative signal peptide and the 756 amino acids of the mature protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data for tryptic fragments of the purified C. puteana CDH were consistent with partial amino acid sequences of the mature protein deduced from the cloned cDNA. Moreover, the sequences contained common characteristics of CDH, i.e., two possible residues for a heme ligand (Met 64 and His 160), a flavin-binding motif, and two glucose-methanol-choline oxidoreductase motifs. This is the first cloning of CDH from a brown-rot fungus, and the results suggest structural and kinetic similarity of C. puteana CDH to white-rot fungal CDHs.     

Cloning and analysis of the AWA1 gene of a nonfoaming mutant of a sake yeast

Almost all sake yeasts form a thick foam layer on sake mash during fermentation. To reduce the amount of foam, nonfoaming mutants were bred from foam-forming sake yeasts. To elucidate the mechanism of this foam formation, we have cloned a gene from a foam-forming sake yeast that confers foam-forming ability to a nonfoaming mutant. This gene, named AWA1, encodes a glycosylphosphatidylinositol (GPI) anchor protein that is localized to the cell wall and is required for cell surface hydrophobicity. In this paper, we describe the genomic analysis of the AWA1 gene in a nonfoaming mutant strain K701 derived from a foam-forming sake yeast strain K7. K701-AWA1 was cloned in a cosmid and its sequence was compared with that of K7-AWA1. Although the 5' half of K701-AWA1 was identical to that of K7-AWA1, the 3' half of K701-AWA1 was different from that of K7-AWA1, resulting in a loss of the C-terminal hydrophobic sequence of Awa1p. Since this sequence is considered to be required for the anchoring of Awa1p to the cell wall, K7-Awa1p could not confer both cell surface hydrophobicity and foam-forming ability to strain K701 cells. Since the change found in K701-AWA1 was not a point mutation but a larger scale event, we analyzed chromosome rearrangement by pulsed-field gel electrophoresis Southern blot analyses. The results suggest that the left subtelomeric region of chromosome IX in strain K7 was translocated to the AWA1 gene in chromosome XV by a nonreciprocal recombination.     

Assessing the association between phenolic compounds and the antioxidant activity of Brazilian red wines using chemometrics

The objective of this study was to evaluate the association among chemical parameters, the commercial value, and the antioxidant activity of Brazilian red wines using chemometric techniques. Twenty-nine samples from five different varieties were assessed. Samples were separated into three groups using hierarchical cluster analysis: cluster 1 presented the highest antioxidant activity towards DPPH (68.51% of inhibition) and ORAC (30,918.64 μmol Trolox Equivalents/L), followed by cluster 3 (DPPH = 59.36% of inhibition; ORAC = 25,255.02 μmol Trolox Equivalents/L) and then cluster 2 (DPPH = 46.67% of inhibition; ORAC = 19,395.74 μmol Trolox Equivalents/L). Although the correlation between the commercial value and the antioxidant activity on DPPH and ORAC was not statistically significant (P = 0.13 and P = 0.06, respectively), cluster 1 grouped the samples with higher commercial values. Cluster analysis applied to the variables suggested that non-anthocyanin flavonoids were the main phenolic class exerting antioxidant activity on Brazilian red wines.     

Eukaryotic translation initiation factor 1 (eIF1), the inspector of good AUG context for translation initiation,has an extremely bad AUG context

The nucleotide sequence surrounding the translation initiation AUG codon (AUG context) is important for the effective translation initiation. A compilation analysis revealed that all the genes of the eukaryotic translation initiation factor 1, which plays a crucial role in the recognition of the optimal AUG context, ironically have extremely bad AUG contexts.     

Comparison of three Chlamydomonas strains which show distinctive oxidative stress tolerance

Methyl viologen (MV) causes severe oxidative stress by generating superoxide in the photosystem. The marine Chlamydomonas strain W80 is highly tolerant to MV (inhibitory concentration 50% [IC??]=110 μM), and another marine Chlamydomonas strain HS5 shows also relatively a high tolerance (IC??=12 μM). These two marine strains and a freshwater Chlamydomonas reinhardtii, which is highly sensitive to MV (IC??=0.03 μM), were compared with respect to their reactive oxygen species (ROS) eliminating enzymes (superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase), intracellular free amino acids, and antioxidant activities of the cell extracts. The marked difference between the marine Chlamydomonas strains and C. reinhardtii is the much higher (more than 5 fold) ascorbate peroxidase (APX) activity in the marine strains. The marine strains also kept the high APX activities (more than 100% of non-stressed condition) under the MV stressed condition, while the APX activity in C. reinhardtii was significantly decreased (36% of non-stressed condition) under the stressed condition, indicating that APX activity potentially contributes to the oxidative stress tolerance in Chlamydomonas. In addition, the levels of intracellular free proline, which is supposed to ameliorate oxidative stress, were several tens of times higher in the marine Chlamydomonas strains than in C. reinhardtii.     

QTL mapping of sake brewing characteristics of yeast

A haploid sake yeast strain derived from the commercial diploid sake yeast strain Kyokai no. 7 showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small-scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in the specification of sake brewing characteristics such as ethanol fermentation and the production of aromatic components.     

Administration of biotin prevents the development of insulin resistance in the skeletal muscles of Otsuka Long-Evans Tokushima Fatty rats

Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for type 2 diabetes mellitus. In the present study, we investigated whether pharmacologic doses of biotin have the potential to abate insulin resistance in the skeletal muscles of OLETF rats. OLETF rats (34 weeks of age) were divided into 2 groups and given distilled water (OLETF-control group) or distilled water containing 3.3 mg L(-1) of biotin (OLETF-biotin group) for 8 weeks. At the end of experimental period, the OLETF-control rats developed severe hyperglycemia and hyperinsulinemia, whereas the OLETF-biotin rats showed significantly smaller responses to oral glucose tolerance test than the OLETF-control rats. The glucose uptake in the hind limbs of the rats was significantly higher in the OLETF-biotin group than in the OLETF-control group. Biotin administration increased the glucose transporter type 4 (GLUT4) protein content in the total membrane fraction but had little effect on the GLUT4 content in the plasma membrane fraction. These results indicate that administration of a pharmacological dose of biotin prevents the development of insulin resistance in the skeletal muscles of OLETF rats presumably via an increase in GLUT4 protein expression but not via GLUT4 translocation.