Biochemical Studies on the Mineral Components in Sake Yeast

The critical concentrations of minerals in a growing medium for maximum fermentation of yeast were as follows: P, 1 mmol/1; Mg, 0.2 mmol/1; and K, 1~2 mmol/1. These values are lower than those for the saturation of the cells with each mineral. The order of the concentration for maximum fermentation (K>P>Mg) is in agreement with that for yeast growth.

Only a small amount of mineral salt was required to increase the fermentative activity. Very small increase of fermentative activity was observed when the starved yeast was enriched with corresponding minerals by incubating cells with the mineral salt and glucose.     

The daidzein- and estradiol- induced anorectic action in CCK or leptin receptor deficiency rats

We investigated the effect of daidzein feeding and estradiol treatment on food intake in cholecystokinin-1 receptor (CCK1R) deficiency, leptin receptor (ObRb) deficiency rats and their wild-type rats. These rats underwent an ovariectomy or a sham operation. For the 5 week experiment, each rat was divided in three groups: control, daidzein (150 mg/kg diet), and estradiol (4.2 μg/rat/day) groups. In both CCK1R+ and CCK1R? rats, daidzein feeding and estradiol treatment significantly decreased food intake. Daidzein feeding significantly reduced food intake in ovariectomized ObRb? rats, although not in ObRb+ rats. Estradiol treatment significantly lowered food intake in ovariectomized ObRb+ and ObRb? rats. In the ovariectomized rats, estradiol treatment significantly increases uterine weight, while daidzein feeding did not change it, suggesting that daidzein might have no or weak estrogenic effect in our experiment. These results suggest that CCK1R and ObRb signalings were not essential for the daidzein- and estradiol-induced anorectic action.     

Regional Cardiac Dysfunction and Dyssynchrony in a Murine Model of Afterload Stress

Small animal models of afterload stress have contributed much to our present understanding of the progression from hypertension to heart failure. High-sensitivity methods for phenotyping cardiac function in vivo, particular in the setting of compensated cardiac hypertrophy, may add new information regarding alterations in cardiac performance that can occur even during the earliest stages of exposure to pressure overload. We have developed an echocardiographic analytical method, based on speckle-tracking-based strain analyses, and used this tool to rapidly phenotype cardiac changes resulting from afterload stress in a small animal model. Adult mice were subjected to ascending aortic constriction, with and without subsequent reversal of the pressure gradient. In this model of compensated hypertrophic cardiac remodeling, conventional echocardiographic measurements did not detect changes in left ventricular (LV) function at the early time points examined. Strain analyses, however, revealed a decrement in basal longitudinal myofiber shortening that was induced by aortic constriction and improved following relief of the pressure gradient. Furthermore, we observed that pressure overload resulted in LV segmental dyssynchrony that was attenuated with return of the afterload to baseline levels. Herein, we describe the use of echocardiographic strain analyses for cardiac phenotyping in a mouse model of pressure overload. This method provides evidence of dyssynchrony and regional myocardial dysfunction that occurs early with compensatory hypertrophy, and improves following relief of aortic constriction. Importantly, these findings illustrate the utility of a rapid, non-invasive method for characterizing early cardiac dysfunction, not detectable by conventional echocardiography, following afterload stress.     

Tyrosinase‐catalyzed oxidation of resveratrol produces a highly reactive ortho‐quinone: Implications for melanocyte toxicity

trans‐Resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene, RES), a naturally occurring polyphenol, has recently attracted increased interest as a health‐beneficial agent. However, based on its p‐substituted phenol structure, RES is expected to be a substrate for tyrosinase and to produce a toxic o‐quinone metabolite. The results of this study demonstrate that the oxidation of RES by tyrosinase produces 4‐(3′,5′‐dihydroxy‐trans‐styrenyl)‐1,2‐benzoquinone (RES‐quinone), which decays rapidly to an oligomeric product (RES‐oligomer). RES‐quinone was identified after reduction to its corresponding catechol, known as piceatannol. RES‐quinone reacts with N‐acetylcysteine, a small thiol, to form a diadduct and a triadduct, which were identified by NMR and MS analyses. The production of a triadduct is not common for o‐quinones, suggesting a high reactivity of RES‐quinone. RES‐quinone also binds to bovine serum albumin through its cysteine residue. RES‐oligomer can oxidize GSH to GSSG, indicating its pro‐oxidant activity. These results suggest that RES could be cytotoxic to melanocytes due to the binding of RES‐quinone to thiol proteins.     

Affinity Improvement of a Cancer-Targeted Antibody through Alanine-Induced Adjustment of Antigen-Antibody Interface

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Structural transition of a 15 amino acid residue peptide induced by GM1

The ganglioside GM1-binding peptide, p3, with a sequence of VWRLLAPPFSNRLLP, displayed a clear structural alteration depending on the presence or absence of GM1 micelles. The three-dimensional structures of the p3 peptide in the free and GM1 bound states were analyzed using two-dimensional NMR spectroscopic experiments with distance-restrained simulated annealing calculations. The NMR experiments for the p3 peptide alone indicated that the peptide has two conformers derived from the exchange of cis and trans forms at Pro(7)-Pro(8). Further study with theoretical modeling revealed that the p3 peptide has a curb conformation without regular secondary structure. On the other hand, the NMR studies for the p3 peptide with the GM1 micelles elucidated a trans conformer and gave a structure stabilized by hydrophobic interactions of beta- and helical turns. Based on these structural investigations, tryptophan, a core residue of the hydrophobic cluster, might be an essential residue for the recognition of the GM1 saccharides. The dynamic transition of the p3 peptide may play an important role in the function of GM1 as a multiple receptor as in the traditional pathway of the infection by cholera toxin.     

Enhanced accumulation of Cd2+ by a Mesorhizobium sp. transformed with a gene from Arabidopsis thaliana coding for phytochelatin synthase

We expressed the Arabidopsis thaliana gene for phytochelatin synthase (PCS(At)) in Mesorhizobium huakuii subsp. rengei B3, a microsymbiont of Astragalus sinicus, a legume used as manure. The PCS(At) gene was expressed under the control of the nifH promoter, which regulates the nodule-specific expression of the nifH gene. The expression of the PCS(At) gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [(gamma-Glu-Cys)(n)-Gly; PCs] in strain B3. A range of PCs, with values of n from 2 to 7, was synthesized by cells that expressed the PCS(At) gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl(2) activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd(2+)/mg (dry weight) of cells. The expression of the PCS(At) gene in M. huakuii subsp. rengei B3 increased the ability of cells to bind Cd(2+) approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of A. sinicus containing the PCS(At) gene. When recombinant M. huakuii subsp. rengei B3 established the symbiotic relationship with A. sinicus, the symbionts increased Cd(2+) accumulation in nodules 1.5-fold.     

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.     

Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation.

BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.