Characterization of developmental colony formation in Corynebacterium glutamicum.

We report that Corynebacterium glutamicum colonies exhibit a developmental transition in culture. When cultured on a routinely used complete medium (CM2B), this bacterium first formed a flat translucent colony. Subsequently, some parts of this colony expanded to form small spherical yellow colonies that finally developed into a single large yellow colony. The small flat colony consisted of long thick cells, which were occasionally V or Y shaped, while the large yellow colony consisted of short small rods. A similar colony development pattern was observed in Corynebacterium ammoniagenes and Corynebacterium callunae. Analysis following shotgun cloning revealed that the introduction of a multi-copy-number plasmid carrying amtR, a global transcriptional regulator for nitrogen metabolism, into C. glutamicum cells induced precocious colony development. An amtR-null C. glutamicum mutant exhibited delayed development. Detailed observations of C. glutamicum cells cultured on CM2B medium containing buffers at various pH values revealed that the colony growth was rapid at a pH value of 6.4 or higher and slow but distinct at a pH of less than 6.4. This pH threshold increased to 6.8 following the addition of 0.1% glucose into the medium.

Oral treatment with L-lysine and L-arginine reduces anxiety and basal cortisol levels in healthy humans.

Dietary supplementation with an essential amino acid L-lysine has been shown to reduce chronic anxiety in humans with low dietary intake of L-lysine. A combination of L-lysine and L-arginine has been documented to normalize hormonal stress responses in humans with high trait anxiety. The present study was carried out in one hundred eight healthy Japanese adults. The aim of study was to find out whether a week-long oral treatment with L-lysine (2.64 g per day) and L-arginine (2.64 g per day) reduces trait and stress-induced state anxiety and basal levels of stress hormones. We confirmed that, without regard to gender, the amino acid treatment significantly reduced both trait anxiety and state anxiety induced by cognitive stress battery. In addition, we found that the treatment with L-lysine and L-arginine decreased the basal levels of salivary cortisol and chromogranin-A (a salivary marker of the sympatho-adrenal system) in male subjects. These results of this double-blind, placebo controlled and randomized study confirm the previous findings in humans and animals and point to a combination of L-lysine and L-arginine as a potentially useful dietary intervention in otherwise healthy humans with high subjective levels of mental stress and anxiety.

Cloning CYP2D21 and CYP3A22 cDNAs from liver of miniature pigs.

To compare the identity of the primary structure of drug-metabolizing cytochrome P450 between miniature pigs and humans, two cDNA clones, coding for miniature pig CYP2D21 and CYP3A22, were isolated. The deduced amino acid sequences of CYP2D21 and CYP3A22 were 78.3 and 75.0% identical to human CYP2D6 and CYP3A4, respectively. These values were nearly the same as those of bovine, dog, and some rodent isoforms, and 12.2 to 18.4% lower than those of nonhuman primates such as cynomolgus monkeys, Japanese monkey, and marmosets. These data indicate that miniature pig P450s are genetically not so close as monkey P450s to human P450s as previously expected. The recombinant CYP2D21 enzyme, however, showed bufuralol 1'-hydroxylase activity, suggesting that miniature pig CYP2D21 is capable of metabolizing some of the same substrates associated with human CYP2D6 despite its low identity to human counterparts.

Transglutaminase in sporulating cells of Bacillus subtilis.

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.