Mother–calf interactions and social behavior development in Commerson’s dolphins (Cephalorhynchus commersonii)

Mother–calf interactions and the behaviors of mothers during separation from their calves were examined in four Commerson’s dolphin (Cephalorhynchus commersonii) mother–calf pairs. Four infants were observed: 56.8 h over 30 days from birth to 263 days of age, 36.9 h over 20 days from birth to 149 days of age, 10.4 h over 3 days from birth to 2 days of age, and 15.0 h over 3 days from birth to 2 days of age. All four pairs shared common characteristics in the rate and frequencies of mother–calf interactions and the behaviors of mothers during the first week of life. After the first week, individual differences in changes in the frequency of each behavior were observed. The three behaviors considered representative of maternal care (parallel swimming, synchronous breathing, and body-to-body contact) were frequently performed in the first week; thereafter, the frequencies declined. Separate behaviors of mothers were infrequent during the first week and increased with an increase in infants’ age. Bumping by infants increased with time, suggesting an increase in soliciting by calves and conflict between mothers and calves. The frequency of flipper-to-body rubbing also changed but in a complex manner, probably because the calves needed to learn how to perform this behavior from their mothers and because initiator and recipient of this behavior can be changed quickly.     

Minor Constituents of Japanese Ho-Leaf Oil

The main component of Japanese Ho-leaf oil has been shown to be (?)-linalool (80~90%), and the following twenty minor constituents newly have been identified; methyl vinyl ketone, methyl isobutyl ketone, mesityl oxide, β-pinene, myrcene, (+)-limonene, cis- and trans-ocimene, n-hexanol, cis-3-hexenol, cis- and trans-linalool oxide, (?)-1-terpinen-4-ol, (+)-cis and (+)-trans-2,6,6-trimethyl-2-vinyl-5-hydroxytetrahydropyran, citronellol, nerol, (+)-β-selinene, (+)-tagetonol and (?)-trans-hotrienol. (+)-Tagetonol and (?)-trans-hotrienol have been demonstrated to be (+)-3,7-dimethyl-3-hydroxy-1-octen-5-one (III) and (3R)-(?)-trans-3,7-dimethyl-3-hydroxy-1,5,7-octatriene (IX), respectively.     

Modification of Proteinase Inhibitor II in Adzuki Beans during Germination

We investigated the effects of compounds isolated from a methanolic extract of rose hips on melanin biosynthesis in B16 mouse melanoma cells and the possible mechanisms responsible for the inhibition of melanin biosynthesis. We found that, among the isolated compounds, quercetin was a particularly potent melanogenesis inhibitor. To reveal the mechanism for this inhibition, the effects on tyrosinase of B16 mouse melanoma were measured. Quercetin decreased the intracellular tyrosinase activity as well as the tyrosinase activity in a cell culture-free system. We also examined the cellular level of tyrosinase protein and found that quercetin dose-dependently inhibited tyrosinase protein expression. We consider from these results that the inhibition of melanogenesis by quercetin was due to the inhibition of both tyrosinase activity and of the protein expression.     

Carotenoid production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> achieved by metabolic engineering

The carotenoid synthetic genes, crtM and crtN, derived from Staphylococcus aureus, were introduced into B. subtilis, resulting in yellow pigmentation. Absorption maxima of pigments and MALDI-TOF mass spectrometry demonstrated that the pigmented strain accumulated two C₃₀ carotenoids, 4,4′-diapolycopene and 4,4′-diaponeurosporene. A survival test using H₂O₂ revealed that the pigmented strain was more resistant to oxidative stress than the strain harboring an empty-vector. These findings indicate that B. subtilis can produce carotenoids, and the strain accumulating the carotenoids, CarotenoBacillus, will become a basal host for production of C₃₀ carotenoids and evaluation of their antioxidative effects.     

Differentiation of Bacillus anthracis, B. cereus, and B. thuringiensis by Using Pulsed-Field Gel Electrophoresis

A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis. A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.     

Differential down-regulation of protein kinase C isozymes

Types I, II, and III protein kinase C have been shown to be products of, respectively, gamma, beta, and alpha genes of this enzyme family (Huang, F. L., Yoshida, Y., Nakabayashi, H., Knopf, J. L., Young, W. S., III, and Huang, K.-P. (1987) Biochem. Biophys. Res. Commun. 149, 946-952). Incubation of the highly purified rat brain protein kinase C isozymes with trypsin (kinase/trypsin (w/w) = 100) under identical conditions results in a preferential degradation of types I and II enzymes, whereas the type III enzyme was relatively resistant to tryptic proteolysis. Degradation of the type III enzyme by trypsin could be facilitated with the addition of Ca2+, phosphatidylserine, and dioleoylglycerol; none of these components alone was effective. Limited proteolysis of the three protein kinase C isozymes generated distinctive fragments for each isozyme, indicating that each isozyme has different trypsin-sensitive sites. Tryptic digestion of the type III protein kinase C was used as a model to determine the effects of various modulators on protein kinase C degradation. While Ca2+ and phosphatidylserine together were sufficient to convert the type III protein kinase C from a trypsin-insensitive to a -sensitive form, addition of dioleoylglycerol greatly reduced the Ca2+ requirement for such a conversion. Among the various phospholipids tested, in the presence of either dioleoylglycerol or phorbol ester, phosphatidylserine, cardiolipin, and phosphatidic acid were the most effective, and phosphatidylcholine and phosphatidylethanolamine were the least effective in supporting the digestion of type III protein kinase. Other acidic phospholipids, such as lysophosphatidylserine and phosphatidylinositol, were also effective in supporting the degradation in the presence of phorbol ester but not in the presence of dioleoylglycerol. The relevance of these proteolytic reactions to physiological responses was assessed with phorbol ester on rat basophilic leukemia RBL-2H3 cells, which contained both types II and III protein kinase C. Immunoblot analysis with the isozyme-specific antibodies revealed that phorbol ester induced a faster degradation of type II than that of type III isozyme in these cells. The results demonstrate that the various protein kinase C isozymes have different susceptibilities to proteolysis in vitro, when tested with trypsin, as well as to endogenous proteases in intact cells.     

Purification and characterization of a protease from Xenopus embryos

A proteolytic enzyme was purified from Xenopus embryos. The purification procedure consisted of fractionation of an extract of embryos with acetone, gel filtration of Sephadex G-75 and chromatography on carboxymethyl-cellulose and hydroxylapatite. The preparation of enzyme appeared to be homogeneous as judged by electrophoresis in polyacrylamide gels. This protease had a molecular mass of 43-44 kDa and was composed of two subunits with molecular masses of 30 kDa and 13 kDa. The optimal pH of the reaction catalysed by the protease was approximately 4.0. This proteolytic activity was inhibited by antipain, leupeptin and iodoacetic acid; it was not affected by phenylmethylsulfonyl fluoride and pepstatin; and it was enhanced by dithiothreitol. In the presence of RNA, the optimal pH was shifted from pH 4.0 to pH 4.5. The protease was activated by addition of total RNA from Xenopus embryos, by poly(rU) or poly(rG). In contrast, after addition of tRNA or poly(rC), no activation of the protease was observed.     

A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P-glycoproteins

Screening for leptomycin B (LMB)-resistant transformants in a gene library constructed in Schizosaccharomyces pombe with the chromosomal DNA of an LMB-resistant mutant of S. pombe and with multicopy plasmid pDB248' as the vector led to the isolation of a gene, named pmd1+, encoding a 1362-amino-acid protein. This protein showed great similarity in amino acid sequence to the mammalian P-glycoprotein encoded by the multidrug resistance gene, mdr, and the Saccharomyces cerevisiae a-factor transporter encoded by STE6. In addition, computer analyses predicted that the protein encoded by pmd1+ formed an intramolecular duplicated structure and each of the halves contained six transmembrane regions as well as two ATP-binding domains, as observed with the P-glycoproteins and the STE6 product. Consistent with this was that S. pombe cells containing the pmd1+ gene on a multicopy plasmid showed resistance not only to LMB but also to several cytotoxic agents. The pmd1 null mutants derived by gene disruption were viable and hypersensitive to these agents. All these data suggest that the pmd1+ gene encodes a protein that is a structural and functional counterpart of mammalian mdr proteins.     

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.