Direct Effect of Sodium Nitrite on Extracted Histones

Irpex lacteus milk-clotting enzyme hydrolyzed the Phe(105)-Met(106) bond of κ-casein, causing the precipitation of para-κ-casein along with other casein fractions in the presence of calcium ions, with a mechanism similar to other milk-clotting enzymes. Furhtermore, Irpex enzyme hydrolyzed at the positions Leu(79)-Ser(80) and Tyr(30)-Val(31) of para-κ-casein.

Degradation patterns of β-casein by Irpex and Mucor miehei enzymes were almost the same by polyacrylamide gel electrophoresis, but Endothia parasitica enzyme showed a different degradation pattern. Under the conditions employed, β-casein appeared to be scarcely hydrolyzed by chymosin.

Comparing the specificity of Irpex enzyme on β-casein with that of chymosin, the common cleaving points were Leu(165)-Ser(166), Ala(189)-Phe(190), and Tyr(192)-Glu(193). The difference in the specificity between the enzymes was exhibited in the cleavage at the Leu(139)-Leu(140) bond by chymosin and of the Ser(142)-Trp(143) bond by Irpex enzyme. Although the cleaving points of β-casein by both enzymes resembled each other, each enzyme exhibited different degradation patterns of β-casein because of thier different order of cleavage.     

IL-17-mediated regulation of innate and acquired immune response against pulmonary Mycobacterium bovis bacille Calmette-Guerin infection

IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against intracellular bacterial infection remains unclear. In the present study, we demonstrate that IL-17 is an important cytokine not only in the early neutrophil-mediated inflammatory response, but also in T cell-mediated IFN-gamma production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that gammadelta T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN-gamma in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection.     

Efficient cross-presentation by heat shock protein 90-peptide complex-loaded dendritic cells via an endosomal pathway

It is well-established that heat shock proteins (HSPs)-peptides complexes elicit antitumor responses in prophylactic and therapeutic immunization protocols. HSPs such as gp96 and Hsp70 have been demonstrated to undergo receptor-mediated uptake by APCs with subsequent representation of the HSP-associated peptides to MHC class I molecules on APCs, facilitating efficient cross-presentation. On the contrary, despite its abundant expression among HSPs in the cytosol, the role of Hsp90 for the cross-presentation remains unknown. We show here that exogenous Hsp90-peptide complexes can gain access to the MHC class I presentation pathway and cause cross-presentation by bone marrow-derived dendritic cells. Interestingly, this presentation is TAP independent, and followed chloroquine, leupeptin-sensitive, as well as cathepsin S-dependent endosomal pathways. In addition, we show that Hsp90-chaperoned precursor peptides are processed and transferred onto MHC class I molecules in the endosomal compartment. Furthermore, we demonstrate that immunization with Hsp90-peptide complexes induce Ag-specific CD8(+) T cell responses and strong antitumor immunity in vivo. These findings have significant implications for the design of T cell-based cancer immunotherapy.     

Behavioral thermoregulation of wild Japanese macaques: comparisons between two subpopulations

We studied the behavioral thermoregulation of Japanese macaques in two troops that live in the coniferous (1,000-1,200 m in elevation) and coastal forests (0-200 m in elevation) of Yakushima. Frequency of sunbathing, huddling, and microhabitat selection during inactivity was compared. The difference in mean annual air temperature between the forests was more than 7 degrees C. In both forests, when the weather was clear, macaques spent more time being inactive in the sunshine in winter than in autumn. In winter, they huddled more often when it was clear than when cloudy. Microhabitat selection to stay in the sunshine during winter differed between the two forests. In winter, macaques spent more time inactive in open habitats in the coniferous forest and in the trees in the coastal forest than in autumn, respectively. This difference is related to the lower crown height in the coastal forest and the large open habitats (logged area) available only in the coniferous forest. In winter, skin temperature measured by temperature-sensitive transmitters was 1.32-1.71 degrees C higher when sunbathing, and 0.83-4.75 degrees C higher when huddling than staying in the shade without huddling. In winter, the proportion with which they stayed in the sunshine or huddled in winter did not differ between the two forests, in spite of the difference in air temperature. This suggests that Japanese macaques respond to seasonal changes in air temperature, not the absolute temperature, and that they acclimatize themselves to thermal conditions that require behavioral thermoregulation only during the season when thermoregulation is most costly.     

RNA Cytidine Acetyltransferase of Small-Subunit Ribosomal RNA: Identification of Acetylation Sites and the Responsible Acetyltransferase in Fission Yeast,Schizosaccharomyces pombe

The eukaryotic small-subunit (SSU) ribosomal RNA (rRNA) has two evolutionarily conserved acetylcytidines. However, the acetylation sites and the acetyltransferase responsible for the acetylation have not been identified. We performed a comprehensive MS-based analysis covering the entire sequence of the fission yeast, Schizosaccharomyces pombe, SSU rRNA and identified two acetylcytidines at positions 1297 and 1815 in the 3′ half of the rRNA. To identify the enzyme responsible for the cytidine acetylation, we searched for an S. pombe gene homologous to TmcA, a bacterial tRNA N-acetyltransferase, and found one potential candidate, Nat10. A temperature-sensitive strain of Nat10 with a mutation in the Walker A type ATP-binding motif abolished the cytidine acetylation in SSU rRNA, and the wild-type Nat10 supplemented to this strain recovered the acetylation, providing evidence that Nat10 is necessary for acetylation of SSU rRNA. The Nat10 mutant strain showed a slow-growth phenotype and was defective in forming the SSU rRNA from the precursor RNA, suggesting that cytidine acetylation is necessary for ribosome assembly.     

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor β and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.     

A PCR-based method to test for the presence or absence of β-conglycinin α′- and α-subunits in soybean

The soybean cultivar Yumeminori, which lacks the α′- and α-subunits of β-conglycinin, carries both naturally occurring and induced mutations. While the cause of the natural mutation resulting in the α′-subunit deficiency has been determined, the induced mutation in the CG-2 gene encoding the α-subunit has not been characterized at the molecular level. In this study, we identified a four base pair insertion in the first exon of CG-2, which introduced a premature stop codon. The insertion co-segregated with the lack of α-subunit, indicating that this mutation is the cause of the α-subunit deficiency. A multiplex PCR method of testing for the presence or absence of α′- and α-subunits was developed based on the sequences of mutated and wild-type alleles. This PCR-based test was also capable of detecting the presence of wild-type genes when Yumeminori DNA samples were contaminated with wild-type DNA at levels of 0.2% or greater. Thus, this method will be useful both for marker-assisted selection in soybean breeding programs, and for seed purity tests in food industries.     

Characterization of the 105-kDa molecular chaperone. Identification, biochemical properties, and localization.

We have characterized the biochemical properties of the testis and brain-specific 105-kDa protein which is cross-reacted with an anti-bovine HSP90 antibody. The protein was induced in germ cells by heat stress, resulting in a protein which is one of the heat shock proteins [Kumagai, J., Fukuda, J., Kodama, H., Murata, M., Kawamura, K., Itoh, H. & Tanaka, T. (2000) Eur. J. Biochem.267, 3073-3078]. In the present study, we characterized the biochemical properties of the protein. The 105-kDa protein inhibited the aggregation of citrate synthase as a molecular chaperone in vitro. ATP/MgCl2 has a slight influence of the suppression of the citrate synthase aggregation by the 105-kDa protein. The protein possessed chaperone activity. The protein was able to bind to ATP-Sepharose like the other molecular chaperone HSP70. A partial amino-acid sequence (24 amino-acid residues) of the protein was determined and coincided with those of the mouse testis- and brain-specific APG-1 and osmotic stress protein 94 (OSP94). The 105-kDa protein was detected only in the medulla of the rat kidney sections similar to OSP94 upon immunoblotting. The purified 105-kDa protein was cross-reacted with an antibody against APG-1. These results suggested that APG-1 and OSP94 are both identical to the 105-kDa protein. There were highly homologous regions between the 105-kDa protein/APG-1/OSP94 and HSP90. The region of HSP90 was also an immunoreactive site. An anti-bovine HSP90 antibody may cross-react with the 105-kDa protein similar to HSP90 in the rat testis and brain. We have investigated the localization and developmental induction of the protein in the rat brain. In the immunohistochemical analysis, the protein was mainly detected in the cytoplasm of the nerve and glial cells of the rat brain. Although the 105-kDa protein was localized in all rat brain segments, the expression pattern was fast in the cerebral cortex and hippocampus and slow in the cerebellum.     

Ribosomal DNA distribution in somatic chromosomes of Stangeria eriopus (Stangeriaceae, Cycadales) and molecular-cytotaxonomic relationships to some other cycad genera

Somatic chromosomes ofStangeria eriopus (Stangeriaceae, Cycadales) were investigated by fluorescentin situ hybridization (FISH) using an 18S ribosomal DNA (rDNA) probe.Stangeria eriopus showed a chromosome number of 2n=16 with a karyotype of 12 median-, 2 subterminal-, and 2 terminal-centromeric chromosomes. FISH study ofS. eriopus revealed 16 signals made up of rDNA sites located on the terminal regions of the long arms of the 7 median- and 2 subterminal-centromeric chromosomes, on terminal region of the short arm of the 1 median-centromeric chromosome, on the terminal regions of the long and the short arms of 1 median- and 2 terminal-centromeric chromosomes. This result suggests that, not only karyomorphologically but also molecular-cytologically, the genusStangeria may be more closely related to the genusCeratozamia than the genusBowenia or the genusMicrocycas previously hypothesized.     

Comparison of energy balance between two different-sized groups of Japanese macaques (<Emphasis Type="Italic">Macaca fuscata yakui</Emphasis>)

Quantifying the energy balance is essential for testing socio-ecological models. To reveal costs and benefits of group living in Japanese macaques from the perspective of feeding competition, Kurihara and Hanya (Am J Primatol 77:986–1000, 2015) previously compared feeding behavior between two different-sized groups of macaques (larger group 30–35 individuals; smaller group 13–15 individuals) in the coastal forest of Yakushima, Japan. The results suggested that the larger group exhibited greater feeding effort because of intragroup scramble competition and that the smaller group suffered from higher travel costs, possibly owing to intergroup contest competition. However, it remained unclear whether the behavioral differences affected their energy budgets. The present study examined energetic consequences of the different feeding behaviors in the two groups. Using behavioral data from 10 to 13 adult females and nutritional composition of food items, we compared ingestion rates, energetic/nutritional content of diet, and energy budgets between the two groups. Ingestion rates and energetic/nutritional content of diet did not differ between the two groups. Despite the higher feeding effort of the larger group, energy intake did not differ between the two groups. Energy expenditure did not differ between the two groups because higher travel costs were negated by lower feeding effort in the smaller group. Consequently, the energy balance did not differ between the two groups. We demonstrated that the behavioral measures of feeding competition were not translated into their energetic condition; moreover, our findings re-emphasize the importance of quantifying behavioral and fitness measures for interpreting variation in feeding behavior properly.