Seasonal change in xylem growth of <Emphasis Type="Italic">Pinus densiflora</Emphasis> in central Japan

This study investigated the seasonal change in xylem growth of Japanese red pine (Pinus densiflora). Wood cores were sampled at 2-week intervals from April to November in 2012 using the microcoring method. Daily increment rates of tracheid number and tree-ring width were compared with seasonal changes in daily mean temperature and photoperiod. Xylem growth started in early to late May and stopped in late October to early November. The maximum daily increment rates of tracheid number and tree-ring width were in early July. The 95 % confidence intervals of the timing of the maximum daily increment rates included the summer solstice (23 June) with the longest photoperiod, but not the warmest day (30 July). The maximum daily increment rate of xylem growth is thought to be controlled by the photoperiod rather than by temperature. The daily mean temperature exceeded 20 °C after the summer solstice, indicating that temperature is not a limiting factor for xylem growth. This study suggests that the timing of maximum daily increment rates of xylem growth of P. densiflora is controlled by the photoperiod.     

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.     

Improvement of neural stem cell survival in collagen hydrogels by incorporating laminin-derived cell adhesive polypeptides

Cell transplantation is a potential methodology for the treatment of Parkinson's disease. However, the therapeutic effect is limited by poor viability of transplanted cells. To overcome this problem, we hypothesized that a dual step approach, whereby providing an adhesive substrate for transplanted cells and, at the same time, by preventing the infiltration of activated microglia into the site of transplantation promotes the cell survival. To establish above conditions, attempts were made to prepare 3-D matrices using collagen hydrogels that incorporated integrin-binding polypeptides derived from laminin-1. Tandem combinations of laminin globular domains as well as a single globular domain 3 were prepared using recombinant DNA technology as a fusion with hexahistidine and bound to metal chelated surfaces to screen for the adhesion and proliferation of neural stem cells (NSCs). In addition, a small peptide derived from laminin γ1 chain was prepared and heterodimerized with the globular domain-containing chimeric proteins to evaluate for the enhancement of integrin-mediated cell adhesion. As a result, a heterodimer consisting of the globular domain 3 of the laminin α1 chain and the peptide from the laminin γ1 chain was selected as the best candidate among the polypeptides studied here for the incorporation into a collagen hydrogel. It was shown that the survival of NSCs was indeed promoted in the collagen hydrogel incorporating the heterodimer compared to the pure collagen hydrogel.     

Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

Induction by mycoplasmas of tumor necrosis factor-alpha from macrophages

Several species of mycoplasmas including M. pneumoniae, the causative agent of human respiratory infection, were investigated for tumor necrosis factor-alpha (TNF-alpha) induction. The cytotoxic activity to Meth A cells of peritoneal macrophages purified from BALB/c mice was enhanced markedly when cultured with either viable or nonviable mycoplasmas. The supernatant of macrophage culture mixed with mycoplasmas, M. pneumoniae or A. laidlawii, showed a potent cytotoxic activity to TNF-alpha-sensitive but not to TNA-alpha-insensitive L cells. Addition of anti-TNA-alpha antiserum inhibited completely the cytotoxic activity of the supernatant, indicating that the cytotoxic activity is due mostly to TNF-alpha. These results strongly suggest that mycoplasmas possess an activity to induce TNF-alpha, which enhances the cytotoxic activity of macrophages and prevent infection with mycoplasmas in vivo.     

Comparison of stress responses between mental tasks and white noise exposure

The purpose of this study was to compare the cardiovascular responses to different types of mental stress. Ten healthy males performed a mental arithmetic task (MA) on one day and were exposed to white noise (WN, 80dB) on another day. Both the MA and the WN were composed of four 5-min consecutive periods with a 3-min rest between them. On each day, the systolic and diastolic blood pressure (SBP and DBP), mean arterial pressure (MAP), cardiac output (CO), and total peripheral resistance (TPR) were measured continually during the entire experimental period. The changes from the baseline (Delta) in all periods were calculated for both mental stresses. As for the results, the DeltaMAP, DeltaCO, DeltaHR, and DeltaTPR in the MA did not significantly change during the task periods. However, in the WN, the DeltaMAP and DeltaTPR showed significant increases over the time of the consecutive periods. In addition, we discuss the response patterns for the two mental stresses. We examine three hemodynamic reactivity patterns: a cardiac pattern characterized by increased CO and decreased TPR, a mixed pattern characterized by a moderate increase in both CO and TPR, and a vascular pattern characterized by increased TPR and decreased CO. The results show that throughout all task/exposure periods, the response pattern remained the same for six subjects in each stress. Furthermore, of these six subjects, half showed the same response pattern in both the MA and the WN. In conclusion, compared to the MA task, consecutive WN exposure showed an accumulation of stress responses. A change in TPR contributed to a gradual increase in MAP in the WN. It is also possible that among the subjects there were different types of response to the MA and WN.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.