Genetic diversity of an endangered plant, <Emphasis Type="Italic">Cypripedium macranthos</Emphasis> var. <Emphasis Type="Italic">rebunense</Emphasis> (Orchidaceae): background genetic research for future conservation

Cypripedium macranthos var. rebunense is an endangered plant endemic to Rebun Island, Japan. A proper understanding of genetic diversity is needed when conducting conservation programs for rare and endangered species. We therefore examined the genetic diversity of C. macranthos var. rebunense using allozyme markers with a view to future conservation. Our study revealed that C. macranthos var. rebunense has relatively high genetic diversity (P was 0.62, n _a and n _e were 1.85 and 1.28 respectively, and H _o and H _e were 0.163 and 0.187, respectively) when compared with other plant taxa. The natural habitats of C. macranthos var. rebunense are geographically separated into northern and the southern populations. Disappearance of alleles and increase in homozygosity expected as a result of the bottleneck effect were observed, particularly in the southern populations composed of a small number of plants. As additional negative effects (inbreeding depression and further genetic drift) due to fragmentation are predicted in these populations, the southern populations may show deterioration of genetic diversity in the near future.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

Exercise ameliorates high-fat diet-induced impairment of differentiation of adipose-derived stem cells into neuron-like cells in rats

Adipose-derived stem cells (ADSCs) can differentiate into neurons under particular conditions. It remains largely unknown whether this differentiation potential is affected by physical conditions such as obesity, which modulates the functions of adipose tissue. In this study, we determined the impact of either a 9-week high-fat diet (60% fat; HFD) or 9-week exercise training on the differentiation potential of ADSCs into neuron-like cells in male Wistar rats. Rats were randomly assigned to a normal diet-fed (ND-SED) group, HFD-fed (HFD-SED) group, or exercise-trained HFD-fed group (HFD-EX). After a 9-week intervention, ADSCs from all groups differentiated into neuron-like cells. Expression of neuronal marker proteins (nestin, βIII-tubulin, and microtubule-associated protein 2 [MAP2]) and the average length of cell neurites were lower in cells from HFD-SED rats than in other groups. Instead, protein expression of COX IV and Cyt-c, the Bax/Bcl-2 and LC3-II/I ratio, and the malondialdehyde level in culture medium were higher in cells from HFD-SED rats. No significant difference between ND-SED and HFD-EX rats was observed, except for the average length of cell neurites in MAP2. Thus, HFD impaired the differentiation potential of ADSCs into neuron-like cells, which was accompanied by increases in apoptotic activity and oxidative stress. Importantly, exercise training ameliorated the HFD-induced impairment of neurogenesis in ADSCs. The adipose tissue microenvironment could influence the differentiation potential of ADSCs, a source of autologous stem cell therapy.     

Disruption of phospholipase B gene, PLB1, increases the survival of baker's yeast Torulaspora delbrueckii

Abstract An uracil auxotrophic mutant of baker's yeast Torulaspora delbrueckii , which is resitant to 5-fluoro-orotic acid, was complemented by transformation with YEp24 which harbors 2 μm origin and URA3 derived from Saccharomyces cerevisiae . The phospholipase B in T. delbrueckii cells is active in both acidic and alkaline conditions. However, activity of phospholipase B gene ( PLB1 ) in cells of disruption mutant ( plbI : : URA3 ) was lost in both conditions, which indicates that all phospholipase B activity is encoded by a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delbrueckii cells showed ∼ 2.5-fold increase in phospholipase B activity, comparing with that in wild-type cells. Cells of plb1 Δ mutant showed increased survival when cells of plb1 Δ mutant and wild-type strain were incubated in water at 30 °C. Cells of PLB1 -over-expressed strain died rapidly even during the cultivation period, indicating that phospholipase B activity may be a determinant for the survival of this yeast.     

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparansulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110–131 and 17–21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin- oligosaccharides in a similar manner.     

Growing and differentiating characterization of aortic smooth muscle cell line, p53LMAC01 obtained from p53 knock out mice

Recently we have established an aortic smooth muscle cell line, p53LMAC01 obtained from p53 knockout mice. This cell line showed some differentiated properties which were accelerated by 5-azacytidine treatment [1]. In this study, further characterization of p53LMAC01 cell line was investigated according to cell growth and differentiation, and especially focused into the changes of cell feature, actin filaments' formation, and changes of intracellular calcium concentrations to sympathetic nerve transmitter, norepinephrine. While the cell feature was changed from flattened shape to extended form during 4 days, actin filaments were developing, arranging in parallel to longitudinal direction, and gathering under the surface membrane. In 11 days many cells died and detached from substrate, while actin filaments became poor except for the surface membrane in the remained cells. Appearance of calcium response to noradrenalin needed several days after passage as well as a morphological change of the cells for the extended form and development of actin filaments. The calcium response was maintained on 11 days, which coincided with the result that the cells hold actin filaments under the surface membrane. These results suggest that p53LMAC01 cell line maintains several differentiated characters of adult smooth muscle cell and that their expression needs several days after passage.     

Wnt-3a and Dvl induce neurite retraction by activating Rho-associated kinase

Dvl is a key protein that transmits the Wnt signal to the canonical beta-catenin pathway and the noncanonical planar cell polarity (PCP) pathway. We studied the roles of Rho-associated kinase (Rho-kinase), which is activated by Dvl in the PCP pathway of mammalian cells. The expression of Dvl-1, Wnt-1, or Wnt-3a activated Rho-kinase in COS cells, and this activation was inhibited by the Rho-binding domain of Rho-kinase. The expression of Dvl-1 in PC12 cells activated Rho and inhibited nerve growth factor (NGF)-induced neurite outgrowth. This inhibition was reversed by a Rho-kinase inhibitor but not by a c-Jun N-terminal kinase inhibitor. Dvl-1 also inhibited serum starvation-dependent neurite outgrowth of N1E-115 cells, and expression of the Rho-binding domain of Rho-kinase reversed this inhibitory activity of Dvl-1. Dvl-1 mutants that did not activate Rho-kinase did not inhibit the neurite outgrowth of N1E-115 cells. Furthermore, the purified Wnt-3a protein activated Rho-kinase and inhibited the NGF-dependent neurite outgrowth of PC12 cells. Wnt-3a-dependent neurite retraction was also prevented by a Rho-kinase inhibitor and a Dvl-1 mutant that suppresses Wnt-3a-dependent activation of Rho-kinase. These results suggest that Wnt-3a and Dvl regulate neurite formation through Rho-kinase and that PC12 and N1E-115 cells are useful for analyzing the PCP pathway.     

Approach for functional analysis of glycan using RNA interference

The elucidation of the biological role of glycan is one of the most important issues to be resolved following the genome project. RNA interference is becoming an efficient reverse genetic tool for studying gene function in model organisms, including C.elegans and Drosophila melanogaster. Our molecular evolutionary study has shown that a prototype of glycosyltransferases, which synthesize a variety of glycan structures in the Golgi apparatus, was conserved between mammals and Drosophila. For analyses of the basic physiological functions of glycans, we established the Drosophila inducible RNAi knockdown system and applied it to one glycosyltransferase and one transporter, proteoglycan UDP-galactose: beta-xylose beta1,4galactosyltransferase I and the PAPS-transporter, respectively. If on the silencing of each gene induced ubiquitously under the control of a cytoplasmic actin promoter, the RNAi knockdown fly died, then the protein was indispensable for life. The expression of the target gene was disrupted specifically and the degree of interference was well correlated with the phenotype. The inducible RNAi knockdown fly obtained using the GAL4-UAS system will pave the way for the functional analysis of glycans.     

Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.