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.     

Involvement of Collagen Synthesis in Tissue Reconstitution by Dissociated Sponge Cells

Cells dissociated from the sponge Haliclona permollis reconstituted a new body by the following three consecutive processes: (1) Reaggregation, cells aggregate as a spheroidal mass. (2) Spreading, aggregates flatten and spread. (3) Reconstruction, aggregates coalesce into a reconstituted body. Cells in the process of reconstitution incorporated ¹⁴C-proline into proteins, converting 17% of it to hydroxyproline and synthesize collagen molecules during this process. Inhibitors of collagen biosynthesis did not affect the reaggregation, but caused incomplete morphogenesis in the processes of spreading and reconstruction: cycloheximide and 2,2'-dipyridyl inhibited spreading while 3-aminopropionitrile induced incomplete reconstruction. These findings suggested that spreading and reconstruction, but not reaggregation require both synthesis and cross-linking of collagens. Three polypeptides with molecular weights of 58 K, 160 K and 180 K were identified in sponge cells as collagens by immunoblot analysis with antibodies against sea urchin collagen and studies on susceptibilities to collagenase and pepsin. The 58 KDa polypeptide appeared in reconstituted bodies but not in dissociated cells, suggesting its importance in tissue reconstitution by dissociated cells.     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.     

Regulation of the starfish sperm acrosome reaction by cGMP, pH, cAMP and Ca2+

In the starfish, Asterias amurensis, three components in the jelly coat of eggs, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to induce the acrosome reaction (AR). Molecular recognition between the sperm surface molecules and the egg jelly molecules must underlie signal transduction events triggering the AR. Asterosap is a sperm-activating molecule, which stimulates rapid synthesis of intracellular cGMP, pH and Ca(2+). This transient elevation of Ca(2+) level is caused by a K(+)-dependent Na(+)/Ca(2+) exchanger, and the increase of intracellular pH is sufficient for ARIS to induce the AR. The concerted action of ARIS and asterosap could induce elevate intracellular cAMP levels in starfish sperm and the sustained increase in [Ca(2+)], which is essential for the AR. The signaling pathway induced by these factors seems to be synergistically regulated to trigger the AR in starfish sperm.     

Production of bioactive compounds based on phylogeny in the genus Penicillium preserved at NBRC

Penicillium strains (n=394) preserved at NBRC (the NITE Biological Resource Center) were compared as to groupings (11 species-clusters) based on phylogeny and the production of bioactive compounds. The strains in two clusters, of which P. chrysogenum and P. citrinum are representative, showed higher rates of positive strains with multi-biological activities.     

Effect of epidermal growth factor on intestinal adaptation after allogeneic small bowel transplantation in rats

We reported that epidermal growth factor (EGF) stimulated graft adaptation in a rat model of syngeneic small bowel transplantation. However, graft rejection is a severe problem with clinical small bowel transplantation, because small intestinal wall contains large amounts of lymphoid tissue. Studies were performed to investigate the effect of EGF on allogeneic graft adaptation after small bowel transplantation in rats treated with an immunosuppressant FK506. The transplanted animals received intraperitoneally EGF or saline (untreated) after surgery and were examined for analysis one week later. EGF-treated group markedly enhanced the water absorption and induction of sodium glucose cotransporter (SGLTI) as compared with EGF-untreated group. EGF-treated group also increased the mucosal crypt depth and its cell proliferating rate, although there was no significant difference in the mucosal villus height between the two groups. These results indicate that EGF accelerates intestinal allograft adaptation in part by the recovery of mucosal structure and function after small bowel transplantation in rats. EGF may have relevance to promote graft function in clinical small intestinal transplantation.     

Oxytocin hypersensitivity in pregnant P-LAP deficient mice

AimsOxytocin (OT) is the strongest uterotonic substance and has been used widely to induce labor. The physiological importance of OT in modulating the initiation and progression of labor remains unclear. In this study, we showed the roles of OT with onset of labor and also the arginine vasopressin (AVP) effect on urine volume in vivo using both wild type (WT) and placental leucine aminopeptidase (P-LAP)-deficient (KO) mice.Main methodsOT (1, 2, 2.5 U/day) or recombinant P-LAP (0.01 U/day) was continuously infused from gestation day 15.5 in WT and P-LAP KO mice. Duration until onset of labor was observed. Before and after administration of AVP (1 U/day) in WT and P-LAP KO mice, urine volume was measured.Key findingsA significant shortening of pregnancy term was observed in P-LAP KO mice. Continuous infusion of OT (1 U/day) revealed that P-LAP KO mice resulted in premature delivery (OT hypersensitivity). We could observe a significant decrease of urine volume in P-LAP KO mice by administration of AVP. Administration of recombinant P-LAP in WT mice resulted in the delay of the onset of labor about 1.5 days compared with control mice.SignificanceOur present study shows that the regulation of the onset of labor mainly depends on OT and its degradation by P-LAP and also the possible role of P-LAP in the regulation of urine output. P-LAP might be involved in the increased OT sensitivity just prior to onset of labor and also in the onset of labor by degradation of OT.     

Faunal turnover at the end of the Cretaceous in the North Pacific region: Implications from combined magnetostratigraphy and biostratigraphy of the Maastrichtian Senpohshi Formation in the eastern Hokkaido Island,northern Japan

A combined magnetostratigraphic and biostratigraphic study has been performed on the Maastrichtian Senpohshi Formation in eastern Hokkaido Island, northern Japan, which is an approximately 1300 m thick section mainly composed of hemipelagic mudstone. The identification of magnetic polarity was possible at 51 horizons, whereby four magnetozones were recognized. These magnetozones were correlatable to geomagnetic polarity chrons C31r to C30n, suggesting that the age of the Senpohshi Formation is spanning from middle to upper part of the Maastrichtian (ca. 69–67 Ma).The magnetostratigraphy of the Senpohshi Formation established in this study enables a direct age correlation to the Maastrichtian successions in other regions. Thus, this detailed chronology of the formation contributes to paleontological studies of the Maastrichtian in the North Pacific region. For instance, this magnetostratigraphic age assessment implies the following: (1) the stratigraphic range of the ammonite Pachydiscus flexuosus contains polarity chrons from the lower part of C31r to the lower part of C31n, (2) the first occurrence (FO) of the calcareous nannofossil Nephrolithus frequens in the North Pacific region is correlatable to polarity chron C30n or below, and (3) the FO of the bivalve “Inoceramus” awajiensis is located within polarity chrons from C31r to the upper part of C31n. This suggests that the inoceramid extinction event in the North Pacific region might have occurred during polarity chrons from C31r to the upper part of C31n (ca. 70.5–67.8 Ma), which is 2.3–5.0 Myr prior to the Cretaceous/Paleogene boundary. The trend of the Maastrichtian faunal turnover in the North Pacific is well consistent with those of other regions, brings a new evidence for understanding the global faunal turnover in the Maastrichtian, just before Cretaceous/Paleogene mass extinction.