The production of a diabetic mouse using constructs encoding porcine insulin promoter-driven mutant human hepatocyte nuclear factor-1alpha

A diabetic mouse model was produced using a mutant human hepatocyte nuclear factor-1alpha gene (HNF1alphaP291fsinsC) regulated by the porcine insulin promoter. The functionality of two different constructs containing HNF1alphaP291fsinsC, termed PD1 and PD2 (cytomegalovirus enhancer minus and plus), were examined in transgenic mice. The blood glucose levels and body weights of the PD1 transgenic mice did not differ from their non-transgenic littermates over the period from 3 to 8 weeks of age. Conversely, the PD2 transgenic mice exhibited hyperglycemia and decreased body weight. Western blot analysis demonstrated that mutant HNF-1alpha protein (HNF1alphaP291), derived from the PD2 transgene, was expressed in the PD2 mice. Morphometric studies of the pancreas of a PD2 mouse revealed that the number of pancreatic islets present was less than that in the non-transgenic mice, indicating disturbed islet neogenesis. These results suggest that impaired insulin secretion in disrupted islets causes hyperglycemia. In addition, the phenotype of PD2 transgenic mice similar to that of the HNF-1alpha gene-deficient mouse, which displays growth retardation and impaired viability. These results indicate that HNF1alphaP291 expression driven by the porcine insulin promoter, together with the cytomegalovirus enhancer, induces a diabetic phenotype in transgenic mice.     

Germination response in wheat grains to dihydroactinidiolide,a germination inhibitor in wheat husks,and related compounds

On the basis of our recent findings that the germination of intact wheat grains with glumes (husks) belonging to dormant varieties was restrained as compared with that of dehusked grains, we have explored the identities of germination inhibitors in the glumes, resulting in the characterization of dihydroactinidiolide (1) and some aromatic compounds. A related natural product, tetrahydroactinidiolide (2), showed similar activity. The present study has demonstrated that the sensitivity in inhibition response of germination of the grains to 1 and 2 declined during after-ripening, in parallel with changes in germinability; the sprouting of after-ripened seeds on a whole spike was preventable by exogenous application of 2 in laboratory conditions, and germination of after-ripened grains was delayed by more than two weeks by the action of 1 or 2. The term "pseudodormancy" is proposed for the phenomenon of delay of germination caused by the inhibitor. After accumulation of additional evidence on inhibition response of actinidiolide-type natural products, structurally related to inhibitor 1, a mechanism concerning germination inhibition by 1 or 2 is proposed on the basis of the concept of nonbonding interaction with the inhibitors at an active site of an acceptor.     

Behaviors of D- and L-lactic acids during the brewing process of sake (Japanese rice wine)

The amounts of D- and L-lactic acids during the brewing process of sake were determined by capillary electrophoresis using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Because L-lactic acid, which prevents the growth of nonuseful microorganisms, is a raw material of sake, the ratio of L-lactic acid to total lactic acid is almost 1.0 at the initial stage of sake brewing. During brewing, the ratio decreased gradually and finally reached 0.39. Yeast (Saccharomyces cerevisiae) for sake brewing produced D-lactic acid, but not L-lactic acid in a culture medium. These results suggest that the decrease in the ratio of L-lactic acid to total lactic acid during sake brewing resulted in D-lactic acid production by yeast. The ratios in 18 brands of sake obtained commercially ranged from 0.23 to 0.78. The levels of D-lactic acid in sake (140-274 mg/L) were in a narrower range than those of L-lactic acid (61-461 mg/L). Although the D-lactic acid level in sake did not correspond to total lactic acid level, the L-lactic acid level correlated well with total lactic acid level (R(2) = 0.867). These results suggest that the ratio of L-lactic acid to total lactic acid in sake reflected the amount of L-lactic acid added at the initial stage of sake brewing.     

Community and trophic structures of abalone <Emphasis Type="Italic">Haliotis diversicolor</Emphasis> habitat in Sagami Bay,Japan

Abstract:   The community and trophic structures of the subtidal habitat of the abalone Haliotis diversicolor were examined in Nagai, Sagami Bay, Japan. Conventional community indices showed no significant differences among three sampling stations. The overall continuum of stable isotope ratios was structured into three different trophic linkages: (i) brown algae-dependent benthic food chain; (ii) red algae-dependent benthic food chain; and (iii) planktonic food chain. Brown algae and red algae likely play different roles with respect to carbon sources in the habitat. Conventional fractionation values indicated that the abalone H. diversicolor (δ¹³C = −12.4 ± 1.0‰, δ¹⁵N = 9.3 ± 0.5‰) feeds on the lamina of Undaria pinnatifida during juvenile and adult stages (8.0–65.6 mm shell length). Stable isotope signatures suggested that the juveniles of other abalone species as well as some amphipods and a sea cucumber Holothuria decorata are competitors of H. diversicolor , whereas some Muricidae gastropods such as Thais bronni and Ergalatax contractus are predators. The isotopic differences among macroalgal species and the subsequent transfer to consumers indicate that stable isotopic analysis is an effective means of studying food webs in an open rocky shore community with little influence from external primary production such as terrestrial vegetation.     

Relationship between the incidence of pine wilt disease and the drainage area

We examined the relationship between the incidence of pine wilt disease and moisture conditions in the stand level ofPinus densiflora Sieb. et Zucc. forests in the warm-temperate zone of the western part of Japan. For this analysis, pine trees killed by pine wilt disease were distinguished from the suppressed trees by their position in the layer of the pine forest stand. The drainage area, which is small in the upper part and large in the lower part of the slope, was adopted for representing the moisture conditions in the soil of the slope. The percentage of the pine trees killed by pine wilt disease increased as the size of the drainage area increased. This result suggested that the incidence of pine wilt disease tended to be high in areas with moist conditions. Pine trees attacked by the pinewood nematode die from extensive water deficit due to tracheid cavitations. Pine wilt disease mainly emerges in the summer when the soil water conditions become especially severe, and the radical water stress is thought to accelerate the disease. It was assumed that pine trees in the plots with the small drainage area resisted the influence of the attack of the pinewood nematode because pine trees in the plots with the small drainage area encountered long-term water stress and acquired drought tolerance. Pine trees in the plots with the large drainage area were presumed to be well established in the moist conditions and not to have acquired drought tolerance. The drought tolerance of pine trees was thought to be an important factor in resistance to pine wilt disease.     

A new <Emphasis Type="Italic">Rhizoctonia</Emphasis> sp. closely related to <Emphasis Type="Italic">Waitea circinata</Emphasis> causes a new disease of creeping bentgrass

Isolates of an unidentified Rhizoctonia sp. (UR isolates) were obtained from creeping bentgrass and Kentucky bluegrass with reddish brown sheath and foliar rots. Because the UR isolates anastomosed with isolates of three varieties of Waitea circinata (var. oryzae, var. zeae, and var. circinata), colony morphology, hyphal growth rate at different temperatures, pathogenicity, sequence analysis of the internal transcribed spacers (ITS) region of ribosomal RNA genes (rDNA) were compared. The colony color of mature UR isolates was distinct from isolates of the other three varieties of W. circinata. In pathogenicity tests on creeping bentgrass, the severity of the disease caused by UR isolates was significantly higher than that caused by the three varieties of W. circinata. Sequence similarities of the rDNA-ITS region between UR isolates and between isolates within each variety were high (97–100%), but they were lower among isolates from UR and the varieties of W. circinata (88–94%). In a phylogenetic tree based on the rDNA-ITS sequences, UR isolates formed a cluster separate from each of the clusters formed by the three varieties of W. circinata. These results indicate that the UR isolates clearly differ from the three varieties of W. circinata. We therefore propose that the UR isolates be classified as new Rhizoctonia sp. that are closely related to W. circinata and that the disease on creeping bentgrass should be called Waitea reddish-brown patch disease (Sekikasshoku-hagusare-byo in Japanese).     

Establishment of embryo transfer in the musk shrew (Suncus murinus)

The present study established techniques to induce pseudopregnancy, in vitro oocyte cultures from pronuclear to 2- to 4-cell stages, and embryo transfer in musk shrews, a reflex ovulator. Offspring were subsequently obtained by transferring in vivo-developed or in vitro-cultured embryos. Female musk shrews received human chronic gonadotropin (hCG), with or without mating stimuli, from vasectomized males to produce pseudopregnant recipients. Embryos at the 2- to 4-cell stage were collected 44–48 h after mating. Another set of embryos was collected 26–27 h after mating and then cultured for 20 h from the pronuclear to 2- to 4-cell stages. Subsequently, embryos were transferred into the oviducts of pseudopregnant recipients 24 or 48 h after the induction of pseudopregnancy. Offsprings were successfully obtained from recipients that received hCG 24 h before embryo transfer, regardless of mating stimuli. These techniques may be valuable for producing transgenic musk shrews.