Vertical distribution and germination ability of Alexandrium spp. cysts (Dinophyceae) in the sediments collected from Kure Bay of the Seto Inland Sea, Japan

The vertical distribution of Alexandrium tamarense/ catenella (hereinafter Alexandrium spp.) cysts was investigated with special attention to living cysts filled with fresh protoplasm and empty cysts. In addition, based on the incubation experiments of Alexandrium spp. cysts, the germination ability of the cysts was examined. A sediment core 63 cm in length, collected from Kure Bay of the Seto Inland Sea, West Japan, in September 2000, was provided for an analysis on the vertical distribution of Alexandrium spp. cysts. Samples from every 1 cm interval depth from the top down to 13 cm depth of the same core were taken to examine the germination ability of the cysts. Results show that Alexandrium spp. cysts were continuously observed from 59 to 60 cm depth to the top. The cyst densities in the upper parts of the core (from 9 to 10 cm depth to the top) were much more abundant those that in the lower parts (below 10–11 cm depth). The relationship between living and empty cysts in each depth did not reveal a positive correlation with the sediment depth. Based on the sedimentation rate of the core sediment (approximately 1.6 cm/year), Alexandrium spp. cysts have been produced since 1962, and a remarkable increase of these cysts was observed from ca 1993. Such a rapid increase of Alexandrium spp. cysts has probably been as a result of dense blooms of A. tamarense occurring since 1992 in Hiroshima Bay, including Kure Bay. The germination of Alexandrium spp. cysts was observed in samples collected from the top to 12–13 cm depth of the core. It suggests that the Alexandrium spp. cysts can keep the germination ability for more than 8 years.     

Characterization of diabetes-related traits in MSM and JF1 mice on high-fat diet

We examined the effect of a high-fat diet on the diabetes-related traits of the Japanese Fancy mouse 1 (JF1), MSM, and C57BL/6J (B6J) mice. MSM and JF1 mice were derived from Mus musculus molossinus. B6J is a commonly used laboratory strain, with the vast majority of genome segments derived from Mus musculus domesticus and Mus musculus musculus, and is susceptible to high-fat diet-induced type 2 diabetes. None of the strains showed symptoms of diabetes or obesity when fed a laboratory chow diet. Under a high-fat diet, JF1 mice developed impaired glucose tolerance, hyperglycemia, hyperinsulinemia, and obesity. B6J mice fed a high-fat diet mildly developed these diabetes-related traits compared to JF1 mice fed a high-fat diet. JF1 mice fed a high-fat diet were classified as having type 2 diabetes and were susceptible to high-fat diet-induced diabetes and obesity. On the other hand, MSM mice were resistant to high-fat diet-induced diabetes. These results indicate that the JF1 strain, with its unique genetic origin, is a useful new animal model of high-fat diet-induced diabetes and obesity. Further investigations using JF1 mice will help to clarify the role of the high-fat diet on human diabetes and obesity.     

Effect of N-terminal residues on the structural stability of recombinant horse L-chain apoferritin in an acidic environment

The denaturation of recombinant horse L-chain apoferritin (rLF), which is composed of 24 L-chain subunits, in acidic solution was studied. Using two rLF mutants, lacking four (Fer4) or eight (Fer8) N-terminal amino acid residues, the effect of N-terminal residues on the protein's stability was investigated. Of the two mutants and wild-type rLF, the tertiary and secondary structures of Fer8 were found to be most sensitive to an acidic environment. The Fer8 protein dissociated easily into subunit dimers at or below pH 2.0. Comparing the crystal structures of the mutant proteins, deletion of the N-terminal residues was found to result in fewer inter- and intra-subunit hydrogen bonds. The loss of these bonds is assumed to be responsible for lower endurance against acidic denaturation in N-terminus-deleted mutants. These results indicated that the inter- and intra-subunit hydrogen bonds of N-terminal residues affect the denaturation, especially oligomer formation of apoferritin subunits and will be of use in designing ferritin-based nanodevices.     

Enzymatic activities of uridine and thymidine phosphorylase in normal and cancerous uterine cervical tissues

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.     

Age-related decrease of miRNA-92a levels in human CD8+ T-cells correlates with a reduction of naïve T lymphocytes

MicroRNA (miR)-17-92a expression plays a crucial role in lymphocyte ontogeny. We therefore set out to determine miR-92a expression levels in peripheral blood lymphocytes from healthy subjects to ascertain any association between these levels and ageing. We found a positive correlation between the miR-92a expression level and the percentages of RO-CD8⁺CD27⁺ (P = 0.0046) and CD3⁺CD8⁺CD62L⁺ (P = 0.0011). This suggests that the majority of miR-92a of CD8⁺ T cells is derived from naïve cells, and the miR-92a expression level in CD8⁺ T cells declines progressively with age. These results indicate that the age-related attrition of naïve T cells is linked to a reduction of miR-92a in human T -lymphocytes. Therefore, we should careful attention when evaluating human miRNA levels in T lymphocytes to use normal control values.     

5a-Carba-β-D-glucopyranose derivatives as novel sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors for the treatment of type 2 diabetes

5a-Carba-β-D-glucopyranose derivatives were synthesized and identified as novel SGLT2-selective inhibitors. These inhibitors exhibited potent SGLT2 inhibition with high selectivity over SGLT1. Among the tested compounds, 6f indicated the most potent hSGLT2 inhibition and the highest selectivity over hSGLT1. Moreover, the pharmacokinetics data also showed that 6h, which had the same aglycon structure as sergliflozin-active (3-active), had a threefold longer half-life time (T(1/2)) than sergliflozin (3) with a high distribution volume in db/db mice. Subsequently, 6h lowered blood glucose levels as much as 3 and showed longer hypoglycemic action than 3 in db/db mice.     

Genetic divergence with ongoing gene flow is maintained by the use of different hosts in phytophagous ladybird beetles genus Henosepilachna

Adaptation to different environments can promote population divergence via natural selection even in the presence of gene flow – a phenomenon that typically occurs during ecological speciation. To elucidate how natural selection promotes and maintains population divergence during speciation, we investigated the population genetic structure, degree of gene flow and heterogeneous genomic divergence in three closely related Japanese phytophagous ladybird beetles: Henosepilachna pustulosa, H. niponica and H. yasutomii. These species act as a generalist, a wild thistle (Cirsium spp.) specialist and a blue cohosh (Caulophyllum robustum) specialist, respectively, and their ranges differ accordingly. The two specialist species widely co‐occur but are reproductively isolated solely due to their high specialization to a particular host plant. Genomewide amplified fragment‐length polymorphism (AFLP) markers and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences demonstrated obvious genomewide divergence associated with both geographic distance and ecological divergence. However, a hybridization assessment for both AFLP loci and the mitochondrial sequences revealed a certain degree of unidirectional gene flow between the two sympatric specialist species. Principal coordinates analysis (PCoA) based on all of the variable AFLP loci demonstrated that there are genetic similarities between populations from adjacent localities irrespective of the species (i.e. host range). However, a further comparative genome scan identified a few fractions of loci representing approximately 1% of all loci as different host‐associated outliers. These results suggest that these three species had a complex origin, which could be obscured by current gene flow, and that ecological divergence can be maintained with only a small fraction of the genome is related to different host use even when there is a certain degree of gene flow between sympatric species pairs.     

Vasoactive-intestinal-polypeptide (VIP)-like immunoreactive cells in the skull base of rats

Summary We investigated the distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactive cell bodies in relation to the major cerebral and internal carotid arteries at the skull base in rats. Acetylcholinesterase (AChE) histochemistry was also applied to investigate the localization of this enzyme. VIP staining revealed a few positive cell bodies in nerves close to the internal carotid artery at the base of the skull as well as in the cerebral arterial wall. Ganglion-like cell bodies were detectable within the greater superficial petrosal (GSP) nerve. AChE activity was observed in VIP-like immunoreactive cell bodies along the whole of the GSP nerve. These cell bodies in the GSP nerve may give rise to at least some of the perivascular VIP-and AChE-containing nerves of the internal carotid arteries at the base of the skull.     

Inhibitory Effect of Ascorbic Acid on the Proliferation and Invasion of Hepatoma Cells in Culture

Effect of ascorbic acid (AsA) on the proliferation and invasion of rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cells and by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells, respectively. AsA suppressed the invasion of AH109A cells in a dose-dependent manner at concentrations of 62.5–500 μM, while it inhibited the proliferation of the cells at higher concentrations of 250 and 500 μM. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. AsA suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with AsA, HX and XO or with AsA and hydrogen peroxide. Furthermore, AsA reduced the intracellular peroxide levels in AH109A cells. These results suggest that the antioxidative property of AsA may be involved in its anti-invasive action on hepatoma cells.