Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.     

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.     

Cdc15 is required for spore morphogenesis independently of Cdc14 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae exit from mitosis requires the Cdc14 phosphatase to reverse CDK-mediated phosphorylation. Cdc14 is released from the nucleolus by the Cdc14 early anaphase release (FEAR) and mitotic exit network (MEN) pathways. In meiosis, the FEAR pathway is essential for exit from anaphase I. The MEN component Cdc15 is required for the formation of mature spores. To analyze the role of Cdc15 during sporulation, a conditional mutant in which CDC15 expression was controlled by the CLB2 promoter was used. Cdc15-depleted cells proceeded normally through the meiotic divisions but were unable to properly disassemble meiosis II spindles. The morphology of the prospore membrane was aberrant and failed to capture the nuclear lobes. Cdc15 was not required for Cdc14 release from the nucleoli, but it was essential to maintain Cdc14 released and for its nucleo-cytoplasmic transport. However, cells carrying a CDC14 allele with defects in nuclear export (Cdc14-DeltaNES) were able to disassemble the spindle and to complete spore formation, suggesting that the Cdc14 nuclear export defect was not the cause of the phenotypes observed in cdc15 mutants.     

Gliclazide inhibits proliferation but stimulates differentiation of white and brown adipocytes

Gliclazide, a second-generation sulfonylurea, has anti-oxidant properties as well as hypoglycemic activities. In the present study, we investigated whether gliclazide affected proliferation and/or differentiation of HW white and HB2 brown adipocyte cell lines. Gliclazide inhibited proliferation of HW and HB2 cells in the medium containing fetal calf serum or epidermal growth factor (EGF). Gliclazide inhibited phosphorylation of EGF receptor and of extracellular signal-regulated kinase (ERK) 1/2 stimulated by EGF. Gliclazide increased lipid accumulation and peroxisome proliferator-activated receptor gamma (PPARgamma) expression in the early stage of differentiation of adipocytes. A K(ATP) channel activator, diazoxide, did not inhibit the increase of lipid accumulation by gliclazide. Furthermore, gliclazide inhibited the DNA-binding activity of PPARgamma in mature adipocytes. On the other hand, glibenclamide, other sulfonylurea, did not show these effects. These results indicate gliclazide inhibits proliferation and stimulates differentiation of adipocytes via down-regulation of the EGFR signalling. Gliclazide may have preventive and therapeutic effects on obesity, as well as on type 2 diabetes.     

Regulation of Wnt signalling by receptor-mediated endocytosis

Wnts compromise a large family of secreted and hydrophobic glycoproteins that control a variety of developmental and adult processes in all metazoan organisms. Recent advances in the field of Wnt signalling have revealed that Wnt activates multiple intracellular cascades, resulting in the regulation of cellular proliferation, differentiation, migration and polarity. However, it is not clear how Wnt activates these pathways after it binds to the receptors. It has been shown that Wnt and its antagonist Dickkopf are internalized with their receptors. This review highlights distinct endocytic pathways correlate with specificity of Wnt signalling events.     

Physiological effects of Shinrin-yoku (taking in the atmosphere of the forest)--using salivary cortisol and cerebral activity as indicators

The purpose of this study is to examine the physiological effects of Shinrin-yoku (taking in the atmosphere of the forest). The subjects were 12 male students (22.8+/-1.4 yr). On the first day of the experiments, one group of 6 subjects was sent to a forest area, and the other group of 6 subjects was sent to a city area. On the second day, each group was sent to the opposite area for a cross check. In the forenoon, the subjects were asked to walk around their given area for 20 minutes. In the afternoon, they were asked to sit on chairs and watch the landscapes of their given area for 20 minutes. Cerebral activity in the prefrontal area and salivary cortisol were measured as physiological indices in the morning at the place of accommodation, before and after walking in the forest or city areas during the forenoon, and before and after watching the landscapes in the afternoon in the forest and city areas, and in the evening at the place of accommodation. The results indicated that cerebral activity in the prefrontal area of the forest area group was significantly lower than that of the group in the city area after walking; the concentration of salivary cortisol in the forest area group was significantly lower than that of the group in the city area before and after watching each landscape. The results of the physiological measurements show that Shinrin-yoku can effectively relax both people's body and spirit.     

Physiological effects of Shinrin-yoku (taking in the atmosphere of the forest) in an old-growth broadleaf forest in Yamagata Prefecture, Japan

The physiological effects of "Shinrin-yoku" (taking in the atmosphere of the forest) were examined by investigating blood pressure, pulse rate, heart rate variability (HRV), salivary cortisol concentration, and immunoglobulin A concentration in saliva. Subjective feelings of being "comfortable", "calm", and "refreshed" were also assessed by questionnaire. The subjects were 12 male university students aged from 21 to 23 (mean+/-SD: 22.0+/-1.0). The physiological measurements were conducted six times, i.e., in the morning and evening before meals at the place of accommodation, before and after the subjects walked a predetermined course in the forest and city areas for 15 minutes, and before and after they sat still on a chair watching the scenery in the respective areas for 15 minutes. The findings were as follows. In the forest area compared to the city area, 1) blood pressure and pulse rate were significantly lower, and 2) the power of the HF component of the HRV tended to be higher and LF/(LF+HF) tended to be lower. Also, 3) salivary cortisol concentration was significantly lower in the forest area. These physiological responses suggest that sympathetic nervous activity was suppressed and parasympathetic nervous activity was enhanced in the forest area, and that "Shinrin-yoku" reduced stress levels. In the subjective evaluation, 4) "comfortable", "calm", and "refreshed" feelings were significantly higher in the forest area. The present study has, by conducting physiological investigations with subjective evaluations as supporting evidence, demonstrated the relaxing and stress-relieving effects of "Shinrin-yoku".     

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.