Low genetic variation and inbreeding depression in small isolated populations of the Japanese rosy bitterling, Rhodeus ocellatus kurumeus

The Japanese rosy bitterling, Rhodeus ocellatus kurumeus, has been affected not only by the invasion of another subspecies, R. o. ocellatus, from China, but also by habitat fragmentation. In this study, the effects of habitat fragmentation on the fitness of R. o. kurumeus were investigated. Owing to exclusion by R. o. ocellatus, R. o. kurumeus in Honshu and Shikoku has disappeared entirely, except for small populations in isolated man-made ponds in Osaka and Kagawa. In Kyushu it still occupies open water systems, into which R. o. ocellatus has only recently invaded. Meristic and genetic data show that the diversity of R. o. kurumeus is significantly lower in the isolated Osaka and Kagawa populations than the non-isolated Fukuoka population. The Osaka population is inferior to the Fukuoka population in terms of viability and growth. The viability of reciprocal inter-population hybrids between the Osaka and Fukuoka populations was, however, as high as that of the Fukuoka population. In addition to the high scores of band sharing index (BSI) in RAPD-PCR analysis, acceptance of transplanted scales among individuals, irrespective of natal pond, indicates that the Osaka population forms a highly inbred line. These results suggest that low genetic variation is associated with inbreeding depression in the small isolated Osaka populations. Consequently, the management of ponds, including the free movement of individuals, in addition to measures to prevent the invasion of R. o. ocellatus, is necessary for the conservation of R. o. kurumeus.     

Localization of interleukin-18 and its receptor in somatotrophs of the bovine anterior pituitary gland

A pro-inflammatory cytokine, interleukin 18 (IL-18), induces intracellular expression of IL-1 and the release of IL-6. IL-1 and IL-6 has been detected in anterior pituitary cells, suggesting that IL-18 is produced in anterior pituitary cells and may serve to aid immuno-endocrine regulation. In the present study, we addressed this hypothesis by investigating the intracellular localization of IL-18 and its receptor in bovine anterior pituitary gland. IL-18 mRNA and its protein were detected in the anterior pituitary gland by RT-PCR and Western blotting. In situ hybridization showed that IL-18 mRNA was expressed in the anterior pituitary cells. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs. Furthermore, the expression of GH mRNA in IL-18 immunoreactive cells was confirmed by immuno-laser microdissection. These results first demonstrated that somatotrophs produced IL-18. Subsequently, the distribution of the IL-18 receptor alpha (IL-18Rα) was investigated in order to understand IL-18 signaling among the anterior pituitary cells. Bovine IL-18Rα cDNA was partially sequenced and detected in the anterior pituitary gland by RT-PCR. Immunohistochemistry of IL-18Rα, IL-18 and GH showed that IL-18Rα was co-localized in IL-18 immunoreactive cells or somatotrophs. These data suggest that IL-18 acts on somatotrophs as an immuno-endocrine mediator through the autocrine pathway. This work was supported by a Grant-in-Aid for Scientific Research (B) (No.13460122) from the Ministry of Education, Science and Culture of Japan     

Gene therapy for chronic myocardial ischemia using platelet-derived endothelial cell growth factor in dogs

Platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP), has been reported to possess angiogenic activity and to inhibit apoptosis. This study was performed to determine whether PD-ECGF/TP can be used to ameliorate chronic myocardial ischemia. Myocardial ischemia was created in 40 mongrel dogs by placement of an ameroid constrictor on the proximal left anterior descending coronary artery (LAD). Plasmid vector encoding human PD-ECGF/TP cDNA (pCIhTP group; n = 12), empty vector pCI (pCI group; n = 12), or saline (Saline group; n = 12) was directly injected into the LAD territory 3 wk after ameroid constrictor implantation. Myocardial blood flow was detected using PET at baseline, 3 wk after ameroid constrictor implantation, and 2 wk after therapeutic treatment. At the end of the experiment, the hearts were isolated for biological and histological analysis. In the pCIhTP group, the transfected heart strongly expressed PD-ECGF/TP. The size of the infarct was smaller in the pCIhTP group than in the pCI or Saline group. The number of apoptotic myocardial cells was decreased in the pCIhTP group compared with the control groups based on triple immunohistochemical staining for von Willebrand factor, alpha-actin smooth muscle cells, and single-strand DNA. The level of proapoptotic protein Bax markedly decreased in the pCIhTP group compared with the other groups. Double immunohistochemical staining for von Willebrand factor and alpha-actin smooth muscle cells demonstrated that angiogenesis and arteriogenesis occurred, and paralleled the changes in myocardial blood flow and myocardial function in the pCIhTP group. We conclude that genetic approaches using PD-ECGF/TP to target the myocardium are effective for alleviating chronic myocardial ischemia.     

Validation of the plasma half-life of 11alpha-deuterium cortisol as a sensitive index for the analysis of human 11beta-HSD2 activity in vivo

This study is concerned with validating the measurement of the plasma half-life of 11alpha-(2)H cortisol in an attempt to accurately assess the in vivo activity of 11beta-HSD2 in man. Oral administration of 5mg of cortisol-(13)C(4),(2)H(1) to a human subject after repeated ingestions of 130mg/day of glycyrrhetinic acid for 5 days resulted in a decrease in the rate constant of the cortisol-(13)C(4),(2)H(1) to cortisone-(13)C(4) conversion, a direct index reflecting 11beta-HSD2 activity. The reduced 11beta-HSD2 activity led to an increase in the elimination half-life of cortisol-(13)C(4),(2)H(1), indicating that the loss of 11alpha-(2)H is a sensitive in vivo means of assessing 11beta-HSD2 activity. A simultaneous oral administration of 3mg each of [1,2,4,19-(13)C(4),11alpha-(2)H]cortisol (cortisol-(13)C(4),(2)H(1)) and 11alpha-(2)H cortisol to another human subject confirmed the bioequivalency of the two labeled cortisols. The information obtained from the kinetic analysis of the 11beta-HSD2-catalyzed conversion of cortisol-(13)C(4),(2)H(1) to cortisone-(13)C(4) indicated that the elimination half-life of 11alpha-(2)H cortisol was a sensitive index of renal 11beta-HSD2 activity. The use of 11alpha-(2)H cortisol as a tracer appears to offer a significant advance in evaluating human 11beta-HSD2 activity in vivo.     

Prostaglandin E2 production in ovarian cancer cell lines is regulated by cyclooxygenase-1, not cyclooxygenase-2

The significance of cyclooxygenase-2 (COX-2) expression in ovarian cancer has been discussed. In this study, we found increased expression of COX-1 mRNA and protein in three out of 10 ovarian cancer cell lines. Prostaglandin E 2 (PGE2) production was elevated in these three cell lines, but not in other seven cell lines. COX-2 protein was not detected in any of the cell lines. Cytosolic prostaglandin E synthase (cPGES) mRNA and protein were detected in all 10 cell lines. Membrane-associated PGES-1 (mPGES-1) was detected in some of the ovarian cell lines, but its presence did not correspond with PGE2 production. In contrast, mPGES-2 mRNA and protein were detected in all 10 cell lines. A nonselective COX inhibitor (indometacin) and a selective COX-1 inhibitor (SC-560) strongly inhibited PGE2 production by the three cell lines, while selective COX-2 inhibitors (NS-398 and rofecoxib) did not inhibit PGE2 production. In addition, increased expression of COX-1, not COX-2 protein was observed in the mass of ovarian cancer tissues from 22 patients when compared with that in normal tissue. These findings suggest that COX-1 might be a major enzyme regulating PGE2 production in ovarian cancer cells.     

Comparative analysis of the cytotoxicity of homopolymeric amino acids

Many human proteins have homopolymeric amino acid (HPAA) tracts, although the physiological significance or cellular effects of their presence is poorly understood. We previously reported that 20 kinds of HPAAs show characteristic intracellular localization and that among those, hydrophobic HPAAs aggregate strongly and form high molecular weight proteins when expressed in cultured cells. In this study, we investigated the cytotoxicity of 20 kinds of HPAAs. HPAA tracts of approximately 30 residues fused to the C-terminus of YFP were expressed in COS-7 cells. Cells expressing homopolymeric-Cys, -Ile, -Leu, and -Val showed low viability in Trypan Blue assay. Caspase-3 activity, which is usually upregulated in dying cells, was determined by measuring the cleavage of the peptide substrate Ac-DEVD-MCA and by detecting the cleaved active form of the caspase-3 by Western blotting. The activity of caspase-3 was drastically elevated in cells expressing those HPAAs which showed low viability in Trypan Blue assay. Interestingly, it was found that there is a correlation between the hydrophobicity of a single amino acid and the cytotoxicity of the corresponding HPAA as a homopolymer. These results indicate that the hydrophobicity of HPAAs may cause cytotoxicity.