Morphological discrimination of two genetic groups of a Japanese salamander, Hynobius naevius (Amphibia, Caudata)

Hynobius naevius, distributed on western Honshu, Shikoku, and Kyushu Islands of Japan, includes two genetically distinct groups (Groups A and B) that have never been delimited morphologically. Using specimens from the entire species range, we investigated the possibility of distinguishing these groups morphologically. Multivariate analyses of morphometric characters resulted in recognition of two groups that corresponded well to the two genetic groups. One (Group A) was characterized by larger body, compressed tail, shallower vomerine tooth series, bluish- or reddish-purple ground color, and pale-white lateral markings. In contrast, another (Group B) was characterized by smaller body, cylindrical tail, longer vomerine tooth series, reddish-brown ground color, and white lateral markings. Group A was composed of populations from the Chugoku District of Honshu and northern Kyushu, and could not be divided into subgroups, while Group B encompassed populations from the Chubu and Kinki Districts of Honshu, Shikoku and Kyushu, and was subdivided into three local subgroups that are geographically separated by marine straits. Morphometric differentiation in Group A is presumed to have been less affected by genetic factors than by other factors, such as ecological relationships with other, coexisting species. Differentiation in Group B is assumed to have been enhanced not only by genetic but also by climatological factors.     

IL-7 exacerbates chronic colitis with expansion of memory IL-7Rhigh CD4+ mucosal T cells in mice

We have previously demonstrated that mucosal CD4(+) T cells expressing high levels of IL-7 receptor (IL-7R(high)) are pathogenic cells responsible for chronic colitis. Here we investigate whether IL-7 is directly involved in the expansion of IL-7R(high) memory CD4(+) mucosal T cells and the exacerbation of colitis. We first showed that CD4(+) lamina propria lymphocytes (LPLs) from wild-type, T cell receptor-alpha-deficient (TCR-alpha(-/-)), and recombinase-activating gene (RAG)-2(-/-)-transferred mice with or without colitis showed phenotypes of memory cells, but only CD4(+) LPLs from colitic mice showed IL-7R(high). In vitro stimulation by IL-7, but not by IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4(+), but not normal CD4(+) LPLs. Importantly, in vivo administration of IL-7 mice accelerated the expansion of IL-7R(high) memory CD4(+) LPLs and thereby exacerbated chronic colitis in RAG-2(-/-) mice transferred with CD4(+) LPLs from colitic TCR-alpha(-/-) mice. Conversely, the administration of anti-IL-7R monoclonal antibody significantly inhibited the development of TCR-alpha(-/-) colitis with decreased expansion of CD4(+) LPLs. Collectively, the present data indicate that IL-7 is essential for the expansion of pathogenic memory CD4(+) T cells under pathological conditions. Therefore, therapeutic approaches targeting the IL-7R pathway may be feasible in the treatment of human inflammatory bowel disease.     

Adenylyl cyclase-cAMP system inhibits thrombin-induced HSP27 in vascular smooth muscle cells

We previously reported that thrombin stimulates the induction of heat shock protein (HSP) 27 via p38 mitogen-activated protein (MAP) kinase activation in aortic smooth muscle A10 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the thrombin-stimulated induction of HSP27 in A10 cells. Forskolin, a direct activator of adenylyl cyclase, reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the HSP27 accumulation. Furthermore, dibutyryl-cAMP (DBcAMP), a permeable analog of cAMP, significantly suppressed the accumulation of HSP27. On the other hand, calphostin C, an inhibitor of protein kinase C (PKC), reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. Moreover, forskolin reduced the p38 MAP kinase phosphorylation induced by the 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, and significantly suppressed the TPA-stimulated accumulation of HSP27. These results indicate that adenylyl cyclase-cAMP system has an inhibitory role in thrombin-stimulated HSP27 induction in aortic smooth muscle cells, and the effect seems to be exerted on the thrombin-induced PKC- p38 MAP kinase signaling pathway.     

Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.     

A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seed length

We have characterized a rice (Oryza sativa) dwarf mutant, dwarf11 (d11), that bears seeds of reduced length. To understand the mechanism by which seed length is regulated, the D11 gene was isolated by a map-based cloning method. The gene was found to encode a novel cytochrome P450 (CYP724B1), which showed homology to enzymes involved in brassinosteroid (BR) biosynthesis. The dwarf phenotype of d11 mutants was restored by the application of the brassinolide (BL). Compared with wild-type plants, the aberrant D11 mRNA accumulated at higher levels in d11 mutants and was dramatically reduced by treatment with BL, implying that the gene is feedback-regulated by BL. Precise determination of the defective step(s) in BR synthesis in d11 mutants proved intractable because of tissue specificity and the complex control of BR accumulation in plants. However, 6-deoxotyphasterol (6-DeoxoTY) and typhasterol (TY), but not any upstream intermediates before these compounds, effectively restored BR response in d11 mutants in a lamina joint bending assay. Multiple lines of evidence together suggest that the D11/CYP724B1 gene plays a role in BR synthesis and may be involved in the supply of 6-DeoxoTY and TY in the BR biosynthesis network in rice.     

Effects of aging on the relation of adenyl purine release with plasma membrane fluidity of arterial endothelial cells

Age-related changes in adenyl purine release from rat arteries and endothelial cell (EC) plasma membrane (PM) fluidity were studied. High performance liquid chromatography-fluorescence revealed that aging significantly decreased the release of adenyl purines. Pyrene-excimer spectroscopy disclosed that EC PM fluidity of aged rats decreased more significantly than that of young rats. An increase in cholesterol content and a decrease in the unsaturation index (USI) of fatty acids in cholesterol-enriched ECs reduced PM fluidity and 5'-nucleotidase (5'-ND) activity (measured by coupled assay of adenosine deaminase and glutamate dehydrogenase). Moreover, a decrease in cholesterol content and an increase in the USI of fatty acyl chains of the PM in docosahexaenoic acid-enriched ECs concurrently increased enzyme activity and extracellular adenosine. Therefore, decreases in PM fluidity, observed with age-dependent increased cholesterol and decreased USI, induce a decrease in 5'-ND activity, decrease extracellular adenosine levels, and might relate to hypertension in aged rats.     

Measurement of homocysteine thiolactone hydrolase activity using high-performance liquid chromatography with fluorescence detection and polymorphisms of paraoxonase in normal human serum

We developed a non-radioactive and sensitive assay method for measurement of the HTL hydrolase (HTLase) activity in biological samples, using OPA as a fluorescent post-labeling agent, l-homocysteine thiolactone (L-HTL) as the substrate, and HPLC to achieve rapid and selective separation of the substrate and product. The method was applied to measure the activity of HTLase in human, rabbit, rat and mouse serum samples. In addition, the correlation between the serum HTLase activity and PON1 polymorphisms in Japanese subjects was also investigated. The serum HTLase activity in humans, as determined by measurement of the enzyme activity in 22 subjects, was found to be in the range of 0.89-2.06 nmol/min mg protein, with a mean activity of 1.44 nmol/min mg protein.