Structural Requirements for Mutagenic Activities of N-Heterocyclic Bases in the Salmonella Test System

The mutagenic activities of quinoline, isoquinoline, phenanthridine, benzo(f)quinoline, benzo(h)quinoline and their α-amino derivatives were compared in relation to the effect of structural changes using the Salmonella typhimurium test system. All mutagenic compounds tested require the liver microsomal fraction for their mutagenic activity. Phenanthridine, two benzoquinolines and quinoline were mutagenic. α-Amination of two benzoquinolines and quinoline resulted to increase their mutagenic activity intensively. Addition of a benzene ring to the benzene moiety of 2-aminoquinoline, so that two carbon atoms are shared, affected distinctly the increase in the mutagenic activity. The co-existence of benzoquinoline series with 2-aminobenzo(f)quinoline showed the clear synergistic action.     

A novel gene family defined by human dihydropyrimidinase and three related proteins with differential tissue distribution

We have isolated cDNA clones encoding dihydropyrimidinase (DHPase) from human liver and its three homologues from human fetal brain. The deduced amino acid (aa) sequence of human DHPase showed 90% identity with that of rat DHPase, and the three homologues showed 57–59% aa identity with human DHPase, and 74–77% aa identity with each other. We tentatively termed these homologues human DHPase related protein (DRP)-1, DRP-2 and DRP-3. Human DRP-2 showed 98% aa identity with chicken CRMP-62 (collapsin response mediator protein of relative molecular mass of 62 kDa) which is involved in neuronal growth cone collapse. Human DRP-3 showed 94–100% aa identity with two partial peptide sequences of rat TOAD-64 (turned on after division, 64 kDa) which is specifically expressed in postmitotic neurons. Human DHPase and DRPs showed a lower degree of aa sequence identity with Bacillus stearothermophilus hydantoinase (39–42%) and Caenorhabditis elegans unc-33 (32–34%). Thus we describe a novel gene family which displays differential tissue distribution: i.e., human DHPase, in liver and kidney; human DRP-1, in brain; human DRP-2, ubiquitously expressed except for liver; human DRP-3, mainly in heart and skeletal muscle.     

Functional characterization of a new human Ad4BP/SF-1 variation, G146A

Ad4BP/SF-1 plays key roles at all levels of the hypothalamic-pituitary-steroidogenic organ axis and its functional disruption causes endocrine disorders of these organs. However, only three human subjects with Ad4BP/SF-1 mutations have been reported to date, suggesting limited clinical significance as a cause of inborn adrenal or sexual abnormalities. We report the first functional characterization of a new variation found in the hinge region of human Ad4BP/SF-1, G146A. Resulting from a single nucleotide shift (GGG-->GCG), G146A bears slightly diminished transactivation activity evidenced by both adrenal specific cyp11A promoter and ovary specific cyp19 promoter II. The variation does not affect protein expression or stability, exhibiting no dominant negative effect. G146A has a normal interaction pattern with standard co-regulators and subnuclear distribution pattern, and can be considered as a nonsynonymous single nucleotide polymorphism, since it occurs in normals and patients with adrenal diseases. In normal Japanese the allele C frequency is 8%, while in a preliminary population of patients with adrenal diseases it is elevated to 30%; suggesting the G146A variation might be of clinical importance.     

Contribution of membrane-associated prostaglandin E2 synthase to bone resorption

This study initially confirmed that, among prostaglandins (PGs) produced in bone, only PGE(2) has the potency to stimulate osteoclastogenesis and bone resorption in the mouse coculture system of osteoblasts and bone marrow cells. For the PGE(2) biosynthesis two isoforms of the terminal and specific enzymes, membrane-associated PGE(2) synthase (mPGES) and cytosolic PGES (cPGES) have recently been identified. In cultured mouse primary osteoblasts, both mPGES and cyclooxygenase-2 were induced by the bone resorptive cytokines interleukin-1, tumor necrosis factor-alpha, and fibroblast growth factor-2. Induction of mPGES was also seen in the mouse long bone and bone marrow in vivo by intraperitoneal injection of lipopolysaccharide. In contrast, cPGES was expressed constitutively both in vitro and in vivo without being affected by these stimuli. An antisense oligonucleotide blocking mPGES expression inhibited not only PGE(2) production, but also osteoclastogenesis and bone resorption stimulated by the cytokines, which was reversed by addition of exogenous PGE(2). We therefore conclude that mPGES, which is induced by and mediates the effects of bone resorptive stimuli, may make a target molecule for the treatment of bone resorptive disorders.     

Plastic changes of leaf mass per area and leaf nitrogen content in response to canopy openings in saplings of eight deciduous broad-leaved tree species

Leaf nitrogen content per area (N_area) is a good indicator of assimilative capacity of leaves of deciduous broad-leaved trees. This study examined the degrees of increase in N_area in response to canopy openings as leaf mass per area (LMA) and leaf nitrogen content per mass (N_mass) in saplings of eight deciduous broad-leaved tree species in Hokkaido, northern Japan. Five of the species were well-branched species with a large number of small leaves (lateral-growth type), and the other three species were less-branched species with a small number of large leaves (vertical-growth type). The degrees of increase in N_area were compared between the two crown types. In closed-canopy conditions, leaves of the vertical-growth species tended to have a lower LMA and higher N_mass than those of the lateral-growth species, which resulted in similar N_area for both. LMA increased in canopy openings in the eight species, and the degrees of increase were not largely different between the lateral- and vertical-growth species. On the contrary, N_mass was unchanged in canopy openings in the eight species. As a result, N_area of each species increased in canopy openings in proportion to the increase in LMA, and the degrees of increase in N_area were similar in the lateral- and vertical-growth species. Therefore, this study showed that the degrees of increase in N_area were not correlated with the crown architecture (i.e., the lateral- and vertical-growth types).     

Comparison of cellular response in bovine intervertebral disc cells and articular chondrocytes: effects .of lipopolysaccharide on proteoglycan metabolism

Lipopolysaccharide (LPS) induces matrix degradation and markedly stimulates the production of several cytokines, i.e., interleukin-1β, −6, and −10, by disc cells and chondrocytes. We performed a series of experiments to compare cellular responses of cells from the bovine intervertebral disc (nucleus pulposus and annulus fibrosus) and from bovine articular cartilage to LPS. Alginate beads containing cells isolated from bovine intervertebral discs and articular cartilage were cultured with or without LPS in the presence of 10% fetal bovine serum. The DNA content and the rate of proteoglycan synthesis and degradation were determined. In articular chondrocytes, LPS strongly suppressed cell proliferation and proteoglycan synthesis in a dose-dependent manner and stimulated proteoglycan degradation. Compared with articular chondrocytes, nucleus pulposus cells responded in a similar, although less pronounced manner. However, treatment of annulus fibrosus cells with LPS showed no significant effects on proteoglycan synthesis or degradation. A slight, but statistically significant, inhibition of cell proliferation was observed at high concentrations of LPS in annulus fibrosus cells. Thus, LPS suppressed proteoglycan synthesis and stimulated proteoglycan degradation by articular chondrocytes and nucleus pulposus cells. The effects of LPS on annulus fibrosus cells were minor compared with those on the other two cell types. The dissimilar effects of LPS on the various cell types suggest metabolic differences between these cells and may further indicate a divergence in pathways of LPS signaling and a differential sensitivity to exogenous stimuli such as LPS.This work was supported in part by NIH grants 2-P50-AR39239 and 1-P01-AR48152.     

Reactive oxygen species mediate shear stress-induced fluid-phase endocytosis in vascular endothelial cells

To elucidate the role of shear stress in fluid-phase endocytosis of vascular endothelial cells (EC), we used a rotating-disk shearing apparatus to investigate the effects of shear stress on the uptake of lucifer yellow (LY) by cultured bovine aortic endothelial cells (BAEC). Exposure of EC to shear stress (area-mean value of 10 dynes/cm2) caused an increase in LY uptake that was abrogated by the antioxidant, N-acetyl-L-cysteine (NAC), the NADPH oxidase inhibitor, acetovanillone, and two inhibitors of protein kinase C (PKC), calphostin C and GF109203X. These results suggest that fluid-phase endocytosis is regulated by both reactive oxygen species (ROS) and PKC. Shear stress increased both ROS production and PKC activity in EC, and the increase in ROS was unaffected by calphostin C or GF109203X, whereas the activation of PKC was reduced by NAC and acetovanillone. We conclude that shear stress-induced increase in fluid-phase endocytosis is mediated via ROS generation followed by PKC activation in EC.     

Responses of crown development to canopy openings by saplings of eight tropical submontane forest tree species in Indonesia: a comparison with cool-temperate trees

BACKGROUND AND AIMS: Growth in trunk height in canopy openings is important for saplings. How saplings increase height growth in canopy openings may relate to crown architectural constraints. Responses of crown development to canopy openings in relation to trunk height growth were studied for saplings (0.2-2.5 m tall) of eight tropical submontane forest tree species in Indonesia. The results of this study were also compared with those of temperate trees in northern Japan. METHODS: The crown architecture differed among the eight tropical species, i.e. they had sparsely to highly developed branching structures. Crown allometry was compared among the eight species in each canopy condition (closed canopy or canopy openings), and between closed canopy and canopy openings within a species. A general linear regression model was used to analyse how each species increases height growth rate in canopy openings. Crown allometry and its plasticity were compared between tropical and temperate trees by a nested analysis of covariance. KEY RESULTS: Tropical submontane trees had responses similar to cool-temperate trees, showing an increase in height in canopy openings, i.e. taller saplings of sparsely branched species increase height growth rates by increasing the sapling leaf area. Cool-temperate trees have a wider crown projection area and a smaller leaf area per crown projection area to avoid self-shading within a crown compared with tropical submontane trees. Plasticity of the crown projection area is greater in cool-temperate trees than in tropical submontane trees, probably because of the difference in leaf longevity. CONCLUSIONS: This study concluded that interspecific variation in the responses of crown development to canopy openings in regard to increasing height related to the species' branching structure, and that different life-forms, such as evergreen and deciduous trees, had different crown allometry and plasticity.     

The effects of understorey dwarf bamboo (Sasa kurilensis) removal on soil fertility in a Betula ermanii forest of northern Japan

We investigated the changes in soil microbial biomass C (MBC), microbial biomass N (MBN) and N mineralization in Sasa kurilensis-present (SP) and S. kurilensis-removed (SR) stands in a Betula ermanii forest. The mean levels of MBC and MBN were significantly higher in the SR stand than in the SP, which may have positively influenced the N-mineralization rate as depicted by a significant positive correlation between these variables and the N-mineralization rate. N immobilization and subsequent N release along with decreased use of available soil N due to S. kurilensis removal may have ensured greater N availability in the SR stand.