Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration

RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.     

The cDNA microarray analysis using an Arabidopsis pad3 mutant reveals the expression profiles and classification of genes induced by Alternaria brassicicola attack

The hypersensitive response (HR) was induced in a wild-type Arabidopsis thaliana plant (Columbia) (Col-wt) by inoculation with Alternaria brassicicola that causes the development of small brown necrotic lesions on the leaves. By contrast, pad3-1 mutants challenged with A. brassicicola produced spreading lesions. The cell death in pad3-1 mutants could not inhibit the pathogen growth and development, although both production of H(2)O(2) and localized cell death were similar in Col-wt and pad3-1 plants after the inoculation. The difference between Col-wt and pad3-1 plants is defense responses after the occurrence of cell death. In other words, PAD3 is necessary for defense response to A. brassicicola. Therefore, we examined the changes in the expression patterns of ca. 7,000 genes by cDNA microarray analysis after inoculation with A. brassicicola. The cDNA microarrays were also done to analyze Arabidopsis responses after treatment with signal molecules, reactive oxygen species (ROS)-inducing compounds and UV-C. The results suggested that the pad3-1 mutation altered not only the accumulation of camalexin but also the timing of expression of many defense-related genes in response to the challenge with A. brassicicola. Furthermore, the plants integrate two or more signals that act together for promoting the induction of multiple defense pathways.     

Cloning of a nitrate reductase inactivator (NRI) cDNA from <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L. and expression of mRNA and protein of NRI in cultured spinach cells

The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.     

Antidiabetic effect of Lactobacillus GG in streptozotocin-induced diabetic rats

Neonatally streptozotocin-induced diabetic (n-STZ) rats were given food containing Lactobacillus GG cells (GG) or a control diet (control), from 9 to 18 weeks of age. The GG cells significantly lowered the blood hemoglobin A(1C) (HbA(1C)) level and improved glucose tolerance in n-STZ rats (p<0.05). In the GG group, the serum insulin level at 30 min after glucose loading was significantly higher than in the control group (p<0.05).     

Characterization of a zebra mutant of rice with increased susceptibility to light stress

The rice zebra mutant TCM248 is a single recessive mutant. This mutant develops transverse-striped leaves with green and white sectors under alternate light/dark growth conditions. Mutants that were grown under a higher light intensity during the light period showed a more intense striped phenotype. The white tissues contained abnormal chloroplasts with few internal membrane structures, while the green tissues in the mutants contained normal chloroplasts. The white tissue contained only trace amounts of Chls and carotenoids, and mRNA accumulation of nuclear genes encoding chloroplast proteins (rbcS, cab) was strongly suppressed compared to that in the wild type plants. A series of growth condition shift experiments demonstrated that the mutant displayed the striped phenotype only if it was exposed to the alternate light/dark growth conditions during a limited stage of early leaf development. These data suggest that the zebra gene is involved in the acquisition of photoprotective capacity of the plants and that this gene functions at an early stage of chloroplast differentiation.     

Analysis of reductant supply systems for ferredoxin-dependent sulfite reductase in photosynthetic and nonphotosynthetic organs of maize

Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide in chloroplasts and root plastids using ferredoxin (Fd) as an electron donor. Using purified maize (Zea mays L.) SiR and isoproteins of Fd and Fd-NADP(+) reductase (FNR), we reconstituted illuminated thylakoid membrane- and NADPH-dependent sulfite reduction systems. Fd I and L-FNR were distributed in leaves and Fd III and R-FNR in roots. The stromal concentrations of SiR and Fd I were estimated at 1.2 and 37 microM, respectively. The molar ratio of Fd III to SiR in root plastids was approximately 3:1. Photoreduced Fd I and Fd III showed a comparable ability to donate electrons to SiR. In contrast, when being reduced with NADPH via FNRs, Fd III showed a several-fold higher activity than Fd I. Fd III and R-FNR showed the highest rate of sulfite reduction among all combinations tested. NADP(+) decreased the rate of sulfite reduction in a dose-dependent manner. These results demonstrate that the participation of Fd III and high NADPH/NADP(+) ratio are crucial for non-photosynthetic sulfite reduction. In accordance with this view, a cysteine-auxotrophic Escherichia coli mutant defective for NADPH-dependent SiR was rescued by co-expression of maize SiR with Fd III but not with Fd I.     

cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana triflora

Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less understood modification reactions during anthocyanin biosynthesis. Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins. A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) ( EC 2.3.1.153 ) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme. The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52 736. The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multifunctional acyltransferases ( St-Pierre et al . (1998 ) Plant J. 14, 703–713). The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT. The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT. Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G. triflora . RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes. Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol.     

Development of glycosylated human interleukin-1α, neoglyco IL-1α, coupled with D-galactose monosaccharide: biological activities in vivo

In our previous study, a galactose monosaccharide with C9 spacer was chemically coupled to recombinant human interleukin 1 (rhIL-1) in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. The glycosylated IL-1 exhibited reduced activities in vitro by 10 to 10 000-fold depending upon different aspects of activities addressed. The affinity to type I and II IL-1 receptors were also reduced. In this study we examined a variety of IL-1 activities in vivo, including upregulation of serum levels of IL-6, 1-acid glycoprotein, NOx, corticosterone, downregulation of serum level of glucose, and recovery of peripheral white blood cells (WBCs) from myelosuppression in 5-fluorouracil-treated mice. In contrast to the biological activities in vitro, these activities in vivo were uniformly reduced by only about 10 to 20-fold compared to untreated IL-1.