Role of the second immunoglobulin-like loop of nectin in cell-cell adhesion

We investigated whether and how rat liver thioredoxin reductase spares alpha-tocopherol in biomembranes. Purified hydroperoxides of beta-linoleoyl-gamma-palmitoylphosphatidylcholine were decreased 35% by treatment with thioredoxin reductase and 54% by thioredoxin reductase plus E. coli thioredoxin. Thioredoxin reductase also halved the amount of hydroperoxides that had been formed during photoperoxidation of liposomes composed of beta-linoleoyl-gamma-palmitoylphosphatidylcholine, and of emulsions of both cholesterol and cholesteryl linolenate. In erythrocyte ghosts, thioredoxin reductase spared alpha-tocopherol from oxidation by both soybean lipoxygenase and ferricyanide. Thioredoxin reductase also decreased F(2)-isoprostanes in ghosts oxidized by ferricyanide, suggesting that its ability to spare alpha-tocopherol relates to reduction of lipid hydroperoxides.     

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.     

OsSEND-1: a new RAD2 nuclease family member in higher plants

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H₂O₂, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.     

Measurement of plasma pristanic, phytanic and very long chain fatty acids by liquid chromatography-electrospray tandem mass spectrometry for the diagnosis of peroxisomal disorders

High pressure liquid chromatography with a narrow bore C8 column has been used to separate pristanic, phytanic and very long chain fatty acids, important in the diagnosis of peroxisomal disorders, for their accurate isotope dilution quantification by tandem mass spectrometry. The fatty acids, isolated from plasma, were analysed as trimethylaminoethyl ester (quaternary ammonium) derivatives. Analysis time was 2.5 h and sample requirement was 10 microl of plasma. Good agreement with GC-MS methods for the levels of pristanic and phytanic acids, C26:0/C22:0 and C24:0/C22:0 ratios were obtained for 12 plasma samples from peroxisomal disorder patients and a set of controls.     

Antimicrobial activity of essential oils against Helicobacter pylori

Background. Helicobacter pylori is an important pathogen responsible for gastroduodenal diseases in humans. Although the eradication of H. pylori using antibiotics often improves gastroduodenal diseases, resistance to the antibiotics is emerging. Materials and Methods. The antimicrobial effect of essential oils and the development of resistance to the essential oils were evaluated in vitro and in vivo. Results. Thirteen essential oils used in this study completely inhibited the growth of H. pylori in vitro at a concentration of 0.1% (v/v). Cymbopogon citratus (lemongrass) and Lippia citriodora (lemon verbena) were bactericidal against H. pylori at 0.01% at pH 4.0 and 5.0. Resistance to lemongrass did not develop even after 10 sequential passages, whereas resistance to clarithromycin developed under the same conditions. In in vivo studies, the density of H. pylori in the stomach of mice treated with lemongrass was significantly reduced compared with untreated mice. Conclusions. These results demonstrate that the essential oils are bactericidal against H. pylori without the development of acquired resistance, suggesting that essential oils may have potential as new and safe agents for inclusion in anti‐H. pylori regimens.     

Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation

We have applied in vivo intracellular antibody capture (IAC) technology to isolate human intrabodies which bind to the oncogenic RAS protein. IAC facilitates the capture of antibody fragments, in this case single-chain Fvs (scFvs), which tolerate reducing environments, such as the cytoplasm of cancer cells. Three anti-RAS scFvs with different affinity, solubility and intracellular binding activity were characterized. The anti-RAS scFvs with highest affinity were expressed relatively poorly in mammalian cells, and greater soluble expression was achieved by mutating the antibody framework to canonical consensus scaffolds, previously derived from IAC, without losing antigen specificity. Mutagenesis experiments showed that the consensus scaffolds are functional as intrabody fragments without an intra-domain disulfide bond. Furthermore, we could convert an intrabody which does not bind RAS in mammalian cells into a high-affinity reagent capable of inhibiting RAS-mediated NIH 3T3 transformation by exchanging VH and VL complementarity-determining regions onto its consensus scaffold. These data show that the consensus scaffold is a robust framework by which to improve intrabody function.     

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility

The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.     

A new method for enzymatic preparation of isopentenyladenine-type and<Emphasis Type="Italic"> trans</Emphasis>-zeatin-type cytokinins with radioisotope-labeling

We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin research, such as receptor–ligand assays and cell metabolism studies.     

A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons

We tested the contribution of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation in cultured cerebellar granule neurons. p160ROCK inhibition, presumably by reducing the stability of the cortical actin network, triggered immediate outgrowth of membrane ruffles and filopodia, followed by the generation of initial growth cone-ike membrane domains from which axonal processes arose. Furthermore, a potentiation in both the size and the motility of growth cones was evident, though the overall axon elongation rate remained stable. Conversely, overexpression of dominant active forms of Rho or ROCK was suggested to prevent initiation of axon outgrowth. Taken together, our data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics.