Dock8 interacts with Nck1 in mediating Schwann cell precursor migration

During embryonic development of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations, where they will myelinate the axons after birth. While the intercellular signals controlling Schwann cell precursor migration are well studied, the intracellular signals controlling Schwann cell precursor migration remain elusive. Here, using a rat primary cell culture system, we show that Dock8, an atypical Dock180-related guanine-nucleotide exchange factor (GEF) for small GTPases of the Rho family, specifically interacts with Nck1, an adaptor protein composed only of Src homology (SH) domains, to promote Schwann cell precursor migration induced by platelet-derived growth factor (PDGF). Knockdown of Dock8 or Nck1 with its respective siRNA markedly decreases PDGF-induced cell migration, as well as Rho GTPase activation, in precursors. Dock8, through its unique N-terminal proline-rich motif, interacts with the SH3 domain of Nck1, but not with other adaptor proteins composed only of SH domains, e.g. Grb2 and CrkII, and not with the adaptor protein Elmo1. Reintroduction of the proline-rich motif mutant of Dock8 in Dock8 siRNA-transfected Schwann cell precursors fails to restore their migratory abilities, whereas that of wild-type Dock8 does restore these abilities. These results suggest that Nck1 interaction with Dock8 mediates PDGF-induced Schwann cell precursor migration, demonstrating not only that Nck1 and Dock8 are previously unanticipated intracellular signaling molecules involved in the regulation of Schwann cell precursor migration but also that Dock8 is among the genetically-conservative common interaction subset of Dock family proteins consisting only of SH domain adaptor proteins.     

Concentrated polymer brushes do not induce the expression of inflammatory and angiogeneic genes in human umbilical vein endothelial cells

Objective

When polymer brushes are applied as the inner coating for artificial blood vessels, they may induce unwanted responses in vascular endothelial cells continuously exposed to the polymer surface. Accordingly, we have examined the in vitro effect of non-biofouling concentrated polymer brushes (CPBs) on pro-inflammatory and angiogenic responses of human umbilical vein endothelial cells (HUVECs).

Results

Micro-patterned CPBs were prepared on silicon wafers using biocompatible polymers, poly(poly(ethylene glycol)methyl ether methacrylate) (PPEGMA) and poly(2-hydroxyethyl methacrylate) (PHEMA). HUVECs were cultured on PPEGMA-CPBs and PHEMA-CPBs with different channel widths (20, 50, and 80 µm) and analyzed for mRNA expression of the pro-inflammatory cytokines IL-6 and IL-8 and angiogeneic vascular endothelial growth factor (VEGF). Irrespective of channel width, PHEMA-CPBs reduced the expression of all target genes, whereas PPEGMA-CPBs reduced VEGF and did not affect IL-6 and IL-8 levels.

Conclusion

Micro-patterned CPBs, irrespective of chemical structure or adhesion area, do not induce the expression of important pro-inflammatory and angiogenic mediators in endothelial cells.

     

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.     

Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells

Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.     

A comparative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and organic hydroperoxide by glutathione and functions in the protection of cells against oxidative damage. Glutathione peroxidase exists in several forms that differ in their primary structure and localization. We have also shown that selenoprotein P exhibits a glutathione peroxidase-like activity (Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K. (1999) J. Biol. Chem. 274, 2866-2871). To understand the physiological significance of the diversity among these enzymes, a comparative study on the peroxide substrate specificity of three types of ubiquitous glutathione peroxidase (cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, and extracellular glutathione peroxidase) and of selenoprotein P purified from human origins was done. The specific activities and kinetic parameters against two hydroperoxides (hydrogen peroxide and phosphatidylcholine hydroperoxide) were determined. We next examined the thiol specificity and found that thioredoxin is the preferred electron donor for selenoprotein P. These four enzymes exhibit different peroxide and thiol specificities and collaborate to protect biological molecules from oxidative stress both inside and outside the cells.     

The slender rice mutant,with constitutively activated gibberellin signal transduction,has enhanced capacity for abscisic acid level

The slender rice (slr1-1) mutant, carrying a lethal and recessive single mutation, has a constitutive gibberellin (GA)-response phenotype and behaves as if it were saturated with GAs [Ikeda et al. (2001) Plant Cell 13, 999]. The SLR1 gene, with sequence homology to members of the plant-specific GRAS gene family, is a mediator of the GA signal transduction process. In the slender rice, GA-inducible alpha-amylase was produced from the aleurone layer without applying GA. GA-independent alpha-amylase production in the mutant was inhibited by applying abscisic acid (ABA). Shoot elongation in the mutant was also suppressed by ABA, indicating that the slender rice responds normally to ABA. Interestingly, shoot ABA content was 10-fold higher in the mutant than in the wild type, while there was no difference in root ABA content. Expression of the Rab16A gene, which is known to be ABA inducible, was about 10-fold higher in shoots of the mutant than in those of the wild type. These results indicate that constitutive activation of the GA signal transduction pathway by the slr1-1 mutation promotes the endogenous ABA level.     

Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone ¹⁵N T ₁/T ₂ data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T ₁/T ₂ ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.     

Involvement of Gi/o in the PAR-4-induced NO production in endothelial cells

We investigated the involvement of G(i/o) protein in NO production following the activation of proteinase-activated receptor-4 (PAR-4) in cultured bovine aortic endothelial cells. AYPGKF-NH(2) (PAR-4 activating peptide), thrombin, and ionomycin induced a concentration-dependent NO production, with the maximal production seen at 30 microM, 0.1U/ml, and 1 microM, respectively. Ionomycin elevated [Ca(2+)](i) in a concentration-dependent manner. However, AYPGKF-NH(2) and thrombin induced no [Ca(2+)](i) elevation. The loading of cells with BAPTA almost completely inhibited both the NO production and [Ca(2+)](i) elevation induced by 1 microM ionomycin, while it had no significant effect on the AYPGKF-NH(2)-induced NO production. Treatment with pertussis toxin inhibited the AYPGKF-NH(2)-induced NO production, while it had no effect on the ionomycin-induced NO production. Our findings thus demonstrate, for the first time, that PAR-4 activation induced NO production in a manner mostly independent of the Ca(2+) signal and also that G(i/o) is involved in such NO production in vascular endothelial cells.     

A functional model for pMMO (particulate methane monooxygenase): Hydroxylation of alkanes with H2O2 catalyzed by beta-diketiminatocopper(II) complexes

The reaction of copper(II) complexes supported by a series of beta-diketiminate ligands ((R1,R2)L, [(Dipp)N-C(R(2))-C(R(1))-C(R(2))-N(Dipp)](-), Dipp=2,6-diisopropylphenyl; see ) and H(2)O(2) has been examined spectroscopically at a low temperature. The beta-diketiminatocopper(II) complexes with R(2)=H (no substituent on the beta-carbon) provided a copper-oxygen intermediate that exhibited the same spectroscopic features as those of the bis(mu-oxo)dicopper(III) complex generated by the reaction of corresponding beta-diketiminatocopper(I) complex and O(2). On the other hand, the beta-diketiminatocopper(II) complexes with methyl substituent on the beta-carbon (R(2)=Me) did not produce such an intermediate in the same reaction. The beta-diketiminatocopper(II) complexes carrying an electron-withdrawing substituent on the alpha-carbon (R(1)=NO(2) or CN) but no beta-substituent (R(2)=H) exhibited a high catalytic activity in the oxygenation reaction of alkanes with H(2)O(2). Mechanism of the catalytic oxygenation reaction as well as the substituent effects of the ligands on the copper(II)-H(2)O(2) reactivity is discussed.