Tetrahymena eukaryotic translation elongation factor 1A (eEF1A) bundles filamentous actin through dimer formation

Eukaryotic translation elongation factor 1A (eEF1A) is known to be a multifunctional protein. In Tetrahymena, eEF1A is localized to the division furrow and has the character to bundle filamentous actin (F-actin). eEF1A binds F-actin and the ratio of eEF1A and actin is approximately 1:1 (Kurasawa et al., 1996). In this study, we revealed that eEF1A itself exists as monomer and dimer, using gel filtration column chromatography. Next, eEF1A monomer and eEF1A dimer were separated using gel filtration column, and their interaction with F-actin was examined with cosedimentation assay and electron microscopy. In the absence of Ca2+/calmodulin (CaM), eEF1A dimer bundled F-actin and coprecipitated with F-actin at low-speed centrifugation, but eEF1A monomer did not. In the presence of Ca2+/CaM, eEF1A monomer increased, while dimer decreased. To examine that Ca2+/CaM alters eEF1A dimer into monomer and inhibits bundle formation of F-actin, Ca2+/CaM was added to F-actin bundles formed by eEF1A dimer. Ca2+/CaM separated eEF1A dimer to monomer, loosened F-actin bundles and then dispersed actin filaments. Simultaneously, Ca2+/CaM/ eEF1A monomer complexes were dissociated from actin filaments. Therefore, Ca2+/CaM reversibly regulates the F-actin bundling activity of eEF1A.     

Cardioprotective role of endogenous hydrogen peroxide during ischemia-reperfusion injury in canine coronary microcirculation in vivo

We have recently demonstrated that endogenous H2O2 plays an important role in coronary autoregulation in vivo. However, the role of H2O2 during coronary ischemia-reperfusion (I/R) injury remains to be examined. In this study, we examined whether endogenous H2O2 also plays a protective role in coronary I/R injury in dogs in vivo. Canine subepicardial small coronary arteries (>or=100 microm) and arterioles (<100 microm) were continuously observed by an intravital microscope during coronary I/R (90/60 min) under cyclooxygenase blockade (n=50). Coronary vascular responses to endothelium-dependent vasodilators (ACh) were examined before and after I/R under the following seven conditions: control, nitric oxide (NO) synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA), catalase (a decomposer of H2O2), 8-sulfophenyltheophylline (8-SPT, an adenosine receptor blocker), L-NMMA+catalase, L-NMMA+tetraethylammonium (TEA, an inhibitor of large-conductance Ca2+-sensitive potassium channels), and L-NMMA+catalase+8-SPT. Coronary I/R significantly impaired the coronary vasodilatation to ACh in both sized arteries (both P<0.01); L-NMMA reduced the small arterial vasodilatation (both P<0.01), whereas it increased (P<0.05) the ACh-induced coronary arteriolar vasodilatation associated with fluorescent H2O2 production after I/R. Catalase increased the small arterial vasodilatation (P<0.01) associated with fluorescent NO production and increased endothelial NOS expression, whereas it decreased the arteriolar response after I/R (P<0.01). L-NMMA+catalase, L-NMMA+TEA, or L-NMMA+catalase+8-SPT further decreased the coronary vasodilatation in both sized arteries (both, P<0.01). L-NMMA+catalase, L-NMMA+TEA, and L-NMMA+catalase+8-SPT significantly increased myocardial infarct area compared with the other four groups (control, L-NMMA, catalase, and 8-SPT; all, P<0.01). These results indicate that endogenous H2O2, in cooperation with NO, plays an important cardioprotective role in coronary I/R injury in vivo.     

CrkL plays a role in SDF-1-induced activation of the Raf-1/MEK/Erk pathway through Ras and Rac to mediate chemotactic signaling in hematopoietic cells

Intracellular signaling mechanisms regulating SDF-1-induced chemotaxis of hematopoietic cells have remained elusive. Here we demonstrate that overexpression of the adaptor molecule CrkL enhances SDF-1-induced chemotaxis of hematopoietic BaF3 and 32Dcl3 cells. Overexpression of CrkL also enhanced SDF-1-induced activation of the Raf-1/MEK/Erk signaling pathway as well as that of the small GTPases Ras, Rap1, and Rac, while a dominant negative mutant of Ras or Rac suppressed CrkL-enhanced Erk activation. SDF-1 stimulation induced tyrosine phosphorylation of CrkL, which was inhibited by the Src family kinase inhibitor PP1 or by dominant negative mutants of Lyn, thus indicating that Lyn mediated SDF-1-induced phosphorylation of CrkL. However, inhibition of the Lyn kinase activity failed to affect SDF-1-induced activation of the small GTPases and Erk. On the other hand, SDF-1-induced activation of the Erk signaling pathway as well as chemotaxis was inhibited by overexpression of a CrkL mutant lacking the N-terminal SH3 domain, which mediates interaction with various signaling molecules including guanine nucleotide exchange factors for the Ras and Rho family GTPases. SDF-1-induced chemotaxis was also inhibited by the dominant negative Ras or Rac mutant as well as by the MEK inhibitor PD98059. These results indicate that CrkL mediates SDF-1-induced activation of the Raf-1/MEK/Erk signaling pathway through Ras as well as Rac in hematopoietic cells and, thereby, plays important roles in the induction of chemotactic response.     

Modulation by the steroid/thyroid hormone superfamily of TGF-beta-stimulated VEGF release from vascular smooth muscle cells

We previously reported that transforming growth factor-beta (TGF-beta) stimulates the release of vascular endothelial growth factor (VEGF) from aortic smooth muscle A10 cells via activation of p38 mitogen-activated protein (MAP) kinase. In the present study, we investigated whether nuclear hormone receptor superfamily members affect TGF-beta-stimulated VEGF release from A10 cells. Retinoic acid or 1,25-dihydroxyvitamin D3 enhanced TGF-beta-induced VEGF release in a concentration-dependent manner, whereas dexamethasone or corticosterone suppressed TGF-beta-induced VEGF release. 1,25-Dihydroxyvitamin D3 and TGF-beta stimulated phosphorylation of p38 MAP kinase in an additive manner. SB203580, an inhibitor of p38 MAP kinase, decreased the VEGF release induced by TGF-beta or 1,25-dihydroxyvitamin D3. However, retinoic acid, dexamethasone, or corticosterone did not affect phosphorylation of p38 MAP kinase. These results indicate that retinoic acid, 1,25-dihydroxyvitamin D3, and glucocorticoids affect TGF-beta-stimulated VEGF release from aortic smooth muscle cells. The stimulatory effect of 1,25-dihydroxyvitamin D3 occurs, in part, via modification of TGF-beta-induced activation of p38 MAP kinase.     

Isolation and characterization of a beta-primeverosidase-like enzyme from Penicillium multicolor

p-Nitrophenyl and eugenyl beta-primeveroside (6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) hydrolytic activity was found in culture filtrate from Penicillium multicolor IAM7153, and the enzyme was isolated. The enzyme was purified as a beta-primeverosidase-like enzyme by precipitation with ammonium sulfate followed by successive chromatographies on Phenyl Sepharose, Mono Q, and beta-galactosylamidine affinity columns. The molecular mass was estimated to be 50 kDa by SDS-PAGE and gel filtration. The purified enzyme was highly specific toward the substrate p-nitrophenyl beta-primeveroside, which was cleaved in an endo-manner into primeverose and p-nitrophenol, but a series of beta-primeveroside as aroma precursors were hydrolyzed only slightly as substrates for the enzyme. In analyses of its hydrolytic action and kinetics, the enzyme showed narrow substrate specificity with respect to the aglycon and glycon moieties of the diglycoside. We conclude that the present enzyme is a kind of beta-diglycosidase rather than beta-primeverosidase.     

Gene transfer effects on various cationic amphiphiles in CHO cells

Gene transfer is an important tool to explore genomic, cell biologic, or gene therapeutic research. In this paper we report that several cationic amphiphiles have the potential to efficiently deliver DNA into CHO cells, which is one of the cell lines considered to be important for production of proteins including therapeutic proteins. We have found that O,O′-ditetradecanoyl-N-(trimethylammonio acetyl) diethanolamine chloride (14Dea2), among 29 types of cationic amphiphiles tested, shows a transfection efficiency of more than 40% in CHO cells. In addition, the results from a series of hydrocarbon chains of varying lengths bound to a connector have shown that an optimal chain length is important for the efficient delivery of DNA into cells. Moreover, flow cytometer analysis has shown that 14Dea2 transfection leads to high levels of expression of the reporter gene (green fluorescent protein) in individual cells. These findings have suggested that 14Dea2 is able to effectively deliver a number of plasmids into a cell nucleus. Thus, our system might be a powerful tool for high efficiency gene transfer and production of high levels of recombinant protein.     

Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice

This study evaluates whether Spirulina, including its components such as phycocyanin, enhances or sustains immune functions by promoting immune competent-cell proliferation or differentiation. The effects of Spirulina of a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall component extract (SpCW) on proliferation of bone marrow cells and induction of colony-forming activity in mice were investigated. The Spirulina extracts, SpHW, Phyc, and SpCW, enhanced proliferation of bone-marrow cells and induced colony-forming activity in the spleen-cell culture supernatant. Granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were detected in the culture supernatant of the spleen cells stimulated with the Spirulina extracts. Bone marrow-cell colony formation in soft-agar assay was also significantly induced by the blood samples and the culture supernatants of the spleen and Peyer's patch cells of the mice which ingested Spirulina extracts orally for 5 weeks in in vivo study. Ratios of neutrophils and lymphocytes in the peripheral blood and bone marrow, consequently, increased in the mice. Spirulina may have potential therapeutic benefits for improvement of weakened immune functions caused by, for example, the use of anticancer drugs.