Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Zinc ions suppress mitogen-activated interleukin-2 production in Jurkat cells

Calcineurin (CN) is thought to play an important role in the immune system by regulating cytokine production, for example, interleukin-2 (IL-2) in T-lymphocytes. We have previously shown that physiological concentrations of Zn2+ inhibit CN activity in vitro [K. Takahashi, E. Akaishi, Y. Abe, R. Ishikawa, S. Tanaka, K. Hosaka, Y. Kubohara, Zinc inhibits calcineurin activity in vitro by competing with nickel, Biochem. Biophys. Res. Commun. 307 (2003) 64-68], in spite of the fact that Zn2+ is an essential element of the CN catalytic domain. In this study, in order to assess whether Zn2+ regulates (suppresses) CN activity in vivo and whether Zn2+ can be used as an anti-inflammatory and/or immunosuppressive drug, we examined the effects of Zn2+ on IL-2 production induced by the mitogen, concanavalin A (ConA), in Jurkat T-cells. Zn2+ at 0.2 mM suppressed ConA-induced IL-2 accumulation in the medium of an in vitro culture of Jurkat cells. Zn2+ at 0.03-0.3 mM dose-dependently suppressed ConA-induced IL-2 mRNA expression in Jurkat cells. Zn2+ also suppressed IL-2 mRNA expression induced by phorbol ester (PMA) and ionomycin. Furthermore, Zn2+ and the immunosuppressant FK506 showed an additive inhibitory effect on ConA-induced IL-2 mRNA expression. These results suggest that exogenously added Zn2+ may disturb (increase) the intracellular Zn2+ concentration and inhibit CN activity, thereby suppressing IL-2 production in Jurkat cells. The present study further indicates that Zn2+ may have therapeutic potential in the treatment of T-cell related inflammation and also that Zn2+ may be utilized as a supplemental drug with FK506.     

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.     

Perchloric acid-soluble protein regulates cell proliferation and differentiation in the spinal cord of chick embryos

The role of perchloric acid-soluble protein (PSP) was investigated in chick embryos. Fluorescently labeled anti-chick liver (CL)-PSP IgG was injected into the yolk sac in ovo at embryonic day 3, and became localized in neuroepithelial cells. Within 12 h, morphological changes were observed in 37.5% of anti-CL-PSP IgG-injected embryos, and the neuroepithelial cells formed a wavy line. No significant changes were observed in embryos injected with non-immune IgG or PBS. Increased expression of PCNA and decreased expression of neuronal class III beta-tubulin were observed in the spinal cord after anti-CL-PSP IgG injection. These results suggest that PSP controls the proliferation and differentiation of neuroepithelial cells in chick embryos.     

Functional co-expression of xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 and UDP-glucuronosyltransferase 1A6, in yeast microsomes

Xenobiotic Phase I and Phase II reactions in hepatocytes occur sequentially and cooperatively during the metabolism of various chemical compounds including drugs. In order to investigate the sequential metabolism of 7-ethoxycoumarin (7EC) as model substrate in vitro, xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 (P450 1A1) and UDP-glucuronosyltransferase 1A6 (UGT1A6) were co-expressed in Saccharomyces cerevisiae AH22. Rat P450 1A1 and yeast NADPH-P450 reductase were expressed on a multicopy plasmid (pGYR1) in the yeast. Rat UGT1A6 cDNA with a yeast alcohol dehydrogenase I promoter and terminator was integrated into yeast chromosomal DNA to achieve the stable expression. Co-expression of P450 1A1 and UGT1A6 in yeast microsomes was confirmed by immunoblot analysis. Protease treatment of the microsomes showed the correct topological orientation of UGT to the membranes. The metabolism of 7EC to 7-hydroxycoumarin (7HC) and its glucuronide in yeast microsomes was analyzed by reverse phase HPLC. In a co-expression system containing 7EC, NADPH and UDP-glucuronic acid, glucuronide formation was detected after a lag phase, following the accumulation of 7HC. In the case of P450 1A1 and UGT1A6, efficient coupling of hydroxylation and glucuronidation in 7EC metabolism was not observed in the co-expression system. This P450 and UGT co-expression system in yeast allows the sequential biotransformation of xenobiotics to be simulated in vitro.     

The mammalian circadian timing system: from gene expression to physiology

Many physiological processes in organisms from bacteria to man are rhythmic, and some of these are controlled by self-sustained oscillators that persist in the absence of external time cues. Circadian clocks are perhaps the best characterized biological oscillators and they exist in virtually all light-sensitive organisms. In mammals, they influence nearly all aspects of physiology and behavior, including sleep-wake cycles, cardiovascular activity, endocrinology, body temperature, renal activity, physiology of the gastro-intestinal tract, and hepatic metabolism. The master pacemaker is located in the suprachiasmatic nuclei, two small groups of neurons in the ventral part of the hypothalamus. However, most peripheral body cells contain self-sustained circadian oscillators with a molecular makeup similar to that of SCN (suprachiasmatic nucleus) neurons. This organization implies that the SCN must synchronize countless subsidiary oscillators in peripheral tissues, in order to coordinate cyclic physiology. In this review, we will discuss some recent studies on the structure and putative functions of the mammalian circadian timing system, but we will also point out some apparent inconsistencies in the currently publicized model for rhythm generation.     

Two independent light signals cooperate in the activation of the plastid psbD blue light-responsive promoter in Arabidopsis

Though phospholipase C PLCgamma2 is known to play an important role in platelet activation by collagen and fibrinogen, its importance in GPIb-mediated platelet activation is less well understood. To better understand the role of PLCgamma2 in GPIb-mediated adhesion and thrombus formation, we examined the ability of wild-type and PLCgamma2- deficient murine platelets to spread on immobilized von Willebrand factor (VWF) under static conditions, and to attach to and form thrombi on VWF under conditions of arterial shear. While absence of PLCgamma2 had only a minimal effect on platelet adhesion to immobilized VWF, its absence impaired spreading and profoundly affected thrombus growth and stability on VWF.     

Musclin, a novel skeletal muscle-derived secretory factor

Skeletal muscle is involved in the homeostasis of glucose and lipid metabolism. We hypothesized that the skeletal muscle produces and secretes bioactive factor(s), similar to adipocytokines secreted by fat tissue. Here, we report the identification of a novel secretory factor, musclin, by signal sequence trap of mouse skeletal muscle cDNAs. Musclin cDNA encoded 130 amino acids, including NH(2)-terminal 30-amino acid signal sequence. Musclin protein contained a region homologous to natriuretic peptide family, and KKKR, a putative serine protease cleavage site, similar to the natriuretic peptide family. Full-length musclin protein and KKKR-dependent cleaved form were secreted in media of musclin cDNA-transfected mammalian cell cultures. Musclin mRNA was expressed almost exclusively in the skeletal muscle of mice. Musclin mRNA levels in skeletal muscle were markedly low in fasted, increased upon re-feeding, and were low in streptozotocin-treated insulin-deficient mice. Musclin mRNA expression was induced at late stage in the differentiation of C2C12 myocytes. In myocytes, insulin increased, while epinephrine, isoproterenol, and forskolin reduced musclin mRNA, all of which are known to increase the cellular content of cyclic AMP, a counter-regulator to insulin. Pathologically, overexpression of musclin mRNA was noted in the muscles of obese insulin-resistant KKAy mice. Functionally, recombinant musclin significantly attenuated insulin-stimulated glucose uptake and glycogen synthesis in myocytes. In conclusion, we identified musclin, a novel skeletal muscle-derived secretory factor. Musclin expression level is tightly regulated by nutritional changes and its physiological role could be linked to glucose metabolism.