Introduction of DPR, an enterostatin fragment peptide, into soybean beta-conglycinin alpha' subunit by site-directed mutagenesis

DPR, a fragment peptide of enterostatin (VPDPR) having hypocholesterolemic activity, was introduced into the three homologous sites, EPR, DYR, and DPI, in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified beta-conglycinin was expressed in Escherichia coli and recovered in the soluble fraction. After purification on ion-exchange HPLC, the modified beta-conglycinin was digested by trypsin to release integrated DPR. The yield of DPR from 1 mole of the modified beta-conglycinin was 1.2 mole.     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

Endogenous Dopamine Maintains Synchronous Oscillation of Intracellular Calcium in Primary Cultured-Mouse Midbrain Neurons

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

Abrogation of autoimmune disease in Lyn-deficient mice by the deletion of IL-5 receptor alpha chain gene

Lyn, the src-family protein tyrosine kinase, plays a crucial role in the regulation of B cell antigen receptor (BCR)- and IL-5-receptor (IL-5R)-mediated signaling. Lyn-deficient mice have been reported to exhibit an increase in B-1 cell numbers, splenomegaly and accumulation of lymphoblast-like cells in the spleen with age, resulting in hyperimmunoglobulinemia and glomerulonephritis caused by the deposition of autoantibody complexes. To elucidate the role of IL-5 in B-1 cell activation, autoantibody production and autoimmune diseases, Lyn-deficient mice were crossed with IL-5Ralpha chain (IL-5Ralpha)-deficient mice and generated Lyn- and IL-5Ralpha-deficient (DKO) mice. In contrast to Lyn-deficient mice, DKO mice showed significantly reduced splenomegaly and lymphoadenopathy and reduced B-1 cell number in the peritoneal cavity. DKO mice also secreted low levels of IgM and IgG autoantibodies. Biochemical and histological analyses revealed that DKO mice showed milder pathogenesis of autoimmune-like disorders than Lyn-deficient mice. These results suggest involvement of IL-5 in enhanced B-1 cell activation, autoantibody production, and development of autoimmune disease in Lyn-deficient mice.     

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.     

beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.     

Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl(2) but not by CdCl(2). These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.     

Protein oxidation by the cytochrome P450 mixed-function oxidation system

This mini-review summarizes results of studies on the oxidation of proteins and low-density lipoprotein (LDL) by various mixed-function oxidation (MFO) systems. Oxidation of LDL by the O₂/FeCl₃/H₂O₂/ascorbate MFO system is dependent on all four components and is much greater when reactions are carried out in the presence of a physiological bicarbonate/CO₂ buffer system as compared to phosphate buffer. However, FeCl₃ in this system could be replaced by hemin or the heme-containing protein, hemoglobin, or cytochrome c. Oxidation of LDL by the O₂/cytochrome P450 cytochrome c reductase/NADPH/FeCl₃ MFO system is only slightly higher (25%) in the bicarbonate/CO₂ buffer as compared to phosphate buffer, but is dependent on all components except FeCl₃. Omission of FeCl₃ led to a 60% loss of activity. These results suggest that peroxymonobicarbonate and/or free radical derivatives of bicarbonate ion and/or CO₂ might contribute to LDL oxidation by these MFO systems.