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.     

Type 2 diabetes mellitus in obese mouse model induced by monosodium glutamate.

The number of diabetic patients is increasing every year, and new model animals are required to study the diverse aspects of this disease. An experimental obese animal model has reportedly been obtained by injecting monosodium glutamate (MSG) to a mouse. We found that ICR-MSG mice on which the same method was used developed glycosuria. Both female and male mice were observed to be obese but had no polyphagia, and were glycosuric by 29 weeks of age, with males having an especially high rate of incidence (70.0%). Their blood concentrations of glucose, insulin, total cholesterol, and triglycerides were higher than in the control mice at 29 weeks. These high concentrations appeared in younger males more often than in females, and were severe in adult males. Also, the mice at 54 weeks of age showed obvious obesity and increased concentrations of glucose, insulin, and total cholesterol in the blood. The pathological study of ICR-MSG female and male mice at 29 weeks of age showed hypertrophy of the pancreatic islet. This was also observed in most of these mice at 54 weeks. It was recognized as a continuation of the condition of diabetes mellitus. From the above results, these mice are considered to be useful as new experimental model animals developing a high rate of obese type 2 (non-insulin dependent) diabetes mellitus without polyphagia.     

Regulation of mitochondrial morphology and cell survival by Mitogenin I and mitochondrial single-stranded DNA binding protein

We found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I in this paper, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology. We demonstrated that overexpression of Mitogenin I or mtSSB increased elongated or fragmented mitochondria in mouse C2C12 myoblast cells, respectively. On the other hand, the silencing of Mitogenin I or mtSSB by RNA interference led to an increase in fragmented or elongated mitochondria in the cells, respectively, suggesting that Mitogenin I and mtSSB are involved in the processes of mitochondrial fusion and fission, respectively. In addition, we showed that the silencing of Mitogenin I resulted in an increase in the number of trypan blue-positive cells and the silencing of mtSSB resulted in an enhancement of the sensitivity of the cells to apoptotic stimulation by etoposide. The present results demonstrated that these proteins play a role in cell survival.     

Inhibition of NADPH oxidase subunits translocation by tea catechin EGCG in mast cell

The inhibitory mechanism of tea catechins for allergy remains undefined. We studied the effect of catechins, mainly EGCG, on the activation of mast cell line canine cutaneous mastocytoma cells (CM-MC). Compound 48/80 induced the degranulation in CM-MC dose dependently, whereas its release of beta-hexosaminidase was inhibited by EGCG and O-methylated EGCG (EGCG-Me). Both catechins were found to inhibit intracellular ROS generation dose dependently together with DPI. Intracellular ROS generation in human polymorphonuclear leukocytes was also inhibited by EGCG. Neither L-NAME, ebeselen nor NAC inhibited ROS generation. From the Western blot analysis of the subunits components of NADPH oxidase, we detected cytosolic subunits; p47(phox), p67(phox), p40(phox), rac2 and membrane subunits; gp91(phox), p22(phox) in CM-MC. Cytosolic subunits were translocated from cytosol to membrane time dependently after stimulation with compound 48/80. EGCG and DPI inhibited cytosolic subunits from translocating into membrane. These data suggest that EGCG inhibits the activation of NADPH oxidase in CM-MC.     

Negative regulation of hematopoiesis by the fused in myeloproliferative disorders gene product

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.     

Crystal structure of serine dehydratase from rat liver

SDH (L-serine dehydratase, EC 4.3.1.17) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent dehydration of L-serine to yield pyruvate and ammonia. Liver SDH plays an important role in gluconeogenesis. Formation of pyruvate by SDH is a two-step reaction in which the hydroxyl group of serine is cleaved to produce aminoacrylate, and then the aminoacrylate is deaminated by nonenzymatic hydrolysis to produce pyruvate. The crystal structure of rat liver apo-SDH was determined by single isomorphous replacement at 2.8 A resolution. The holo-SDH crystallized with O-methylserine (OMS) was also determined at 2.6 A resolution by molecular replacement. SDH is composed of two domains, and each domain has a typical alphabeta-open structure. The active site is located in the cleft between the two domains. The holo-SDH contained PLP-OMS aldimine in the active site, indicating that OMS can form the Schiff base linkage with PLP, but the subsequent dehydration did not occur. Apo-SDH forms a dimer by inserting the small domain into the catalytic cleft of the partner subunit so that the active site is closed. Holo-SDH also forms a dimer by making contacts at the back of the clefts so that the dimerization does not close the catalytic cleft. The phosphate group of PLP is surrounded by a characteristic G-rich sequence ((168)GGGGL(172)) and forms hydrogen bonds with the amide groups of those amino acid residues, suggesting that the phosphate group can be protonated. N(1) of PLP participates in a hydrogen bond with Cys303, and similar hydrogen bonds with N(1) participating are seen in other beta-elimination enzymes. These hydrogen bonding schemes indicate that N(1) is not protonated, and thus, the pyridine ring cannot take a quinone-like structure. These characteristics of the bound PLP suggest that SDH catalysis is not facilitated by forming the resonance-stabilized structure of the PLP-Ser aldimine as seen in aminotransferases. A possible catalytic mechanism involves the phosphate group, surrounded by the characteristic sequence, acting as a general acid to donate a proton to the leaving hydroxyl group of serine.     

Why does avian influenza A virus hemagglutinin bind to avian receptor stronger than to human receptor? Ab initio fragment molecular orbital studies

Influenza A viruses attach to alpha-sialosides on the target cell surface by their hemagglutinins, which strictly recognize the difference in sialic acid-galactose linkage. Why does avian virus H3 subtype bind to avian receptor Neu5Ac(alpha2-3)Gal stronger than to human receptor Neu5Ac(alpha2-6)Gal? Why does avian H3 mutated Gln226 to Leu preferentially bind to human receptor? In this paper, we theoretically answer the questions by molecular mechanics and ab initio fragment molecular orbital (FMO) calculations. The binding energy between avian H3 and avian receptor is 8.2kcal/mol larger than that of the avian H3-human receptor complex estimated at the FMO-HF/STO-3G level, which is a reason that avian H3 binds to avian receptor stronger than to human receptor. Avian Leu226 H3 clashes to Gal unit on the avian receptor to quite decrease its binding affinity. In contrast, Gal unit on the human receptor forms intermolecular hydrophobic interaction with avian Leu226 H3 to afford moderate binding affinity.     

Synthesis and binding affinities of methylvesamicol analogs for the acetylcholine transporter and sigma receptor

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (Ki=6.7 nM), as well as (-)-vesamicol (Ki=4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki=3.0 nM), as well as SA4503 (Ki=4.4 nM), reported as a sigma-1 mapping agent for positron emission tomography (PET), had a high affinity for the sigma-1 receptor. The binding affinity of (+)-PMV (16) for the sigma-1 receptor (Ki=3.0 nM) was about 13 times higher than that for the sigma-2 (sigma-2) receptor (Ki=40.7 nM). (+)-PMV (16) (Ki=199 nM) had a much lower affinity for VAChT than SA4503 (Ki=50.2 nM) and haloperidol (Ki=41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the sigma-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of sigma receptor ligand, respectively, but may also become a new target compound of VAChT and the sigma-1 receptor radioligand for PET, respectively.