A single amino acid mutation at position 170 of human parainfluenza virus type 1 fusion glycoprotein induces obvious syncytium formation and caspase-3-dependent cell death

An escape mutant of human parainfluenza virus type 1 (hPIV1), which was selected by serial passage in the presence of a sialidase inhibitor, 4-O-thiocarbamoylmethyl-2-deoxy-2,3-didehydro-N-acetylneur-aminic acid (TCM-Neu5Ac2en), exhibited remarkable syncytium formation and virus-induced cell death in LLC-MK2 cells but no difference in susceptibility for the sialidase inhibitor TCM-Neu5Ac2en from that of wild-type hPIV1 strain C35 (WT). The mutant virus also had higher replication and plaque formation abilities. The mutant virus acquired two amino acid mutations, Glu to Gly at position 170 and Ala to Glu 442 in fusion (F) glycoprotein, but no mutations in haemaggulutinin-neuraminidase (HN) glycoprotein. Using cells co-expressing F and HN genes with site-specific mutagenesis, we demonstrated that a point mutation of Glu to Gly at position 170, which was estimated to be located in hPIV1 F glycoprotein heptad repeat 1, was required for obvious syncytium formation and caspase-3-dependent cell death. In contrast, wild-type F glycoprotein induced no synctium formation or cell death. The findings suggest that a single amino acid mutation of hPIV1 F glycoprotein promotes syncytium formation that is followed by caspase-3-dependent cell death.     

Regulation of the expression of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium cells.

Lysyl oxidase, an extracellular amine oxidase, controls the maturation of collagen and elastin. We examined the regulation of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium (RPE) cells in relation to the changes in subretinal fluid transport and phenotype of RPE cells. The level of the mRNA in cells grown on microporous membranes was markedly increased by application of hyperosmotic mannitol solution on the apical side (191% of control), implying that RPE cells express more lysyl oxidase in the condition which may cause the accumulation of subretinal fluid. Platelet-derived growth factor increased the mRNA level in subconfluent cells in culture (137% of control) and basic fibroblast growth factor decreased it (79% of control). In addition, exposure of cells to retinoic acid alone or in combination with dibutyryl cAMP for 22 days markedly decreased the level of lysyl oxidase mRNA (52 or 35% of control) while increasing the level of mRNA of N-acetylglucosaminidase (NAG), a marker enzyme for lysosomes (162 or 142% of control). Moreover, the level of lysyl oxidase mRNA in cells grown on microporous membranes was lower than that in cells grown on plastic dishes, while the level of NAG mRNA in the former cells was higher than that in the latter. Taken together, the expression of lysyl oxidase seemed to increase during proliferation of RPE cells and decrease toward differentiation. beta-Aminopropionitrile, an inhibitor of lysyl oxidase, significantly inhibited the contraction of collagen gels by fetal calf serum, suggesting that lysyl oxidase may be involved in pathogenesis caused by RPE cells.     

Endogenous neurotoxic dopamine derivative covalently binds to Parkinson's disease‐associated ubiquitin C‐terminal hydrolase L1 and alters its structure and function

Parkinson's disease (PD) is a common neurodegenerative disease, but its pathogenesis remains elusive. A mutation in ubiquitin C‐terminal hydrolase L1 (UCH‐L1) is responsible for a form of genetic PD which strongly resembles the idiopathic PD. We previously showed that 1‐(3′,4′‐dihydroxybenzyl)‐1,2,3,4‐tetrahydroisoquinoline (3′,4′DHBnTIQ) is an endogenous parkinsonism‐inducing dopamine derivative. Here, we investigated the interaction between 3′,4′DHBnTIQ and UCH‐L1 and its possible role in the pathogenesis of idiopathic PD. Our results indicate that 3′,4′DHBnTIQ binds to UCH‐L1 specifically at Cys152 in vitro. In addition, 3′,4′DHBnTIQ treatment increased the amount of UCH‐L1 in the insoluble fraction of SH‐SY5Y cells and inhibited its hydrolase activity to 60%, reducing the level of ubiquitin in the soluble fraction of SH‐SY5Y cells. Catechol‐modified UCH‐L1 as well as insoluble UCH‐L1 were detected in the midbrain of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐treated PD model mice. Structurally as well as functionally altered UCH‐L1 have been detected in the brains of patients with idiopathic PD. We suggest that conjugation of UCH‐L1 by neurotoxic endogenous compounds such as 3′,4′DHBnTIQ might play a key role in onset and progression of idiopathic PD.

     

Early development in utero of bovine nuclear transfer embryos using early G1 and G0 phase cells

Bovine somatic cell nuclear transfer (NT) embryos can develop to normal calves, but the success rates are still quite low. Recently, enhanced development of bovine NT embryos to full term has been achieved using fibroblasts at the early G1 phase instead of cells at the quiescent (G0) phase. In the present study, we examined the morphological development in utero of NT embryos using early G1 phase cells (eG1-NT embryos) and G0 phase cells (G0-NT embryos). We produced eG1- and G0-NT blastocysts, and then they were transferred to recipient heifers for transient development in utero up to day 14 of gestation. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The rate of formation of embryonic disks of the recovered embryos was the same among the groups of eG1-NT, IVF, and AI embryos (p>0.05). The formation rate in eG1-NT embryos was significantly higher than that in G0-NT embryos (p<0.05). The lengths of eG1-NT embryos were the same as those of IVF, parthenogenetic, and AI embryos (p>0.05), but significantly shorter than those of G0-NT embryos (p<0.01). We conclude that the morphological development of day 14 embryos derived from eG1-NT embryos was mostly similar to that of AI embryos, but that the morphological development of G0-NT embryos was abnormally large and different from that of AI and eG1-NT embryos.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

Metabolic Fate of O-Ethyl S,S-Diphenyl Phosphorodithiolate (Hinosan®) in Rice Plant

Metabolism and residual fate of O-ethyl S,S-diphenyl phosphorodithiolate (Hinosan®) applied on rice plant was examined by using ³⁵S-labeled or ³²P-labeled compound. Ion exchange chromatography, thin-layer chromatography and gas-liquid chromatography with flame thermionic detector or flame photometric detector were applied for identification of water soluble and toluene soluble metabolites of Hinosan. Degradation of Hinosan at the initial stage of metabolism was mainly the cleavage of P-S linkage, and a large portion of phenyl dihydrogen phosphorothiolate and a minor portion of O-ethyl S-phenyl hydrogen phosphorothiolate were found as water soluble metabolites. Phenylthio radical released on the production of the above mentioned metabolites was recovered as diphenyl disulfide, which was finally converted to sulfuric acid through benzenesulfonic acid. Triphenyl phosphorotrithiolate and O,O-diethyl S-phenyl phosphorothiolate were produced by transesterification between molecules of Hinosan at the initial stage of metabolism. Examination of metabolites in rice grains showed that sulfur and phosphorus atoms in Hinosan were incorporated into neutral or cationic substances probably after several steps of chemical transformation.     

Neuronal nitric oxide synthase (NNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: implication for oxidative stress

To determine the mechanism of 2,4,6-trinitrotoluene (TNT)-induced oxidative stress involving neuronal nitric oxide synthase (nNOS), we examined alterations in enzyme activity and gene expression of nNOS by TNT, with an enzyme preparation and rat cerebellum primary neuronal cells. TNT inhibited nitric oxide formation (IC(50) = 12.4 microM) as evaluated by citrulline formation in a 20,000 g cerebellar supernatant preparation. A kinetic study revealed that TNT was a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. It was found that purified nNOS was capable of reducing TNT, with a specific activity of 3900 nmol of NADPH oxidized/mg/min, but this reaction required CaCl(2)/calmodulin (CaM). An electron spin resonance (ESR) study indicated that superoxide (O(2)(.-)) was generated during reduction of TNT by nNOS. Exposure of rat cerebellum primary neuronal cells to TNT (25 microM) caused an intracellular generation of H(2)O(2), accompanied by a significant increase in nNOS mRNA levels. These results indicate that CaM-dependent one-electron reduction of TNT is catalyzed by nNOS, leading to a reduction in NO formation and generation of H(2)O(2) derived from O(2)(.-). Thus, it is suggested that upregulation of nNOS may represent an acute adaptation to an increase in oxidative stress during exposure to TNT.     

Modulation of functional properties of KCNQ1 channel by association of KCNE1 and KCNE2

The KCNE proteins (KCNE1 through KCNE5) function as beta-subunits of several voltage-gated K(+) channels. Assembly of KCNQ1 K(+) channel alpha-subunits and KCNE1 underlies cardiac I(Ks), while KCNQ1 interacts with all other members of KCNE forming complexes with different properties. Here we investigated synergic actions of KCNE1 and KCNE2 on functional properties of KCNQ1 heterologously expressed in COS7 cells. Patch-clamp recordings from cells expressing KCNQ1 and KCNE1 exhibited the slowly activating current, while co-expression of KCNQ1 with KCNE2 produced a practically time-independent current. When KCNQ1 was co-expressed with both of KCNE1 and KCNE2, the membrane current exhibited a voltage- and time-dependent current whose characteristics differed substantially from those of the KCNQ1/KCNE1 current. The KCNQ1/KCNE1/KCNE2 current had a more depolarized activation voltage, a faster deactivation kinetics, and a less sensitivity to activation by mefenamic acid. These results suggest that KCNE2 can functionally couple to KCNQ1 even in the presence of KCNE1.