Overexpression of Newcastle disease virus (NDV) V protein enhances NDV production kinetics in chicken embryo fibroblasts

Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established. The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strains.     

MT2 Melatonin Receptor Immunoreactivity in Neurons is Very High in the Aged Hippocampal Formation in Gerbils

Melatonin exerts many physiological functions via its G protein-coupled receptors. In the present study, we investigated age-related changes in MT2 melatonin receptor immunoreactivity and its levels in the gerbil hippocampus during normal aging. In the postnatal month 1 (PM 1) group, MT2 immunoreaction was well observed in neurons in all subregions of the gerbil hippocampus. In the PM 3 and 6 groups, MT2 immunoreactivity in neurons was decreased compared to that in the PM 1 group. Thereafter, MT2 immunoreactivity in neurons was increased. In the PM 18 and 24 groups, MT2 immunoreactivity in neurons was strong in all subregions of the gerbil hippocampus. In addition, the number of MT2 immunoreactive cells was lowest at PM 3 and highest at PM 24. From western blot analysis, age-dependent change pattern in MT2 level in the gerbil hippocampus was similar to the immunohistochemical result. These results indicate that MT2 immunoreactivity and levels are altered in the gerbil hippocampus during normal aging; lowest at young adult stage and highest at aged stage.     

Proteomic profiling of human bone marrow mesenchymal stem cells under shear stress

Diets containing 8% salt or 4% fructose (FR) cause insulin resistance and increase tissue methylglyoxal and advanced glycation end products (AGEs), platelet cytosolic-free calcium, and systolic blood pressure (SBP) in rats. In WKY rats, we have shown that moderately high salt, 4% NaCl (MHS) alone in diet does not cause hypertension, and when given along with 4% FR it does not have an additive effect. N-acetylcysteine (NAC) or l-arginine (ARG), treatment alone does not prevent hypertension in this model. The objectives of this study were to investigate the effect of NAC plus ARG in diet on SBP, platelet cytosolic-free calcium in a MHS + FR model, and to measure the plasma levels of methylglyoxal and the AGE, methylglyoxal-derived hydroimidazolone (MGH). At 7 weeks of age, WKY rats were divided into three groups: control group was given regular rat chow (0.7% NaCl) and water; MHS + FR group, diet containing 4% NaCl and 4% FR in drinking water; and MHS + FR + NAC + ARG group, MHS diet supplemented with 1.5% N-acetylcysteine (NAC) and 1.5% l-arginine (ARG), and 4% FR in drinking water, and followed for 6 weeks. NAC + ARG prevented the increase in platelet cytosolic-free calcium and SBP in MHS + FR treated rats. There was no difference in mean values of plasma methylglyoxal and MGH among the groups. In conclusion, NAC + ARG treatment is effective in preventing hypertension in a moderately high salt + FR-induced animal model. Plasma methylglyoxal and MGH may not represent tissue modification or, alternatively, other tissue AGEs, derived from methylglyoxal or other aldehydes, may be involved in hypertension in this model.     

Genetic and functional interactions between Mus81–Mms4 and Rad27

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81–Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81–Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81–Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81–Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81–Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein–protein interaction between the two proteins. Our in vitro data indicate that Mus81–Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81–Mms4 in context of DNA replication.     

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity and its protein levels in the gerbil hippocampus during normal aging

Phosphorylated extracellular signal-regulated kinase (pERK) mediates neuronal synaptic plasticity, long-term potentiation, and learning and memory in the hippocampus. In this study, we examined pERK1/2 immunoreactivity and its protein level in the gerbil hippocampus at various ages. In the postnatal month 1 (PM 1) group, very weak pERK1/2 immunoreactivity was detected in the hippocampus. In the CA1 region, pERK1/2 immunoreactivity was considerably increased in the stratum pyramidale in the PM 6 group. Thereafter, pERK1/2 immunoreactivity was decreased. In the CA2/3 region, pERK1/2 immunoreactivity increased in an age-dependent manner until PM 12. Thereafter, numbers of pERK1/2-immunoreactive neurons were decreased. However, in the mossy fiber zone, pERK1/2 immunostaining became stronger with age. In the dentate gyrus, a few pERK1/2-immunoreactive cells were observed until PM 12. In the PM 18 and 24 groups, numbers of pERK1/2-immunoreactive cells were increased, especially in the polymorphic layer. In Western blot analysis, pERK1/2 level in the gerbil hippocampus was increased with age. These results indicate that total pERK1/2 levels are increased in the hippocampus with age. However pERK1/2 immunoreactivity in subregions of the gerbil hippocampus was changed with different pattern during normal aging.     

Helical image reconstruction of the outward-open human erythrocyte band 3 membrane domain in tubular crystals

The C-terminal membrane domain of erythrocyte band 3 functions as an anion exchanger. Here, we report the three-dimensional (3D) structure of the membrane domain in an inhibitor-stabilized, outward-open conformation at 18 Å resolution. Unstained, frozen-hydrated tubular crystals containing the membrane domain of band 3 purified from human red blood cells (hB3MD) were examined using cryo-electron microscopy and iterative helical real-space reconstruction (IHRSR). The 3D image reconstruction of the tubular crystals showed the molecular packing of hB3MD dimers with dimensions of 60 × 110 Å in the membrane plane and a thickness of 70 Å across the membrane. Immunoelectron microscopy and carboxyl-terminal digestion demonstrated that the intracellular surface of hB3MD was exposed on the outer surface of the tubular crystal. A 3D density map revealed that hB3MD consists of at least two subdomains and that the outward-open form is characterized by a large hollow area on the extracellular surface and continuous density on the intracellular surface.     

Reducing Pectobacterium virulence by expression of an N-acyl homoserine lactonase gene Plpp-aiiA in Lysobacter enzymogenes strain OH11

An N-acyl homoserine lactonase gene aiiA, transcribed by a strong and constitutive Escherichia coli promoter P_lpp (Accession No. EU723847), was transformed into Lysobacter enzymogenes strain OH11, creating strain OH11A. The N-acyl-homoserine lactone (AHL)-degradation assay showed that transformant OH11A acquired the ability to degrade AHL molecules produced by Agrobacterium tumefaciens, Pectobacterium carotovorum, Pseudomonas syringae pv. tomato strain DC3000 and Acidovorax avenae subsp. citrulli. Pathogenicity tests showed that while the parental strain OH11 did not reduce P. carotovorum infection, the transformant OH11A caused a strong reduction of Pectobacterium virulence on Chinese cabbage and cactus, whereas strain OH11A did not seem to interfere with the normal growth of this pathogen in cabbages. In antimicrobial activity assays, strain OH11A and OH11 showed similar antimicrobial activity against Phytophthora capsici and Sclerotinia sclerotiorum. This work provided a new strategy for developing genetically engineered multi-functional L. enzymogenes strains that possessed the ability to biologically control fungal pathogens and reduce bacterial pathogenicity.