Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A.

Two proteins, a 56-kDa protein (p56) and a 58-kDa protein (p58), are produced from the hepatitis C virus (HCV) nonstructural region 5A (NS5A). Recently, we found that both proteins are phosphorylated at serine residues and that p58 is a hyperphosphorylated form of p56. Furthermore, hyper-phosphorylation depends on the production of an intact form of the HCV NS4A protein. To clarify the nature of NS5A phosphorylation, pulse-chase analysis was performed with a transient protein production system in cultured cells. The study indicated that basal and hyperphosphorylation of NS5A occurred after proteolytic production of NS5A was complete. In an attempt to identify the location of the hyperphosphorylation sites in p58, proteins with sequential deletions from the C-terminal region of NS5A and with mutations of possible phosphorylated serine residues to a neutral amino acid, alanine, were constructed. The deleted or mutated proteins were then tested for hyperphosphorylation in the presence of the NS4A product. Here, we report that serine residues 2197, 2201, and/or 2204 are important for hyper-phosphorylation. Important sites for basal phosphorylation were identified in the region from residues 2200 to 2250 and in the C-terminal region of the NS5A product. A subcellular localization study showed that most of the NS5A products were localized in the nuclear periplasmic membrane fraction.     

Circular DNA Plasmid in the Phytopathogenic Fungus Alternaria Alternata: Its Temperature-Dependent Curing and Association with Pathogenicity

We found the presence of plasmid DNA in strain T88-56 of the Japanese pear pathotype of Alternaria alternata, which causes black spot of certain cultivars of Japanese pear by producing host-specific AK-toxin. The plasmid, designated pAAT56, was identified to be an ~5.4-kilobase (kb) circular molecule by electron microscopic observation and restriction endonuclease mapping. Southern blot analysis showed that pAAT56 DNA had no homology with either nuclear or mitochondrial DNA. Cultures of strain T88-56 grown at 26° showed markedly reduced plasmid levels relative to those grown at lower temperatures. The strain was completely cured of pAAT56 during growth at 29°. Temperature-dependent curing of pAAT56 was confirmed by using single-protoplast isolates from mycelia grown at 23°, most of which maintained the plasmid, and from mycelia grown at 29°, most of which had lost the plasmid. Northern blot analysis detected the presence of three RNA species (~1.7, 2.7 and 5.4 kb) transcribed from pAAT56. The biological function of pAAT56 was observed using single-protoplast isolates from mycelia that either contained or had been cured of pAAT56. The plasmid-containing isolates tended to be reduced in AK-toxin production and pathogenicity compared with the plasmid-cured isolates.     

Hepatitis C virus envelope proteins bind lactoferrin.

Hepatitis C virus (HCV) has two envelope proteins, E1 and E2, which form a heterooligomer. During dissection of interacting regions of HCV E1 and E2, we found the presence of an interfering compound or compounds in skim milk. Here we report that human as well as bovine lactoferrin, a multifunctional immunomodulator, binds two HCV envelope proteins. As determined by far-Western blotting, the bacterially expressed E1 and E2 could bind lactoferrin in human milk directly separated or immunopurified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bindings of lactoferrin and HCV envelope proteins in vitro were confirmed by another method, the pull-down assay, with immunoprecipitated lactoferrin-bound protein A resin. By the same assay, mammal-expressed recombinant E1 and E2 were also demonstrated to bind human lactoferrin efficiently in vitro. Direct interaction between E2 and lactoferrin was proved in vivo, since anti-human lactoferrin antibody efficiently coimmunoprecipitated with secreted and intracellular forms of the E2 protein, but not glutathione S-transferase (GST), from lysates of HepG2 cells transiently cotransfected with the expression plasmids of human lactoferrin and gE2t-GST (the N-terminal two-thirds of E2 fused to GST) or GST. The N-terminal loop of lactoferrin, the region important for the antibacterial activity, has only a little role in the binding ability to HCV E2 but affected the secretion or stability of lactoferrin. Taken together, these results indicate the specific interaction between lactoferrin and HCV envelope proteins in vivo and in vitro.     

Multiple glucose 6-phosphate dehydrogenase-deficient variants correlate with malaria endemicity in the Vanuatu archipelago (southwestern Pacific).

In studying the relationship between genetic abnormalities of red blood cells and malaria endemicity in the Vanuatu archipelago in the southwestern Pacific, we have found that of 1,442 males tested, 98 (6.8%) were G6PD deficient. The prevalence of GdPD deficiency varied widely (0%-39%), both from one island to another and in different parts of the same island, and generally correlated positively with the degree of malaria transmission. The properties of G6PD from GdPD-deficient subjects were analyzed in a subset of 53 samples. In all cases the residual red-blood-cell activity was < 10%. There were three phenotypic patterns. PCR amplification and sequencing of the entire coding region of the G6PD gene showed that the first of these patterns corresponded to G6PD Union (nucleotide 1360C-->T; amino acid 454Arg-->Cys), previously encountered elsewhere. Analysis of samples exhibiting the second pattern revealed two new mutants: G6PD Vanua Lava (nucleotide 383T-->C; amino acid 128Leu-->Pro) and G6PD Namoru (nucleotide 208T-->C; amino acid 70Tyr-->His); in three samples, the underlying mutation has not yet been identified. Analysis of the sample exhibiting the third pattern revealed another new mutant: G6PD Naone (nucleotide 497G-->A; amino acid 166Arg-->His). Of the four mutations, G6PD Union and G6PD Vanua Lava have a polymorphic frequency in more than one island; and G6PD Vanua Lava has also been detected in a sample from Papua New Guinea. G6PD deficiency is of clinical importance in Vanuatu because it is a cause of neonatal jaundice and is responsible for numerous episodes of drug-induced acute hemolytic anemia.     

In vitro study on release of thyroid hormone in solitary autonomously functioning thyroid nodules using cell culture method

In vitro release of thyroid hormone was investigated under basal and TSH-stimulated conditions in the solitary autonomously functioning thyroid nodules (AFTN). A small portion (0.5 g of wet weight) of the nodules and adjacent thyroid tissues removed surgically from five patients with solitary AFTN were prepared for the dispersed cell culture. In the experiment on non TSH-stimulated (basal) conditions, those culture media which were totally replaced on the 5th day after primary culture were utilized for the determination of thyroxine (T4) and triiodothyronine (T3) by radioimmunoassay. T4 and T3 levels in culture media of the functioning nodules were 1.15 +/- 0.33 microgram/dl (mean +/- SEM) and 2.72 +/- 0.68 ng/ml, contrasted with levels of 0.67 +/- 0.09 microgram/dl and 1.24 +/- 0.22 ng/ml in the paranodular tissues. The mean ratios of T3/T4 of the nodules and paranodular tissues were 0.25 +/- 0.02 and 0.19 +/- 0.02, respectively (p less than 0.05). Meanwhile, in another experiment under TSH stimulatory conditions employing 40 and 80 microU/ml of human TSH, there were no significant differences in T4 and T3 releases when the two groups were compared.     

Alteration in two enzymatically active forms of valyl-tRNA synthetase during the sporulation of Bacillus subtilis.

Two enzymatically active forms of valyl-tRNA synthetase [EC 6.1.1.9] were found in the cells of Bacillus subtilis. The aminoacylation activities of the two forms were altered during the sporulation of B. subtilis. The tRNA'S acylated by these enzymes were analyzed by methylated albumin-Kieselguhr column chromatography.     

Formation of porphyrin pi cation radical in zinc-substituted horseradish peroxidase

Zinc-substituted horseradish peroxidase is oxidized by K2IrCl6 to a characteristic state which retains one oxidizing equivalent more than the zinc peroxidase. The oxidized enzyme gives an optical absorption spectrum similar to that of compound I of peroxidase and catalase, and a g = 2 electron paramagnetic resonance signal which has an intensity corresponding to the porphyrin content. It is reduced back to the zinc peroxidase by a stoichiometric amount of ferrocyanide or by a large excess of K3IrCl6. From the equilibrium data, the value of E0' for the zinc peroxidase couple is estimated to be 0.74 V at pH 6. The oxidized zinc peroxidase is also formed by the addition of H2O2 or upon illumination with white light. The rate constants for the oxidation by K2IrCl6 and H2O2 at pH 8.0 are 8 x 10(5) and 8 x 10(2) M-1 s-1, respectively. No essential spectral change can be observed when K2IrCl6 is added to the metal-free peroxidase (protoporphyrin--apoperoxidase complex) or to zinc-substituted sperm whale myoglobin.