Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.     

Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity

A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1–150] and the lysozyme (aa 401–624) domains but not the linker domain (aa 151–400) caused efficient lysis of S . aureus . Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Study on the interaction between soybean beta-amylase and substrate by the stopped-flow method.

The hydrolysis of substrates (maltoheptaose, maltopentaose, and maltotetraose) catalyzed by soybean beta-amylase [EC 3.2.1.2] at pH 5.4 and 25 degrees C was followed by monitoring small changes in the quenching of fluorescence due to tryptophan residues by the stopped-flow method. By analysis of whole time course, the dissociation constants, KdS, of enzyme-substrate and enzyme-product complexes were reasonably evaluated; and the difference in fluorescence intensities per mol between the enzyme-complex (ES or EP) and the free enzyme, delta F, was determined. The molecular activity, k0, was also determined by a new method of half time analysis. The KdS and k0 values are in good agreement with our kinetic data reported previously. The delta Fs of substrates were of smaller magnitude than those of products (G2 and G3), which means that the higher the binding affinity of the ligand is, the smaller the delta F value is. This indicates that at least two tryptophan residues must be located in the active site if the enzyme is rigid, or that if there is only one, the active site must undergo a structural change caused by the binding of ligand.     

Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

Contents of Free Adenine in Soybeans from Several Countries

Methionine auxotrophic mutants of Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine, and mutated lysE (lysE24) encoding the L-lysine exporter from Corynebacterium glutamicum 2256, produced higher amounts of L-lysine from methanol as sole carbon source than did other amino acid auxotrophic mutants. Especially, the M. methylotrophus 102 strain, carrying both dapA24 and lysE24, produced L-lysine in more than 1.5 times amounts higher than the parent. A single-base substitution was identified in this auxotroph in codon-329 of the open reading frame of metF, encoding 5,10-methylene-tetra-hydrofolate reductase. We constructed a metF disruptant mutant carrying both dapA24 and lysE24, and confirmed increases in L-lysine production. This is the first report to the effect that metF deficient increased L-lysine production in methylotroph.     

Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system.

Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion. Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.     

Comparative studies of the haemagglutination of adult and umbilical cord erythrocytes by animal lectins

1. The sugar specificities of four lactose-binding lectins were studied through the agglutination of adult and/or umbilical cord human erythrocytes (AHRBC and/or CHRBC). 2. Rana catesbeiana egg lectin specifically agglutinated both intact blood group A-AHRBC and intact blood group A-CHRBC. 3. Rana catesbeiana liver lectin agglutinated intact A-AHRBC much more strongly than intact A-CHRBC. 4. Xenopus laevis skin lectin nonspecifically agglutinated AHRBC and CHRBC. 5. Plecoglossus altivelis egg lectin specifically agglutinated intact B-AHRBC, but weakly agglutinated intact B-CHRBC. 6. Comparative studies of lectin-induced AHRBC or CHRBC agglutination clarified the sugar-binding specificities of these lectins.     

Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes

Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.