Adsorption of polyhedra of a cytoplasmic-polyhedrosis virus by soil particles

Tests on the quantitative adsorption of polyhedra of a cytoplasmic-polyhedrosis virus of Bombyx mori to soil showed an increase in the adsorption of polyhedra with an increase in the amount of soil, but the number of polyhedra adsorbed per unit weight of soil decreased. The number of polyhedra adsorbed to a fixed quantity of soil was in direct proportion to the polyhedron concentration, and the amount of adsorption increased with acidity but decreased with the addition of reagents which masked the polyvalent cations exposed on the surfaces of soil particles. The polyhedra applied to the top of a soil column were detected microscopically within 4 cm from the surface after the equivalent of 1,120 cm depth of water was passed through the column. The polyhedra occurred on the upper surface of the soil column and on the walls of voids formed by random packing of the soil particles.     

Amino acid composition of proteins extracted from virions of cytoplasmic-polyhedrosis virus

The ratio of basic to acidic amino acids in protein from free virions of CPV of Malacosoma disstria was 0.26, while that of occluded virions liberated from the polyhedra was 0.43. The ratio in free virions of CPV of Orgyia leucostigma was 0.39. This suggests that the virions of O. leucostigma reach a more advanced stage of maturity before occlusion than do those of M. disstria. By comparison, the composition of the free virions of O. leucostigma CPV was remarkably similar to that of the midgut protein of host cells. On the other hand, the composition of ribosomes of M. disstria was significantly different from either free or occluded virions of M. disstria CPV. Protein amino acids from virions of Bombyx mori differed from those of virions of other hosts in content of basic amino acids yielding a ratio of 0.68. These characteristics help to identify the viral strains and support previous serological studies reported with these proteins.     

Ultrastructural investigation on a strain of a cytoplasmic-polyhedrosis virus with nuclear inclusions

A strain of a cytoplasmic-polyhedrosis virus causes the formation of crystalline inclusions almost entirely in the nucleus, and only rarely in the cytoplasm, of the midgut epithelial cells of the silkworm Bombyx mori. It also differs from the typical strain in causing the hypertrophy of the nucleoli and the formation of dense reticulum and spherical bodies in the nucleus. The virus particles and the virogenic stromata of the new strain resemble those of the typical strain. The cytoplasmic inclusions contain virus particles, while the nuclear inclusions do not. When the infected larvae are kept at 30°C for 15 hr or at 35°C for 3–15 hr, the nuclear inclusions break up into particles of 70–250 nm in diameter. The particles are dispersed in the cells but not present in the spaces previously occupied by the decomposed inclusions.     

Purification and properties of the Bombyx mori nuclear polyhedrosis virus liberated from polyhedra by dissolution with silkworm gut juice

Occluded virions of the Bombyx mori nuclear polyhedrosis virus were efficiently liberated from polyhedra by dissolution with the silkworm gut juice. The liberated virions were purified by sucrose density gradient centrifugation and the bands of enveloped virions were observed in the gradients. There was no functional difference between the gut juice-liberated and the carbonate-liberated virions. Disruption of enveloped virions by the gut juice was observed, but the formation of nucleocapsids from the degradation of the occluded virions was not detected. High yields of the enveloped virions from the polyhedra dissolved by the gut juice was obtained by separating the virions through sucrose density gradient centrifugation immediately after the dissolution of the polyhedra. Many factors, e.g., rearing seasons, silkworm strains, and rearing conditions, affect the polyhedra-dissolving property of the larval gut juice.     

Insect virus polyhedra, infectious protein crystals that contain virus particles

High-resolution atomic structures have been reported recently for two types of viral polyhedra, intracellular protein crystals produced by ubiquitous insect viruses. Polyhedra contain embedded virus particles and function as the main infectious form for baculoviruses and cypoviruses, two distinct classes of viruses that infect mainly Lepitoptera species (butterflies and moths). Polyhedra are extremely stable and protect the virus particles once released in the environment. The extensive crystal contacts observed in the structures explain the remarkable stability of viral polyhedra and provide hints about how these crystals dissolve in the alkaline midgut, releasing embedded virus particles to infect feeding larvae. The stage is now set to answer intriguing questions about the in vivo crystallization of polyhedra, how virus particles are incorporated into polyhedra, and what determines the size and shape of the crystals. Large quantities of polyhedra can be obtained from infected larvae and polyhedra can also be produced using insect cell expression systems. Modified polyhedra encapsulating other entities in place of virus particles have potential applications as a means to stabilize proteins such as enzymes or growth factors, and the extremely stable polyhedrin lattice may provide a framework for future engineered micro-crystal devices.     

Purification and characterization of a nitroreductase from the soil bacterium Streptomyces mirabilis

A NADH-dependent nitroreductase from an efficient nitro-reducing soil bacterium, Streptomyces mirabilis DUT001, was isolated and characterized. The enzyme was purified to near homogeneity using ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography. The native enzyme was estimated by gel filtration to have a molecular weight of 68 kDa, and its subunit molecular weight determined by SDS-PAGE was about 34 kDa, which indicated this enzyme was a dimer. Polycyclic nitroaromatic compounds were preferred substrates for this enzyme. The purified enzyme exhibited maximum activity at pH 7.5 and 40 °C. The addition of various chemicals such as reducing agents, metal ions, and chelating agents, had effects on enzyme activity. Mg²⁺, Ca²⁺, Sr²⁺, and 1% (w/v) Triton X-100 increased activity. However, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺, and SDS reduced activity. The maximum reaction rate (V_max) was 64 μM min^?1 mg^?1 enzyme and the apparent Michaelis–Menten constants (K_m) for 4-nitro-1,8-naphthalic anhydride and NADH were 276 and 29 μM, respectively. Menadione, bimethylenebis, sodium benzoate, and antimycin A were inhibitors of the purified nitroreductase with apparent inhibition constants (K_is) of 20, 36, 44 and 80 μM, respectively.     

Purification and properties of benzoate-4-hydroxylase from a soil pseudomonad.

Benzoate-4-hydroxylase from a soil pseudomonad was isolated and purified about 50-fold. Polyacrylamide gel electrophoresis of this enzyme preparation showed one major band and one minor band. The approximate molecular weight of the enzyme was found to be 120,000. Benzoate-4-hydroxylase was most active around pH 7.2. The enzyme showed requirements for tetrahydropteridine as the cofactor and molecular oxygen as the electron acceptor. NADPH, NADH, dithiothreitol, β-mercaptoethanol, and ascorbic acid when added alone to the reaction mixture did not support the hydroxylation reaction to any significant extent. However, when these compounds were added together with tetrahydropteridine, they stimulated the hydroxylation. This stimulation is probably due to the reduction of the oxidized pteridine back to the reduced form. This enzyme was activated by Fe²⁺ and benzoate. It was observed that benzoate-4-hydroxylase could catalyze the oxidation of NADPH in the presence of benzoate,p-aminobenzoate, p-nitrobenzoate, p-chlorobenzoate, and p-methylbenzoate, with only benzoate showing maximum hydroxylation. Inhibition studies with substrate analogs and their kinetic analysis revealed that the carboxyl group is involved in binding the substrate to the enzyme at the active center. The enzyme catalyzed the conversion of 1 mol of benzoate to 1 mol of p-hydroxybenzoate with the consumption of slightly more than 1 mol of NADPH and oxygen.     

RNP-complex stoichiometry of Bombyx mori nuclear polyhedrosis virus polyhedra

By gel-filtration through Sephacryl S-300 it was shown that RNP A complex present in polyhedra of Bombyx mori nuclear polyhedrosis virus has molecular weight (M(w)) about 700 kDa. It was shown that RNP A with M(w) 788 kDa is composed of two polyhedrin 13S-associates with M(w) 342 kDa, two p14 polypeptide with M(w) 14 kDa, two 21 kDa small non-coded RNAs and two 17 kDa small non-coded RNAs. The model of RNP A formation from components making it is proposed. The complex role in the course of polyhedron formation and its role in the course of infection are discussed.