Electron tomography reveals polyhedrin binding and existence of both empty and full cytoplasmic polyhedrosis virus particles inside infectious polyhedra

Previous studies have described the structure of purified cytoplasmic polyhedrosis virus (CPV) and that of polyhedrin protein. However, how polyhedrin molecules embed CPV particles inside infectious polyhedra is not known. By using electron tomography, we show that CPV particles are occluded within the polyhedrin crystalline lattice with a random spatial distribution and interact with the polyhedrin protein through the A-spike rather than as previously thought through the B-spike. Furthermore, both full (with RNA) and empty (no RNA) capsids were found inside polyhedra, suggesting a spontaneous RNA encapsidating process for CPV assembly in vivo.     

A comparison of dogfish and bovine chymotrypsins

Bovine and dogfish chymotrypsins were compared to determine if chymotrypsin from a poikilothermic organism (spiny dogfish (Squalus acanthias] adapted to low temperatures possessed catalytic properties different from those of the same enzyme from a warm-blooded animal. An improved procedure was developed for isolating dogfish pancreatic chymotrypsin. The least hydrophobic and smallest substrate used, p-nitrophenyl acetate, had similar enthalpies of association (delta Ha) with both enzymes, whereas larger, more hydrophobic substrates had delta Ha values that were of opposite sign for the two enzymes. As the temperature increased, the association constants (1/Ks) for p-nitrophenyl valerate and p-nitrophenyltrimethyl acetate increased for dogfish chymotrypsin and decreased for bovine chymotrypsin, while the free energies of association (delta Ga) remained relatively constant. Acylation of chymotrypsin was 1.5-2.5 times slower in the dogfish enzyme than in the bovine enzyme except below 15 degrees C with p-nitrophenyltrimethyl acetate. delta H++ for acylation by p-nitrophenyltrimethyl acetate were 2.0 kcal/mol for the dogfish enzyme and 10.2 kcal/mol for the bovine, whereas delta H++ values were only slightly lower in the dogfish enzyme for the other two substrates. For all substrates, the deacylation rate constant (kcat) was greater with dogfish chymotrypsin than bovine. However, the free energies of activation (delta G++) for deacylation were nearly equal between the two enzymes for each of the substrates.     

Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.     

A comparison of bovine lymphocyte antigens

In Canberra, 31 antigens have been described on the surface of bovine lymphocytes. Seven antigens are subgroups of other antigens. Eleven antigens are similar to the eleven antigens which have been described in Melbourne. Fourteen antigens are similar to twelve international-workshop antigens and two European-workshop antigens.     

A comparison of bovine lymphocyte antigens

In Canberra, 31 antigens have been described on the surface of bovine lymphocytes. Seven antigens are subgroups of other antigens. Eleven antigens are similar to the eleven antigens which have been described in Melbourne. Fourteen antigens are similar to twelve international-workshop antigens and two European-workshop antigens.     

Fungal degradation of pine and straw alkali lignins

Summary Kraft pine and straw lignins were fractionated into aqueous soluble and organic soluble-ether insoluble parts. Chemical analysis, UV characteristics, and gel permeation chromatograms of crude and fractionated lignins were studied. Using pure and mixed, N-limited and non N-limited standing cultures of several fungal species, the biodegradability of curde and fractionated lignins was compared. Straw lignins, especially the aqueous fraction were degraded by most of the fungi studied. Except for Sporotrichum pulverulentum, nitrogen limitation did not seem to favour degradation. The best fungi for degradation under conditions of N-limitation were S. pulverulentum, Humicola fuscoatra, and Aspergillus wentii, under sufficient nitrogen: A. wentii, Chaetomium cellulolyticum and H. fuscoatra. The greatest percentage degradation, 55%, was obtained with S. pulverulentum under nitrogen limited conditions from 1 gl^–1 organic soluble-ether insoluble kraft lignin. Gel chromatography showed that the degradation was over the complete molecular size range.     

Molecular identification and characterization of a new type of bovine enterovirus

Bovine enteroviruses belong to the family Picornaviridae. Little is known about their pathogenic potential; however, they cause asymptomatic infections in cattle and are excreted in feces. In the present study, viruses isolated from environmental samples were sequenced. According to phylogenetic analyses and standard picornavirus nomenclature, these isolates constitute a new type of bovine enterovirus serogroup A.     

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.     

Non-oxidative and oxidative degradation of d-galacturonic acid with alkali

Degradation products from non-oxidative and oxidative treatment of d-galacturonic acid with alkali have been analysed by g.l.c.-m.s.; 13 hydroxy monocarboxylic acids and 26 dicarboxylic acids were identified in addition to several isomerisation products. In the absence of oxygen, the main degradation products were 3-deoxy-lyxo-hexaric, 3-deoxy-xylo-hexaric, malic, tartonic, C-methyltartronic, lactic, and 3-deoxytetronic acids. In the presence of oxygen, arabinaric, threaric, malic, tartronic, and glycolic acids were formed in large proportions. The routes of formation for most of the compounds are outlined.     

Expression of rotavirus VP2 produces empty corelike particles.

The complete VP2 gene of bovine rotavirus strain RF has been inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. Cotransfection of Spodoptera frugiperda 9 cells with wild-type baculovirus DNA and transfer vector DNA led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. Infection of S. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the authentic rotavirus VP2. The protein binds double-stranded RNA and DNA in an overlay protein blot assay. Expressed VP2 assembles in the cytoplasm of infected cells in corelike particles 45 nm in diameter. These corelike particles were purified by sucrose gradient centrifugation and found to be devoid of nucleic acid. Coexpression of VP2 and VP6 from heterologous rotavirus strains (bovine and simian) resulted in the formation of single-shelled particles. These results definitively show the existence of an innermost protein shell in rotavirus which is formed independently of other rotavirus proteins. These results have implications for schemes of rotavirus morphogenesis.     

Expression and self-assembly of empty virus-like particles of hepatitis E virus.

Hepatitis E virus (HEV) is a pathogenic agent that causes fecally-orally transmitted acute hepatitis. The genome, a single-stranded positive-sense RNA, encodes three forward open reading frames (ORFs), in which an approximately 2-kb structural protein is located in the 3' end. To produce HEV-like particles the structural protein, with its N terminus truncated (amino acid residues 112 to 660 of ORF2), was expressed in insect Tn5 cells by a recombinant baculovirus. In addition to the primary translation product with a molecular mass of 58 kDa, a large amount of a further-processed molecule with a molecular mass of 50 kDa was generated and efficiently released into the culture medium. Electron microscopic observation of the culture medium revealed that the 50-kDa protein self-assembled to form empty virus-like particles (VLPs). The buoyant density of the VLPs in CsCl was 1.285 g/cm3 and their diameter was 23.7 nm, a little smaller than the 27 nm of native HEV particles secreted into the bile or stools of experimentally infected monkeys. The yield of the VLPs was 1 mg per 10(7) cells as a purified form. The particles possess antigenicity similar to that of authentic HEV particles and, consequently, they appear to be a good antigen for the sensitive detection of HEV-specific immunoglobulin G (IgG) and IgM antibodies. Furthermore, the VLP may be the most promising candidate yet for an HEV vaccine, owing to its potent immunogenicity.     

A comparison of six methods for the concentration of a porcine enterovirus.

Single-step concentration of porcine enterovirus strain T80 by adsorption to the polyelectrolyte PE60 gave virus concentration factors of 35- to 88-fold in terms of plaque-forming units, with recovery rates of 22 to 75% of total virus present in the original virus suspension. Concentration by separation in an aqueous polymer two-phase system gave virus concentration factors of 56- to 105-fold and recovery rates of 37 to 107%. In the latter procedure, sodium dextran sulfate appeared to have no effect on plaque numbers, although plaques were sharper and clearer when this substance was incorporated in the overlay. The failure of sonication of virus concentrated by either procedure to increase plaque numbers indicated the absence of virus aggregates in the concentrates. T80 virus was not effectively concentrated by cobalt chloride or polyethylene glycol precipitation or by adsorption to either aluminium hydroxide or calcium hydrogen phosphate.     

Microbial degradation of tyre rubber particles

Degradation of rubber particles from tyre treads, having diameters from 0.8 to 2.3 mm, was achieved using Nocardia sp. 835A-Rc, a mutant strain with strong rubber-degrading ability. The entire surface of the particles was uniformly attacked by the organism either without stirring of the culture medium or at a very low stirring rate of 40 rpm. At a higher rate of stirring, however, a small number of large microbial colonies were formed on the rubber surface and separate deep semi-spherical cavities were observed after the removal of microbial cells by washing. The number of microbial colonies decreased with increasing stirring rate but each one of the colonies became larger at the same time. As the result of these two counteracting effects of stirring on microbial activity, the weight loss of the particles increased when the stirring rate was raised from 0 to 40 rpm but decreased when the rate was increased from 40 to 70 or 150 rpm. At the stirring rate of 40 rpm, the weight losses of the particles with mean diameters of about 0.8, 1.1 and 2.3 mm were 57, 50 and 36%, respectively, after 8 weeks. The rate of microbial degradation increased again when the stirring was raised from 150 to 300 rpm.     

A comparison between the full diallel cross and the simplified triple-test cross

Summary The simplified triple-test cross (sTTC) is a mating design that, because of its economic use of the experimental material as compared with other designs, seems very attractive. In theory, its power is almost equal to that of more elaborate designs such as the diallel cross. To evaluate the merits of both designs in a genetic analysis of mouse behavior, the results of a previous replicated 4×4 diallel cross (Crusio and van Abeelen 1986) were reanalyzed as a sTTC. We found that, at least with the fairly low number of strains employed, the sTTC analysis is clearly inferior to the diallel cross. This finding, in combination with some theoretical considerations, leads to the conclusion that the sTTC design is not a very useful one for such studies.