Properties of flacherie virus of the silkworm, Bombyx mori

The flacherie virus of the silkworm, Bombyx mori, was isolated from infected larvae reared under aseptic conditions. Two types of infectious particles, tentatively designated FVS I and FVS II, were separated by density gradient centrifugation. Some properties of the separated particles were investigated. Electron micrographs showed that FVS I and FVS II were spherical particles with diameters of 27 ± 2 nm and 22 ± 2 nm, respectively. The sedimentation coefficients of FVS I and FVS II were 180 S and 134 S, respectively. It was concluded from experiments of incorporation of ³H-uracil inoculated into diseased larvae at late stage of flacherie disease that the nucleic acid of FVS II was RNA. The two types of particles were present in Sakaki and Wadayama strains of flacherie virus.     

THE MACROMOLECULAR PARACRYSTALLINE LATTICE OF INSECT VIRAL POLYHEDRAL BODIES DEMONSTRATED IN ULTRATHIN SECTIONS EXAMINED IN THE ELECTRON MICROSCOPE

Thin sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus and from silkworm larvae infected with B. mori virus revealed viral particles contained within a pseudohexagonal, macromolecular, paracrystalline lattice. The gipsy moth virus occurs in bundles of one to eight rods enclosed by a limiting membrane. The particles of the silkworm virus, although generally occurring singly, also possess a limiting membrane. The macromolecules appear to be dense, discrete particles when cross-sectioned and to form dense bands by superimposition when longitudinally or obliquely sectioned at certain angles. Calculations of macromolecular size have been made.     

Functional differences between occluded and nonoccluded viruses of a nuclear polyhedrosis of the silkworm, Bombyx mori.

Comparative infectivity and virus neutralization studies on occluded and nonoccluded viruses of Bombyx mori nuclear polyhedrosis revealed that the infectious unit causing peroral infection differed from that causing hemocoelic infection. There were functional differences between the occluded (mainly virons with envelopes) and the nonoccluded virus (mainly virions without envelopes) preparations. The peroral infection was largely due to the virion with an envelope (peroral infectious unit), and the hemocoelic infection was due largely to the virion without an envelope (hemocoelic infectious unit). The apparent change of the virions with envelope to those without envelopes was detected as a slight increase in hemocoelic infectivity when the occluded virus was diluted and incubated at 4°C for more than 6 days.     

Freeze-Etch Study of Pseudomonas aeruginosa: Localization Within the Cell Wall of an Ethylenediaminetetraacetate-Extractable Component

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

Ultrastructure of Mitochondria and Crystal-containing Bodies in Mature Ballistospores of the Fungus Basidiobolus ranarum as Revealed by Freeze-etching

By using the freeze-etch technique, a regular pattern on both sides of the outer mitochondrial membrane can be demonstrated in mature spores of Basidiobolus ranarum. Bands consisting of 5 to 10 parallel ridges, each of which is 20 nm wide, envelop the outer as well as the inner side of this membrane. Hexagonal crystals, probably representing stored protein, are enclosed in special bodies with a very smooth surrounding unit membrane. The crystals are formed by parallel rods which consist of globular subunits, 6.5 to 7.0 nm wide.     

An esterase in relation to yolk cell lysis at diapause termination in the silkworm,Bombyx mori

Esterase A is one of the esterase isozymes in eggs of Bombyx mori. The effect of this esterase A on the yolk cells of diapause eggs was examined with a hanging-drop culture in order to discover the mechanism of diapause termination in silkworm eggs.The culture of yolk cells in diapause eggs shows spherical forms with dark fine grains in the central parts, large translucent granules in the outer parts, and a membrane on the exterior. When such yolk cells were incubated with yolk materials of acid-treated or diapause-terminated eggs, they were damaged and cell lysis occurred. This suggested that substance(s) causing the cell lysis were present in diapause-terminated eggs. When esterase A separated electrophoretically from non-diapause eggs and diapause-terminated eggs was added to hanging-drop cultures of yolk cells of diapause eggs, the yolk cells were also greatly affected. That is, a part of the yolk cell membrane was dissolved or disappeared, and the central dark fine grains diffused over the cell causing the whole cell to become dark. A few cells lost almost all of their contents and collapsed. Other esterase fractions and fractions without esterase activity in the electrophoresis exerted little effect on the yolk cells. Furthermore, a parallelism between esterase activity to hydrolyse 2-naphthyl acetate as substrate and the lytic activity on the yolk cell membrane was observed in this esterase A fraction from different sources.From these results it is highly probable that the substance responsible for cell lysis is the esterase A enzyme itself. Diapause termination of silkworm eggs is discussed in relation to the lysis of yolk cells.     

USE OF SERIAL SECTIONS TO DELINEATE THE STRUCTURE OF PORTHETRIA DISPAR VIRUS IN THE ELECTRON MICROSCOPE

Consecutive serial sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus revealed bundles of viral rods scattered and oriented at random within the polyhedral body. Each bundle was entirely surrounded by a dense, sharply defined membrane. The rods measured 18 to 22 mµ in diameter and averaged 280 mµ in length. No spherical viral particles were encountered. The effects of variable compression and periodic distortion of the sections on the appearance of the virus are discussed.     

Mitochondrial protein BmPAPI modulates the length of mature piRNAs

PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons in animal germlines. The molecular mechanisms and components responsible for piRNA biogenesis remain elusive. PIWI proteins contain conserved symmetrical dimethylarginines (sDMAs) that are specifically targeted by TUDOR domain-containing proteins. Here we report that the sDMAs of PIWI proteins play crucial roles in PIWI localization and piRNA biogenesis in Bombyx mori-derived BmN4 cells, which harbor fully functional piRNA biogenesis machinery. Moreover, RNAi screenings for Bombyx genes encoding TUDOR domain-containing proteins identified BmPAPI, a Bombyx homolog of Drosophila PAPI, as a factor modulating the length of mature piRNAs. BmPAPI specifically recognized sDMAs and interacted with PIWI proteins at the surface of the mitochondrial outer membrane. BmPAPI depletion resulted in 3′-terminal extensions of mature piRNAs without affecting the piRNA quantity. These results reveal the BmPAPI-involved piRNA precursor processing mechanism on mitochondrial outer membrane scaffolds.     

Crystallization of belladonna mottle virus

Belladonna mottle virus belongs to the turnip yellows mosaic virus group of the small spherical plant viruses. It contains 180 protein subunits, which are arranged in a T = 3 icosahedral surface lattice. The top and bottom viral components crystallize isomorphously in hexagonal space group R3 ($\text{a = b = 296}\text{A}\text{?}\text{, c = 729}\text{A}\text{?}$). The unit cell contains three virus particles, while the crystallographic asymmetric unit consists of only one-third of a particle. X-ray diffraction data from the crystals extend to at least 3.8 Å resolution.     

Characterization of an orthorhombic crystal form of iron-containing superoxide dismutase from Escherichia coli B.

Human erythrocytes were treated with the diazonium salt of oligodeoxythymidylic acid 5′-p-aminophenylphosphate, a reagent that does not penetrate the plasma membrane. Ghosts were isolated, and the oligomers, covalently linked at their 5′ ends to the outer surface of the membrane, were extended by treatment with terminal deoxynucleotidyl transferase in the presence of deoxythymidine triphosphate. The membranes were dissolved in sodium dodecyl sulfate, and complexes containing cell surface components were isolated by hybridization to polyriboadenylic acid-agarose. The cell surface components were regenerated by treatment with nuclease P₁ in the presence of Triton X100. Sodium dodecyl sulfate/polyacrylamide gels of the regenerated material showed bands III, PAS-1, PAS-2, and PAS-3, i.e. the major proteins known to be accessible at the outer surface of the human erythrocyte. The method should be useful for the isolation of surface components in other cell types.     

Fine Structure of Thermus aquaticus, an Extreme Thermophile

Electron microscopic studies using thin sections revealed that Thermus aquaticus has a structure similar to that of most other gram-negative bacteria. The cell envelope is tripartite: plasma membrane, thin middle layer, and a thicker and irregular outer layer. The outer layer appears to be joined to the plasma membrane by a series of connections and, when seen in tangential section, the outer layer appears as a series of parallel bands. The cell division mechanism resembles that of typical gram-negative bacteria. Large spherical bodies designated “rotund bodies” are formed as a result of the association of a number of separate cells. In this association the outer envelope layers of the cells fuse and pull away from the middle layer. The rotund body thus appears as a series of rods, usually lying in parallel around the periphery of the sphere, completely connected by means of the fused outer layer.     

Subdominant Antigens in Bacterial Vaccines: AM779 Is Subdominant in the Anaplasma marginale Outer Membrane Vaccine but Does Not Associate with Protective Immunity

Identification of specific antigens responsible for the ability of complex immunogens to induce protection is a major goal in development of bacterial vaccines. Much of the investigation has focused on highly abundant and highly immunodominant outer membrane proteins. Recently however, genomic and proteomic approaches have facilitated identification of minor components of the bacterial outer membrane that have previously been missed or ignored in immunological analyses. Immunization with Anaplasma marginale outer membranes or a cross-linked surface complex induces protection against bacteremia, however the components responsible for protection within these complex immunogens are unknown. Using outer membrane protein AM779 as a model, we demonstrated that this highly conserved but minor component of the A. marginale surface was immunologically sub-dominant in the context of the outer membrane or surface complex vaccines. Immunologic sub-dominance could be overcome by targeted vaccination with AM779 for T lymphocyte responses but not for antibody responses, suggesting that both abundance and intrinsic immunogenicity determine relative dominance. Importantly, immunization with AM779 supports that once priming is achieved by specific targeting, recall upon infectious challenge is achieved. While immunization with AM779 alone was not sufficient to induce protection, the ability of targeted immunization to prime the immune response to highly conserved but low abundance proteins supports continued investigation into the role of sub-dominant antigens, individually and collectively, in vaccine development for A. marginale and related bacterial pathogens.     

Proteolytic cleavage of polyhedral protein during dissolution of inclusion bodies of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella under alkaline conditions

Inclusion bodies (IB) of nuclear polyhedrosis viruses (NPV) of Bombyx mori and Galleria mellonella were dissolved in 67% acetic acid and in sodium carbonate solution at pH 11.0. The polyhedral protein preparations obtained in this way were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by ultracentrifugation. The polyhedral proteins of B. mori and G. mellonella NPV obtained by dissolving IB in acetic acid were shown to have mainly one component with a molecular weight of about ± 28,000 and sedimentation coefficients in 0.1 M NaOH of 1.8 and 2.1 S, respectively. During dissolution of IB in alkaline condition, both the proteins are cleaved and reveal several components. The quantity and the ratio of the components depend on the duration of storing and on the temperature during the dissolution of IB and on the stage of insect development at the time of IB isolation.It is suggested that the cleavage of IB protein is a result of alkaline proteinase(s) activity.     

Chemotactic attraction of Crassostrea virginica hemolymph cells to Staphylococcus lactus.

The nuclear polyhedrosis virus (NPV) of Porthetria dispar was isolated and purified through a two-step zonal centrifugation procedure. The LD₅₀ of the purified NPV was determined by a dose-response assay. Quantitative analyses were made of whole polyhedra and of separated fractions of polyhedral protein, virus rods, and denatured material, i.e., the pellet obtained from low speed centrifugation of dissolved polyhedra, to determine the protein, DNA, and “RNA” (orcinol-positive material) present in this NPV. Approximately one-half the “RNA” was present in the denatured material. Trace elements were also determined, and four, Fe, Mg, Cu, and Zn, of the ten assayed were present in the polyhedral protein fraction, while only Mg and Zn were in the virus rod fraction.     

The intestinal barrier in lepidopteran larvae: permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides

Endogenous peptide regulators of insect physiology and development are presently being considered as potential biopesticides, but their efficacy by oral delivery cannot be easily anticipated because of the limited information on how the insect gut barrier handles these kind of molecules. We investigated, in Bombyx mori larvae, the permeability properties of the two components of the intestinal barrier, the peritrophic membrane (PM) and the midgut epithelium, separately isolated and perfused in conventional Ussing chambers. The PM discriminated compounds of different dimensions but was easily crossed by two small peptides recently proposed as bioinsecticides, the neuropeptide proctolin and Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF), although their flux values indicated that the permeability was highly affected by their steric conformation. To date, there is very little functional data available on how peptides cross the insect intestinal epithelium, but it has been speculated that peptides could reach the haemocoel through the paracellular pathway. We characterized the permeability properties of this route to a number of organic molecules, showing that B. mori septate junction was highly selective to both the dimension and the charge of the permeant compound. Confocal images of whole-mount midguts incubated with rhodamine(rh)-proctolin or fluorescein isothiocyanate (FITC)-Aea-TMOF added to the mucosal side of the epithelium, revealed that rh-proctolin did not enter the cell and crossed the midgut only by the paracellular pathway, while FITC-Aea-TMOF did cross the cell apical membrane, permeating also through the transcellular route.     

A mycosis of the American lobster, Homarus americanus, caused by Fusarium sp.

Successful inoculations of citrus red mites, Panonychus citri, with the nonoccluded virus were made by feeding healthy mites through membranes on contaminated lemon fruit. Manipulation of the membranes showed that healthy mites pick up the virus from contaminated surface substrates rather than from within plant cells. Electron microscopical studies substantiated that feces of infected mites should be highly infectious since the hindgut contains numerous virus rods.     

Isolation of a factor, from the capsule of a granulosis virus, synergistic for a nuclear-polyhedrosis virus of the armyworm

When the capsules of a granulosis virus are fed together with the polyhedra of a nuclear-polyhedrosis virus to larvae of the armyworm, Pseudaletia unipuncta, the former enhances the infectivity of the latter virus, a synergistic interaction. The enhancement of infectivity depends upon the concentration of the polyhedra and the capsules. The factor responsible for the synergistic activity in the capsule can be dissolved in alkaline solution, separated from the virus particles by centrifugation, and further purified by Sephadex G-200 gel filtration with 4 m urea. The fraction obtained from Sephadex filtration and containing the synergistic factor can be separated into two components by disc-electrophoresis with 8 m urea. Both components possess synergistic activity. The ID₅₀ of the synergistic factor corresponds to 0.0015 OD₂₈₀. Its optimum pH is 8.5. Synergism is most evident when the factor is fed to larvae together with the polyhedra or is fed 24 hr prior to the ingestion of the polyhedra. The factor appears to be a simple or a conjugated protein of the capsule.     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE : III. Continuity between Plasma Membrane and Nuclear Envelope in Androgonial Cells of Blasia

An ultrastructural study of late-stage androgonial cells of Blasia pusilla, a thallose liverwort, showed the nearly spherical nuclei often lying close or appressed to the cell walls. In some cells the two membranes comprising the nuclear envelope separated, the inner membrane continuing intact as a limiting boundary of the nucleus and the membrane on the outer, cytoplasmic side recurving away from the nucleus to continue without evident interruption around the periphery of the cell as the plasma membrane. It is believed that Blasia offers the first completely convincing demonstration of the heretofore problematic continuity of cytoplasmic membranes. A possible sequence of events leading to this unusual relationship between nucleus and cytoplasm is suggested. The sequence includes blebbing of the outer membrane of the nuclear envelope and subsequent membrane proliferation, apparent isolation of cytoplasmic ground substance, fusion of internal membrane with the ectoplast, and migration that finally brings the nucleus into flat contact with the wall. While this manifestation of membrane continuity may be anomalous, it is not presently considered the result of cell injury.