Ultrastructural organization of recombinant Marburg virus nucleoprotein: comparison with Marburg virus inclusions

HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP) have been studied by immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral genome and other nucleocapsid-associated proteins.     

Purification of polyhedra of a cytoplasmic-polyhedrosis virus from soil

Polydedra of a cytoplasmic-polyhedrosis virus of the silkworm, Bombyx mori, were purified from soil by multistep procedures consisting of desorption of the polyhedra with sodium pyrophosphate, aqueous two-phase separation with the dextran-polyethylene glycol system, and CsCl density-gradient centrifugation. The overall recovery of polyhedra was 7% and there was a 170-fold increase in the number of polyhedra per gram minerals.     

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 and cytochemical examination of the nuclear crystalloid inclusions of Olea europaea L. mesophyll cells

Summary The nuclei of mesophyll cells of olive trees contain numerous sizeable crystalloid inclusions. Cytochemical examination using epoxy resin-embedded, semithin-sectioned tissue indicated the presence of proteins and oligoor polysaccharides in these inclusions. Their electron microscopical analysis revealed a crystalline substructure consisting of intersected subunits of high order. The spacing of the lattice fibrils and the angles of intersection were determined and used to establish a model of the unit cell of crystallization. It is suggested that the nuclear crystalloids of olive trees consist of glycoprotein molecules. They differ from the intranuclear crystalloids observed in other species predominantly in the high density of their subunit arrangement.     

Ultrastructural and cytochemical investigations on the functional role of proteinaceous nuclear inclusions (PNIs) in chlorenchyma plant cells

Summary— Detailed investigations on the fine ultrastructural organization of different forms of proteinaceous nuclear inclusions (PNls) in chlorenchyma plant cells suggest they consist of the same elementary subunits. Previous high magnification of TEM micro-graphs had shown that the amorphous type of inclusion (A) was mainly composed of elementary fibrils measuring 3.0–3.5 nm in diameter, with no orderly spatial arrangement. New computer image treatments of electron micrographs allowed us to establish that the 8.0–13.0 nm thick filaments — forming the fibrillar (F), crystalline (C) and lamellar (L) inclusions — consist of two elementary fibrils which are coiled in a helix with variable pitch, depending on the type of inclusion. A further secondary coiling of two filaments, about 8.0–9.0 nm in diameter, gives the 20.0–25.0 nm thick tubules which form the characteristic tubular inclusion (T). Correlating the distributive data of PNIs with observations on their ultrastructural morphology and with micrographs of partial aggregation or disgregation patterns of the inclusions, led to the hypothesis that the different forms are not different classes of proteins, but simply different stages of structural complexity of the same protein. To determine whether the intranuclear inclusion protein is nucleolar or nucleolus-associated, cytochemical and immunocytochemical tests were performed on ultrathin sections or leaf lamina tissue in block. These techniques proved that PNIs do not belong to the class of argyrophilic proteins (AgNOR-proteins), and particularly not nucleolin and fibrillarin, two of the major nucleolar proteins. Structural similarities to other plant inclusions, especially P-proteins, and to animal and plant intermediate cytoskeletal filaments (IFs) are discussed with regard to the functional role of PNIs.     

Ultrastructural and X-ray analytical studies on intranuclear bismuth inclusions

Parenteral administration of bismuth subnitrate produced intranuclear inclusions in the rabbit kidney tubules. In glutaraldehyde fixed material these inclusions had a homogeneous appearance but in osmicated material some of these inclusions showed a granular and fibrillar substructure. In relatively thick unstained sections prepared from unosmicated tissues, it was found that the inclusions were electron-dense, but they vanished from view when the electron beam was focussed on them. This phenomenon is thought to be due to the presence of bismuth, which is a mental with a high electrical resistance and low thermal conductivity. With the aid of electron-probe analysis the presence of bismuth was demonstrated in these inclusions and it seems possible that sulphur may be present here.     

Unusual polyphosphate inclusions observed in a marine Beggiatoa strain

Sulfide-oxidizing bacteria of the genus Beggiatoa are known to accumulate phosphate intracellularly as polyphosphate but little is known about the structure and properties of these inclusions. Application of different staining techniques revealed the presence of unusually large polyphosphate inclusions in the marine Beggiatoa strain 35Flor. The inclusions showed a co-occurrence of polyphosphate, calcium and magnesium when analyzed by scanning electron microscopy and energy dispersive X-ray analysis. Similar to polyphosphate-enriched acidocalcisomes of prokaryotes and eukaryotes, the polyphosphate inclusions in Beggiatoa strain 35Flor are enclosed by a lipid layer and store cations. However, they are not notably acidic. 16S rRNA gene sequence-based phylogenetic reconstruction showed an affiliation of Beggiatoa strain 35Flor to a monophyletic branch, comprising other narrow vacuolated and non-vacuolated Beggiatoa species. The polyphosphate inclusions represent a new type of membrane surrounded storage compartment within the genus Beggiatoa, distinct from the mostly nitrate-storing vacuoles known from other marine sulfide-oxidizing bacteria of the family Beggiatoaceae.