Electron Microscopic Observations on Virus-Like Particles Associated with SH Antigen

The structural aspects of SH antigen-containing particles were investigated. These studies confirmed the existence of a large spherical particle (ca. 43 nm) and smaller (ca. 20 nm) rod- and sphere-shaped particles. The large particle consists of an outer and inner membrane and a core of "nucleic acid" as seen by positive staining techniques. The outer membrane of the large particle appears to be similar to that of the 20-nm diameter spheres and rods known to possess the SH antigen.     

DNA Polymerase Associated with Human Hepatitis B Antigen

DNA polymerase activity was detected in each of eight preparations of concentrated human hepatitis B antigen (HBAg) rich in Dane particles prepared by high-speed centrifugation of antigen-positive human plasma and in none of seven control preparations prepared in the same way from HBAg-negative plasma. The incorporation of (3)H-thymidine-methyl-5'-triphosphate into DNA was dependent on four deoxyribonucleoside triphosphates and MgCl(2). Treatment of the concentrated HBAg preparations with the nonionic detergent Nonidet P-40 (NP40) more than doubled the enzyme activity. Fractionation of the concentrated HBAg preparation in sucrose density gradients after treatment with NP40 revealed that the enzyme activity appeared within the density range of Dane core antigen but at a slightly higher density than the average for core antigen. The only particles observed by electron microscopy in this region of the gradient were typical 28-nm cores, suggesting that the DNA polymerase activity was associated with a subpopulation of cores. No DNA polymerase activity was found in purified 20-nm HBAg particles. The DNA product of the reaction remained associated with the 110S core and was not susceptible to DNase digestion when associated with the core. Inhibition of the reaction by actinomycin D and daunomycin suggested that the reaction was dependent on a DNA template associated with the core.     

Virus-like particles in a positive case of canine parvovirus enteritis

Virus-like particles measuring approximately 110 nm in diameter were found, along with parvoviruses, in a fecal specimen of a parvovirus-infected dog. Under the electron microscope, the outer component of the particles was found to be composed of what appeared to be numerous fine spikes or a solid coat perforated by many regular small slits. The inner ‘core’ of the particle had an irregular border, measured approximately 60 nm in diameter and was occasionally eccentrically located.     

DNA Polymerase in the Core of the Human Hepatitis B Virus Candidate

Experiments were done to show that the human hepatitis B antigen (HBAg)-associated DNA polymerase is a component of Dane particles and their antigenically distinct cores prepared by Nonidet P-40 detergent treatment of Dane particles. Before detergent treatment, the DNA polymerase was precipitated by serum containing anti-HB surface antigen (anti-HB(s)) but not with serum containing anti-HB core antigen (anti-HB(c)). After detergent treatment, the enzyme was precipitated by anti-HB(c)- and not by anti-HB(s)-containing serum. Highly purified 16- to 25-nm HBAg particles blocked only the precipitation of DNA polymerase in untreated HBAg preparations. The 110S structure with which the DNA reaction product remains associated in Nonidet P-40-treated preparations was identified as Dane particle core by immunoprecipitation with serum containing anti-HB(c). The DNA polymerase and the radioactive DNA reaction product were used as markers for core in immunoprecipitation tests for anticore. In such assays, 8 of 11 human sera with anti-HB(s) activity and all of 10 sera from chronic HBAg carriers were found to contain anti-HB(c) activity.     

Association of different kinds of bacilliform particle with vein chlorosis and mosaic diseases of raspberry (Rubus idaeus)

Thin sections of diseased raspberry (Rubus idaeus) were examined by electron microscopy. Plants of the cv. Baumforth's B and of an aphid (Amphorophora rubi)-resistant breeding selection (6820/54), both infected with raspberry vein chlorosis virus (RVCV) but not with other detectable viruses, contained large bacilliform particles c. 430 × 65 nm. Particles occurred in the cytoplasm and perinuclear space of a small proportion of xylem parenchyma cells. They had an inner core c. 25–30 nm in diameter with cross-banding of periodicity 4·5 nm, and were bounded by an outer membrane. They are probably the particles of RVCV. Plants of cv. Mailing Jewel and of a selection (M14) both showing symptoms of raspberry mosaic (veinbanding) disease contained smaller bacilliform particles c. 125 × 30 nm, which occurred singly or in clusters in the cytoplasm of a small proportion of vascular parenchyma cells. It is not known which, if any, of the viruses associated with raspberry mosaic are represented by the particles.     

Morphology of proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli

Proteoliposomes reconstituted with purified lac carrier protein from Escherichia coli were ultra-rapidly frozen and examined by freeze-fracture-etch electron microscopy. The proteoliposomes are greater than 95% unilamellar, and the majority are 30-150 nm in diameter. Fracture faces of proteoliposomes (at a protein:lipid molecular ratio of about 1:2500) display 7.0-nm diameter globular intramembrane particles uniformly distributed on convex and concave surfaces. Calculations of particle composition suggest that each intramembrane particle probably contains one or two molecules of the 46.5-kDa transmembranous lac carrier protein, depending on the correction factor for the thickness of the metal deposited to form the platinum/carbon replicas. Etched surfaces of the proteoliposomes are smooth. Incubation of the proteoliposomes with monoclonal antibody 4B1, which binds to an epitope in the lac carrier on the exterior of the proteoliposomes, dramatically alters the intramembrane particle distribution. After incubation with antibody, the convex (inner monolayer) fracture faces are nearly devoid of intramembrane particles, and an overall 4-fold reduction in the total number of intramembrane particles is observed.     

HBcAg基因在甲醇型酵母中的表达、产物纯化及其性质的初步研究

为研究乙肝核心抗原蛋白(HBcAg)在甲醇型酵母(PichiaPastoris)中的表达和性质,用PCR方法将HBcAg基因(L)克隆到P.Pastoris胞内表达载体pPIC3k中,并利用电击和同源重组法,将重组质粒pPIC3kL转化感受态的甲醇型GS115酵母菌株。经过筛选得到阳性P.Pastoris重组子。重组菌株经甲醇诱导培养,表达产物的Western印迹结果表明,HBcAg蛋白能在甲醇型酵母(PichiaPastoris)中诱导表达,产物为一215kDa的蛋白。经蔗糖密度梯度超离心和CsCl密度梯度超离心纯化后,ELISA和密度测定结果表明重组HBcAg蛋白主要分布在密度为12576gml和13013gml的2个峰值处。电镜观察表明,该重组HBcAg蛋白能自主装配成大小不同的2种颗粒(即核心颗粒),大颗粒直径约34nm左右,小颗粒直径约30nm左右。同时,我们还观察到,该核心蛋白颗粒在体外可发生集聚现象。     

Extramembraneous particles and structural variations of tubular myelin figures in rat lung surfactant

Tubular myelin figures of pulmonary surfactant were examined by electron microscopy after fixation in glutaraldehyde and postfixation in an osmium tetroxide-ferrocyanide mixture. Bilayered membranes were seen as parallel arrays or as lattices with spacings varying from about 36 to 50 nm. This method also produced good visualization of drumstick-like particles, 5 nm in diameter and about 15 nm in length. The particles were regularly spaced at intervals of 16 nm in rows along the rectangular angles of myelin membranes. Depending on the size of the tubules the particles contacted each other in the center of the tubules at low diameters (tubular diameter less than 40 nm) and formed a continuous filamentous central core, or they were separated from one another (tubular diameter greater than 40 nm). In the latter case the central core had a hollow appearance. Based on further findings employing tannic acid, lipid extraction with 2,2-dimethoxypropane, and a ruthenium red-osmium tetroxide technique for the demonstration of polyanionic proteins it is suggested that these particles are protein in nature and that they are involved in the formation and maintenance of the structure of tubular myelin. A new concept of the ultrastructure of tubular myelin figures is proposed.     

Localization of hepatitis A antigen in liver tissue by peroxidase-conjugated antibody method: light and electron microscopic studies.

Hepatitis A antigen (HAAG) was localized in liver tissue from marmosets inoculated with human hepatitis A virus (HAV) by light and electron microscopy by using a peroxidase-conjugated antibody method. The fine granular peroxidase staining was scattered throughout the cytoplasm of liver cells when viewed with the light microscope. The distribution of HAAg-positive cells was focal. Virus-like particles, 24 to 27 nm in diameter, were observed in the cytoplasm of hepatocytes and smaller cells, resembling Kupffer cells, by standard thin-section electron microscopy (thin section EM). By immunoperoxidase electron microscopy (immunoperoxidase EM), HAAg was detected on the particles, which were aggregated within cytoplasmic vesicles of the hepatocyte. The surrounding membrane of the vesicles was also HAAg- positive. Similar HAAg particles were observed in the cytoplasm of smaller cells adjacent to hepatocytes as well. Thus, immunoperoxidase EM revealed that the 24- to 27-nm virus-like particles in the cytoplasm of liver cells obtained from marmosets were infected with HAV contained HAAg.     

Electron microscopy of hepatitis B virus core antigen expressing yeast cells by freeze-substitution fixation

We have used the freeze-substitution fixation technique for electron microscopy of yeast cells that express the hepatitis B virus core antigen (HBcAg) following transformation with the cloned gene. Abundant spherical particles were found within the transformed cells. These particles had a uniform size and shape, measured about 21 nm in diameter, had electron-lucent centers, and consisted of many subunits. They were localized in both the cytoplasm and the nucleus. None of these particles was found in the cells of the parent strain. Comparison of the HBcAg particles isolated from the yeast cells and the particles within the yeast cells demonstrated that the 21-nm particles were in fact ultrastructurally superimposable on HBcAg. Thus, the HBcAg particles within the yeast cells were similar to the HBcAg particles in human liver tissues infected with hepatitis B virus, not only in their size and appearance, but also in their intracellular localization. These results suggest that the yeast cell has the same machinery for synthesis and intracellular translocation of the HBcAg polypeptides as the human cell.     

西伯利亚旱獭中类人乙肝病毒的研究

对111份西伯利亚旱獭血清标本进行血清学,形态和组织病理学检测,HBsAg阳性22份,阳性率19.8％;抗-HBs1份,阳性率0.9％;HBV-DNA核酸杂交,阳性斑点16份,阳性率14.4％。IEM观察在3份标本中发现以22—24nm球形颗粒为主,其中有42—45mm的Dane样颗粒。20.7％的肝脏标本有组织病理改变,其中19份为急性肝类,4份为慢性肝炎。超薄切片肝细胞核内有20nm左右的HBeAg颗粒,结果进一步证实我国西伯利亚旱獭中有嗜肝病毒感染。     

Ultrastructural changes occurring during germination and outgrowth of spores of the thermophile Bacillus acidocaldarius.

Spores of the thermophilic, acidophilic, Bacillus acidocaldarius were covered by a thick outer coat and a laminated inner coat (5.5 nm periodicity). Small membranous vesicles were present in the spore core and they disappeared as germination proceeded. After depolymerization of the cortex, and a 30% increase in spore diameter a localized gap appeared in the laminated inner coat only. This inner coat gap was narrow and could be the whole length of the spore. The germ cell appeared to grow, or to be pushed towards the inner coat gap, at which stage the outer coat disappeared in the same localized area. As the vegetative cell grew out the spore coat fell away, with loose cortical material still attached to it. The young germ cell developed a large spherical electron dense inclusion body in the cytoplasm, at the same time as the ribosomal and nuclear areas became distinct.     

Tubular structures occurring in cells infected with herpesviruses

Certain viruses belonging to the Herpesviridae family generate tubular structures of three distinct size ranges late in the growth cycle. The smallest tubules are about 10 to 20 nm in diameter, and are restricted to the cell nucleus, tending to form palisades of lattice-like structures. The medium sized tubular structures are about the width of a core particle, measuring 55 to 65 nm in diameter, while the larger tubular structures are the same diameter as viral capsids, about 80 to 100 nm. Both are rigid capsomered structures, which are sometimes encountered outside the cell nucleus budding onto spherical particles. The available data on some properties of the various tubular structures, as well as speculations concerning their nature and significance are briefly reviewed. Their importance is emphasized for antigen and vaccine production, as well as in differentiating herpesviruses by electron microscopy.     

Microvoids in Bombyx mori silk. An electron microscope study.

The size and distribution of microvoids in Bombyx mori silk were examined by transmission electron microscopy of silver sulphide 'stained' filaments. Silver sulphide deposited in voids and accessible regions of molecular structure appears as dense particles in thin transverse and longitudinal sections of silk filaments. Small particles (about 8 nm or less in diameter) occur around or adjacent to the periphery of the filaments. Larger particles (around 10-15 nm in diameter) occur in the form of dendritic arrays in the core region of the filaments. The leading edges of the dendritic arrays are oriented towards the fibre periphery. The particles (microvoids) appear to be either spherical or rod-like in shape and are aligned parallel to the long axis of the filament. A skin/core structure is proposed.     

Specific Lymphocyte Stimulation by Purified,Heat-inactivated Hepatitis-B Antigen

The ability of purified, heat-inactivated hepatitis-B antigen (HBAg) to stimulate sensitized lymphocytes in vitro was investigated with the lymphocyte stimulation test on lymphocytes from three groups of individuals. Stimulation was minimal in the lymphocytes of two out of 15 normal controls, whereas lymphocytes from nine out of 12 patients who had recovered from hepatitis B showed stimulation, as did lymphocytes from five out of 12 laboratory technicians who had been regularly exposed to HBAg but who had no history of hepatitis or signs of it in the previous two years. No differences were observed in the responses to phytohaemagglutinin of lymphocytes from persons in the three groups. HBAg and HBAg inactivated by heat were shown in immunodiffusion to be immunologically identical. Inactivated HBAg stimulated antibody production in guinea-pigs. These findings suggest that not only humoral but also cell-mediated immunity might be induced by vaccination with purified, heat-inactivated HBAg.     

Ultrastructural changes occurring during germination and outgrowth of spores of the thermophile Bacillus acidocaldarius

Spores of the thermophilic, acidophilic, Bacillus acidocaldarius were covered by a thick outer coat and a laminated inner coat (5.5 nm periodicity). Small membranous vesicles were present in the spore core and they disappeared as germination proceeded. After depolymerization of the cortex, and a 30% increase in spore diameter, a localized gap appeared in the laminated inner coat only. This inner coat gap was narrow and could be the whole length of the spore. The germ cell appeared to grow, or to be pushed towards the inner coat gap, at which stage the outer coat disappeared in the same localized area. As the vegetative cell grew out the spore coat fell away, with loose cortical material still attached to it. The young germ cell developed a large spherical electron dense inclusion body in the cytoplasm, at the same time as the ribosomal and nuclear areas became distinct.     

Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain

DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.     

Construction of biotinylated peptide nanotubes for arranging proteins

Three kinds of biotinylated peptides with different linkers between biotin and beta-sheet peptide were designed and synthesized. The transmission electron microscopy revealed that the biotinylated peptides self-assembled to form a tubular structure with external diameter of ca. 60 nm and inner diameter of ca. 30 nm in an aqueous solution. The anti-biotin antibody effectively bound to biotin groups in the peptide nanotubes. The binding of antibody was regulated by not only the concentration of the protein in the solution but also the properties of biotinylated peptides forming the tubes. The antibody preferentially bound to the biotinylated peptide tubes assembled from the peptide with the most hydrophilic linker, suggesting that the surface properties and functions of the tubular structure were modulated and engineered by the design of the peptides.