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PubMed1981

Hollow cylinder protein in the cytoplasm of human erythrocytes.

A "Hollow Cylinder Protein' (HCP) similar to the protein originally isolated by Harris from human erythrocyte membranes (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534--537) is present in the cytosol of erythrocytes at a concentration of more than 15 micrograms/ml packed erythrocytes. When negatively stained and examined in the electron microscope, cytosol HCP is similar in morphology to the HCP associated with erythrocyte ghost membranes. Cytosol HCP can be purified by isoelectric precipitation at pH 5.2 followed by repeated sucrose gradient centrifugation at alkaline pH. Negatively stained purified cytosol HCP appears as a hollow cylinder with apparent dimensions of 18.0 nm in length by 11.8 nm in diameter and contains a hollow core. Purified cytosol HCP migrates as a single band by non-denaturing polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis shows that it is composed of five peptides having apparent molecular weights 21 500, 23 500, 26 000, 27 500 and 29 000. Chymotryptic peptide maps of each of these bands indicate that each is a unique polypeptide chain. These results indicate that erythrocyte cytosol HCP is a macromolecular complex composed of multiple copies of five non-identical subunits arranged as a hollow cylinder.

Blood Proteins↗
PubMed1967

Fine structure of the cytomembranes of Nitrosocystis oceanus.

Thin-sectioned, negatively stained, and freeze-etched preparations of Nitrosocystis oceanus cytomembranes were compared. The cytomembranes in freeze-etched cells were covered with 80- to 120-A particles. When cells were disrupted and differentially centrifuged, various membrane and particle fractions were obtained. Negatively stained membrane fragments from the pellet centrifuged at 3,000 x g showed 70- to 80-A stalked particles, whereas those from the pellet centrifuged at 39,000 x g exhibited a crystalline array of subunits with a 30- to 40-A periodicity. High-speed supernatant and pellet fractions centrifuged at greater than 39,000 x g contained 40- to 120-A free particles but no membranes. In chemically fixed cells, 40-A particles were found embedded in the matrix of membranes. Results suggest that the larger 80- to 120-A particles are enzyme complexes, whereas the smaller 30- to 40-A particles represent a structural protein or a lipoprotein of the membrane.

Bacteria↗
PubMed1978

Identification of type C viruses by electron microscopy.

The cores of a small proportion of the particles in purified preparations of type C viruses are characterized by two concentric shells that have a "double-ring" appearance in specimens prepared for electron microscopy by negative staining or thin sectioning techniques. This distinctive feature allows such particles to be recognized as viral in impure preparations. Detectability of double-ring particles is considerably enhanced by treating the specimens with a non-ionic detergent before fixation and negative staining. The limit of detectablity of type C virus (identified by the double-ring feature) in the presence of a large excess of nonviral particulates of similar size and density was assessed.

Detergents↗
PubMed1981

Ultrastructure of hemoglobin-depleted human erythrocyte resealed ghosts.

Both negative-stain and freeze-fracture electron microscopic techniques revealed that the ultrastructure of resealed white ghosts prepared at high dilution during the hemolysis step is very different from that of resealed ghosts prepared at low or moderate dilution (pink ghosts). The negative-stained resealed white ghosts showed light halo substructures on membrane surfaces and protrusions at the edge of the ghosts. Freeze-fracturing of these ghosts showed that membrane blebbing had occurred and that fragments of the membranes resealed to form small right-side-out vesicles ranging from 0.1 to 0.3 micrometer in diameter.

Erythrocyte Membrane↗
PubMed1973

Morphology, ultrastructure, and bacteriophage infection of the helical mycoplasma-like organism (Spiroplasma citri gen. nov., sp. nov.) cultured from "stubborn" disease of citrus.

The mycoplasma-like organism Spiroplasma citri gen. nov., sp. nov., isolated from citrus infected with "Stubborn" disease and carried in serial cultures in several media, was examined by dark-field microscopy and electron microscopy of negatively-stained and shadowed preparations and of sections. It grows as motile, helical filaments in liquid, but as nonmotile, nonhelical filaments and round bodies in agar cultures. Helicity and motility are lost in old broth cultures and upon addition of a variety of negative stains, fixatives, and other solutions. No organelles accounting for motility are present, but a layer of surface projections is present on the surface of the single, bounding membrane. The mycoplasma produces a tailed, type B bacteriophage which appear to attach to the outer layer. Helical filaments are preserved in ammonium molybdate, but not in sodium phosphotungstate, and by fixation in Formalin or glutaraldehyde made up in medium, but not by osmium nor by glutaraldehyde in cacodylate buffer. This mycoplasma appears similar to the noncultured helical microorganism in corn stunt-diseased tissues and is probably a representative of a new group of mycoplasmas which are in possession of surface projections, rotary motility, and bacteriophage infection.

Agar↗
PubMed1973

Membrane lesions in immune lysis: surface rings, globule aggregates and transient openings.

It is known that there are 100 A-wide circular structures associated with the erythrocyte membrane in immune lysis. To determine whether these structures were functional holes extending through the membrane, freeze-etch electron microscopy was carried out. Sheep erythrocytes incubated with either rabbit complement or rabbit antibody (anti-sheep erythrocyte antibody) did not hemolyze and did not reveal any abnormalities in freeze-etch or negative-stain electron microscopy. Erythrocytes incubated with both complement and antibody revealed rings on the extracellular surface (etch face) of the cell membrane. Allowing for the 30 A-thick Pt/C replica, the dimensions of the surface rings were similar to those seen by negative staining. The ring's central depression was level with the plane of the membrane; some rings were closed circles, others were crescent shaped. The cleavage face of the extracellular leaflet revealed globule aggregates, each aggregate appearing to be composed of about four fused globules. The cleavage face of the cytoplasmic leaflet was normal. When immune lysis was carried out in the presence of ferritin, ferritin was subsequently detected in all lysed erythrocytes. If ferritin was added after immune lysis was complete, only 15% of the cells were permeated by ferritin, indicating that transient openings exist in the cell membrane during immune lysis. No abnormal structures were detected when C6-deficient rabbit serum was used as a source of complement. It is concluded that antibody and complement produce surface rings, prelytic leakage of K(+), colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.

Animals↗
PubMed1964

Ultrastructure of Treponema microdentium and Borrelia vincentii.

Bladen, Howard A. (National Institute of Dental Health, Bethesda, Md.), and Edward G. Hampp. Ultrastructure of Treponema microdentium and Borrelia vincentii. J. Bacteriol. 87:1180-1191.-A small oral Treponema (FM) and Borrelia vincentii (N9) were harvested after 3 to 7 days of incubation and either embedded in Vestopal W or negatively stained with phosphotungstate. The protoplasmic cylinders of both strains were identical except for size, and had a triple-structured cell wall as well as intracellular concentric laminations. Protoplasmic cylinders of both strains were enclosed in a cell envelope which appeared amorphous in negatively stained preparations, but which had a triple-structured wall when viewed in thin sections. The cell envelope of strain FM also acted as an envelope for the terminal filament; no filament envelope was evident in strain N9. Large structures which contained variable numbers of organisms and which were representative of spirochetal granules were observed. Protoplasmic cylinders contained within such granules frequently were devoid of cell envelopes. The axial filament consisted of several individual fibers which usually terminated in small end knobs. Occasionally, a fiber of the axial filament became a fiber of the terminal filament. Fibers of the terminal filament originated in end knobs similar to, but separate from, those to which the axial filament was attached. A periodicity of 60 A was occasionally observed in the terminal filament envelope of strain FM. A microperiodicity of approximately 20 A was also observed. The fibers of the terminal filament of strain N9 were composed of a large number of fibrils approximately 15 A wide. The periodicity and fibrillar structure of the terminal filament is discussed with reference to proposed models of bacterial flagella suggested by X-ray diffraction data.

Borrelia↗
PubMed1991

[Electron microscopy of alpha-latrotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus].

Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.

Crystallization↗
PubMedDate not supplied

Differences in the distribution of acidic mucins between flat tubular adenomas and flat serrated adenomas of the colorectal mucosa.

Two consecutive sections from 72 endoscopically detected flat colorectal polyps (flat tubular adenomas n = 40, flat serrated adenomas n = 18, flat hyperplastic polyps n = 7 and normal colonic mucosa n = 7) were stained with Alcian blue pH 2.5 (AB) or with high iron diamine (HID) without counterstain, to evidence the most important attribute of the colorectal cells i.e. their capacity to produce acidic mucins (sialomucins and sulphomucins respectively). Intense AB and HID staining (++) was found in the upper part of the crypts of all (100%) sections with normal colonic mucosa or with flat hyperplastic polyps, in 13 of the 18 (72%) flat serrated adenomas (with low grade dysplasia = LGD, n = 16; or high grade dysplasia = HGD, n = 2) but in none (0%) of the 40 tubular adenomas. Slight (+) or negative staining of the upper part of the crypts were recorded in all 40 (100%) flat tubular adenomas (LGD n = 32; HGD, n = 8), but in only 5 of the 18 (27,7%) flat serrated adenomas (LGD n = 3; HGD n = 2) and in none (0%) of the sections with normal colonic mucosa or with flat hyperplastic polyps. Intense AB and HID staining (++) was found in the lower part of the crypts in all sections (100%) with normal mucosa or with flat hyperplastic polyps, in 36 of the 40 flat (90%) tubular adenomas (LGD n = 32; HGD n = 8), but in none (0%) of the flat serrated adenomas. Slight (+) or negative stainings in the lower part of the crypts were recorded in all 18 (100%) flat serrated adenomas, but in only 4 of the 40 (10%) flat tubular adenomas and in none of the sections (0%) with normal mucosa or with flat hyperplastic polyps. Thus, the mucosal localization of the depletion of mucin production differs in flat serrated adenomas and in flat tubular adenomas. The difference in topographic distribution of the depletion of acidic mucins in epithelial cells is a new argument in favour of the identity of flat serrated adenomas as an independent neoplastic phenotype in the colorectal mucosa.

Adenoma↗
PubMed2006

Metastasis-associated protein S100A4--a potential prognostic marker for colorectal cancer.

BACKGROUND AND OBJECTIVES: Expression of S100A4, a small calcium-binding protein, in breast, oesophagus and gall bladder cancers is shown to be associated with adverse clinical outcome. We retrospectively examined the correlation of S100A4 expression and outcome in patients with colorectal cancer. METHODS: Tissue sections from 54 patients with Dukes B, C and D cancers operated on between 1995 and 1998 were stained with anti-S100A4 antibody. The S100A4 expression profile was correlated to the clinico-pathological details. RESULTS: There were 31 males and 23 females (mean age 65.94 years +/- 12.29). Dukes stage, >4 positive lymph node status and S100A4 expression were significantly associated with poorer survival. The 3 years survival of patients whose tumour stained positive for S100A4 was 62.85% compared to 93.75% for those stained negative (P < 0.012). In patients with <4 involved nodes, S100A4 expression led to poorer survival (57 months vs. 74 months; P < 0.0052). Within a particular Dukes stage, S100A4 expression was associated with poorer outcome. The 5 years survival of Dukes B patients whose tumour stained negative for S100A4 was 92% compared to 54.6% for those with positive tumours. CONCLUSION: Our results suggest that S100A4 expression is associated with adverse clinical outcome. Inclusion of S100A4 expression status may enhance our accuracy to prognosticate in patients with colorectal cancer.

Adult↗
PubMed1982

Electron microscopic investigations on the upper respiratory tracts of infants and children for detecting viral infections.

The upper respiratory tract of 20 infants and children, suffering from viral diseases, have been investigated cytochemically and electron microscopically. We employed the electron microscope negative staining and the concanavalin-A-peroxidase methods in our investigations, besides the usual serological and immunological ones. Concanavalin-A-peroxidase reaction showed a characteristic damaging of the cell-surface which may play an important role in viral infection. The electron microscope negative staining investigations showed adeno-, herpes- and influenza-virions which are referring to viruses. These morphological methods are useful and they increase the efficiency of diagnosing viral infections.

Cell Membrane↗
PubMed1991

Macromolecular structure and aggregation states of Helicobacter pylori urease.

Urease purified from Helicobacter pylori by differential ultracentrifugation and fast pressure liquid chromatography was composed of subunits with apparent molecular weights (MrS) of 66,000 and 30,000. Electron microscopy of this purified material demonstrated that it formed disc-shaped macromolecular aggregates that were approximately 13 nm in diameter and 3 nm thick. Images of both negatively stained and shadowed preparations indicated that the discs tended to stack to form pairs and then these pairs further aggregated to form four-disc stacks. This stacking of subunits explains the heterogeneity observed previously in the molecular weight of urease preparations. In some negatively stained preparations there were also some smaller (approximately 8-nm-diameter) annular units present, which may represent individual urease units or possibly an aggregate of one of the two subunits from which urease is constructed.

Helicobacter pylori↗
PubMed1967

Morphogenesis of type 2 parainfluenza virus examined by light and electron microscopy.

The development of type 2 parainfluenza virus in HeLa and stable human amnion cells was examined by use of antisera labeled with fluorescein and ferritin. Serum containing antibody predominantly to soluble viral antigen gave specific fluorescence which was first detectable in small cytoplasmic foci 8 to 10 hr after initiation of infection. By 20 to 24 hr, when the production of infective virus and hemagglutinin was maximal, large perinuclear aggregates of fluorescence were observed which corresponded in distribution and time of appearance to the eosinophilic inclusions seen in similar preparations stained with azure eosin. The inclusions, examined by electron microscopy, were composed of fibrils, presumably viral ribonucleoprotein, which specifically bound the antibody labeled with ferritin. With antiserum to concentrated virus, on the other hand, specific fluorescence was most marked at the surface of infected cells. Foci of fluorescence at the surface represented segments of membrane which had become differentiated morphologically and antigenically to resemble the viral envelope. These were the sites where mature virions appeared. The latter exhibited marked pleomorphism; in some instances, particles were formed which lacked recognizable internal fibrils but which possessed an enclosing membrane bearing viral antigen. Filamentous forms showing an organized internal structure were also observed at the cell surface, but were never encountered in negatively stained preparations. No clear relationship between these filaments and the spherical or oval forms could be established. In negatively stained preparations, nucleocapsid released by rupture of viral particles was similar in appearance to that reported for other paramyxoviruses. It seems probable that this component has a helical configuration.

Amnion↗
PubMed1983

Bacteriophage phi W-14: the contribution of covalently bound putrescine to DNA packing in the phage head.

Bacteriophage phi W-14 is unusual because its DNA contains 12 mol% of the hypermodified pyrimidine, alpha-putrescinylthymine. The phi W-14 virion is similar in morphology to T4, except that the phi W-14 head is isometric rather than prolate, there is no collar-whisker structure associated with the neck, the tail fibers are short (approximately 15 nm), and the base plate terminates in small plates or knobs rather than spikes. The contractile tail sheath of phi W-14 appears to have a right-handed helical arrangement of subunits with a pitch in the extended form of approximately 20 nm. The "stacked disk" appearance of the tail sheath visible on negatively stained particles has a periodicity of 3-4 nm. The protein shell of the head has a similar thickness (2-3 nm) to that of T4. The phi W-14 virion contains at least 17 different polypeptide species. Based on measurements from electron micrographs of negatively stained phage particles on the same grid square, the volume of the phi W-14 head was estimated to be approximately 72% that of the T4 head. Surprisingly, however, the lengths of the DNA molecules released from phi W-14 and T4 heads by osmotic shock were 59.6 +/- 1.9 and 62.1 +/- 2.4 microns, respectively. am42 is an amber mutant of phi W-14 in which there is only 5 mol% putThy in the DNA made in the nonpermissive host. am42 virions are morphologically normal, but the length of the DNA released from these virions is only 53.1 +/- 3.1 microns. We conclude that phi W-14 DNA is packed much more compactly than T4 DNA into a virion of similar morphology and comparable complexity and that the tight packing is a consequence of, and dependent upon, the presence of putThy in phi W-14 DNA.

Bacteriophages↗
PubMed1984

Reconstructions of tubulin protofilaments: different appearances of the same structure.

We compare the structures of tubulin protofilaments obtained by image reconstruction of tubulin sheets and hoops, and by X-ray diffraction of microtubules. Negatively stained specimens yield up to eight different appearances of protofilaments. They all represent the same intrinsic structure. The effects are explained by the packing of the protein subunits and the stain distribution resulting from it. They cannot be interpreted directly in terms of protein structure or composition; in particular, differences in staining cannot be attributed to microtube-associated proteins (MAPs). None of the reconstructions reproduce the X-ray structure faithfully. The discrepancies between the reconstructions are seen at resolutions of 4 nm or less. This appears to be the limit of structural fidelity of negatively stained tubulin specimens, even when the nominal resolution of the micrographs is better. Most of the differences may be defined in terms of the diffraction patterns and are therefore genuine. Additional differences become apparent after computing the 3D reconstruction and may be genuine and/or due to the data treatment. Several factors affecting the appearances are discussed.

Crystallography↗
PubMed2004

Appearance of the bona fide spiral tubule of ORF virus is dependent on an intact 10-kilodalton viral protein.

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.

Animals↗
PubMed1983

Isolation and characterization of invertebrate smooth septate junctions.

Using modifications of techniques used for the isolation of macula type intercellular junctions (gap junctions and desmosomes) the arthropod smooth septate junction has been isolated from insect midgut tissue. Midguts from cockroaches or mealworms were used and membrane fractions were obtained by sucrose gradient and ultracentrifugation techniques. Preparations with reasonable concentrations of septate junction were obtained and have been studied by thin-section, negative-stain and freeze-fracture electron microscopy. The junctions appeared to be well preserved, although there was evidence that the junction strands were able to slide within the plane of the membrane. Septa were seen to have a cross-striated appearance when viewed after negative staining but their exact structure remained difficult to determine. Polyacrylamide gel electrophoretic studies demonstrated the reproducibility of the isolation procedure and showed that septa may have a 47 000 molecular weight glycoprotein component. Gel electrophoresis also gave some indication of the intramembrane biochemistry of the smooth septate junction, with proteins of 31 000 and 32 000 molecular weight always occurring in the junction fractions. The junctions were, however, very sensitive to both mechanical and chemical treatments, the septa were destroyed by rough homogenization or by treatment with urea at a concentration as low as 1 M. Freeze-fracture of untreated, isolated junctions demonstrated no differences from junctions in intact tissue, while replicas of urea-treated material were more difficult to interpret as the component parts of the junctions became separated once the septa had been destroyed. Gap junctions were also obtained and resisted both mechanical and chemical treatment, which destroyed the septate junctions. Their major protein component appeared to have a molecular weight of 36 000. Attempts to isolate pleated septate junctions (from insects, molluscs and annelids) by the same techniques failed, implying a significant difference in the structures of the two types of septate junction.

Animals↗
PubMed1995

Lamellar bodies coexist with vesicles and micelles in human gallbladder bile. Ursodeoxycholic acid prevents cholesterol crystal nucleation by increasing biliary lamellae.

The aggregative forms of lipids in human gallbladder bile and their relation to cholesterol crystallization are controversial. Using combined chemical, gel-chromatographic, optical/electron microscopic and quasielastic light-scattering methods, we investigated this issue in native gallbladder bile obtained from nine untreated cholesterol gallstone patients and eight cholesterol gallstone patients treated for 1 week with 600 mg/day of ursodeoxycholic acid. Bile obtained at cholecystectomy was ultracentrifuged for 2 h at 150,000 g to obtain isotropic samples. The conventional cholesterol crystal observation time was 3.1 +/- 4.1 (SD) days in controls and 19.0 +/- 1.9 days in the ursodeoxycholic acid-treated group (p < 0.001). Bile was analyzed by high-resolution gel-chromatography using 7 mM sodium taurocholate in the elution buffer. Biliary lipids eluted in four chromatographic zones: zone #I, corresponding to the column void volume, contained only minimal amounts of lipids; zone #II (apparent m.w. 100-220 kDa) comprised 29.1 +/- 12.4% of biliary cholesterol in the untreated group and 8.3 +/- 4.3% in the ursodeoxycholic acid-group (p < 0.001). At negative staining electron microscopy, this region was composed of roundish vesicles ranging from 7 to 20 nm in diameter. Zone #III (apparent m.w. 50-100 kDa) carried 59.1 +/- 2.1% of cholesterol in untreated patients and 81.2 +/- 9.5% in ursodeoxycholic acid-rich biles, respectively (p < 0.001). At negative staining electron microscopy, this region was composed of lamellar stacks of variable length, usually with 5 nm interspaces and up to 30 nm in width. In ursodeoxycholic acid-rich biles, lamellae often appeared in the form of concentric fingerprint-like images. Quasielastic light-scattering measurements in this region were compatible with the size estimates obtained at electron microscopy. Zone #IV (apparent m.w. 6-50 kDa) carried 11.8 +/- 9.4% and 11.6 +/- 9.0% of cholesterol, respectively (not significant). Since this region comprised a considerable fraction of endogenous bile salts and had no distinct morphological structures, it was interpreted as mixed micelles. The cholesterol crystal observation time showed a significant inverse correlation (r = -0.85, p < 0.001) with percent cholesterol carried by vesicles (zone #II) and a direct correlation (r = 0.86, p < 0.001) with percent cholesterol carried by lamellar bodies (zone #III). Vesicles and lamellae identical to those observed in isolated gel-chromatographic fractions were observed also on direct electron microscopic examination of unfractionated isotropic native biles. Similar findings were observed also in matched model biles.(ABSTRACT TRUNCATED AT 400 WORDS)

Bile↗