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PubMed1988

The cell wall structure of a magnesium-dependent halobacterium, Halobacterium volcanii CD-2, from the Dead Sea.

Cell wall preparations from the magnesium-dependent halophilic bacterium, Halobacterium volcanii, were studied by high-resolution electron microscopy complemented with image analysis and processing. For ultrastructural studies, specimens were prepared by a variety of methods, including negative staining, and metal shadowing after air-drying, freeze-drying, or freeze-fracturing and etching. All methods revealed the cell wall to be composed of a near-hexagonal lattice of unit cells having a center-to-center spacing of 15.5 nm. While negatively stained samples yielded two types variably revealed the unit cell to be composed of six protomers surrounding a central mass depression. This low-resolution unit cell morphology appears very similar to that of other bacterial cell wall S-layers studied to date.

Cell Wall↗
PubMed1984

Purification and properties of native titin.

A procedure has been developed for the extraction and purification of the massive myofibrillar protein titin without exposing it to denaturing conditions. The form of the molecule that has been isolated is soluble at high ionic strength and alkaline pH, but precipitates in low salt or at pH values below 7. Sedimentation velocity experiments indicate that titin is a highly asymmetric molecule with a sedimentation coefficient of 13.4 S. This asymmetry is confirmed by electron microscopy of rotary-shadowed specimens, which shows string-like structures of diameter 40 A and lengths up to 8000 A. Significant differences were observed depending on whether the electron microscope specimens were prepared by spraying or by layering of the titin onto a mica substrate; we tentatively attribute these differences to elasticity in the titin, revealed by the high shearing forces that accompany spraying. In accord with this, the circular dichroism spectrum of titin indicates that its secondary structure is largely random coil, a conformation characteristic of elastic proteins such as elastin. Negative staining of titin again shows long string-like structures, but these can now be seen to have an appearance similar to a string of beads, where the spacing between successive beads is about 40 A. Very similar beaded strings have been observed also associated with negatively stained separated native thick filaments; these are found running alongside the cross-bridge regions and in coils near the filament ends. Since the periodicity of the strings is similar to that of end-filaments, recently identified structures at the tips of thick filaments, it is likely that end-filaments are formed from titin. Titin comprises approximately 9% of the myofibrillar mass, which means that it is the third most abundant protein in muscle. The possible role of titin in forming elastic filaments within myofibrils is discussed.

Animals↗
PubMed1979

Ultraviolet irradiation disrupts somatic pili structure and function.

Three piliated bacterial species were exposed to ultraviolet light (7 X 10(3) microW/cm2), and the effect of increasing duration of irradiation on the integrity of the somatic pili was quantitated by negative-stain electron microscopy. Heavily piliated Proteus mirabilis became devoid of pili after 20 min of irradiation, but Escherichia coli and Neisseria gonorrhoeae required 40 min for complete depiliation. Partially purified proteus pili underwent progressive loss of structural integrity with increasing doses of irradiation as determined by negative staining and nephelometry, suggesting that ultraviolet light exerted an effect directly on the pili themselves. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that new, small molecular weight fragments appeared after irradiation of purified E. coli pili, suggesting that cleavage of the peptide chain rather than disassociation of pilin monomers accounted for the loss of pili structure. Ultraviolet irradiation also inhibited the ability of piliated bacteria to bind to human buccal epithelial cells. These observations indicate that the ultrastructural integrity and function of pili can be disrupted by ultraviolet light.

Dose-Response Relationship, Radiation↗
PubMed2004

Spontaneous vesicle formation and vesicle-tubular microstructure transition in aqueous solution of a poly-tailed cationic and anionic surfactants mixture.

Spontaneous vesicle formation has been observed in aqueous mixtures of tri-(N-dodecyldimethylhydroxypropylammonium chloride) phosphate (PTA) and bis-(2-ethylhexyl) sulfosuccinate (Aerosol OT), which is supported by negative-staining TEM and dynamic light scattering. The range of vesicle formation in the PTA/AOT mixtures is wide and monodisperse vesicles are obtained. The vesicle diameter increases with the total surfactant concentration. Tubular microstructures, vesicle fusion, and vesicle-tubular microstructure transition have been also observed by negative-staining TEM. The vesicle formation mechanism is discussed from the viewpoint of molecular geometry, conformation, and the interaction between surfactant molecules.

Journal Article↗
PubMed1981

[Electron microscope characteristics of the nuclear matrix and its fractions].

The rat liver nuclear matrix retains the shape of the nucleus and reveals a sponge-like structure in negative staining and scanning electron microscopy. A fibrous layer (dense lamina) with associated pore complexes are preserved on the surface of the nuclear matrix. The cytoplasmic face of the nuclear matrix is perceived as a network consisting of cells (or units) of 10-30 nm in diameter in negative staining as well as in high resolution scanning electron microscopy. In sections, a fibrous layer, 15-30 nm in width with granules of 7-10 nm in diameter, can be observed. In pore complexes associated with the fibrous layer granular and fibrillar components rather than central granules are observed. The pore complexes differ in arrangement of the annular granules. Structures similar to pore complexes are revealed in close proximity to the nucleoli. The biogenesis of the pore complexes is discussed. A few morphologically different structures could be derived be fractionation of the nuclear matrix. A fraction rich in pore complexes, and a fraction retaining the shape of the nucleus with spongy or alveolar structure were isolated. The latter fraction is regarded to form a protein framework or skeleton of the nucleus.

Animals↗
PubMed1992

A new bacterial staining method involving Gram stain with theoretical considerations of the staining mechanism.

In order to investigate the mechanism of Gram staining of bacteria, tests with anionic dyes followed by treatment with cationic octyltrimethylammonium (OTMA) were carried out. The study revealed that tetrabromophenolphthalein ethylester (TBPE) gave the most reliable staining of Gram-negative bacteria with negative staining of Gram-positive bacteria. Tests on many species of bacteria showed that TBPE positive bacteria were Gram-negative and vice versa, without exception.

Bacteria↗
PubMed1983

Substructure of the postacrosomal sheath of bovine spermatozoa.

The substructure of the postacrosomal sheath and its relationship to the plasma membrane and nuclear membrane complex were examined in thin-section, negative-stain, surface-replica, and freeze-fracture preparations. The matrix of the postacrosomal sheath contains a single layer of closely associated 10- to 12-nm filamentous elements aligned parallel to the long axis of the sperm. A precise lateral interaction of the filaments is suggested from negative-stain images which reveal a second set of parallel striations extending over the surface of the sheath at 60 degrees relative to the filament long axis. Several structural differences between the posterior and anterior segments and the outer and inner surface of the postacrosomal sheath were identified. Data on structural specializations of the plasma membrane and nuclear membrane complex which relate to the asymmetric structure are presented and their potential significance in fertilization events discussed.

Acrosome↗
PubMed2002

Bacteroides fragilis adherence to Caco-2 cells.

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76-100%) to non- or weakly adherent (0-25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.

Journal Article↗
PubMed1997

How tubulin subunits are lost from the shortening ends of microtubules.

Microtubules exhibit dynamic instability, switching between persistent states of growth and shortening at their ends. The switch between growth and shortening has been proposed to depend on end conformation where growing ends have "straight" tubulin protofilaments stabilized by a terminal cap of GTP-tubulin, while-shortening ends have lost their GTP-tubulin cap, allowing terminal GDP-tubulin dimers to curve inside-out and peel rapidly away from the microtubule lattice. This "conformational cap" model predicts that tubulin dissociation from shortening ends is a two-step process where the average lengths of curved GDP-tubulin protofilaments at a depolymerizing end will depend on the ratio of the rate of peeling to the rate of breakage of the longitudinal bonds between adjacent curved dimers. We have tested this model for the plus and minus ends of microtubules assembled with pure porcine tubulin off the ends of axoneme fragments in standard assembly buffer. Individual microtubule ends were imaged using video-enhanced differential interference contrast light microscopy. The rate of rapid shortening was systematically increased by isothermal dilution into assembly buffer containing various concentrations of Mg2+ or Ca2+ ions. At 1 mM Mg2+ and no Ca2+, shortening occurred at 20 (plus) and 45 (minus) microns/min. The ends appeared similar in contrast to growing ends and the core of the microtubule and the ends appeared blunt or slightly frayed by negative stain electron microscopy. Above 20 mM Mg2+ or above 5 mM Ca2+, microtubule shortening occurred at 60 (plus) and 115 (minus) microns/min or faster and "knobs" were distinctly visible at depolymerizing ends, particularly at the faster minus ends, and knob contrast remained constant during many micrometers of rapid shortening. Negative stain electron microscopy revealed that these knobs were "blossoms" of inside-out curved protofilaments, some extending for several helical turns (30 to 60 dimers in length) at constant curvature from the ends. At these high shortening velocities, the peeling of curved protofilaments was confined to within several dimers of the end of the microtubule cylinder, suggesting that dimer curling and protofilament peeling is constrained to the tip by interactions between adjacent straight protofilaments. Depolymerization is produced by conformational changes in GDP-tubulin since microtubules assembled with a slowly hydrolizable analog of GTP, GMPCPP, are stable even at 20 mM Mg2+ or 5 mM Ca2+. Monte Carlo simulations show that the ratio of the peeling to breakage rate constants can control the steady-state average length of curved GDP-tubulin protofilaments at the depolymerizing end.

Animals↗
PubMed1985

Electron microscopic and solution X-ray scattering observations on the structure of hepatitis B surface antigen.

The structure of the small, spherical hepatitis B surface antigen was studied by negative staining, freeze-fracture and freeze-etching electron microscopy and solution X-ray scattering techniques. The protein appears to be organized at the surface into a small number of morphological subunits which display two- and threefold axes of symmetry. The mean particle size was 18.3 nm by negative staining and 19.6 nm by freeze-fracture electron microscopy. The diameter of the individual subunits was about 7.5 nm with an intersubunit distance of about 10.0 nm. The lipid is distributed more homogeneously. Some heterogeneity of the particle structure is apparent which may be due to a slightly variable lipid-protein composition or incomplete or defective particle formation.

Freeze Etching↗
PubMedDate not supplied

EM single particle analysis of the ATP-dependent BchI complex of magnesium chelatase: an AAA+ hexamer.

BchI, belonging to the AAA+ -protein family, forms the enzyme magnesium chelatase together with BchD and BchH. This enzyme catalyses the insertion of Mg2+ into protoporphyrin IX upon ATP hydrolysis. Previous studies have indicated that BchI forms ATP-dependent complexes and it is a member of the AAA+ -protein family (ATPases associated with various cellular activities) and it was suggested based on structural homology that the BchI formed hexameric complexes. AAA+ -proteins are Mg2+ -dependent ATPases that normally form oligomeric ring complexes in the presence of ATP. Single particle analysis of fully formed ring complexes of BchI observed by negative staining EM indicate that the BchI has strong 6- and 2-fold rotational symmetries and a weaker 4-fold rotational symmetry which are reminiscent of DNA helicase. A 2D average of the fully formed BchI-ATP ring complex is presented here from images of the complex obtained from negative staining EM. Other complexes are also observed in the EM micrographs and the class averages of these are indicative of the fragility and dynamic nature of the BchI complex which has been reported and they are suggestive of partially circular complexes with six or less protomers per particle. The resolution of the average circular complex is estimated at approximately 30A and it is similar in shape and size to an atomic resolution hexameric model of BchI rendered at 30A.

Adenosine Triphosphatases↗
PubMed1984

Three-dimensional structure of bladder membrane protein.

The naturally occurring crystals of the mammalian bladder membrane belong to the two-sided plane group p6 and have a lattice parameter of 160 A. The 3D reconstruction in negative stain at a resolution of 35 A in-plane and 45 A out-of-plane shows each motif to consist of twelve subunits elongated perpendicular to the crystal plane to a total height of about 50 A. Comparison with shadowed specimens shows that the protein particles revealed by negative stain are exclusively on one side of the membrane.

Animals↗
PubMedDate not supplied

Ultrastructural morphology of the hnRNP C protein tetramer.

The C protein tetramer is one of three heterotetramers which comprise the majority of the protein mass of mammalian 40S nuclear ribonucleoprotein particles (hnRNP particles). The events of RNA processing occur while the nascent transcripts are packaged in these structures. The C protein tetramer contains three monomers of C1 and one C2 monomer [i.e., (C1)3C2]. The tetramer's mass (129.2 kDa), approximate sedimentation coefficient (5.8S), and Stokes radius (6.2 nm) suggest that the tetramer may be either highly anisotropic or may possess an unusually large hydration shell in solution. The tetramer binds approximately 235 nucleotides of pre-mRNA. Electron microscopy of purified individual RNA-free C protein tetramers has revealed the overall morphology of this important pre-mRNA binding complex. In negatively stained preparations, the tetramer clearly displays a nonlinear, three- or four-lobed appearance with a diameter of 8.5 +/- 0.5 nm. A detailed comparison of the substructure seen in individual images suggests a tetrahedral arrangement of the four polypeptides. Rotary-shadowed images confirm the size of the tetramer observed in negatively stained preparations. This study provides the first demonstration of the overall arrangement of polypeptides in the C protein tetramer.

Animals↗
PubMed1989

Molecular architecture of Escherichia coli F1 adenosinetriphosphatase.

The structure of the E. coli F1 ATPase (ECF1) has been studied by a novel combination of two specimen preparation and image analysis techniques. The molecular outline of the ECF1 was determined by three-dimensional reconstruction of images of negatively stained two-dimensional crystals of ECF1. Internal features were revealed by analysis of single particles of ECF1, preserved in their native state in a thin layer of amorphous ice, and examined by cryoelectron microscopy. Various projections of the unstained ECF1 were interpreted consistently with the three-dimensional structure in negative stain, yielding a more informative description of the enzyme than otherwise possible. Results show that the ECF1 is a roughly spherical complex approximately 90-100 A in diameter. Six elongated protein densities (the alpha and beta subunits, each approximately 90 A X approximately 30 A in size) comprise its hexagonally modulated periphery. At the center of the ECF1 is an aqueous cavity which extends nearly or entirely through the length of the complex. A compact protein density, located at one end of the hexagonal barrel and closely associated with one of the peripheral subunits, partially obstructs the central cavity.

Crystallography↗
PubMed1979

Correlation of ultrastructure of reconstituted sarcoplasmic reticulum membrane vesicles with variation in phospholipid to protein ratio.

We have previously described the reconstitution of functional membrane vesicles with lipid content similar to that of the normal sarcoplasmic reticulum membrane (approximately 1.0 mumol of phospholipid/mg of protein). The present study describes methodology to prepare reconstituted membrane vesicles with defined phospholipid to protein ratio, both lower and higher than that of the original membrane. The Ca2+ loading rate and efficiency are greatest in the membranes of highest protein content (0.38 mumol of phospholipid/mg of protein), decline slowly as the lipid content is quadrupled, and decrease markedly as the lipid content is quadrupled again. Such membranes of defined composition can be used to study lipid-protein interaction and to correlate membrane structure with composition. The number of particles observed by freeze-fracture electron microscopy can be correlated with protein content, whereas the percentage of smooth domain is proportional to the lipid content of the reconstituted membrane. Since 90% or more of the protein of the reconstituted membrane is the calcium pump protein, the number of particles observed by freeze-fracture is directly proportional to the amount of calcium pump protein in the membrane. The number of pump molecules calculated to be in the membrane is greater by a factor of two than the number of particles which we observed. This multiplicity ratio could be greater depending upon the assumptions made regarding the width of the membrane (see "Appendix"). Thus, it would appear that the particles consist of two or more molecules of pump protein. The change in protein concentration of the membrane is reflected also in thin sections and by negative staining. In thin sections, the broad inner and outer 70 A bands become discontinuous and patchy and, in the limit, approach a symmetrical 20,20,20 A trilayer as the protein content of the membrane becomes small. In an analogous fashion, the concentration of particles at the surface of the membrane, observed by negative staining, decreases with increasing lipid concentration in the membrane. Thus, the correlation of composition with structure can be observed by each of the three methods of sample preparation for electron microscopic analysis.

Adenosine Triphosphate↗
PubMed1983

Structure of the actin molecule determined from electron micrographs of crystalline actin sheets with a tentative alignment of the molecule in the actin filament.

Electron microscopy and image processing of negatively stained crystalline sheets induced from Acanthamoeba actin have been used to yield a three-dimensional reconstruction of the actin molecule, including data to a maximum resolution of 15 A. This model shows actin to be an asymmetric, wedge-shaped molecule. A three-dimensional reconstruction of an averaged, polar actin filament from negatively stained polylysine-induced actin filament paracrystals has also been computed. We show two possible ways in which the wedge-shaped actin molecule from the sheets can be placed into such a filament reconstruction. In both, the major intermolecular contacts are formed on complementary surfaces of the actin subunit and follow the left-handed genetic helix of the filament, a feature also found in the filament reconstruction.

Actins↗
PubMed1995

Crystalline surface protein of Peptostreptococcus anaerobius.

The surface ultrastructure of three anaerobic Gram-positive cocci frequently encountered in oral infections, Peptostreptococcus micros, P. magnus and P. anaerobius, was studied. The type strains of P. micros (DSM 20468) and P. anaerobius (ATCC 27337), several clinical isolates of both species and the type strain of P. magnus (DSM 20470) were included. Thin-sectioned cells studied by electron microscopy revealed a homogeneous layer outside the peptidoglycan layer in P. anaerobius. In P. micros and P. magnus a more amorphous layer was present. No periodic structures were seen in negatively stained whole cells of these three species. However, in freeze-etched cells of P. anaerobius a crystalline surface protein layer (S-layer) was detected. No periodicity was seen in any of the P. micros strains or the P. magnus type strain by the methods used, but a periodic pattern was observed in negatively stained specimens of cell wall fragments of sonicated P. anaerobius cells. No capsular material was visible outside the S-layer in P. anaerobius. The cells of the Peptostreptococcus spp. were extracted for 30 min with detergents and urea. One per cent SDS and M urea both extracted a major 78 kDa protein from all strains of P anaerobius. Extraction of P. micros and P. magnus cells did not reveal any major protein bands comparable to that of P. anaerobius. Surface biotinylation of cells followed by Western blotting and detection by alkaline-phosphatase-conjugated extravidin showed strong staining of the 78 kDa band in P. anaerobius, further indicating that this molecule is located on the surface of the cell and is the S-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1993

Presence of papova-like viral particles in cerebrospinal fluid of AIDS patients with progressive multifocal leukoencephalopathy. An additional test for "in vivo" diagnosis.

An "in vivo" diagnosis of progressive multifocal leukoencephalopathy (PML), a neurological opportunistic viral infection in AIDS patients, can be made only by brain biopsy. In order to identify viral particles, we examined the cerebrospinal fluid (CSF) of 15 AIDS patients with focal neurological signs by electron microscopy using negative staining technique. In 2 out of 3 patients with clinical and neuroradiological presumptive diagnosis of PML, the CSF examination revealed papova-like viral particles. Our results support the hypothesis that the severe cell-mediated immunodeficiency reactivates papovavirus from a latent state in the brain, leading to PML. Therefore, the CSF study by negative staining might be a useful test for an "in vivo" diagnosis of PML.

Acquired Immunodeficiency Syndrome↗