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PubMedDate not supplied

Three-dimensional immunoelectron microscopy of scorpion hemocyanin labeled with a monoclonal Fab fragment.

An immunocomplex of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis with the monoclonal Fab fragment L104 was reconstructed from electron micrographs of a negatively stained specimen, using the double-carbon-layer technique. The resulting structure enables a clear visualization of the Fab fragments bound to the four copies of the Aa6 subunit and directly confirms a previous localization of the L104 epitope deduced from two-dimensional image processing. Despite a strong flattening effect produced by the negative-staining technique the orientations of the Fab fragments are well characterized. Moreover, the observation of a central hole within the elbow bends of the Fab fragments provides information about the disposition of the Fabs around their main axis.

Animals↗
PubMed1981

Catalytically active monomer and dimer forms of rat liver carbamoyl-phosphate synthetase.

Purified carbamoyl-phosphate synthetase of rat liver is shown to exist in a state of rapid, reversible monomer-dimer equilibrium. The allosteric activator N-acetyl-L-glutamate displaces the equilibrium toward monomer formation. This effect is observed over a range of initial protein concentrations of 0.02-5 mg/mL. Measurements of Stokes radii by analytical gel chromatography indicate that at concentrations less than 0.1 mg/mL at 25 degrees C in the presence of all the substrates the enzyme exists as a monomer of 160000 molecular weight. A gel chromatographic method was developed to identify the active form of carbamoyl-phosphate synthetase. On the basis of analysis of the ADP boundary formed during gel chromatography, the monomer is established to be catalytically active. Active enzyme centrifugation studies confirm that the monomer is a reactive species and suggest that the dimer also functions catalytically. Under the conditions of the usual enzyme assay, carbamoyl-phosphate synthetase is mainly in the monomer form. Activation by acetylglutamate can occur at the level of the monomer and is not coupled to dissociation since the enzyme dissociates at low concentrations even in the absence of acetylglutamate. The stoichiometry of the association is observed directly in the electron microscope. The dimensions of the negatively stained particles of the enzyme in the presence or absence of substrates correspond to monomers and dimers, assuming the molecule to be a prolate ellipse. The number of monomers observed in the presence of substrate represents 86% of the total number of enzyme molecules. The average molecular weight calculated from the numbers of particles seen in negatively stained specimens of carbamoyl-phosphate synthetase is 182000. Electron microscope studies provide independent evidence for monomer--dimer interactions and show that under the conditions examined the enzyme is mainly in the monomer form.

Adenosine Diphosphate↗
PubMed1988

Ultrastructure of human immunodeficiency virus type 2.

The ultrastructure of human immunodeficiency virus type 2 (HIV-2) was determined by negative stain and thin section electron microscopy (EM). Some virus particles had surface projections about 10 nm in length which were evenly spaced. Nonidet P40-treated particles which were penetrated by stain revealed a distinctive off-centre cone-shaped core and, in addition, free-lying cores were also seen in detergent-treated preparations. The surface of the cores was composed of a layer of small subunits. The structure of HIV-2 determined by thin section EM was the same as that deduced by negative stain EM.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Hierarchical assembly of beta2-microglobulin amyloid in vitro revealed by atomic force microscopy.

The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.

Amyloid↗
PubMed1985

Detection of papillomaviruses in cutaneous fibromas of white-tailed and mule deer.

Naturally occurring cutaneous fibromas affecting white-tailed deer (Odocoileus virginianus) and mule deer (O hemionus), and cutaneous fibropapillomas of domestic cattle were tested for papillomavirus using indirect immunofluorescence (IF), peroxidase-antiperoxidase (PAP), and negative-stain electron microscopic techniques. Papillomavirus was consistently detected using rabbit antiserum against papillomavirus group-specific antigen in all mule deer fibromas and bovine fibropapillomas; only 16 of 28 white-tailed deer fibromas tested by IF and 9 of 15 tested by PAP were detected. Normal skin from white-tailed deer or cattle was consistently negative for virus. Similar results were obtained by negative-stain electron microscopic examination of partially purified tumor homogenates. Using deer fibroma virus or bovine papillomavirus type 1-specific antisera, viruses were typed by IF, PAP, and immunoelectron microscopy.

Animals↗
PubMed1963

THE FINE STRUCTURE AND MODE OF ATTACHMENT OF THE SHEATHED FLAGELLUM OF VIBRIO METCHNIKOVII.

The sheathed flagellum of Vibrio metchnikovii was chosen for a study of the attachment of the flagellum to the bacterial cell. Normal and autolysed organisms and isolated flagella were studied by electron microscopy using the techniques of thin sectioning and negative staining. The sheath of the flagellum has the same layered structure as the cell wall of the bacterium, and in favourable thin sections it appears that the sheath is a continuation of the cell wall. After autolysis the sheath is usually absent and the core of the flagellum has a diameter of 120 A. Electron micrographs of autolysed bacteria negatively stained with potassium phosphotungstate show that the core ends in a basal disc just inside the plasma membrane. The basal disc is about 350 A in diameter and is thus considerably smaller than the "basal granules" described previously by other workers.

Bacteria↗
PubMed1991

Surface layers of Eubacterium yurii subsp. yurii and their possible role in test-tube brush formation and iron acquisition.

Eubacterium yurii subsp. yurii is an anaerobic, gram-positive rod. On isolation E. yurii forms cellular arrangements resembling test-tube brushes (TTB). Although TTB decreased in size and number on repeated laboratory subculture in enriched media, media poor in available iron enhanced formation of these. Plasmids were not demonstrated, even after chloramphenicol enhancement. To characterise the nature and possible physiological roles of the structures of the TTB, they were examined by transmission electronmicroscopy (TEM) with thin-section, negative-staining, shadow-casting, freeze-etching and freeze-fracturing techniques, and by scanning electronmicroscopy (SEM). Previous studies by phase-contrast microscopy revealed an amorphous core, the size of which varied in direct proportion to the number of associated bacterial cells. Thin sections of the TTB showed a gram-positive cell wall with additional surface layers. Negative staining, shadow casting and freeze etching revealed a surface layer comprising subunits in tetragonal array (P4 symmetry). Shadow casting showed also that the outermost layer of the cells was composed of fibrillar structures closely associated with but distinct from, the tetragonal layer. The fibrils extended from the cell surface in clumps or strands. The presence of these fibrils was confirmed by the freeze-fracture technique and SEM. Chemical analysis of the core material of the TTB showed it to be low in carbohydrate (0.06%) and protein (0.2%). Energy-dispersive X-ray spectrometry showed that the core was composed mostly of iron.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Adhesion↗
PubMedDate not supplied

Structure of acrosomal matrix domains of rabbit sperm.

The structural organization of different domains of the acrosomal matrix of intact and detergent-extracted rabbit spermatozoa was characterized utilizing both thin sectioning and negative staining techniques. A matrix component with a crystalline substructure was present exclusively within the apical segment of the acrosome. The crystalline matrix remained intact following Triton X-100 extraction and appeared composed of aggregates of plaque-like crystalloids. These crystalloids retained a structural association with a detergent-insoluble coat of the outer acrosomal membrane, termed the acrosomal lamina. Negatively stained specimens demonstrate that the matrix crystalloids are composed of globular subunits, spaced 8-10 nm center-to-center, which are assembled into a two-dimensional lattice. The potential functions of the detergent-insoluble acrosomal lamina-matrix complex are discussed.

Acrosome↗
PubMed1984

The basement membrane of bovine corneal endothelial cells in culture with beta-aminopropionitrile: biosynthesis of hexagonal lattices composed of a 160 nm dumbbell-shaped structure.

Bovine corneal endothelial cells maintained in culture produced an extracellular matrix on their basal surface. In the presence of beta-aminopropionitrile (beta-APN) (100-500 micrograms/ml), an inhibitor of cross-linking, this extracellular matrix contained hexagonal lattices of 150 nm periodicity. The lattice was composed of round densities 60 nm in diameter comprising the nodes of the lattice and rod-like structures 40 nm in thickness comprising its sides. This structure is almost identical to that of bovine Descemet's membrane in situ. The lattice was produced only in the presence of beta-APN. By freeze-etch replica and negative staining of 1 day cultures, the unit component of the lattice appeared to be a novel 160 nm long dumbbell-shaped symmetrical structure. The central rod of the dumbbell-shaped structure was 80 nm long and 20 to 50 nm in diameter with round ends (40-50 nm). In the rod 2 nm fibrillar substructures were discerned by negative staining. The rod-like central portion was resistant to trypsin digestion whereas the round ends were digested by it. Both structures disappeared after bacterial collagenase digestion. By immunoblotting the extracellular matrix contained 160kd and 180kd bands which bound anti-type IV collagen antibodies and a 140kd band which bound anti-type III collagen antibodies. The lattice area was also positively stained with these antibodies by immunoperoxidase. The findings strongly suggest that the hexagonal lattice of bovine Descemet's membranes in situ is composed of this collagenous structure.

Aminopropionitrile↗
PubMed1991

Developmental study of the expression of dystrophin in cultured human muscle aneurally and innervated with fetal rat spinal cord.

So far there have been no developmental studies including the influences of innervation and contractile activity on the expression of dystrophin in cultured human muscle. We performed immunocytochemical studies of the localization of dystrophin on aneurally cultured non-contracting (AMs) and innervated continuously contracting cross-striated human muscle fibers (ICMs) with fetal rat spinal cord from normal and Duchenne muscular dystrophy (DMD) biopsied muscles. In normal AMs, myoblasts and some immature AMs showed negative staining of dystrophin, but many AMs had a patchy (discontinuous) distribution of dystrophin in the subplasmalemmal region and with some granularity near the sarcolemma and in the deeper cytoplasm. In normal ICMs, dystrophin was localized continuously at the inner aspect of the sarcolemmal membrane and some periodic dense patterns were detected in some areas. Both AMs and ICMs from DMD had negative staining of dystrophin. To investigate the muscle contractile activity on the distribution of dystrophin, we paralyzed ICMs with tetrodotoxin (TTX) for two weeks from the first appearance of muscle contractions. In paralyzed innervated muscles (PIMs), dystrophin remained in a patchy (discontinuous) pattern at the inner aspect of the plasmalemma similar to that in AMs. It is strongly suggested that muscle contractile activity plays an important role in the continuous and even distribution of dystrophin at the sarcolemma during development.

Adult↗
PubMed1969

The "9+1" pattern of microtubules in spermatozoa of Mesostoma (Platyhelminthes, Turbellaria).

The living spermatozoa of the flatworm Mesostoma georgianum have a sperm body about 100 mu long and 0.5 mu wide, and two motile free flagella, ca. 200 mu long. In sections examined with the electron microscope, these flagella have the usual nine pairs of peripheral doublet microtubules and have a single central core unit which is connected to the A members of the doublets by spokelike structures. There are also short connections between the doublets and the flagellar membrane. In material negatively stained with phosphotungstic acid, the doublet microtubules seem to have very different elastic properties than the core; they tend to fall on the copper grids in coils of rather uniform diameter (2-4 mu), while the core is much more rigid and is often found extending alone, along a relatively straight course, for very long distances (up to 73 mu). After negative staining, the core has a striking appearance with a dense center around which are wound two hollow structures in a double helix of 45 degrees pitch. The center-to-center distance of each gyre is approximately 650 A, and the hollow structures are ca. 180 A in diameter.

Animals↗
PubMed1995

Ultrastructure of human astrovirus serotype 2.

The ultrastructure of human astrovirus serotype 2 (H-Ast2) grown in cell culture was analysed by electron microscopy of thin sections and negatively stained preparation. Infected LLCMK2 cells, as visualized in thin sections, contained cytoplasmic aggregates of dense or hollow-cored particles that aggregated in quasicrystalline arrays and were specifically labelled using a rabbit polyclonal anti-Ast2 antiserum. H-Ast2 particles from the supernatant of infected LLCMK2 cells in thin sections after flat- embedding were similar in size to intracellular virions. In negatively stained preparations, these virus particles had an external diameter of 41 nm and exhibited a well defined layer of surface spikes. Pentagonal and hexagonal contours were occasionally visible, and probably correspond to the projections of icosahedral structures. Star-like morphologies and particles with surface triangular hollows were seen in dark areas of the preparations only after a short treatment of the viruses of pH 10. Incubation of the viruses at pH 10.5 induced a rapid disassembly of the virus particles. The finding that the particles with icosahedral geometry and surface spikes are fully infective allows an alternative morphological model to the traditional one for astroviruses to be proposed.

Cell Line↗
PubMed1992

Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains.

The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.

Amino Acid Sequence↗
PubMed1991

Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).

Two related forms of the respiratory chain NADH dehydrogenase (NADH:ubiquinone reductase or complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large form that consists of 25 subunits encoded by nuclear DNA and six to seven subunits encoded by mitochondrial DNA. Cells grown in the presence of chloramphenicol, however, make a smaller form comprising only 13 subunits, all encoded by nuclear DNA. When the large enzyme is dissected by chaotropic agents (such as NaBr), all those subunits of the large form that are missing in the small form can be isolated as a distinct, so-called hydrophobic fragment. The small enzyme and the hydrophobic fragment make up, with regard to their redox groups, subunit composition and function, two complementary parts of the large-form NADH dehydrogenase. Averaging of electron microscope images of single particles of the large enzyme was carried out, revealing an unusual L-shaped structure with two domains or "arms" arranged at right angles. The hydrophobic fragment obtained by the NaBr treatment corresponds in size and appearance to one of these arms. A three-dimensional reconstruction from images of negatively stained membrane crystals of the large-form NADH dehydrogenase shows a peripheral domain, protruding from the membrane, with weak unresolved density within the membrane. This peripheral domain was removed by washing the crystals in situ with 2 M-NaBr, exposing a large membrane-buried domain, which was reconstructed in three dimensions. A three-dimensional reconstruction of the small enzyme from negatively stained membrane crystals, also described here, shows only a peripheral domain. These results suggest that the membrane protruding arm of the large form corresponds to the small enzyme, whereas the arm lying within the membrane can be identified as the hydrophobic fragment. The two parts of NADH dehydrogenase that can be defined by the separate genetic origin of (most of) their subunits, their independent assembly, and their distinct contributions to the electron pathway can thus be assigned to the two arms of the L-shaped complex I.

Crystallography↗
PubMed1988

Ultrastructure of the calcium release channel of sarcoplasmic reticulum.

This study is concerned with the characterization of the morphology of the calcium release channel of sarcoplasmic reticulum (SR) from fast-twitch skeletal muscle, which is involved in excitation-contraction coupling. We have previously purified the ryanodine receptor and found it to be equivalent to the feet structures, which are involved, in situ, in the junctional association of transverse tubules with terminal cisternae of SR. The receptor is an oligomer of a single high molecular weight polypeptide and when incorporated into phospholipid bilayers, has channel conductance which is characteristic of calcium release in terminal cisternae of SR. The purified channel can be observed by electron microscopy using different methods of sample preparation, with complementary views being observed by negative staining, double staining, thin section and rotary shadowing electron microscopy. Three views can be observed and interpreted: (a) a square face which, in situ, is junctionally associated with the transverse tubule or junctional face membrane; (b) a rectangle equivalent to the side view; and (c) a diamond shape equivalent to the side view, of which the base portion appears to be equivalent to the transmembrane segment. Negative staining reveals detailed substructure of the channel. A computer averaged view of the receptor displays fourfold symmetry and ultrastructural detail. The dense central mass is divided into four domains with a 2-nm hole in the center, and is enclosed within an outer frame which has a pinwheel appearance. Double staining shows substructure of the square face in the form of parallel linear arrays (six/face). The features of the isolated receptor can be correlated with the structure observed in terminal cisternae vesicles. Sections tangential to the junctional face membrane reveal that the feet structures (23-nm squares) overlap so as to enclose smaller square spaces of approximately 14 nm/side. We suggest that this is equivalent to the transverse tubule face and that the terminal cisternae face is smaller (approximately 17 nm/face) and has larger alternating spaces as a consequence of the tapered sides of the foot structures. Image reconstruction analysis appears to be feasible and should provide the three-dimensional structure of the channel.

Animals↗
PubMed1982

Formyltetrahydrofolate synthase from Clostridium thermoaceticum. An electron microscopic study and specific interaction of the enzyme with ATP and ADP.

Formyltetrahydrofolate synthetase (EC 6.3.4.3) from Clostridium thermoaceticum is a 240 000-molecular-weight, tetrameric enzyme composed of identical subunits. When negatively stained with uranyl acetate or sodium phosphotungstate the enzyme appears as two distinct projections when viewed in the electron microscope. One of these, resembling the letter "H", exhibits four flexible arms, while the other, called a "twin", has a compact projection. The twin projection appears to represent the native enzyme in solution. It consists of two outstanding intensity maxima separated by a narrow bridge of dense stain, with each of these intensity maxima being divided into the two submaxima by a second bridge of stain running perpendicular to the first one. The outstanding intensity maxima represent dimers, whereas the submaxima represent a single subunit. The volume calculated from the electron microscopy projections for the dimer is 157 nm3, which corresponds to a Mr of 127 000 or 254 000 for the tetrameric enzyme. The H projections reflect enzyme particles artificially flattened and "opened" during the drying of the negatively stained enzyme. The four arms of the H each represent a subunit. The appearance of H projections is prevented by either using a deep-stain procedure, which offers a mechanical preservation of the native enzyme structure, or by the binding of ATP, ADP or adenosine 5'-[beta, gamma-methylene]triphosphate. These nucleotides, when binding to the enzyme, cause a conformational change. This was observed by ultraviolet and visible circular dichroism and absorption spectrometry with the native enzyme and an enzyme labeled with fluorescein mercuric acetate attached to a sulfhydryl group of each subunit.

Adenosine Diphosphate↗
PubMed2000

Crystallisation of CP43, a chlorophyll binding protein of photosystem II: an electron microscopy analysis of molecular packing.

Microcrystals of the chlorophyll binding protein, CP43, isolated from spinach thylakoid membranes have been studied by electron microscopy both in negative stain and in vitreous ice. Image analyses of three characteristic views show that the crystals are built of five different layers perpendicular to the c-axis. Each layer consists of different orientations of the CP43 protein. The unit cell derived from the end-on view (looking down the c-axis) shows an angle of 120 degrees, suggesting a threefold rotational symmetry. Both negative staining and cryo data are consistent with a hexagonal crystal lattice. Interpretation of the arrangement of the CP43 protein within this crystal lattice can be made based on 8- and 9-A electron crystallographic structures previously published that provide a model for the organisation of the transmembrane helices of CP43. Overall the analysis presented is consistent with X-ray diffraction data obtained from larger CP43 crystals and forms a framework on which to base further structural studies of this chlorophyll binding protein.

Crystallization↗
PubMed1984

Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle.

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.

Animals↗