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PubMed1991

Image analysis of mineralized and non-mineralized type I collagen fibrils.

Turkey leg tendons at an early stage of mineralization have been thin sectioned and imaged by electron microscopy. At this stage collagen-associated mineral apatite was found to be present within both the gap and overlap zones. The earliest apatite occurs in a microcrystalline form which gives a rather generalized and characteristic density to both the gap and overlap zones; with subsequent development larger defined apatite crystals arise which span gap/overlap zones. Fourier transformation of such images revealed the major 67 nm axial repeat of the gap/overlap zone plus four other maxima corresponding to repeat spacings of 22, 16, 13, and 11 nm respectively. Computer imaging techniques were used to reconstruct images by using selected spatial frequencies from such transforms. In this manner the subperiodic distributions of mineral were visually enhanced. These subperiodicities are positioned in an asymmetric fashion over the entire D unit repeat aligning with the molecular orientation of the fibril. Analyses of both negatively stained collagen and computer-generated maps of collagen hydrophobicity were compared to the mineral distribution of collagen. Densitometric comparisons showed a positional correlation between the axial banding patterns of mineralized fibrils and those of negatively stained non-mineralized fibrils. Comparable spatial frequencies were also present in transforms between hydrophobic maps and mineral distribution of collagen. These results suggest that the lateral clusterings of hydrophobic residues which span the fibril at specific sites in both the gap and overlap zones serve to prohibit early mineral deposition. This observed hydrophobic influence in combination with the gap space appear as contributing factors in the observed axial distribution of mineral within collagen.

Amino Acids↗
PubMed1995

Towards the molecular architecture of the asymmetric unit membrane of the mammalian urinary bladder epithelium: a closed "twisted ribbon" structure.

The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm. Bovine AUM plaques negatively stained with anionic sodium silicotungstate revealed structural detail to 3.1 nm resolution. Correlation averaging resolved each particle into 12 stain-excluding domains arranged in two concentric rings (inner ring radius (rm) = 3.7 nm, outer ring radius (rout) = 6.6 nm), each with six domains which were rotated by roughly 30 degrees relative to each other. Negative staining with cationic uranyl formate increased the resolution to 2.2 nm and unveiled distinct connections between adjacent AUM particles. These connections may provide a molecular basis for the observed insolubility of the plaques in many detergents. Examination of the luminal face of freeze-dried/unidirectionally metal-shadowed AUM plaques established a left-handed vorticity of the 16 nm protein particles, whereas the cytoplasmic face exhibited no significant surface corrugations. Three-dimensional reconstruction from sodium silicotungstate-stained specimens revealed the AUM particles to be built of six "V-shaped" subunits anchored upright in the membrane. The mass density distribution within uranyl formate-stained AUM particles was similar except that the inner tip of each V was bridged to the outer tip of an adjacent V, so that the 16 nm AUM particle appeared as a continuous, "twisted ribbon" embracing a central cavity. Finally, mass measurements of unstained/freeze-dried plaques by scanning transmission electron microscopy yielded a total mass of 1,120 kDa per membrane-bound AUM particle. By imposing constraints on the possible uroplakin stoichiometries within AUM plaques, these data provide a first glimpse of the molecular architecture of the 16 nm particles constituting the plaques.

Animals↗
PubMedDate not supplied

Human erythrocyte catalase: 2-D crystal nucleation and production of multiple crystal forms.

Negatively stained electron microscope images are presented, showing the nucleation of two-dimensional (2-D) crystals of human erythrocyte catalase produced on mica by the negative staining-carbon film technique. Examples of the formation of partially ordered 2-D arrays and more ordered 2-D crystals are shown and the conditions required for the production of large well-ordered 2-D crystals discussed. The structural transformation of one flexuous 2-D paracrystal into a p2 2-D crystal is considered. The crystallographic 2-D image average of this p2 crystal form is presented (lattice parameters a = 9.0 nm, b = 18.6 nm, gamma = 90.8 degrees). It is shown that transmission electron microscopy provides the possibility of defining 2-D crystal nucleation, growth of intermediate forms, and low-resolution crystallographic structural analysis of 2-D crystals of human erythrocyte catalase. Comparison of the various electron microscopical negatively stained images with the peptide backbone of the X-ray structure of bovine liver catalase at different tilt and rotation positions correlates with and emphasizes the multiple intermolecular contacts and orientations that can be adopted by human erythrocyte catalase, leading to various 2-D arrays and 2-D crystals. Alignment of the surface groups involved in the protein-protein interactions that occur during 2-D crystal nucleation and crystal growth may ultimately be determined. From this approach, when taken together with detailed consideration of protein-solvent and protein-solute electrostatic interactions in solution, and at the fluid-air interface, it is considered that a more general theory of crystal nucleation and growth may eventually emerge.

Aluminum Silicates↗
PubMed1993

Fine-needle aspiration biopsy of subcutaneous fat necrosis of the newborn.

This report provides the first description of the fine-needle aspiration biopsy findings in a case of subcutaneous fat necrosis of the newborn. While initial interpretation of the aspirate smears was suspicious for a neoplastic process, subsequent incisional biopsy confirmed the benign nature of the lesion. Review of the cytologic material revealed rare, negatively stained, needle-shaped crystals within histiocytes and multinucleate giant cells. In the appropriate clinical setting, and given a mixture of lymphocytes, histiocytes, and multinucleate giant cells in the aspirate smears, we believe the finding of negatively stained needle-shaped crystals can strongly suggest the diagnosis of subcutaneous fat necrosis of the newborn.

Adipose Tissue↗
PubMed1990

Mitochondria-cytoskeleton interactions in the sperm midpiece.

The mitochondrial sheath of mammalian spermatozoa is adherent to an underlying organized network of electron-dense material termed the submitochondrial reticulum (SMR). In this manuscript we further characterize the substructure of the SMR and the outer mitochondrial membrane and provide new information on their structural interaction. The SMR resists solubilization by detergent and once partially released from the midpiece of extracted spermatozoa, it appears in negatively stained preparations as a network of longitudinally oriented ribons of fibrillar material which are laterally interconnected. In detergent-extracted specimens the SMR remains attached to the outer mitochondrial membrane thereby suggesting a firm structural interaction. Negatively stained specimens also demonstrate that the outer mitochondrial membrane possesses a paracrystalline substructure and it is suggested that ordered arrays of membrane-associated proteins are involved in the structural interaction with the SMR. The potential roles of this cytoskeletal complex during spermiogenesis and in mature sperm are discussed.

Animals↗
PubMed1984

Reversible structure transition in gap junction under Ca++ control seen by high-resolution electron microscopy.

Deoxycholate-extracted rat liver gap junction was studied by high-resolution low-dose electron microscopy. Communicating channels between two adjoining cells supposedly form along the common axis of two apposed hexameric trans-membrane protein assemblies. These double hexamers are often arranged in large plaques on an ordered hexagonal net (8-9 nm lattice constant) and seem able to undergo structural alteration as a possible permeability control mechanism. Calcium is widely reported to uncouple gap junction, and we observed this alteration on exposure to Ca++ down to 10(-4) M concentration. When EGTA was added at matching concentrations, the alteration was reversible several times over one hour, but with considerable variability. It was imaged in the absence of any negative stain to avoid ionic and other complications. The resulting lack of contrast plus low-dose "shot" noise required digital Fourier filtering and reconstruction, but no detail was recovered below 1.8 nm. In other experiments with negative stain at neutral pH, gap junction connexons were apparently locked in the "closed" configuration and no transition could be induced. However, recovery of repeating detail to nearly 1.0 nm was possible, reproducibly showing a fine connective matrix between connexons . Whether this was formed by unfolded portions of the 28,000-dalton gap junction protein is not known, but its existence could explain the observed lattice invariance during the connexon structural transition.

Animals↗
PubMed1985

Polymorphism of reconstituted human epidermal keratin filaments: determination of their mass-per-length and width by scanning transmission electron microscopy (STEM).

We have determined the mass-per-length (MPL) and the width of unstained freeze-dried reconstituted human epidermal keratin filaments by scanning transmission electron microscopy (STEM). Filaments were reassembled from keratins extracted from four different sources: cultured human epidermal cells (CHEC), human callus (CAL), and the living layers (LL) and stratum corneum (SC) of normal human epidermis. MPL histograms of all four keratin filament types could be fitted by a superposition of two or three Gaussians, with their respective major peaks located between 17 and 20 kDa/nm. We interpreted the multiple MPL peaks to represent different polymorphic forms of the reconstituted filaments. The number of subunits per filament cross section calculated from MPL peak positions, average subunit molecular weight, and an axial repeat of the subunits within the filament of 46.5 nm revealed an average difference between polymorphic variants of 7.5 +/- 0.9 subunits. These data suggest that reconstituted human epidermal keratin filaments are made of two to four 8-stranded "protofibrils" (i.e., made of two laterally aggregated 4-stranded protofilaments), in agreement with earlier observations. The average widths of unstained freeze-dried keratin filaments were larger than those of negatively stained filaments: 12.6 nm (9.6 nm) for CHEC, 12.3 nm (9.7 nm) for CAL, 11.6 nm (8.3 nm) for LL, and 11.3 nm (7.9 nm) for SC keratin filaments, with the values in brackets corresponding to negatively stained samples. Assuming the MPL to be proportional to the square of the filament width, there is a good correlation between the MPL and width measurements both for filaments within a given type as well as among those reconstituted from different types of keratin extracts.

Cytoskeleton↗
PubMed1995

Interaction between rose bengal and different protein components.

Bindings of rose bengal to several proteins were determined by Sephadex G-75 chromatography. Their respective blocking effect against dye uptake was demonstrated in an assay using a rabbit corneal epithelial cell layer. The total binding capacity of nonmucin proteins was measured using fluorometry and Scatchard analysis. The results showed that albumin, lactoferrin, transferrin, and lysozyme could--but serum prealbumin, IgA, carboxymethyl cellulose (CMC), and Sepharose 4B-purified porcine stomach mucin (PSM) could not--bind rose bengal. Lysozyme formed precipitates with rose bengal. Sufficient concentrations of albumin, lactoferrin, transferrin, or lysozyme premixed with rose bengal could block dye uptake by cells, but IgA and serum prealbumin could not. Premixed PSM was not as effective as precoated PSM in blocking dye uptake. The dissociation constant (Kd) was 1.2 x 10(-7) M, 3.6 x 10(-7) M, 3.9 x 10(-7) M, and 1.6 x 10(-6) M for albumin, transferrin, lactoferrin, and lysozyme, respectively. Based on these values, the total maximal binding capacity of nonmucin proteins in normal 7-microliters tears was extrapolated to be 0.249 micrograms rose bengal, which is too small to explain the negative staining of rose bengal on the normal ocular surface. Rose bengal, but not fluorescein, could interact with carbohydrate-containing Sephadex, CMC, and PSM to slow down its elution via Sephadex column chromatography. Therefore, the normal negative staining to rose bengal might be caused by the blocking effect of preocular mucus tear layer, which serves as a diffusion barrier. Rose bengal remains a unique dye for detecting the protective function of the preocular mucus tear.

Animals↗
PubMed1988

pH-dependent accumulation of the vesicular stomatitis virus glycoprotein at the ends of intact virions.

The electron microscope was employed to examine the effect of pH on the distribution of the vesicular stomatitis virus glycoprotein (VSV G-protein) on the surface of intact virions. Both rotary shadowed and negatively stained preparations demonstrated that whereas at neutral pH the glycoprotein was evenly distributed along the virus membrane, at pH 5.5 it was concentrated at the ends giving the overall virion a distinct bilobed or bipolar morphology. Negatively stained specimens showed that the shape of individual glycoprotein spikes was not greatly different at pH 5.5 compared to pH 7.5. The most dramatic effect was in their distribution along the virus membrane. The uniform glycoprotein distribution observed at neutral pH changed rapidly (t1/2 less than 1 min at 21 degrees) to the bipolar state when the pH of the medium was shifted from pH 7.5 to pH 5.5, and it reappeared somewhat more slowly (t1/2 = 2 min at 21 degrees) when the virus was returned to pH 7.5. Our observations support the view that the VSV glycoprotein is able to move laterally in the plane of the virion membrane to produce the terminal G-protein clusters found at low pH. They further suggest that the virion ends may be preferred as sites for the low pH-dependent membrane fusion process involved in initiation of infection by VSV.

Cell Compartmentation↗
PubMed2001

Two forms of phycobilisomes in the Antarctic red macroalga Palmaria decipiens (Palmariales, Florideophyceae).

The phycobilisomes (PBS), the light-harvesting antennae, from the endemic Antarctic red macroalga Palmaria decipiens were isolated on discontinuous sucrose gradients in two discrete bands and not in one as expected. To exclude methodical faults, we also isolated PBS from the temperate Palmaria palmata and the unicellular red algae Porphyridium cruentum and Rhodella violacea. In P. palmata the PBS were separated in two discrete bands, whereas the PBS from Porphyridium and Rhodella were found in one band. The double-banded PBS (PBSup and PBSlow) from P. decipiens were further characterized by absorption and fluorescence spectroscopy, native and SDS-PAGE as well as by negative staining. The phycobiliproteins RIII-phycoerythrin, RI-phycocyanin and allophycocyanin were identified and 3 gamma-subunits were described. The PBSup and PBSlow showed no significant differences in their absorption spectra and phycobiliprotein ratios although the negative stained PBSlow were smaller. Differences were found in their low molecular mass subunit complexes, which are assumed to be r-phycoerythrin. The polypeptide pattern of the PBSup and PBSlow showed no differences in the molecular masses of their subunits and linker polypeptides, but in their percentage distribution. The results suggest that the PBSlow is a closer packed and PBSup a little more loosely aggregated hemiellipsiodal PBS form. We discuss the ecophysiological function of two PBS forms in P. decipiens and suggest advantages in the rapid acclimation to changes in environmental light conditions.

Journal Article↗
PubMed1998

Expression of nerve growth factor and its high-affinity receptor Trk-A in the rat pancreas during embryonic and fetal life.

The expression of functional receptors for nerve growth factor in insulin-producing cell lines grown in vitro has recently been demonstrated. The possible importance of signals transduced via these receptors in the control of islet maturation has been proposed based on data obtained using an in vitro culture system. To further support this hypothesis, we have studied the expression of Trk-A, the high-affinity receptor for NGF, in vivo during the embryonic and fetal development of the rat pancreas. We have also examined the expression of NGF during the same period. Immunohistological analysis shows that at embryonic day 11 (E11), Trk-A is expressed by the epithelial cells of the presumptive pancreas. The few pancreatic endocrine cells present at that stage express Trk-A. At E12 and E16, Trk-A expression was detected in the developing ductal network. The endocrine cells located in the ducts express Trk-A while those that have migrated into the surrounding mesenchyme now stain negative for Trk-A. By E20, Trk-A expression by ductal cells has considerably decreased and can be detected only in small ducts closely associated with islet-like structures. These islet-like structures stain negative for Trk-A. After birth, insulin-positive cells arranged into islets re-express Trk-A. During the same period, NGF mRNA is found to be expressed in the developing pancreas. The expression of Trk-A and its ligand NGF in the pancreas during embryonic and fetal life suggests that NGF and its receptor could play an important role in the development of the pancreas.

Animals↗
PubMed1991

Crystallization of mitochondrial creatine kinase. Growing of large protein crystals and electron microscopic investigation of microcrystals consisting of octamers.

Mitochondrial creatine kinase isolated from chicken cardiac muscle was crystallized by vapor diffusion techniques. Depending on the growth conditions, fine needles and platelets as well as large single crystals appeared after a few days. Large crystals were shown to diffract to at least 3.2 A resolution (Schnyder, T., Winkler, H., Gross, H., Sargent, D., Eppenberger, H. M., and Wallimann, T. (1990) Biophys J. 57, 420 and thus are suited for a detailed X-ray analysis in the future. The relatively high density of single crystals measured by a linear organic solvent density gradient indicates a tight packing of mitochondrial creatine kinase molecules within the crystals. Microcrystals, however, were subjected to electron optical examination either after prefixation with glutaraldehyde followed by conventional negative staining or by freeze-fracturing crystals in mother liquor and heavy metal replication with platinum/carbon. In both cases the crystals exhibited a square lattice with parameters of a = b = 139 A and a = b = 132 A in negatively stained and replicated crystals, respectively. No other lattice parameters were found, suggesting that these microcrystals represent a quasi-cubic three-dimensional lattice, which is in accordance with the finding that the building blocks of the crystals are the cube-like octamers described (Schnyder, T., Engel, A., Lustig, A., and Wallimann, T. (1988) J. Biol. Chem. 263, 16954-16962). Digital image processing applied to electron micrographs of crystals clearly revealed the arrangement of mitochondrial creatine kinase octamers in the crystal lattice as well as the subdivision of the octamer into its subdomains at a resolution of 23 A.

Animals↗
PubMed1977

Ultrastructure of elastin coacervates.

Electron micrographs of negatively stained coacervates of supernatant from sonicated-elastin showed the presence of filamentous structure with the center to center distance typical of tropoelastin, synthetic polypentapeptide as in peptide T1 of tropoelastin, and alpha-elastin. Electron micrographs of negatively stained coacervates of the polypentapeptide, alpha-elastin and sonicated elastin exhibit banded fibers when coacervates are formed, stained and dried at temperatures greater than 50 degrees C.

Elastin↗
PubMed1996

[Vimentin and Nup 180 in vitro binding assay].

In order to investigate relationship between vimentin and nuclear pore complex, we examined binding ability of vimentin, expressed in E. coli, with nucleoporin, isolated from rat liver nuclei, in vitro. Negative staining electron microscopy showed that the vimentin expressed in bacteria assembled 10 nm filament in vitro. SDS-PAGE and western blotting showed that Nup 180 bind to vimentin in binding assay in vitro. Combining immunogold labeling and negative staining electron microscopy techniques, we showed that Nup 180 bind on the 10 nm vimentin filaments. The experiment results indicated that vimentin filament may be anchored on nuclear pore complex in vivo by binding with Nup 180.

Animals↗
PubMed2004

Amylopectin: a major component of the residual body in Cryptosporidium parvum oocysts.

Amylopectin is used for carbohydrate storage in different life-stages of a number of apicomplexan parasites. We have performed an ultrastructural analysis of amylopectin granules from the oocyst residual body and sporozoites of Cryptosporidium parvum. Amylopectin granules were studied in situ and after isolation from 'French' press disrupted parasites, by conventional transmission electron microscopy (TEM) of sectioned oocysts and various negative staining and cryoelectron microscopy techniques. Within the membrane-enclosed oocyst residuum large amylopectin granules (0.1-0.3 microm) can be found besides a characteristic large lipid body and a crystalline protein inclusion. Smaller granules were detected in sectioned sporozoites. Negative staining of isolated amylopectin granules revealed some ultrastructural features not readily visible in sectioned material. The large amylopectin granules had a smooth surface with a 'ball of string'-like inner structure. Granules isolated from sporozoites were more irregularly shaped and showed a rod-like particulate composition. With the exception of alpha-amylase, which led to some degree of damage of the surface of the particles, treatment of amylopectin granules with other glycohydrolases had little effect on the overall structure. However, granules adhered to one another. Only when the granules were boiled did the 'ball of string' structure gradually dissolve.

Amylopectin↗
PubMed1982

Lysates of turkey-grown Pasteurella multocida: examination of vaccine preparations by electron microscopy.

The effects of differential centrifugation, density gradient centrifugation, freeze-thawing, and chemical lysis on the morphology of Pasteurella multocida from the blood of infected turkeys were examined by electron microscopy. Morphologic differences were not seen between thin-section preparations of P multocida after differential and density gradient centrifugation procedures. The internal ultrastructure of in vivo-grown cells was different from that observed previously for P multocida grown in vitro. Large membranous blebs were also observed on the surface of negative-stained preparations of organisms from the plasma, but not in thin sections of cells from the same preparation. Freeze-thaw of bacterial suspensions in sucrose resulted in partial lysis, revealing bacteria in different phases of degradation. Complete lysis (but not solubilization) was enhanced by treatment with EDTA, lysozyme, and Triton X-100. Centrifuged lysate pellets were thin-sectioned or negative-stained. Pellets consisted of vesicles, ranging in size from 0.05 to 1.0 micrometer, that had a characteristic trilaminar membranous appearance similar to those reported from other gram-negative bacteria grown in vitro and treated similarly.

Animals↗
PubMed2005

Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling.

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.

Antigens, Bacterial↗
PubMed1992

Structural modelling of P-type ion pumps.

Over forty sequences of P-type ion pumps have been determined. They fall into five families showing between 20% and 50% identity in sequence. The conserved residues are concentrated in several regions which are found in all the pumps. All the defined functional sites are associated with conserved segments and provide a basis for subdivision into domains, to which tentative secondary and tertiary structures can be assigned. The domains have been assembled into a structure consisting of a conserved core with variable loops and deletions on the surface, which accommodates site mutants, affinity labels and known epitopes. This model has been correlated with the results of an electron crystallographic study of the Ca++ pump. Two types of crystal have been examined in negative stain and in amorphous ice; thin plates which diffract to 4A and long helical tubes which diffract to 15A. The plates have given a 6A projection map, some of which can be interpreted by difference from the map of the negatively stained crystal, as transmembrane helices. Three dimensional interpretation will require a tilt series. In the meantime, analysis of the tubes has given a 14A 3D map which clearly defines the cytoplasmic domains and their relation to the transmembrane region (Stokes and Toyoshima in preparation). Although it is not yet possible to assign specific functions to the cytoplasmic lobes, the structure at this resolution is consistent with the model.

Animals↗