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PubMed1994

Light microscopy techniques for the demonstration of silicone gel.

Because of the difficulty in identifying silicone gel in histologic and cytologic specimens by means of conventional light microscopy, we investigated alternative light microscopy techniques, as well as specimen staining and preparation. Specimens from six periprosthetic capsules, one silicone granuloma specimen, and one synovial biopsy specimen obtained from women with silicone breast implants were cut at 4-, 10-, 20-, and 30-microns sections, stained with a variety of common stains, and correlated with electron probe microanalysis. In addition, a commercial silicone gel and silicone gel extracted from a previously implanted silicone breast prosthesis were smeared and examined unstained or stained with Papanicolaou and Diff Quik. Silicone was noted to be refractile, nonpolarizable, and nonstainable. Thicker sections prevented silicone "dropout" during processing and increased the contrast between stained tissue and unstained silicone (negative staining). The relative ease of silicone identification was greatly increased with non-Koehler, phase contrast, and darkfield microscopy. Staining the mounting media of hematoxylin-eosin-stained sections with a nonparticulate commercial ink allowed enhanced negative staining detection of silicone gel.

Breast↗

PubMed1999

Evaluation of transmission electron microscopy for examination of components of acellular pertussis and combination vaccines.

New acellular pertussis and combination vaccines vary in the concentration and presence or absence of specific components and in extent of adsorption to adjuvants. There is a pressing need to develop new control methods for acellular pertussis vaccines. Negative staining electron microscopy has been evaluated as a method for assessing the purity of individual vaccine components and the amount of adsorption to aluminium gels. Negative staining showed the characteristic morphology of vaccine components and permitted detection of contaminants and morphological changes. Reproducible results were obtained by use of a standardized negative staining technique and confidence in the technique was increased by comparison of previously unexamined specimens with a specimen that had been characterized by repeated observations. The degree of adsorption to aluminium adjuvants could only be assessed by observation of the amount of non-adsorbed material in the specimen. Negative staining electron microscopy can be used as one of a number of techniques for control of acellular pertussis combination vaccines.

Adsorption↗

PubMed2001

Expression of L-type amino acid transporter 1 in a rat model of liver metastasis: positive correlation with tumor size.

The purpose of this study was to examine whether L-type amino acid transporter 1 (LAT1), one of the amino acid transporters, is related closely to tumor size in liver metastasis. Rat colon cancer cells (RCN-9) were injected into the capsule of the spleen of 12 male rats (inbred F344/DuCrj). Conversely, four rats received only phosphate-buffered saline (control group 1), and four rats underwent only laparotomy (control group 2). In each rat, the metastatic tumors, areas surrounding tumor nodules, or the livers of the control groups were immunostained with the antibodies to LAT1 C peptide antigen. In control groups 1 and 2, normal hepatocytes did not indicate a staining of LAT1. A total of 90 metastatic nodules were found in 12 livers with metastasis. Of the 90 metastatic nodules, 43 nodules indicated a positive staining of LAT1. Conversely, the remaining 47 metastatic nodules had a negative staining of LAT1. The average size in metastatic nodules in the group with positive staining of LAT1 was 1.6 +/- 0.4 mm2, which was significantly higher than that of the group with negative staining of LAT1 (0.6 +/- 0.2 mm2; P = .0007). The ratio of the average area of the metastatic nodule against the average largest section of the left lobe of the lateral segment (RML, %) was measured. The RML in the group with positive staining of LAT1 was 1.2 +/- 0.3%, whereas the RML in the group with negative staining of LAT1 was 0.4 +/- 0.1%. A significant difference was noted between the two groups (P = .0004). We concluded that LAT1 plays an important role in tumor cell growth of liver metastatic tumors.

Animals↗

PubMed1994

The structure of F-actin. Results of global searches using data from electron microscopy and X-ray crystallography.

The structure of F-actin was investigated by fitting the crystallographically determined actin monomer structure to F-actin electron microscopy data sets obtained by a variety of methods. A reciprocal-space global search procedure was applied to non-equatorial reciprocal-space amplitudes and phases of the microscopy data to locate minima. Fits were performed over a range of cross-sectional radii-of-gyration encompassing values obtained from X-ray solution scattering measurements. Five data sets from four laboratories were investigated: one from frozen-hydrated single filaments, three from negatively stained single filaments, and one from negatively stained single-layer paracrystals. In this last case the paracrystal data were straightened to improve resolution. The best fits nearly unanimously favored a monomer orientation having long-pitch connectivity that was close to that obtained by fitting X-ray fiber diffraction patterns. In certain cases where the resolving power was low, competitive fits were obtained with a quite different orientation, one having only protomer connectivity along the genetic helix. Using a running 10-residue deletion from the monomer in the Holmes-type orientation, subtle differences between the monomer structure and the protomer structure in F-actin could be detected. In particular, differences in the "DNase loop" (residues 41 to 50) and the "hydrophobic loop" (residues 264 to 273) were seen in single-filament data. In addition, a perturbation of the structure was seen in a region near residues 81 to 90. A rearrangement within the protomer structure reported in the literature did not produce fits as good as those obtained when using the undistorted monomer crystallographic structure without 10-residue deletions. These results, taken as a whole, provide strong support for a structure of Mg(2+)-ADP F-actin similar to that originally suggested by Holmes et al. but with alterations of the hydrophobic loop, the DNase loop and in the region near residues 81 to 90. These latter two regions have been proposed as secondary binding sites for myosin heads. The available evidence from electron microscopy and from other sources suggests that residues 40 to 49 are disordered in Mg(2+)-ADP F-actin.

Actins↗

PubMed1984

[HLA antigens in choriocarcinoma].

Choriocarcinoma can be regarded as transplanted cancer, because its origin is in trophoblast which is fetal tissue. It is of interest whether HLA antigens are expressed on choriocarcinoma cells or not, since HLA antigens may play an important role if the patient's body could recognize choriocarcinoma as "not self" and initiate its immune response. In this report choriocarcinoma tissue in the uterus, obtained after hysterectomy, is stained by an indirect immunofluorescence technique using monoclonal antibodies to HLA-A,B,C and HLA-DR. We believe this is the first immunohistological study in which choriocarcinoma in utero is available for study and monoclonal antibodies to HLA antigens are applied. In the staining of HLA-A,B,C, and of HLA-DR, host cells, such as myometrial cells, show positive staining but choriocarcinoma cells show negative staining. It is thought that choriocarcinoma cells are viable and show negative staining of HLA antigens, on the basis of finding following HE staining and hCG staining using anti-hCG antibody and the clinical remarks of the patient such as her high urinary hCG titer before operation and no history of chemotherapy. These results agree with those of previous studies about HLA antigens on choriocarcinoma cell lines.

Animals↗

PubMed2002

The value of MG7 antigen in predicting cancerous change in dysplastic gastric mucosa.

The aim of this study was to ascertain whether MG7Ag is a useful predictor of evolution of gastric dysplasia to carcinoma. A total of 1090 patients with confirmed dysplasia were stained immunohistochemically with MG7 monoclonal antibody by the ABC method. A prospective follow-up study was undertaken on 19 patients with MG7Ag positive staining and 16 with MG7 negative staining over a period of 10-78 months. The expression of MG7Ag was also compared in another two groups by conducting retrospective studies. One group showed an evolution into gastric cancer over 2-4 years, the other did not. Quantitative analysis of MG7Ag expression was carried out on the last two groups. The receiver operating characteristic curve and Youden index were used to assess the best critical value for MG7Ag. MG7Ag was found positive in 456/1090 cases (41.8%) with dysplasia. Prospective follow-up of 35 patients showed that 6/19 patients with MG7Ag positive staining developed gastric cancer, but there were no carcinomatous changes in 16 patients with MG7 negative staining. The results of MG7Ag expression in 72 cases with retrospective follow-up showed there were 24 with positive immunostaining among 34 cancerous cases (70.6%), and only 7 in 38 non-cancerous cases (18.4%) (p<0.01). Image analysis showed that an average MG7Ag density index ++0.19 could be regarded as the critical value for high risk of gastric mucosa with dysplasia evolving to cancer. Positive MG7Ag expression in gastric mucosa of patients with dysplasia, especially in cases with a density index ++0.19, was an indicator of high risk of malignant change.

Adult↗

PubMed1993

Expression of nucleoside diphosphate kinase/nm23 gene product in human pancreatic cancer: an association with lymph node metastasis and tumor invasion.

The expression of nucleoside diphosphate (NDP) kinase/nm23 has been reported to be inversely related to metastasizing potential of experimental cells and human breast cancer. In the present study, levels of NDP kinase/nm23 gene product in curatively resected human pancreatic adenocarcinomas were examined immunohistochemically using anti-NDP kinase antibody. Immunoreactivity for NDP kinase varied between tumors. Of 31 pancreatic tumors examined, 17 (55%; positive staining group) showed strong immunoreactivity for the NDP kinase, while 14 (45%; negative staining group) showed low or no immunoreactivity. Positive staining was associated with higher incidence of lymph node metastasis (13/17; 77%) and perineural invasion (13/17; 77%) than negative staining (5/14, 36%, P < 0.03; 4/14, 29%, P < 0.01, respectively). Positive staining was also associated with shorter overall survival and relapse-free survival than negative staining (P < 0.01, P < 0.01, respectively). No significant difference in age, sex, size, location of tumor, serum carcinoembryonic antigen (CEA) level, or histological type was found between the two groups. These results showed that, in contrast to the reports on breast cancer, NDP kinase/nm23 expression in human pancreatic cancer is positively associated with lymph node metastasis or perineural invasion and with poor prognosis. These, together with other previous reports, suggest that NDP kinase may play an important role in cancer progression or aggressiveness by altering its expression in a tissue-specific manner.

Adenocarcinoma↗

PubMed1975

The morphology of murine oncornaviruses following different methods of preparation for electron microscopy.

The effect of different preparative procedures for electron microscopy on the size and shape of murine oncornaviruses has been studied. With conventional negative staining procedures using neutral sodium phosphotungstate, both murine mammary tumor virus and murine leukemia virus appeared in head-and-tail forms, with a peak head diameter of 122 and 130 nm, respectively. Negative staining with uranyl accetate gave round virions with peak diameters of 148 and 130 nm. Prefixed virus was round with peak diameters of 141 and 130 nm, respectively, in phosphotungstate, and 148 and 117 nm, respectively, in uranyl acetate. With thin sections, the peak diameters were 143 and 123 nm. The preservation of the spherical shape of the virus was obtained by glutaraldehyde fixation dehydration in alcholic solutions of uranyl acetate, and critical point drying. Under these conditions the viruses had peak diameters of 99 and 82 nm, respectively. The size of murine mammary tumor virus has always been found to be larger than murine leukemia virus in all preparations except for negative staining with neutral sodium phosphotungstate. Shadowing of the virion preparations revealed considerable flattening of the particles in all cases except for critical point drying. Negatively stained preparations did not cast any shadow, and thus thethickness of the particles could not be evaluated. Virus can be reversibly converted from spherical to head-and-tail forms by altering osmotic strength. Under most of the conditions used, murine mammary tumor virus gave a bimodal size distribution with significant numbers of particles that were smaller than the major virus size.

Acetates↗

PubMed2005

Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques.

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.

Carbon↗

PubMed1984

Biochemical and ultrastructural characterization of a high molecular weight soluble Mg2+ -ATPase from human erythrocytes.

The isolation and purification of a 600,000 Mr cytosolic Mg2+ -ATPase from human erythrocytes is described. The electrophoretic properties of the native and sodium dodecyl sulphate-dissociated protein are presented and compared with those of the erythrocyte protein cylindrin . The Mg2+-ATPase has a single subunit of Mr 100,000 and it has an isoelectric point of 4.9. From transmission electron microscopy of negatively stained specimens, it is proposed that the Mg2+-ATPase is hexameric, containing two superimposed trimers of the 100,000 Mr subunit, which gives rise to a 13 nm pseudohexagonal particle with a central 3 nm cavity. Varying the orientation of the protein in the negative stain also produces images that are not hexagonal. When orientated on-edge, the protein produces a double-disc image, which is most clearly defined under acidic negative staining conditions with uranyl acetate, when some aggregation of the protein is produced. The ultrastructure of the Mg2+-ATPase is shown to be distinctly different from that of cylindrin . A comparative discussion of the negatively stained transmission electron microscopical images of the Mg2+-ATPase, mitochondrial F1-ATPase and several other oligomeric proteins and enzymes is presented.

Adenosine Triphosphatases↗

PubMed2004

An immunohistochemical analysis of ovarian small cell carcinoma of hypercalcemic type.

Ovarian small cell carcinoma of hypercalcemic type (OSCCHT) is a rare neoplasm with an aggressive behavior, broad differential diagnosis, and unknown histogenesis. To add to knowledge concerning the possible aid of immunohistochemistry in resolving problems in differential diagnosis and to further explore whether that modality points to any specific histogenesis, we undertook an immunohistochemical study of this neoplasm. Fifteen OSCCHTs (including four of the ''large cell" variant) were stained with a range of antibodies, some of which have not been investigated previously in this neoplasm. Cases were stained with AE1/3, EMA, BerEP4, CK5/6, calretinin, WT1, chromogranin, CD56, synaptophysin, CD99, NB84, desmin, S100, CD10, alpha inhibin, TTFI, and p53. Staining was classified as 0 (negative), 1+ (<5% cells positive), 2+ (5% to 25% cells positive), 3+ (26% to 50% cells positive), or 4+ (>50% cells positive). All cases were positive with p53 (two 1+, five 3+, eight 4+), 14 of 15 cases were positive with WT1 (one 1+, thirteen 4+), 14 of 15 with CD10 (three 1+, four 2+, two 3+, five 4+), 13 of 15 with EMA (three 1+, three 2+, two 3+, five 4+), 11 of 15 with calretinin (nine 1+, one 3+, one 4+), 9 of 15 with AE1/3 (eight 1+, one 2+), 4 of 15 with CD56 (one 1+, two 2+, one 4+), 3 of 15 with BerEP4 (two 2+, one 4+), 2 of 15 with synaptophysin (two 1+), and 1 of 15 with S100 (4+). All cases were negative with CK5/6, chromogranin, CD99, NB84, desmin, alpha inhibin, and TTF1. The only noticeable difference in the immunophenotype between typical OSCCHT and the large cell variant was that there was 4 +EMA positivity in three of four cases of large cell variant compared with two of 11 cases of typical OSCCHT. OSCCHT is characteristically positive with AE1/3, EMA, CD10, calretinin, WT1, and p53. Combined EMA and WT1 positivity, the latter usually intense and diffuse, may be of diagnostic value, inasmuch as only a few of the neoplasms in the differential diagnosis are positive with both antibodies. Negative staining with CD99, desmin, NB84, alpha-inhibin, and TTF1 may aid in the cases in which primitive neuroectodermal tumor, rhabdomyosarcoma, intraabdominal desmoplastic small round cell tumor, neuroblastoma, a sex cord-stromal tumor, and metastatic pulmonary small cell carcinoma are in the differential. Calretinin positivity precludes its use in the differential with granulosa cell tumors. The results of this investigation do not settle the issue of histogenesis, which remains enigmatic. The typical age distribution, follicle formation, and calretinin positivity are consistent with a sex cord origin. On the other hand, WT1 and EMA positivity and negative staining with alpha-inhibin would be unusual in a sex cord-stromal neoplasm and can be used as an argument for a surface epithelial origin. Germ cell and neuroendocrine origins seem highly unlikely.

Biomarkers, Tumor↗

PubMed1993

"GA-banding": a new terminology and a study of the glutaraldehyde-induced band pattern of type I collagen fibrils.

The negative staining D-band patterns of glutaraldehyde-reacted collagen fibrils were compared to those of fresh collagen fibrils. Negative staining was obtained by using 1% phosphotungstic acid (PTA) diluted in phosphate buffer 0.1 M, pH 7.4. The stain was dripped onto grids where native type I collagen fibrils, isolated from bovine dermis, were collected. Ultrastructural pictures were digitized to form microdensitometric traces. The glutaraldehyde-induced patterns showed fifteen light bands (micrographs) or negative peaks (microdensitograms), whose D-locations were constant and characteristic. In order to make this ultrastructural feature a precise reference parameter, these bands were called "GA-bands" and numbered. When comparing this averaged microdensitogram with that of negatively stained fresh fibrils, peak "GA1" and peak "GA7" were observed to correspond to peak "X2" (known as N-terminal telopeptide region) and peak "X3" (known as C-terminal telopeptide region) respectively, while there was no correspondence between the other peaks of the two traces. It means that the regions where preexistent crosslinks exist are unaffected by interaction with glutaraldehyde, while in the other regions, where new glutaraldehyde-crosslinks occur, the band pattern modifies. The unchanged D-location of peaks "GA1" and "GA7" leads to the conclusion that the D-shortening induced by glutaraldehyde is not due to shifting of tropocollagen molecules but to changes in their orientation with respect to fibril long axis or in secondary-tertiary structure of collagen.

Animals↗

PubMed1988

Further evidence for the correlation between the primary structure and the stain exclusion banding pattern of the segment-long-spacing crystallites of collagen.

Recently, we have noted the direct correlation between the primary structure of type I collagen and the electron microscopical banding pattern of the negatively stained segment-long-spacing (SLS) crystallites (K. Kobayashi, T. Ito, and T. Hoshino (1986), J. Electr. Microsc. 35, 272-275). In this paper, we examined the correlation in the other types of collagen. Unstained light bands (stain excluding bands) of the negatively stained SLSs of type II and type III collagens were located at the clusters of large hydrophobic amino acid residues along the respective molecules. Photographic averaging of the pattern improved the visual comparison of the correlation. We also noted a few occasions of discrepancy from the above-mentioned correlation. Preliminary computer simulation experiments revealed that, among amino acid parameters so far reported, only the hydrophobicity values of G. D. Rose and S. Roy (1980, Proc. Natl. Acad. Sci. USA 77, 4643-4647) explained the ability of amino acids for the negative staining (stain exclusion) of the collagen SLSs.

Amino Acids↗

PubMed1998

Fine structure of hepatitis B virus surface antigen produced by recombinant yeast: comparison with HBsAg of human origin.

The ultrastructure of hepatitis B virus surface antigen (HBsAg) particles produced by recombinant yeast cells was examined using high-resolution negative staining, and ice embedding, electron microscopy. With negative staining, the HBsAg particles were spherical to slightly ovoid with a mean diameter of 27.5 nm and consisted of many subunits each 4 nm in diameter. Subunits were marked with a minute central pore. With ice embedding, particles were mostly spherical to ovoid, with a mean diameter of 23.7 nm and a 7-8 nm thick cortex surrounding an electron translucent core. Human HBsAg particles, examined using the same methods, were smaller, apparently because of molecular differences in polypeptide structure.

Electrophoresis, Polyacrylamide Gel↗

PubMed1970

Electron microscopic observations on the large subunit of the rat liver ribosome.

Active large subunits obtained by urea treatment of rat liver ribosomes, 59S, were compared with large subunits in intact ribosomes and with the 50S subunits obtained by EDTA treatment. For electron microscopy the specimens were negatively stained or shadow cast. The negatively stained 59S subunits had a slightly ovoidal form; their average dimensions, 244 +/- 17 x 207 +/- 18 A, were very close to the dimensions of the large subunits in intact ribosomes, and lay between the theoretical dimensions for anhydrous and fully hydrated particles that were calculated from the physical properties of the subunits in solution. The shadow-cast preparations showed particles of similar shape. The 50S subunits, which had lost their 5S RNA, were shadow cast at the same time. They appeared to be more spread out than the 59S subunits and had threadlike extensions. In the positively stained regions of uranyl oxalate-stained preparations the 50S particles varied greatly in shape and size, with average dimensions of 330 +/- 21 x 276 +/- 33 A, and showed threadlike extensions like those of the shadow-cast particles. For 50S particles in solution the frictional drag of these extensions probably accounts for their low sedimentation coefficient.

Animals↗

PubMed1970

Structure of Escherichia coli after freeze-etching.

Survival of Escherichia coli, quick-frozen under conditions similar to those employed for freeze-etching, is close to 100%. For determination of cell shrinkage, the diameters of freeze-etched E. coli cells (average, 0.99 mum) were compared with those of preparations after negative staining and after ultrathin sectioning. Negatively stained cells measured from 0.65 to 1.0 mum in diameter, and ultrathin sections showed average cell diameters of 0.70 mum. Freeze-etched replicas of logarithmically growing, as well as stationary, E. coli B cells revealed a smooth, finely pitted cell surface in contrast to cell surfaces seen with other preparative methods. The frozen cell wall may cleave in two planes, exposing (i) a smooth fracture face within the lipid layer and (ii) in rare instances an ill-defined particulate layer. Most frequently, however, cleavage of the envelope occurred between wall and protoplasmic membrane; large areas of the membrane were then exposed and showed a surface studded with predominantly spherical particles, an appearance which did not significantly change when the cells were fixed in formaldehyde and osmium tetroxide before freeze-etching. The distribution of these particles differed between logarithmically growing cells and stationary cells.

Cell Membrane↗

PubMed1990

Correlation analysis of gap junction lattice images.

Fourier averages of connexon images computed from low-irradiation electron micrographs of isolated negatively stained gap junction domains exhibited differences in stain distribution and connexon orientation. To analyze these polymorphic structures, correlation averaging methods were applied to images from negatively stained and frozen-hydrated specimens. For the negatively stained specimens, separate averages over two subsets of connexons with differing degrees of stain accumulation in the axial channel were obtained. Two populations of connexons with opposite skew orientations were distinguishable within a single junctional domain of a frozen-hydrated specimen. Correlation maps calculated using the left- and right-skewed references showed that the selected connexons tend to locally cluster. Using correlation methods to analyze packing disorder in a typical connexon lattice, we estimated the root-mean-square variation in the nearest neighbor pair separation to be approximately 11% of the lattice constant. Displacements of the connexons relative to each other increased with increasing pair separation in the lattice, rather like a liquid, although long-range orientation order was conserved as in a crystal. These results support the hypothesis that the hexagonal ordering of the connexons results from short-range repulsive forces.

Biophysical Phenomena↗

PubMed1966

Physical studies of phospholipids. 3. Electron microscope studies of some pure fully saturated 2,3-diacyl-DL-phosphatidyl-ethanolamines and phosphatidyl-cholines.

On heating pure, fully saturated 2,3-diacyl-DL-phosphatidyl-ethanolamines and 2,3-diacylphosphatidyl-cholines (lecithins) in water to the transition temperature at which large endothermic heat changes occur, they are observed, by light microscopy, to form myelin figures. This result is discussed in terms of the large difference in the transition temperature for "melting" of the hydrocarbon chains of unsaturated and saturated phospholipids and is illustrated by means of differential thermal analysis (D.T.A.) curves. These structures have been examined by electron microscopy after negative staining and after reaction with osmium tetroxide. Typical phospholipid lamella structures are seen in the phosphatidylcholines after negative staining, and in the phosphatidyl-ethanolamines after both negative staining and osmium fixation. The distances across these lamellae have been measured. Some preliminary investigations of the nature of the osmium tetroxide reaction with the phosphatidyl-ethanolamines have been made.

Chromatography, Thin Layer↗