Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Negative Staining”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,027 records · Page 57Linked to original sources
PubMed1994

Fullerene-like organization of HIV gag-protein shell in virus-like particles produced by recombinant baculovirus.

Virus-like particles produced by a recombinant baculovirus containing the HIV gag gene were examined by negative staining after delipidization. This technique demonstrated that the gag-protein shell consisted of radially arranged short rods which formed a network of ring-like structures. Similar structures were observed at the plasma membrane of infected cells which had been opened by wet-cleaving. Occasionally five or six subunits were observed forming a ring. These findings suggest that the gag-encoded precursor (pr55) is a rod-like molecule about 34 A in diameter and 85 A in length. A protein cylinder of such dimensions would have a molecular weight of 56K. The center-to-center distance of two neighboring rings formed by the rods was 66 +/- 8 A (N = 200) by direct measurements and 65 A as obtained from averaged images. This morphology and these dimensions indicate that the virus-like particles contain the gag precursor in the form of a near-spherical "fullerene-like" icosahedral shell. Our data indicate that the triangulation number of the rings equals 63. However, since one rod of pr55 is shared by two rings, the number of copies of the precursor will be 1890 as opposed to 2522 if the molecules were closely packed. The particle diameter of 102 nm deduced from the proposed model was close to the diameter obtained from thin sections of low-temperature-embedded specimens (103-108 nm).

Animals↗
PubMed2000

Histidine residues underlie Congo red binding to A beta analogs.

The binding mechanism of Congo red (CR) to Alzheimer's disease (AD) amyloid fibrils (A beta) in terms of binding affinity and number of sites was quantitated from absorption spectroscopy (at 200-700 nm) by measuring the concentration of CR bound (CR-B) to AD A beta assemblies as a function of CR concentration and pH in 80% ethanol. The rationale for the use of this high concentration of ethanol derives from its use in histological screens for amyloid in tissue sections. Moreover, free CR can be separated from bound CR by filtration in ethanolic but not aqueous medium. The A beta analogs studied here included: (1) peptides having different lengths: A beta1-40, A beta11-28, A beta13-28, A beta19-28, A beta11-25; (2) wildtype, control sequences of A beta1-40 and sequences having different natural amino acid substitutions: primate Pr1-40, rodent Ro1-40, hereditary cerebral haemorrhage with amyloidosis, Dutch type (HCHWA-D) Du1-40, primate reverse sequence Pr40-1; and (3) A beta11-25 sequences having different substitutions: H13D, H14D, and D23K. Negative-staining showed that A beta1-40 fibrils in buffer were indistinguishable from those in buffered ethanolic medium. For all amyloid analogs except A beta19-28, which has no histidine residues and showed no CR binding over the entire pH range 4.0-9.5, CR-B decreased as a function of increasing pH. The decrease was steepest at about pH 5 and became zero above pH 7. For analogs having the same number of histidines, CR-B fell on the same binding curve, indicating that histidine residues are the likely binding sites for CR in this medium. The pH titration of the binding was parameterized by the stoichiometry of dye to the sites, the number of histidines per molecule, the binding dissociation constant Kd, and the apparent proton dissociation constant pK of the histidine; and the calculated pH-titration curves were found to fit the observed ones. For the peptides having 1-3 histidines the average pK was 5.0-5.5, which was similar to the expected pK of histidine in low dielectric medium (80% ethanol), and the Kd's were 2.8-5.9 microM. That histidine residues underlie CR binding in A beta amyloid is consistent with previous findings that A beta peptides sediment as fibrillar assemblies at pH-3-7 and bind Congo red over the same pH range in aqueous medium. Further, the conformation near the binding motif His13-His14-Gln15-Lys16 in A beta assemblies is not greatly altered in 80% ethanol.

Amino Acid Sequence↗
PubMed1997

Morphologic and immunocytochemical characterization of four human uveal cell lines (melanoma- and melanocytes-derived).

PURPOSE: To characterize three cell lines from human uveal melanomas and one ocular melanocyte cell line to study the specificity of several antigens in the malignant transformation of melanocytic uveal cells. METHODS: Light microscopy (LM), transmission electron microscopy (TEM), and immunocytochemical techniques were used in the characterization of OCM-1, SP 6.5, and MKT-BR human uveal melanoma cell lines and UW-1 normal melanocyte cell line from human uvea. Several monoclonal antibodies (MoAbs) S-100, HMB-45, MNF-116, PAL-M1, NK1/C-3, IND-1, and MAAMA were used. RESULTS: All cell lines showed an epithelioid/spindle morphology with occasional multinucleated cells, and nuclear pleomorphism. TEM showed intracytoplasmatic premelanosomes. Incubation with HMB-45 MoAb was positive in all cell lines. PAL-M2, NK1/C-3, MAAMA, and IND-1 MoAbs stainings were positive with variable intensity. MNF-116 MoAb showed negative staining in the four lines, and S-100 MoAb was also negative except for the UW-1 cell line. CONCLUSIONS: Human uveal melanoma cell lines OCM-1, SP 6.5, and MKT-BR and the ocular melanocyte cell line UW-1 exhibited maintenance of some structural and ultrastructural characteristics of melanocytic cells. All four MoAbs, PAL-M2, NK1/C3, IND-1, and MAAMA against cutaneous melanoma-associated antigens stained positively all melanoma cell lines as well as the melanocytic cell line, suggesting that in vitro proliferation of melanocytes could modify their antigenic expression.

Antibodies, Monoclonal↗
PubMed1992

Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity.

The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.

Actin Cytoskeleton↗
PubMed1995

Envelope structure of four gliding filamentous cyanobacteria.

The cell walls of four gliding filamentous Oscillatoriaceae species comprising three different genera were studied by freeze substitution, freeze fracturing, and negative staining. In all species, the multilayered gram-negative cell wall is covered with a complex external double layer. The first layer is a tetragonal crystalline S-layer anchored on the outer membrane. The second array is formed by parallel, helically arranged surface fibrils with diameters of 8 to 12 nm. These fibrils have a serrated appearance in cross sections. In all cases, the orientation of the surface fibrils correlates with the sense of revolution of the filaments during gliding, i.e., clockwise in both Phormidium strains and counterclockwise in Oscillatoria princeps and Lyngbya aeruginosa. The lack of longitudinal corrugations or contractions of the surface fibrils and the identical appearances of motile and nonmotile filaments suggest that this structure plays a passive screw thread role in gliding. It is hypothesized that the necessary propulsive force is generated by shear forces between the surface fibrils and the continuing flow of secreted extracellular slime. Furthermore, the so-called junctional pores seem to be the extrusion sites of the slime. In motile cells, these pores exhibit a different staining behavior than that seen in nonmotile ones. In the former, the channels of the pores are filled with electron-dense material, whereas in the latter, the channels appear comparatively empty, highly contrasting the peptidoglycan. Finally, the presence of regular surface structures in other gliding prokaryotes is considered an indication that comparable structures are general features of the cell walls of gliding microbes.

Cell Movement↗
PubMed1997

Reduced expression of syndecan-1 in human hepatocellular carcinoma with high metastatic potential.

Syndecans comprise a gene family of transmembrane proteoglycans that regulate cellular behavior through interactions with various effectors, including heparin-binding growth factors and insoluble matrix components. Syndecan-1, the most extensively studied, localizes in epithelial cells and has been shown to present in normal hepatocytes. However, little is known about the change of syndecan-1 expression in human hepatocellular carcinoma (HCC). We investigated syndecan-1-protein expression by immunohistochemistry in 57 HCC tissue samples. Syndecan-1 gene expression was also determined. Syndecan-1 protein was expressed in cytoplasm and cell membrane of the hepatocytes and in the bile duct epithelial cells of liver with underlying hepatitis and cirrhosis. Conversely, among 57 HCC tissues, 39 HCC (68.4%) showed negative staining; 50% of well-differentiated HCC showed positive staining, whereas 82.4% of poorly differentiated HCC were negative. Loss of syndecan-1-protein expression was more prevalent in HCC with intra-hepatic metastasis (85.2%) than those without metastasis (48.0%). Similarly, syndecan-1 expression was significantly reduced in HCC with extra-hepatic metastasis (91.7%) as compared with the HCC without extra-hepatic metastasis (62.2%). The gene expression of syndecan-1 was significantly lower in HCC tissue than that in non-tumoral liver tissue. In 2 human HCC cell lines with poorly differentiated phenotype, HLE and HLF, syndecan-1 expression was markedly decreased both at the mRNA and the protein levels. These results suggest that the loss of syndecan-1 expression is a characteristic feature of HCC with high metastatic potential.

Antibody Specificity↗
PubMed1985

Effect of sodium butyrate on actin distribution in rat 3Y1 fibroblasts in monolayer culture.

We studied the effect of sodium butyrate, a potent G1/G2-arresting agent, on actin distribution in rat 3Y1 fibroblasts in monolayer culture by fluorescence microscopy of cells stained with 7-nitrobenz-2-oxa-1, 3-diazole phallacidine (NBD-Ph). When randomly proliferating cells were arrested mainly in G1 phase with butyrate, a reversible overaccumulation of cellular net protein occurred. In the G1-arrested cells, actin markedly accumulated at the margin of cells, and a network structure of actin stress fibers appeared. When density-arrested cells were replated sparsely and rearrested in the G1, early S, and G2 phases with butyrate or hydroxyurea, the actin network was observed extensively in the cells arrested in the G1 and G2 phases with butyrate. These results agree with our previous results indicating the existence of some physiological similarity between cells in the G1 and G2 phases and suggest that actin distribution somehow depends on the phases of the cell cycle. The actin profiles observed by the NBD-Ph staining were confirmed by transmission electronmicroscopy (TEM) of negatively stained whole cells. TEM further revealed that electron-dense amorphous materials were present at crossing points in the network but rarely present on interconnecting microfilament bundles.

Actins↗
PubMed1996

Expression and mutation of H-ras in uterine cervical cancer.

In cervical cancer, abnormalities of ras genes have not been fully investigated. We studied the expression and mutation of H-ras oncogene in cervical cancer to investigate their relationship and usefulness as an independent prognostic indicator. Twenty-seven paraffin-embedded resection specimens of cervical cancer (21 squamous, 3 adeno, 2 adenosquamous, and 1 small cell) were examined by immunohistochemistry using a mAb H-ras p21 and by PCR and allele-specific oligonucleotide hybridization using H-ras codon 12 and 61 amplimers and oligonucleotide probes. A strong immunoreaction was noted in 10 cases (37%) and weak immunoreaction in an additional 6 cases (22%). H-ras codon 12 mutations were detected in 6 of 27 cases (22%) and all of the mutations were guanine to adenine transitions. There was no mutation in codon 61. Cases with codon 12 mutations included all 3 squamous, 2 adeno, and I adenosquamous carcinoma. Only 3 of 16 (19%) cases with positive staining and 3 of 11 (27%) cases with negative staining showed mutations. No correlation was found between ras gene alterations and patient survival time. Our findings indicate that expression and mutation of H-ras oncogene occur in cervical cancer but their determination adds no useful prognostic information.

Adult↗
PubMed1995

Identification of apoptotic hepatocytes in situ in rat liver after lead nitrate administration.

Apoptosis plays a major role in the regression of mitogen (lead nitrate)-induced hepatic hyperplasia. We compared the in situ end-labeling (ISEL) technique with the conventional detection of apoptotic bodies in this process. In hematoxylin and eosin (H&E) sections, apoptosis is usually recognizable by the presence of apoptotic bodies (apoptosis phase 2). Although the early phase of apoptosis (apoptosis phase 1) can be detected as a prekaryorrhectic appearance in H&E sections, it is difficult to detect and is easily overlooked. On the other hand, ISEL presents intense staining mainly in phase 1 and weak or negative staining in phase 2. Thus, simultaneous investigation by these two methods in two serial sections is the most reliable way to calculate the incidence of apoptosis and gives us precise information on the stages of apoptosis in situ. Since the colorized signals of ISEL are much easier to detect than apoptotic bodies in H&E sections, ISEL is particularly useful for liver tissues, where the incidence of apoptosis is low.

Animals↗
PubMed1991

Progress in outgrowth culture from rabbit tracheal explants: balance between proliferation and maintenance of differentiated state in epithelial cells.

Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively.

Animals↗
PubMed1985

Partial cytochrome oxidase (aa3) deficiency in chronic progressive external ophthalmoplegia. Histochemical and biochemical studies.

Biochemical and histochemical studies were carried out on 2 patients with chronic progressive external ophthalmoplegia (CPEO). Histological examination revealed prominent ragged-red fibres in the Gomori trichrome stain and cytochrome oxidase staining revealed partial depletion of cytochrome oxidase with negative staining in some fibres with prominent subsarcolemmal mitochondrial aggregations. Polarographic studies with isolated intact skeletal muscle mitochondria revealed low State III respiration rates with NAD- and FAD-linked substrates. Cytochrome aa3 levels were depressed in the one case where a cytochrome difference spectra was recorded. Cytochrome oxidase levels were greatly depressed in muscle homogenate, whereas monoamine oxidase levels were in the normal range, indicating a selective depletion of the former enzyme complex. It is possible that deficiency of cytochrome oxidase may arise as an epiphenomenon in degenerating mitochondria rather than a primary deficiency.

Adolescent↗
PubMed2004

Primary villoglandular adenocarcinoma of cloacogenic origin of the vulva.

Primary villoglandular adenocarcinomas of the vulva are rare, and only seven cases have been described to date. Hypotheses about the origin of this neoplasm remain highly speculative. We report the case of a 58-year-old woman who developed this very uncommon malignant tumor. Immunohistochemical studies of the tumor revealed positive staining for cytokeratins 7 and 20 and a negative staining for estrogen and progesterone receptors. Special stains showed the presence of intracellular mucin. No other primary lesion was found on clinical evaluation and extensive investigation. The patient was first treated by radical hemivulvectomy, followed by ipsilateral inguinal lymph node dissection and laparoscopic right salpingo-oophorectomy for an incidental finding of an ovarian cyst. The patient remained free of disease 16 months after the initial surgery.

Adenocarcinoma↗
PubMed1995

Heterogeneity of smooth muscle cells in embryonic human aorta.

Cellular composition of aortas from 5- to 12-week and 18- to 28-week-old human embryos were investigated using immunocytochemistry, scanning and transmission electron microscopy. The aorta of the 5- to 12-week-old embryos consisted of three sublayers differing in cellular composition. The inner sublayer adjacent to the endothelium contained round and ovoid cells with synthetic phenotype. In the intermediate sublayer, spindle-like cells ultrastructurally similar to smooth muscle cells were found. Cells of the outer sublayer resembled fibroblasts or poorly differentiated mesenchymal cells. There were not definite morphological borders between sublayers. In the 18- to 28-week-old embryo aorta the intima was separated from media by internal elastic lamina. Intimal and innermost medial cells had predominately stellate shape and synthetic phenotype. The outer part of media contained spindle-like cells that had well developed contractile structures. Both the 5- to 12-week-old and the 18- to 28-week-old embryo aortic cells were positively stained for alpha-actin and myosin and negatively stained for macrophage antigens. Thus, the majority of embryo aortic cells appeared smooth muscle cells, however there was a regional difference in shape and synthetic state of these cells.

Aorta, Thoracic↗
PubMed1993

Mucin-producing poorly differentiated adenocarcinoma of the thyroid. A case report.

A rare case of mucin-producing poorly differentiated adenocarcinoma of the thyroid is reported in a 58-year-old man. Light microscopically, the tumor composed of columnar cells, cuboidal cells and spindle cells and these tumor cells showed a various growth pattern; glandular growth with columnar cells, microfollicular growth with cuboidal cells and solid growth with spindle cells. Mucin histochemistry showed scattered foci of mucin production. Immunohistochemistry showed a focal positive staining for thyroglobulin and carcinoembryonic antigen, but a negative staining for calcitonin throughout the tumor. The patient had cervical and axillary lymph node recurrences after the radical operation. The histologic finding of this tumor is peculiar; we report this case briefly.

Adenocarcinoma, Mucinous↗
PubMed1972

Early biochemical consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation.

1. One week after denervation several biochemical characteristics of the fast extensor digitorum longus and slow soleus muscles from adult rats were investigated and compared with the characteristics of the corresponding unoperated contralateral muscles. 2. After these short periods of denervation-induced atrophy, the isolated myosins showed unchanged ATPase (adenosine triphosphatase) activities, but there was the expected difference between fast and slow muscle. 3. The specific activities of several soluble enzymes and their characteristic patterns were found to be only slightly modified in both the extensor and soleus muscles after denervation, as were most of the activities measured in the isolated mitochondria. 4. The most significant modifications were in the isolated sarcoplasmic reticulum, and appeared to be specific to either slow or fast muscle. 5. Denervation of slow muscle led to a marked increase of Ca(2+)-transport rates, and of the specific activity of the Mg(2+)-activated K(+)-modulated Ca(2+)-stimulated ATPase, together with changes in the polyacrylamide-electrophoretic profiles of the microsomal membrane protein. Transformation of these several properties of slow muscle sarcoplasmic reticulum to those of fast muscle sarcoplasmic reticulum was further substantiated by electron-microscopic analysis after negative staining. Control experiments with tenotomized soleus muscle gave negative results. 6. The isolated sarcoplasmic reticulum from fast muscle showed a slight diminution of ATPase-linked Ca(2+)-transport activity and a selective increase of rotenone-insensitive NADH-cytochrome c reductase activity, in addition to a greater emphasis on slow-type electrophoretic components of the structural membrane protein. 7. The significance of these results in relation to specific differentiating influences from motor nerves is discussed.

Adenosine Triphosphatases↗
PubMed2002

Gastrointestinal metastasis to the breast.

BACKGROUND: Although primary breast cancer is common, metastatic disease to the breast, especially primary gastrointestinal cancer, is rare. Routine pathologic examination may be helpful in determining the true diagnosis, but can be misleading. METHODS: To determine whether a signet ring carcinoma was a primary malignancy of the gastrointestinal tract metastatic to the breast or vice versa, histochemical analysis was performed for Her-2/NEU, gross cystic disease fluid protein-15, estrogen receptor, progesterone, carcinoembryonic antigen, cytokeratin 7, and cytokeratin 20. RESULTS: Positive staining for carcinoembryonic antigen and cytokeratin 20 (and negative staining for the breast cancer antigens), and the clinical criteria favors the diagnosis of gastrointestinal carcinoma metastatic to the mammary gland. CONCLUSIONS: Because the prognosis of therapy for metastatic cancer to the breast differs from that of primary breast cancer, it is imperative that the correct diagnosis be established. Immunohistochemistry for carcinoembryonic antigen and cytokeratin 20 are particularly useful. Metastatic gastrointestinal carcinoma to the breast is a rare lesion but needs to be at least included in the differential diagnosis of breast masses, especially in patients with a history of gastrointestinal cancer.

Adult↗
PubMed1989

Use of keratin 903 as an adjunct in the diagnosis of prostate carcinoma.

The identification of basal cells is often helpful in excluding a diagnosis of prostate carcinoma. However, it can be difficult to distinguish basal cells from underlying fibroblasts or an artifactual two-cell layer in neoplastic glands. To determine the usefulness of anti-keratin antibody 903 for identifying basal cells in glandular patterns sometimes confused with carcinoma, we examined frozen sections from radical prostatectomy specimens and formalin-fixed needle biopsy, radical prostatectomy, and transurethral resection specimens. Atrophic glands, basal cell hyperplasia, intraductal severe dysplasia and various grades of carcinoma were examined. Also evaluated were cases of atypical adenosis, defined as clusters of small glands that mimic low-grade carcinoma yet focally appear to have a basal cell layer and merge with more recognizable benign glands. Almost all normal glands showed some staining, although it was often discontinuous with formalin fixation. Intraductal dysplasia stained in a manner similar to normal glands. Ninety-two percent of atrophic glands and 88% of glands in basal cell hyperplasia stained. Sixty-one percent of the glands in atypical adenosis stained intensely but discontinuously. All grades of adenocarcinoma lacked any immunoreactivity. These results indicate that keratin 903 is useful in the diagnosis of prostatic carcinoma because positive staining identifies a questionable focus as benign whereas negative staining helps to substantiate the diagnosis of carcinoma.

Adenocarcinoma↗
PubMed1998

Peritumoral and nodal muciphages.

Three patients who underwent surgery for cancer showed Alcian blue positive cells in the sinusoids of the regional lymph nodes and, in one case, extensive infiltration in the soft tissues around the tumor. The cells contained variably sized vacuoles that occasionally indented the nuclei, imparting a signet-ringlike appearance. They were interpreted as muciphages based on morphology, reactivity with mucin stains, positive immunohistochemical staining for KP-1 and Mac-387, and negative staining for cytokeratin, S-100, and leukocyte common antigen (LCA). The material contained in these cells was Alcian blue positive at pH 2.5 and pH 1, and was strongly periodic acid-Schiff positive after diastase digestion. Hyaluronidase only slightly reduced the intensity of the Alcian blue stain. These results indicate that the bulk of the material was epithelial-type mucin. To our knowledge, this is the first report of muciphages in lymph nodes. Careful attention to morphology and immunohistochemical findings is necessary to avoid confusion with metastatic or infiltrating signet ring or mucinous adenocarcinoma.

Adenocarcinoma↗