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 595 records · Page 33Linked to original sources
PubMed1980

Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration.

The conformations of the helical nucleocapsids of the paramyxoviruses Sendai virus and simian virus 5, and of a rhabdovirus, vesicular stomatitis virus, have been found to vary extensively with changes in salt concentration. In 10 mM sodium phosphate buffer at pH 7.2, the nucleocapsids are loosely coiled or almost completely extended; with increasing concentrations of NaCl they become more tightly coiled and less flexible. Under isotonic conditions (150 mM) the Sendai virus nucleocapsid is moderately tightly coiled but still curved and apparently flexible, whereas at 400 mM or higher it is very tightly coiled, with the appearance of a rigid rod. These salt-dependent changes in conformation were also found with nucleocapsids composed of proteolytically cleaved protein subunits. Because of the effect of salt concentration, and the fact that it may change during the preparation of negatively stained samples of electron microscopy, it was necessary to fix that nucleocapsids before negative staining to preserve their original conformation. The striking changes in nucleocapsid conformation in response to the ionic milieu indicate the plasticity of its helical structure and suggest that changes in the microenvironment of the nucleocapsid could influence its conformation during viral RNA transcription and replication or during virus assembly by budding, processes in which changes in the coiling of the nucleocapsid or its flexibility could be important.

Capsid↗
PubMed1987

The grid-cell-culture technique: the direct examination of virus-infected cells and progeny viruses.

We describe a method for the structural analysis and identification of viruses, without purification or concentration steps which could alter virus morphology. Virus-infected cells grown on carbon-Parlodion-coated electron microscope grids release large numbers of progeny viruses which adsorb to the surface of the grid and are revealed by negative staining. The technique is rapid, sensitive and can be used at three levels. Negative staining of whole cell preparations revealed both extracellular and intracellular viruses or nucleocapsids beneath the plasma membrane; non-ionic detergent extraction of cells infected with certain viruses reveals cytoskeleton-associated, virus-specific structures normally only observed after thin sectioning; cultures prepared by either procedure are suitable for colloidal gold immunological studies. Extracellular and cytoskeletal-associated viruses were heavily and specifically labelled with gold. The results indicate that the technique may be used to rapidly identify unknown viruses on the basis of size, topography, morphology and mode of maturation from the infected cell, as well as the presence of characteristic intracellular cytoskeletal-associated structures. The technique also has potential use in the sero-grouping and sero-typing of viruses with specific monoclonal antibodies.

Animals↗
PubMed1976

Effect of alkali on the structure of cell envelopes of Chlamydia psittaci elementary bodies.

Suspensions of isolated cell envelopes of infectious elementary bodies (EB) of Chlamydia psittaci at alkaline pH showed a rapid, extensive decrease in absorbance, accompanied by the release of a cell envelope component in a sedimentable form. This phenomenon was observed both at 0 C and with envelopes which had been previously heated to 100 C. Monovalent and divalent cations effectively inhibited the turbidity loss, whereas ethylenediaminetetraacetate (EDTA) caused an accelerated decrease in turbidity. The turbidity loss observed after incubation of the envelopes at alkaline pH could be reversed to the level of the initial value by dialysis against distilled water containing Mg2+. Thin-section electron photomicrographs of purified EB exposed to alkaline buffer with EDTA revealed the loss of the internal contents of cells, but these cells still maintained their round shapes. The cell surface of treated EB appeared pitted in negatively stained preparations, whereas intact EB had a smooth surface. Electron microscopic studies on negatively stained preparations of the clear supernatant obtained after the treatment of the envelope with alkaline buffer containing EDTA demonstrated the presence of spherical particles, approximately 6 to 7 nm in diameter, and rodlike particles, which appeared to be made up of two or more spherical particles.

Alkalies↗
PubMed2005

Clinical significance of vascular endothelial growth factor-C and vascular endothelial growth factor receptor 3 in patients with T1 lung adenocarcinoma.

BACKGROUND: Vascular endothelial growth factor-C (VEGF-C) plays an important role in lymphangiogenesis and activates VEGF receptor-3 (VEGFR-3). Lymphatic spread is an important prognostic factor in patients with lung adenocarcinoma. The aim of the current study was to determine whether the expression of VEGF-C and VEGFR-3 correlates with clinicopathologic factors and prognosis in patients with TNM classification T1 lung adenocarcinoma. METHODS: The authors conducted a retrospective review of 129 consecutive patients who underwent complete resection for T1 lung adenocarcinoma. Immunohistochemical staining for VEGF-C, VEGF, VEGFR-3, CD34 (microvessels), tryptase (mast cells), and CD68 (macrophages) was performed to statistically analyze clinicopathologic implications of VEGF-C and VEGFR-3 status. RESULTS: Of 129 patients with T1 lung adenocarcinoma, 56 (43.3%) patients were positive for tumor-cell VEGF-C and 73 (56.6%) and 69 (53.5%) patients were positive for tumor-cell and endothelial-cell VEGFR-3, respectively. Patients with positive staining for tumor-cell VEGF-C showed significantly less favorable survival rates than patients with negative staining (P = 0.031). The survival rates of patients with positive staining for tumor-cell and endothelial-cell VEGFR-3 were significantly lower than those with negative staining (P = 0.0034 and P = 0.0020, respectively). Patients with positive staining for both tumor-cell VEGF-C and endothelial-cell VEGFR-3 exhibited the most unfavorable prognoses. Multivariate analysis demonstrated that coexpression of tumor-cell VEGF-C and endothelial-cell VEGFR-3 was an independent negative prognostic factor (P = 0.0129) as well as N factor (P = 0.0020). CONCLUSIONS: VEGF-C and VEGFR-3 status may be indicative of survival rates for patients with T1 lung adenocarcinoma.

Adenocarcinoma↗
PubMed1992

Ultrastructure of lymphocystis disease virus (LDV) as compared to frog virus 3 (FV3) and chilo iridescent virus (CIV): effects of enzymatic digestions and detergent degradations.

Ultrastructure of fish lymphocystis disease virus (LDV), the largest of all known icosahedral viruses, has been studied under electron microscopy using enzymatic digestions and detergent degradations. LDV structure appeared roughly the same as those of frog virus 3 (FV3) and chilo iridescent virus (CIV), two other well known viruses of the family Iridoviridae, although the great flexibility of its capsid as observed on negatively stained and shadow cast particles, and its three electron dense layers visualized in ultrathin sections, differed from observations made with the two other viruses. Specific degradation of the virions with enzymes or detergents revealed that the composition of the three iridoviruses was very much alike. In fact, their capsid was composed of two layers as observed in negative staining: an external one, which was removed following digestion with proteinase K, and an internal one which could be digested with phospholipase A2. Thus, the outermost layer is probably made of surface protein units, more or less tightly bound to each other, while the internal one would be a lipoprotein membrane. Consequently, these three iridoviruses appeared structurally related.

Animals↗
PubMedDate not supplied

A rare case of primary malignant mesothelioma originating from the rectovaginal tissue.

A 35 year-old female patient with pelvic malignant mesothelioma is described. The patient underwent total pelvic exenteration due to a pelvic tumor. Macroscopically, the resected tumor was located in the rectovaginal lesion with invasion into the rectal and vaginal wall, and around the internal urethral ostium. Light microscopically, the tumor predominantly consisted of sheets of plump round cells with acidophilic cytoplasm, and focally of tumor cells showing papillary growth pattern. The tumor cells showed remarkable cellular pleomorphism, and were both alcian blue and periodic acid-Schiff stain negative. Electron microscopically, these tumor cells had numerous long bush-like microvilli on their surface with increased length/width ratios. Positive staining with epithelial membrane antigen, cytokeratin, and vimentin, and negative staining with the carcinoembryonic antigen and S-100 protein were observed immunohistochemically. Based on these histological and immunohistochemical estimations, the tumor was diagnosed as a primary malignant mesothelioma originating from the rectovaginal tissue. Review of the literature confirmed the rarity of pelvic malignant mesothelioma. The possibilities of the pathogenesis of the tumor include the tumor's arising from the peritoneal remnant in the rectovaginal tissues, or from the epithelium of the secondary Mullerian system, which shares the same ancestry with the peritoneum.

Adult↗
PubMed1993

Correlation between amino acid composition and ultrastructural features of type I and type II native collagen fibrils.

The D-band patterns of native collagen fibrils negatively stained with phosphotungstic acid (PTA) of type II (bovine cartilaginous nasal septum) and type I (bovine reticular dermis) were compared in order to find differences and correlate these with the different amino acid composition of these two collagen types. By analyzing the averaged microdensitograms of the two types of D-banding, differences in type II fibrils were observed: a) the overlap zone was more electron-transparent on average; b) there was a different hierarchy of positive peaks (intraperiod dark bands) b1, b2 and c1; c) a more marked negative peak (intraperiod light band X3) corresponded to the C-terminal extrahelical region; d) there was a different D-location of one negative peak (intraperiod light band Y3). At the same time, different values were calculated for type I and type II tropocollagen molecule total amino acid "bulkiness" (molecular volume/length ratio) and "hydrophobicity", on the basis of literature-reported data. Consistency was found between these values and the differences of the two types of D-banding. The assumption of a correlation between the above two amino acid parameters and negative staining band patterns was supported.

Amino Acids↗
PubMed1987

Immunohistochemical demonstration of metallothionein in the rat prostate.

Metallothionein in the rat prostate was demonstrated by using an indirect immunoperoxidase technique. Strong staining was found mainly in the supranuclear area of the cytoplasm of the glandular epithelium in the dorsal and the lateral glands. Diffuse cytoplasmic staining and nuclear staining were found with less frequency. Secretory granules were found in the lumen with various intensities of staining. Most of the epithelial cells in the ventral lobes and the coagulating glands were negatively stained. Immunoreactive cells were found sporadically. Two types of immunoreactive cells, round cells and spindle-shaped cells, were scattered in the stroma. Vascular endothelial cells, red blood cells, and smooth muscle cells were negatively stained. These results suggest the synthesis and secretion of metallothionein in the rat prostate.

Animals↗
PubMed1986

The microfibrils of connective tissue: II. Immunohistochemical detection of the amyloid P component.

Immunohistochemical methods were used for the detection of the amyloid P component in the microfibrils of two regions: the zonule of the eye and the connective tissue of the foot pad in 20- to 50-gm mice. Following fixation by immersion in 4% formaldehyde, the eyes and foot pads were embedded in paraffin, and sections were immunostained for light microscopy by using antiamyloid P component antiserum followed by peroxidase-antiperoxidase procedure. For electron microscopy, formaldehyde-fixed tissues were immunostained for the amyloid P component with protein A-gold by using either thin Lowicryl sections or frozen sections which were then embedded in Epon for thin sectioning. In the zonule of the eye, the light microscope showed that zonular fibers were strongly immunostained for the amyloid P component; there was also weak staining of the nonpigmented ciliary epithelium at the distal end of the fibers and of the zonular lamella at their proximal end. The electron microscope revealed clear-cut immunolabeling of the microfibrils making up zonular fibers as well as of individual microfibrils. In the foot pad, the light microscope detected a weak diffuse staining of connective tissue, whereas the electron microscope showed immunolabeling restricted to microfibrils. It was concluded that the amyloid P component was present in, or associated with, microfibrils. Purified amyloid P component was prepared and examined in the electron microscope after either negative staining or routine processing. After negative staining, it appeared as flat pentagonal units, frequently associated into columns. After routine processing, the units looked like cross sections of microfibrillar tubules. The dimensions of the units matched those of the hypothetical segments of the tubules. It was concluded that this tubule consisted of a column of amyloid P units. The cohesion of the units within the column was likely to be reinforced by the bands present at the surface of microfibrils.

Amyloid↗
PubMedDate not supplied

Differential diagnosis between normal endometrium and endometrial hyperplasia with immunostaining cytology using anti-LeY monoclonal antibody.

We have previously reported that both endometrial cancer and endometrial hyperplasia stain positively for the anti-LeY monoclonal antibody, whereas normal endometrium does not. Endometrial hyperplasia is a premalignant change associated with the eventual development of endometrial carcinoma. However, it can be difficult to differentiate hyperplasia from normal endometrium in cytology. This study illustrates the use of immunocytochemical cytology using anti-LeY monoclonal antibody to differentiate between endometrial hyperplasia and normal endometrium. Immunostaining using anti-LeY monoclonal antibody was performed on cytologic specimens obtained from 17 normal endometria, 25 endometria with endometrial hyperplasia, and 13 endometria with endometrial carcinoma. All normal endometria displayed negative staining for anti-LeY monoclonal antibody, whereas all endometria with endometrial carcinoma displayed positive staining. Of the endometrial hyperplasia cases, 21 displayed positive staining. However, four displayed negative staining due to the small number of cells available for diagnosis. We believe that immunostaining cytology using anti-LeY monoclonal antibody is a useful method for differentiating between normal endometrium and endometrial hyperplasia.

Antibodies, Monoclonal↗
PubMed1995

Improved methods for determination of rotational symmetries in macromolecules.

Rotational symmetries of macromolecules are most clearly perceived in the en face projection and may be assessed by inspection of rotational power spectra calculated from electron micrographs of individual particles. However, if the symmetry is not contrasted strongly, this procedure may be inconclusive since the relevant peak may not be convincingly higher than other spectral components. To some extent, this is a sampling problem since the number of repeating elements involved is usually small. We have devised more sensitive statistical tests for rotational symmetry that pool the information contents of entire populations of particles. Both tests involve combining the rotational spectra of many particles and comparing them with the spectra of surrounding background areas. One method is based on the well known t-test which estimates whether two populations differ at a given significance level. In the second test, the ratio between the intensity of each component of the rotational spectrum and the average corresponding intensity for background areas is calculated, and thence, the cumulative product of these ratios over all particles in the data set. If a symmetry is present, this product gradually diverges; otherwise, it converges to zero. As a practical trial, the tests were applied to micrographs of negatively stained hexons of herpes simplex virus and confirmed their 6-fold symmetry. Applied to negatively stained "connector" proteins of bacteriophage T7 purified from a plasmid expression system, both algorithms detected polymorphism with distinct subpopulations of both 13-fold and 12-fold connectors.

Algorithms↗
PubMedDate not supplied

Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine.

Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.

Cell Nucleus Structures↗
PubMed2005

Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase.

Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (alphabetagammadelta)(4). PhK comprises two arched octameric (alphabetagammadelta)(2) lobes that are oriented back-to-back with overall D(2) symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 A. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90 degrees instead of 68 degrees, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.

Animals↗
PubMed1997

Abnormal bcl-2 and pRb expression are independent correlates of radiation response in muscle-invasive bladder cancer.

The objective of this study was to determine whether the overexpression of bcl-2, a key protein governing the apoptotic response to radiation, adds to pRb status in estimating the propensity for radiation response in patients with muscle-invasive bladder cancer. Archival formalin-fixed, paraffin-embedded, pretreatment bladder tumor samples were available in 109 of 301 patients treated preoperatively with 50 Gy in 25 fractions followed by radical cystectomy 4-6 weeks later. Radiation response was assessed by clinical-to-pathological tumor downstaging or upstaging. Altered expression of bcl-2 (47% of 107 patients), p53 (56% of 109 patients), and pRb (30% of 98 patients) was assessed by immunohistochemical staining. Morphological criteria were used to calculate the percentage of apoptotic cells. bcl-2 staining correlated with tumor grade; all grade 2 tumors (n = 7) displayed normal bcl-2 expression (negative staining). No correlations between bcl-2 staining and pretreatment apoptosis levels, p53 staining, and pRb staining were observed. In terms of the radiation response parameters, univariate analyses revealed that bcl-2 overexpression was the only factor associated with upstaging. The main predictor of downstaging was the loss of pRb expression (negative staining). Multivariate logistic regression confirmed these findings and also showed that normal pRb expression (positive staining) was significantly related to upstaging. Patient outcome was adversely affected by bcl-2 overexpression, because these patients experienced significantly increased actuarial local failure rates. No difference in distant metastasis or survival rates by bcl-2 staining was seen. The strongest independent correlates of radiation response thus far identified in muscle-invasive bladder cancer are from bcl-2 and pRb immunohistochemical staining. The overexpression of bcl-2 and the normal expression of pRb seem to thwart the apoptotic response to radiation via independent mechanisms. Abnormalities in the expression of proteins that regulate apoptosis may prove to establish a molecular phenotype to characterize which patients should receive radiotherapy.

Actuarial Analysis↗
PubMed1996

Visualization of the unwinding of long DNA chains by the herpes simplex virus type 1 UL9 protein and ICP8.

UL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding protein, ICP8. Here, electron microscopy has been applied to examine the mechanics of this reaction. Negative staining of the proteins revealed particles consisting primarily of ICP8 monomers and UL9 protein dimers. When the binding of UL9 protein to double strand (ds) DNA containing ss tails was examined by shadowcasting methods, UL9 protein was seen bound to the ss tails or ss/ds junctions; addition of ATP led to its appearance internally along the ds segment. When UL9 protein and ICP8 were incubated together with the tailed dsDNA in the presence of ATP, a highly ordered unwinding of the DNA was observed by negative staining that appeared to progress through four distinct stages: (1) binding of ICP8 to the ss tail and progressive coverage of the ds portion by UL9 protein; (2) formation of highly condensed regular filaments; (3) relaxation of the condensed structures into coiled-coils; and (4) unwinding of the coils and release of ICP8-covered linear ssDNAs. This process represents a mechanism of unwinding that is very different from ones that proceed by a progressive unwinding at Y-shaped forks that move along the DNA.

Adenosine Triphosphate↗
PubMed2002

Diagnostic utility of immunohistochemical staining for p63, a sensitive marker of prostatic basal cells.

Diagnostically reliable identification of prostatic basal cells has depended on staining for high molecular weight cytokeratin. The diagnosis of malignancy is often based on the absence of basal cells. False-negative staining is occasionally observed. Thus, a second method of identifying basal cells might prove useful. Selective expression of p63, a homologue of p53, has been demonstrated in prostatic basal cells. We investigated the diagnostic utility of p63 staining in 70 consecutive specimens for which the differential diagnosis included prostatic adenocarcinoma: 68 needle biopsies and 2 transurethral resection blocks. High molecular weight cytokeratin staining was the gold standard when material was available. A total of 61 specimens were diagnosed as carcinoma, 4 as atrophy, 2 as high-grade prostatic intraepithelial neoplasia, 2 as unclassified collections of benign glands, and 1 as carcinoma versus high-grade prostatic intraepithelial neoplasia. Sections mounted on charged slides were used for p63 staining for 14 specimens. Sections previously hematoxylin and eosin stained on uncharged slides were used for 56 specimens. In every case in which there was successful p63 staining (55 specimens), basal cells in benign lesions were properly marked and other cell types were not stained. Uninformative staining in the remaining 15 specimens was due to failure of tissue adherence in 14 specimens in which sections were on uncharged slides and, in 1 specimen, to poor positive internal control staining of benign glands. Thus, p63 staining was informative in 55 of 56 specimens (98%) for which there was material for examination. No case with satisfactory p63 and high molecular weight staining showed disagreement between the two stains. An additional group of 21 transurethral resection specimens was stained (p63 and high molecular weight cytokeratin). There was less false-negative staining for p63 compared with the case of high molecular weight cytokeratin. No false-positive staining was seen. We conclude that p63 staining is at least as sensitive and specific for the identification of basal cells in diagnostic prostate specimens as is high molecular weight cytokeratin staining.

Adenocarcinoma↗
PubMed1992

Location of the basal disk and a ringlike cytoplasmic structure, two additional structures of the flagellar apparatus of Wolinella succinogenes.

The basal body of Wolinella succinogenes consists of a central rod, a set of two rings (L and P rings), a basal disk from 70 to 200 nm in diameter, and a terminal knob. In negatively stained preparations of flagellar hook-basal body complexes, some disks remain fixed perpendicularly to the grid and show that such a disk is located on the distal side of the P ring. The basal disks have been isolated with and without the P ring; in both cases there is a hole in the center of the disk. The diameter of the disk is smaller in the presence of the P ring. The L-P ring complex is therefore assumed to be a bushing for the rod. Thin sections of whole bacteria and spheroplasts reveal that the disk is attached to the inner surface of the outer membrane. At the insertions of the flagellar hook-basal body-basal disk complexes, depressions are visible in negatively stained preparations of whole bacteria and spheroplasts. A new ringlike structure is connected to an elongation of the basal body into the cytoplasm in both preparations. Its diameter (60 nm) is larger than that of the M ring. A heavily stained compartment can be seen in between the new ringlike structure and the basal disk, which may be formed by the energy transducing units.

Cytoplasm↗
PubMed1992

A new electron microscope positive staining method for viruses in suspension.

A new procedure for the positive staining of viruses in suspension, the Tokuyasu staining procedure (TSP), was evaluated using a non-enveloped virus, rotavirus; an enveloped virus, rubella virus and two glutaraldehyde-treated enveloped viruses, Human T Cell Lymphotropic Virus Type I (HTLV-I) and Human Immunodeficiency Virus Type 1 (HIV-1) as models. The TSP involves an initial staining of the virus with uranyl acetate (UA) followed by thin embedding in a mixture of UA and polyvinyl alcohol (PVA). Using aqueous UA for the TSP, a combination of positively and negatively stained particles was seen for both rotavirus and rubella virus. With glutaraldehyde-fixed HTLV-I and HIV-1, stain penetration did not occur and only negative staining was observed. The substitution of methanolic UA for aqueous UA in the TSP resulted in only positive staining of rotavirus and rubella virus. The change in procedure also resulted in stain penetration of the glutaraldehyde-fixed HTLV-I and HIV-1 to give positively stained particles. Some novel morphological features of rotavirus and rubella virus structure were observed by the TSP.

Glutaral↗