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 847 records · Page 47Linked to original sources
PubMed1994

Gramicidin A/short-chain phospholipid dispersions: chain length dependence of gramicidin conformation and lipid organization.

Gramicidin-lipid interactions were investigated using diacylphosphatidylcholines that contained two identical acyl chains of varying length, between 6 and 14 carbons. The gramicidin A (gA) conformation was monitored by circular dichroism (CD) spectroscopy and high-performance size-exclusion chromatography, and the lipid organization was investigated using 31P and 1H NMR spectroscopy and negative-stain electron microscopy. Diacylphosphatidylcholine (PC) lipids with chain lengths between 4 and 8 carbons have been previously shown to have a micellar organization in aqueous solution [Lin, T.-L., et al. (1986) J. Am. Chem. Soc. 108, 3499-3507]. CD spectra of aqueous gA/lipid dispersions, at a ratio of 1:28, demonstrated that the channel conformation of gA can be readily obtained when the acyl chain length is > or = 10, but not when the chain length is < or = 7. Size-exclusion chromatography revealed that the fraction of gA that could easily be dissociated into monomers in the dispersions increased with increasing acyl chain length, in agreement with the CD results. For a chain length of 8, the results were intermediate. The formation of the channel structure was found to depend on the "solvent-history", the temperature, the gA and lipid concentrations, the gA:lipid ratio, and consequently on the method of sample preparation. 1H and 31P NMR results suggest that codispersed gA increases the size of dioctanoyl-PC aggregates, but not of dihexanoyl-PC micelles. Negative-stain electron microscopy directly supports these findings. Dihexanoyl-PC (28 mM) was able to solubilize 1 mM gA in H2O, but the gA was not in the "channel" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis.

Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain.

Amino Acid Sequence↗
PubMed1966

Basal bodies of bacterial flagella in Proteus mirabilis. I. Electron microscopy of sectioned material.

Years ago (16, 18, 19), in a study of shadowed preparations of Proteus vulgaris that had been autolyzed in the cold, the observation was made that the flagella arose from basal bodies. However, recently (3, 7, 24, 33) doubt has been cast on the conclusion that the flagella of bacteria emerge from sizable basal bodies. This problem has, therefore, been reinvestigated with actively developing cultures of Proteus mirabilis, the cell walls of which had been expanded slightly by exposure to penicillin. Two techniques were applied: ultramicrotomy, and negative staining of whole mount preparations. This paper deals with the thin sections of bacteria after the usual fixation technique had been altered slightly: the cells were embedded in agar prior to their fixation and further processing. The flagella then remained attached to the cells and were seen to extend between the cell wall and the plasma membrane. Occasionally, the flagella appeared to be anchored in the cell by means of a hook-shaped ending. In sections of cells rich in cytoplasm, the basal bodies are particularly difficult to visualize due to their small size (25 to 45 mmicro) and the lack of properties that would enable one to distinguish them from the ribonucleoprotein structures; in addition, their boundary appears to be delicate. However, when the cytoplasm is sparse in the cells, either naturally or as a result of osmotic shocking in distilled water, the flagella can be observed to emerge from rounded structures approximately 25 to 45 mmicro wide. Contrary to a previous suggestion (21), the flagella do not terminate in the peripheral sites of reduced tellurite, i.e. the chondrioids. The observations in this part of the study agree with those described in the following paper (15) dealing with negatively stained preparations.

Cell Membrane↗
PubMedDate not supplied

Structural and physicochemical analysis of the contractile MM phage tail and comparison with the bacteriophage T4 tail.

The three-dimensional (3-D) structure of the bacteriophage MM extended tail has been determined from electron micrographs of negatively stained specimens and compared with 3-D models of coprocessed extended bacteriophage T4 tails. Accordingly, the phage MM extended tail exhibits an axial repeat of 3.8 nm and can be indexed according to the integer helical selection rule l = -3n + 7m (n = 6n') compared to 4.1 nm and l = -2n + 7m (n = 6n') for the T4 phage tail. Compared to the T4 tail sheath, which reveals a stacked-disk-like appearance, the MM tail exhibits a more open structure, yielding an arrow-head-like appearance. Although the phage MM extended tail sheath is more stable than the T4 tail sheath under low-ionic-strength conditions, various chemical treatments of the MM tail sheath revealed responses, notably disassembly and contraction, similar to those previously described for the T4 tail sheath. Extended tails and their structural components contained in phage lysates or prepared by chemical degradation were compared in the EM, and the mass-per-length values of extended tails and tail tubes were determined by quantitative scanning transmission electron microscopy and compared to the corresponding values computed from the respective 3-D mass density maps. Accordingly, masses of 111 and 135 kDa/nm were obtained for the MM and T4 phage tail sheaths, respectively, with the corresponding tail tubes calculated at 19.3 and 25.5 kDa/nm, respectively. Although negative staining and freeze drying/metal shadowing of the two tails revealed different extended tail sheath structures, freeze-dried/metal-shadowed specimens of their contracted tails revealed very similar 6-fold symmetric axial repeats, with the subunits arranged on a pseudo-12-fold symmetric surface lattice following the integer helical selection rule l = n + 11m. In both cases tail contraction started at the baseplate and propagated headward as a wave forming a contraction gradient with a sharp boundary.

Bacteriophage T4↗
PubMed1994

Coiled-coil packing in spermine-induced tropomyosin crystals. A comparative study of three forms.

Electron microscope images of highly ordered spermine-induced microcrystals of tropomyosin have been analyzed to determine the packing of the molecular filaments. Negatively stained microcrystals terminate in a distinctive "double fringe", which reveals the location of the molecular ends. This information, together with the symmetry of the structure in projection, shows that the microcrystals can be accounted for by a packing scheme of four layers of molecules in the unit cell. Knowing the position of the symmetry elements relating the layers then allows the three-dimensional space group of the microcrystals to be established as C222(1). Using cryo-electron microscopy and simulation studies, the run of the filaments and their packing in the C222(1) form have been shown to be related to those in the spermine-induced C2 crystal of tropomyosin whose structure has been solved to 9 A by X-ray crystallography. This result allows us to infer the location of the molecular ends in the C2 crystal as well, and this inference has been confirmed by analysis of thin sections of the C2 crystal. The C222(1) microcrystal has also been shown to be closely related to the classical divalent cation tropomyosin paracrystal. Based on knowledge of the molecular packing in the divalent cation paracrystal, the polarity of the molecules has been deduced in the other two crystal forms. The tropomyosin filament packing in all these forms may be accounted for by coiled-coil close packing and specific cationic bridging of negatively charged zones on the molecule. Taken together the results reveal a hierarchy of interactions in these close-packed crystalline forms, whose principles may apply to the packing in other fibrous proteins. This study also shows the usefulness of co-ordinating results from cryo-electron microscopy with negative staining in the structure analysis of such ordered arrays, and how these findings can complement the results of low resolution X-ray crystallographic studies.

Animals↗
PubMed1979

Reovirus serotypes 1 and 3 differ in their in vitro association with microtubules.

Utilizing negative-stain electron microscopy in which similar concentrations of reovirus types 1 and 3 are incubated with a carbon support film containing chick brain, rabbit brain, or HeLa cell microtubules, 81% of the type 1 and 56% of type 3 exhibited an association with the apparent "edge" of the microtubule. This implies that there is a high level of specific affinity for type 1 but not for type 3 to microtubules, since it has previously been determined that only 50% of randomly associated particles would be associated with the edge. The high edge binding of reovirus type 1 is virtually independent of the origin of microtubule, or of whether microtubules or virus has been initially adhered to the support film. On the other hand, reovirus type 1-specific antiserum reduced the edge binding or reovirus type 1 to 45%, whereas type 3 specific antiserum caused no less (within the variability of the assay) of the edge binding of reovirus type 1 to microtubules (76% edge bound). High edge binding of reovirus type 1 to microtubules is correlated with the presence of type 1 or sigma 1 polypeptide. This minor outer capsid polypeptide is encoded in the S1 double-stranded RNA segment and is the viral hemagglutinin and neutralization antigen. Recombinant reovirus clones containing the S1 double-stranded RNA segment of type 1 (80 and 802) show about 85% edge binding, as compared to a value of 42% for clones and the S1 gene of type 3 (204. Electron microscopy of purified reovirus types 1 and 3 by negative staining reveals that type 1 and 802 capsomers are distinctly visualized, whereas those of type 3 and 204 appear diffuse. Thus, the greater in vitro binding of type 1 to microtubules may reflect an increased accessibility of certain of its outer capsomers, and thereby, sigma 1 polypeptides to microtubules. Examination of its outer sections of reovirus type 1- and 3-infected cells at 24 to 48 h postinfection at 31 degrees C showed that about eight times as many viral factoris in type 1-infected cells exhibited an extensive association of virus particles with microtubules, as compared to viral factories of type 3-infected cells. Thus, both in vivo and in vitro there appears to be a greater specificity for the association of reovirus type 1 particles with microtubules, as compared to reovirus type 3 particles.

Animals↗
PubMed1976

Regular structures in membranes: the lumenal plasma membrane of the cow urinary bladder.

The ultrastructure of the lumenal plasma membrane of the cow urianry bladder has been studied in thin sections of glutaraldehyde- and glutaraldehyde-H2O2-fixed specimens, by negative staining and freeze fracture. A regular hexagonal array of particles confined to polygonal plaques 0-1-0-4-mum in diameter and separated by 0-02-mum interplaque areas is revealed by all 3 techniques. Cross-sections through particulate areas fixed with glutarayldehyde-H2O2 display a tetralaminar structure consisting of the usual approximately 8-nm-thick trilamellar unit membrane structure, on the external dense leaflet of which is located an additional approximately 4-nm-thick stratum which is occasionally resolved into a row of regulrly spaced approximately 4-nm-diameter particles. Non-particulate areas feature only the approximately 8-nm-thick trilamellar structure. Tangential sections reveal an hexagonal array of particles with a unit cell of approximately 16 nm. Four membrane faces can be revealed by freeze fracture and etching of membranes of the cow urinary bladder; 2 complementary split inner membrane faces (A and B) revealed by the cleaving process and the lumenal and cytoplasmic membrane surfaces exposed by etching. Face B, which belongs to the external membrane leaflet and faces the cytoplasm, displays plaques of particles arranged in a hexagonal lattice with a unit cell of approximately 16 nm. Face A, which belongs to the cytoplasmic membrane leaflet and faces the lumen, displays a complementary array of hexagonally packed pits. The hexagonally arranged particles also protrude into the lumenal membrane surface where they can occasionally be resolved into 6 approximately 5-nm-diameter subunits; the cytoplasmic surface appears smooth. Six approximately 5-nm-diameter subunits are also revealed in negatively stained preparations. The data are consistent with a model for the membrane in which the particles forming the hexagonal structure protrude above the lumenal membrane surface and also bridge most of the thickness of the membrane.

Animals↗
PubMed2000

Haliotis tuberculata hemocyanin (HtH): analysis of oligomeric stability of HtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2.

The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31-41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43-56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329-339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100mM concentrations of calcium and magnesium ions in Tris-HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.

Animals↗
PubMed1993

Immunohistochemical localization of transforming growth factor alpha in the major salivary glands of male and female rats.

The three major salivary glands of normal male and female Fischer 344 rats of different ages were examined for the localization of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) by immunohistochemical staining. EGF was demonstrated only in the granulated convoluted tubule (GCT) cells of the submandibular gland, the results confirming the previous reports, and most abundantly in adult males and pregnant females. TGF alpha stain was localized in all three glands and was found throughout the entire duct system, excluding acinar cells. The myoepithelial cells of the sublingual gland were also reactive with the TGF alpha antibody. The specificity of the staining was confirmed by negative staining reaction with the absorbed antibody and by radio-immunoassay and Western blot methods. This is the first report describing the presence of TGF alpha in the rat salivary glands.

Animals↗
PubMed1992

Case report 749: Primary glomus tumor of bone.

We report a case of primary intraosseous glomus tumor in a 30-year-old man who was found to have an expanding, lytic lesion in the distal phalanx of his left thumb. The histological appearance was atypical in that areas of myxoid stroma resembled chondroid material. The unusual location and microscopic appearance caused diagnostic problems. Immunohistochemical studies, including strong positive staining for MSA and negative staining for keratin and S-100 protein, were helpful in establishing the correct diagnosis.

Adult↗
PubMed1989

In vivo and in vitro staining of acidophilic neurons as indicative of cell death following kainic acid-induced lesions in rat brain.

An in vivo method for positively staining dead neurons was developed and compared with an in vitro staining method using acid fuchsin. Neurons previously killed by intracerebral injections of kainic acid were selectively stained by trypan blue within 15 min of its injection in vivo into the central nervous system of rats. Such staining persisted for at least 4 days in vivo, and there was no evidence that trypan blue itself was toxic to the remaining cells. Intense staining of neurons with acid fuchsin in vitro was first observed in brain sections of rats killed about 6h after kainic acid injection. This time was approximately 2-3 h before trypan blue, in vivo, was able to stain neurons. Thus, the loss of transport mechanisms (at least for trypan blue) apparently occurs subsequent to the development of basic products stainable with acidic dyes. At the earliest times, acid fuchsin stained neurons which had not yet lost Nissl substance, whereas the majority of trypan blue-stained neurons were not stained with Nissl dyes. After 24 h the majority of neurons stained with either acid fuchsin or trypan blue were Nissl-stain negative. The combination of staining with trypan blue in vivo with subsequent counterstaining of brain sections with acid fuchsin in vitro may have a potential use in the determination of the time of neuronal death in vivo.

Animals↗
PubMed1982

Mutagen treatment of single Chinese hamster ovary cells produces colonies mosaic for glucose-6-phosphate dehydrogenase activity.

When populations of single Chinesee hamster ovary (CHO) cells were exposed to the mutagen ethyl methane sulfonate (EMS), allowed to grow into colonies, and stained for glucose-6-phosphate dehydrogenase (G6PD) activity, two types of unstained colonies were observed at a frequency of about on per thousand stained colonies. These negative-staining colonies consisted of (1) colonies uniformly deficient in staining activity (pure); and (2) colonies containing both stained and unstained sectors (mosaic) in various relative sizes and patterns. Unstained cells isolated from mosaic colonies were genetically stable and had significantly reduced or absent G6PD activity. Random cell aggregation or chromosome segregation from tetraploid cells is not a significant cause of the sectoring phenomenon. Also, mosaic colonies are not principally caused by mutation at one of two replicated G6PD genes and their subsequent segregation during division. The simplest explanation for this phenomenon is that EMS induces a mutational change in one of the two DNA strands and DNA replication then produces normal and mutant double-stranded DNAs which segregate into wild-type and G6PD-deficient cell types, producing a mosaic colony.

Animals↗
PubMedDate not supplied

Histopathological study of orbital lymphangioma in an infant.

BACKGROUND: There has been controversy over whether orbital lymphangioma is a true entity in the orbit or various venous malformations. CASE: A 1-year-old girl was referred to us with subcutaneous induration of the right upper eyelid. Rapid enlargement of the eyelid mass with blepharoptosis and dislocation of the eyeball were noted within 1 week. A diagnosis of an orbital lymphangioma was made from the clinical course and findings of magnetic resonance imaging. Surgical resection of the tumor was performed to prevent amblyopia. OBSERVATIONS: Histopathologically, the lumen of the expanded cystic tumor consisted of a single layer of endothelium and was filled with erythrocytes as well as lymph. Immunohistochemical study revealed positive staining for factor VIII-related protein and CD 31, weak positive staining for thrombomodulin, and negative staining for CD 34 at the endothelium of the lymphangioma. The endothelium of the capillary vessels around the lymphangioma was positive for factor VIII-related protein, CD 31, and CD 34. Although a part of the tumor remained in the orbit postoperatively, orthophoria was achieved and no recurrence was observed for 2 years. CONCLUSION: Immunohistochemical study suggested that the cellular composition of the endothelium of the orbital lymphangioma was different from that of the surrounding capillary vessels.

Female↗
PubMed2006

[Cutaneous metastases from adenocarcinoma of unknown primary site].

Metastatic cancer of unknown primary site appears in 5-10% of oncologic patients. The primary tumor is usually discovered at autopsy and only in 27% of patients alive. Metastases from cancer of unknown primary site may be located in the skin and subcutaneous tissue and it is the dermatologist the first in evaluating these patients. We present a case of cutanoeus metastases from moderately-differentiated adenocarcinoma of unknown primary site. The immunohistochemical study revealed positive staining for CEA and negative staining for PSA. The primary tumor could not be identified in spite of the imaging and endoscopic studies performed. Based on these studies we excluded a colorectal or prostatic origin and considered a pancreatic adencarcinoma as the possible primary tumor. Even though a minority of these patients will have a curable disease, the appropriate use of pathological diagnosis and selected imaging studies for an optimal management of patients with a tumor of unknown primary site should not be ignored.

Abdominal Neoplasms↗