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Inflammatory pseudotumor of the urinary bladder with an aberrant expression of cytokeratin.

A case of inflammatory pseudotumor of the urinary bladder in a 47 year old Japanese male patient is presented. Inflammatory pseudotumor of the urinary bladder is a benign but rare proliferative lesion of the submucosal stroma, easily mistaken for a malignant neoplasm. Based on the clinical diagnosis of bladder cancer by cystoscopy and magnetic resonance imaging (MRI), urologists started chemotherapy before results of the histological report were available which described inflammatory pseudotumor on the biopsy. Biopsied materials showed marked proliferation of irregularly bundled spindle cells, varied in size and shape and separated in severe loose myxoid stroma with moderate infiltration of the inflammatory cells and capillary proliferations. At a glance, these findings resemble the sarcomatous pattern. However neither severe nuclear atypism nor atypical mitoses were present. Immunohistochemically, these spindle cells, which were positive for vimentin and alpha-smooth muscle actin, showed a diffuse aberrant expression of cytokeratin. Some of them were positive for phosphotungstic acid hematoxylin. Electron microscopy revealed only the fibroblasts. No recurrence has been observed for 10 months. These findings indicate that inflammatory pseudotumor is a benign mesenchymal lesion that must be discriminated from true sarcoma to avoid subjecting the patient to unnecessary therapy. Only careful histological examination can enable a successful diagnosis.

Granuloma, Plasma Cell↗

Effects of total asphyxia on the development of synaptic junctions in the brains of mice.

Using ethanolic phosphotungstic acid staining, we studied the development of synaptic junctions in the brains of mice subjected to total asphyxia. One-day-old mice, Std:ddY strain, were put into a chamber continuously flushed with 100% CO2-gas for 35 min. The surviving mice (28.1%) were used as experimental models, while unasphyxiated littermates served as controls. The synaptic junctions in the frontal motor cortex and hippocampal cortex were studied at 20 and 60 days of age using E-PTA staining, and the numbers of synaptic junctions in both areas were counted. In comparison with the control animals, the frontal motor cortex of the subject mice showed a smaller increase in the number of synaptic junctions, both at 20 and at 60 days of age. In the hippocampal cortex, the number of synaptic junctions seen in the experimental mice brains was similar to that in the controls at 20 days of age; however, the number of synaptic junctions in the treated mice brains showed a slower rate of increase than in the controls at 60 days of age.

Animals↗

Fine structure and cytochemistry of the nucleus and the kinetoplast of epimastigotes of Trypanosoma cruzi.

Ultrastructural cytochemical techniques were used to analyze the nucleus and the kinetoplast of epimastigotes of Trypanosoma cruzi. With the use of ethanolic phosphotungstic acid, which detects basic proteins, reaction product was seen in the chromatin and at the periphery of the kinetoplast. Thallium alcoholate, which interacts with DNA, stained strongly the whole kinetoplast and the chromatin. With the use of a silver impregnation method that detects acidic nucleolar proteins, silver granules were seen preferentially located in the central region of the nucleolus. With the EDTA method, which reveals the presence of ribonucleoproteins, staining was observed in the nuclear pores. Also 6-8 nm fibrils, 25 nm and 40 nm granules, which correspond to the perichromatin fibers, interchromatin granules and the perichromatin granules, respectively, were identified in the nucleus. The EDTA method also revealed the presence of 40 nm granules in the kinetoplast. These granules were seen mainly at the two extremities of the kinetoplast. Freeze-fracture images indicate that the nuclear membrane contains ca. 9 pores/microns2 of nuclear surface area. The mean diameter of the pores was 80 nm. All these results suggest that epimastigotes of T. cruzi have a very active nucleus and a high rate of nucleocytoplasmic interchange.

Animals↗

Study of neutralizing monoclonal antibodies to bovine herpes virus type-1 (Cooper strain) by immunoperoxidase and immunoelectron microscopy.

Three neutralizing monoclonal antibodies (mAbs) that are specific against bovine herpes virus Type-1 (BHV-1) were studied as to their viral specificity by immunoperoxidase and immunoelectron microscopy. Microscopic examination of GBK BHV-1 infected cells revealed peroxidase activity represented by red-brown granular deposits in the nucleus and cytoplasm. No immunoperoxidase activity was observed in negative controls. For the ultrastructural observations, two approaches were used. Firstly we tested a pre-embedding technique using GBK infected cells, mAbs and gold conjugated-protein A. Gold particles were observed linked to the viral envelopes and to the host cell membrane. Alternatively, a second technique employed BHV-1 purified by potassium tartrate gradients, mAbs and gold conjugated-protein A. After performing the immune reaction, the samples were adsorbed to formvar-coated grids, stained with phosphotungstic acid and observed in a transmission electron microscope. Gold particles were mainly attached to the virion envelope.

Animals↗

Cell surface appendages of lactobacilli.

Seventeen vaginal isolates of lactobacillus were negatively stained using 1% phosphotungstic acid and examined by transmission electron microscopy. One or two flagella were observed in a polar or, less frequently, sub-polar position on 14 of the 17 isolates. The structures, measuring 12 nm in diameter and up to 12 microns long, were seen on cells cultured in both solid and liquid MRS medium. Fimbriae, measuring 3.5 nm in diameter and 0.1 micron to 1.0 micron in length, extended from the cell surface of 15 of the 17 lactobacillus isolates. Production of fimbriae was maximal when the lactobacilli were cultured on solid medium in an anaerobic atmosphere. Fimbriation significantly increased adhesion of lactobacilli to vaginal epithelial cells in vitro.

Adhesins, Bacterial↗

Surface ultrastructure of some oral bacteria.

Adhesion of Streptococcus sanguis, Fusobacterium nucleatum and an Actinomyces sp. to enamel and epon and their interspecies cohesion was studied with scanning and transmission electron microscopy. For adhesion studies enamel or epon was coated with salivary macromolecules and then cells of S. sanguis and in some experiments also with F. nucleatum or Actinomyces sp. Cells of S. sanguis were seen scattered over the surface of a thin "pellicle" that was heavily stained, and F. nucleatum and Actinomyces sp. adhered to S. sanguis or directly to the "pellicle". For studies of cohesion S. sanguis was brought to cohere with F. nucleatum or Actinomyces sp. and then processed for transmission electron microscopy. The morphology of the cell surface structures involved was studied in negatively stained preparations or in thin sections of material treated with ruthenium red or poststained with uranyl and lead salts, phosphotungstic acid or periodic acid-thiocarbohydrazide-osmium tetroxide. S. sanguis demonstrated a fuzzy coat of fimbriae that seemed to unfold in areas of contact with other cells, while cells of F. nucleatum had 6-10 polar pilus-like fimbriae, which appeared to be instrumental in cohesion, as did a dense coat of long, slender fimbriae that covered cells of Actinomyces sp.

Actinomyces↗

A combined immunohistochemical and electron microscopic study of the second cell type in the developing sheep pineal gland.

Ultrastructural and immunohistochemical techniques were used to study the second cell type in sheep embryo pineal glands. Thirty-two embryos were studied from day 54 of development through birth. Specimens were arranged in four age groups, defined in terms of the most relevant histological features: Group 1 (54-67 days of prenatal development), Group 2 (71-92 days), Group 3 (98-113 days), and Group 4 (118-150 days). At 98 days, a second cell type was observed which differed from pinealoblasts and showed uniform ultrastructural characteristics similar to those of astrocytes in the central nervous system. Ultrastructural homogeneity was not matched by the results of histochemical and immunohistochemical analysis: while all Type II cells stained positive to phosphotungstic acid hematoxylin, only 50% expressed glial fibrillary acidic protein. In the course of ovine intrauterine development, the vascular affinity of this second cell population, composed of glial-like or astrocytic cells at varying stages of maturity, leads to the formation of a limiting pineal barrier. This barrier may constitute the morphological expression of a hypothetical functional involvement in the exchange of substances between blood and pineal parenchyma.

Animals↗

Zygotene-pachytene substaging and synaptonemal complex karyotyping of boar spermatocytes.

Synaptonemal complex analysis, by electron microscopy of spread spermatocytes after phosphotungstic acid staining, made possible description of the chromosomal synaptic patterns and the synaptonemal complex karyotype of the pig (Sus scrofa domestica). The autosomal synaptic pattern in conjunction with the sex chromosome morphology and pairing behaviour can serve as a reference for the meiotic cell progression from the zygotene stage to the pachytene. The autosomes started terminal synapsis at early zygotene (Z1) and at mid-zygotene (Z2) some of the small bivalents were completely paired. The extension of pairing between the X and the Y chromosome, and the differentiation of their axes, disclosed seven types of sex bivalent (Types 0-VI). The Type 0 sex bivalent occurred in late zygotene (Z3), at which the X and Y axes began terminal synapsis by their short arms. Each one of the pachytene substages, early, mid-, and late, included two sex bivalent types. By early pachytene (P1-P2) the entire Y chromosome was paired with the X axis. Subsequently, progressive desynapsis and differentiation of the sex chromosome axes defined the mid- (P3-P4) and late pachytene substages (P5-P6). At mid-pachytene, the unpaired XY segments were associated end-to-end and showed differing degrees of complexity (thickening, splitting, despiralization and strandedness). These axial complexities were replaced at late pachytene by fine excrescences along the sex chromosome axes, which still formed a short SC stretch. Additionally, an attempt to construct an SC karyotype for the pig is presented. There was general agreement between the autosomal SC karyotype and the autosomal somatic karyotype when comparisons were made, considering the relative lengths and arm ratios of individual chromosome pairs.

Animals↗

The fine structure of cells of Trichomonas vaginalis Donné obtained from the exponential phase of growth and from stationary cultures.

In cells of Trichomonas vaginalis in logarithmic phase of growth (log. T. vaginalis) the number of free cytoplasmic ribosomes was higher than that in cells from stationary cultures (stat. T. vaginalis). Polyribosomes closely surrounding the chromatic granules were present in equal amounts in both categories fo cells, which indicates that they have specific functions and are not directly involved in the protein synthesis of the growing cell. The volume of the Golgi region and of the food vacules were approximately twice as large in log. T. vaginalis cells as in stat. T. vaginalis cells. The round and slightly ovoid chromatic granules were smaller in log. T. vaginalis cells than in stat. T. vaginalis cells, whereas elongated granules were more numerous in the former than in the latter cell type. The elongated chromatic granules presented a coarser texture than ordinary round granules. The chromatic granules probably replicate by binary fission a process which seem to follow an internal reorganization of the granules. Polysaccharides were demonstrated on the cell surface and on the membranes of the Golgi and endocytotic vesicles if sectioned cells were stained with phosphotungstic acid after inert dehydration and embedding in a water-missible methacrylate. Ruthenium red and colloidal iron only showed affinity for the filamentous cell coat.

Cell Membrane↗

Eosinophilic globules in pheochromocytoma of the adrenal medulla. A histochemical, immunohistochemical and ultrastructural study.

Histochemical, immunohistochemical, and ultrastructural characteristics of eosinophilic globules in pheochromocytoma of the adrenal medulla are described. The globules were observed in 7 (63.5%) out of 11 cases of pheochromocytoma of the adrenal medulla. These globules were eosinophilic, PAS-positive with and without diastase predigestion, phosphotungstic acid hematoxylin (PTAH) positive, and autofluorescent under ultraviolet illumination. Although almost all globules were not stained with neuron specific enolase (NSE) using the immunoperoxidase method, a few globules were stained positive. The lectins of Triticum vulgaris (WGA) and Ricinus communis (RCA-120) were weakly bound to most of the eosinophilic globules. Immunohistochemical reactions for alpha-fetoprotein (AFP), alpha-l-antichymotrypsin (ACT), and human chorionic gonadotropin (HCG) revealed negative reaction in these globules. An ultrastructural study revealed no relationship between these globules and chromaffin secretory granules. These findings suggested that the eosinophilic globules in pheochromocytoma of the adrenal medulla were not related to the chromaffin secretory granules and might be some kind of complex protein.

Adrenal Gland Neoplasms↗

In vivo muscle stiffening under bone compression promotes deep pressure sores.

Pressure sores (PS) in deep muscles are potentially fatal and are considered one of the most costly complications in spinal cord injury patients. We hypothesize that continuous compression of the longissimus and gluteus muscles by the sacral and ischial bones during wheelchair sitting increases muscle stiffness around the bone-muscle interface over time, thereby causing muscles to bear intensified stresses in relentlessly widening regions, in a positive-feedback injury spiral. In this study, we measured long-term shear moduli of muscle tissue in vivo in rats after applying compression (35 KPa or 70 KPa for 1/4-2 h, N = 32), and evaluated tissue viability in matched groups (using phosphotungstic acid hematoxylin histology, N = 10). We found significant (1.8-fold to 3.3-fold, p < 0.05) stiffening of muscle tissue in vivo in muscles subjected to 35 KPa for 30 min or over, and in muscles subjected to 70 KPa for 15 min or over. By incorporating this effect into a finite element (FE) model of the buttocks of a wheelchair user we identified a mechanical stress wave which spreads from the bone-muscle interface outward through longissimus muscle tissue. After 4 h of FE simulated motionlessness, 50%-60% of the cross section of the longissimus was exposed to compressive stresses of 35 KPa or over (shown to induce cell death in rat muscle within 15 min). During these 4 h, the mean compressive stress across the transverse cross section of the longissimus increased by 30%-40%. The identification of the stiffening-stress-cell-death injury spiral developing during the initial 30 min of motionless sitting provides new mechanistic insight into deep PS formation and calls for reevaluation of the 1 h repositioning cycle recommended by the U.S. Department of Health.

Animals↗

Role of colonization in the virulence of Actinomyces viscosus strains T14-Vi and T14-Av.

Germfree rats fed a high-sucrose diet were inoculated with Actinomyces viscosus strain T14-Vi (virulent) or T14-Av (avirulent). The mean recovery of strain T14-Vi from six extracted finely ground molars of rats sacrificed after 90 days was 1.1 x 10(8) colony-forming units (CFU). The mean recovery of strain T14-Av was 5.7 x 10(7) CFU, which was significantly less. Strain T14-Vi caused severe alveolar bone loss, but only minimal bone loss occurred in rats infected with strain T14-Av. Scanning electron microscopy of teeth of germfree rats revealed that strain T14-Vi colonized in the fissures as well as on tooth surface areas near the gingiva; strain T14-Av also colonized in fissures but was unable to colonize the teeth near the gingiva. In studies with conventional rats fed a high-sucrose diet, streptomycin-resistant strain T14-Vi colonized on the teeth of all rats inoculated with in the order of 10(8) or 10(7) CFU and on the teeth of about half of the rats inoculated with 10(6) or 10(5) CFU. In contrast, streptomycin-resistant strain T14-Av could not be detected on the teeth of any of the rats in groups similarly inoculated. In vitro "resting" cells of both strains suspended in conventional or germfree rat saliva survived to comparable degrees. [(3)H]thymidine-labeled T14-Vi cells adhered well to hydroxyapatite (HA) beads and to HA beads pretreated with saliva obtained from germfree or conventional rats. In contrast, T14-Av cells adhered less well than did T14-Vi cells to HA, whereas their adherence to saliva-coated HA was negligible. Transmission electron microscopy of negatively stained T14-Vi and T14-Av cells repeatedly passed in 1% phosphotungstic acid revealed fibrils on cells of both strains. T14-Av cells were covered by large amounts of extracellular material which was presumably heteropolysaccharide; little extracellular material was present on the surface of T14-Vi cells. T14-Vi cells had a relatively low affinity for the heteropolysaccharide synthesized by strain T14-Av. Other evidence also suggested that this polysaccharide had a relatively low affinity for saliva-coated HA. Collectively, the evidence indicates that the difference in periodontopathic potential between strains T14-Vi and T14-Av results from their different abilities to colonize teeth. This difference is probably due to the lower adherence of T14-Av cells to teeth rather than to their ability to grow in the mouth. The low affinity of T14-Av cells for tooth surfaces may be due, in part, to the presence of large amounts of cell-surface-associated polysaccharide.

Actinomyces↗

Morphological and biochemical comparison of virulent and avirulent isolates of Haemophilus pleuropneumoniae serotype 5.

Capsular structure and biochemical composition varied between two isolates (virulent and avirulent) of Haemophilus pleuropneumoniae serotype 5. The presence of capsule was determined by transmission electron microscopy with glutaraldehyde-osmium, ruthenium red, alcian blue, and phosphotungstic acid staining procedures. The virulent isolate of H. pleuropneumoniae had a distinct, adherent capsule. The avirulent isolate had a fragile, easily removed capsule. Capsular material (CM) and a lipopolysaccharide (LPS) were isolated from each bacterial isolate and were compared biochemically and biologically. CM from both isolates contained carbohydrates, no detectable protein, and no detectable to trace amounts of lipid A. Each LPS contained heptose, hexose, galactose, glucosamine, 2-keto-3-deoxyoctonate, and lipid A. Biological responses to CM and LPS from both isolates were demonstrated in the proclotting enzyme of Limulus polyphemus amebocyte lysate activation and in serological cross-reactions by immunofluorescence and immunodiffusion precipitation. The virulent isolate contained approximately 10 mg of LPS per g more on an original dry weight basis than the avirulent isolate. LPS from the virulent isolate contained approximately 13 times more galactose than LPS from the avirulent isolate. The differences of capsular structure and biochemical composition may contribute to the role of CM in porcine H. pleuropneumoniae infections.

Animals↗

Three-dimensional morphology, ultrastructure, and replication of Mycoplasma felis.

The morphology and replication of Mycoplasma felis in relation to growth phase in culture were studied by electron microscopy. The organisms showed 1.0 to 1.45-hr doubling times with typical bacterial-type growth curves when grown in dialysate broth supplemented with horse serum. Organisms were fixed for electron microscopy by using Veronal acetate-buffered 0.8% OsO(4) (pH 6.1) in 20% sucrose. The morphology of exponential-phase organisms differed markedly from that of stationary or death-phase organisms, which were essentially large round forms with either dispersed or abnormally aggregated cytoplasm. Plasticine models prepared from serial sections of organisms in exponential phase showed the organisms to be either disc-shaped, triangular, horseshoe-shaped, or multilobular. A central "hole" was frequently present in these structures and could be visualized in the lobular forms as an interconnecting circular membrane. The inner surface of this membrane often showed contact with a small membranous body about 0.12 mum in diameter. The significance of this body is unknown. The morphology of the various shapes was confirmed by using the phosphotungstic acid and critical point methods. When the ratios of the various forms in exponential-phase cultures were determined, it was found that a replication sequence could be proposed which accounted for not only the volume increase required to accommodate deoxyribonucleic acid (DNA) replication but also the distribution of that DNA. Although it is likely that DNA replication in M. felis is a binary process, it appears that the mechanism for production of new cells need not be a binary process.

Cell Division↗

Electron Microscopic Observations on the Fine Structure of Cell Walls of Chlamydia psittaci.

L-cell cultures were infected with elementary bodies (EB) of meningopneumonitis organisms. Cell walls were prepared from reticulate bodies (RB), which are the intracellular developmental forms into which EB are converted, and from EB at appropriate times after infection. When fragmented EB cell walls were shadowcast with platinum palladium alloy, about one-half of the fragments were seen to be composed of hexagonally arrayed structures on the inner side of the cell wall. When EB cell walls were negatively stained with phosphotungstic acid, they all showed this fine structural array. These macromolecular units were estimated to be about 18 nm in diameter. RB cell walls, harvested at various times after infection, were similarly stained; about 20% of RB walls at 15 hr after infection showed traces of these regular structures, but only 2% of them had the structures at 24 hr. When RB cell walls prepared from penicillin-containing culture were examined, they were observed to be similar to RB without penicillin. When EB cell walls were treated with formamide at 160 C, and then centrifuged in a 10 to 40% potassium tartrate density gradient, hexagonal particles about 20 nm in diameter were obtained as a middle band in the gradient column. These particles were not obtained from RB cell walls harvested from cultures with or without penicillin. It is concluded that the particles are macromolecular subunits located on the inner side of the EB cell walls, that the subunits probably provide the structural rigidity found in the EB, and that their synthesis is inhibited by penicillin.

Journal Article↗

Morphology and ultrastructure of Mycoplasma pneumoniae spherules.

During growth in fluid medium, most strains of Mycoplasma pneumoniae produce free-floating granules which become larger with time. We have called these granules "spherules." This study describes the morphological and ultrastructural features of M. pneumoniae strain AP-164 spherules, both free and in association with HeLa cells in cell culture. In thin section, spherules were composed of lobulated cells, connected together by membranes, and ring-shaped cells. The two-dimensional morphology observed varied according to the plane of section and to the age of the culture. In HeLa cell cultures, mycoplasmata were found attached to plasma membranes of cells; in sections, individual mycoplasmata were often aligned in radial apposition to the membranes. Mycoplasmata were not found intracellularly. The three-dimensional morphology of spherules was examined by the critical point method and by scanning electron microscopy. Both methods revealed lobulated forms, ring-shaped forms, and star-shaped forms. Treatment of the spherules with crude porcine pancreatic lipase effectively released large numbers of free organisms. Phosphotungstic acid preparations of these uncentrifuged forms revealed a morphology in agreement with the other methods used. Lobulated ring forms with "beaded" filaments were prominent. In respect to morphology, M. pneumoniae under our conditions resembles that of the type species M. mycoides.

Animals↗

Naturally occurring R factor, derepressed for pilus synthesis, belonging to the same compatibility group as the sex factor F of Escherichia coli K-12.

A naturally occurring R factor with constitutive pilus synthesis is described which resembles the sex factor F in compatibility and in restricting coliphage T7. Unlike F, it is not cured during growth with acridine orange. Results suggest that the R factor produces repressor of pilus synthesis, to which the operator is insensitive (i(+)o(c)). In this respect it differs from both the F factor (i(-)o(+)) and wild-type F-like R factors (i(+)o(+)).

Acridines↗

Structure and chemical composition of prospheroplast envelopes of Saccharomyces cerevisiae and Hansenula anomala.

Cells of Saccharomyces cerevisiae and Hansenula anomala were digested with snail enzyme under conditions yielding prospheroplasts. Surrounding envelopes were isolated after lysis of prospheroplasts in distilled water. The envelope material was embedded and sectioned for electron microscopy, and thin, hollow structures still retaining the elongated form of the original cells were seen. The envelopes were of low electron density in sections stained with uranyl magnesium acetate and lead citrate, but were more electron-dense when stained with phosphotungstic acid. Shadowed preparations of prospheroplast envelopes revealed structures resembling ghosts. These "ghosts" were similar to the original cells in form and size but seemed to be very thin. Varying numbers of anular structures (bud scars) were found on them. Chemical analyses of the envelope indicated that an alkali-soluble glucan was a major constituent. The results show that the prospheroplast envelope is part of the original cell wall of the yeast and is located in close apposition to the cytoplasmic membrane.

Ascomycota↗