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At least 199 records · Page 11Linked to original sources
PubMed1986

Isolation of highly purified fractions of plasma membrane and tonoplast from the same homogenate of soybean hypocotyls by free-flow electrophoresis.

A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K(+)-stimulated, vanadate-inhibited Mg(2+) ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%.

Journal Article↗
PubMed1979

Serum uric acid.

Data from the 1977 College of American Pathologists (CAP) Comprehensive Chemistry Survey and other programs have been analyzed in an interlaboratory comparison of the measurements of serum uric acid. The majority of clinical laboratories utilize some form of phosphotungstic acid (PTA) oxidation-reduction technic for the measurement of uric acid. Methods based on uricase are most often performed on some automated device. In this Survey, in which specimens containing minimal potential interfering compounds were used, there are no significant differences in the results obtained by phosphotungstic acid and uricase methods.

Chemistry, Clinical↗
PubMed1989

A comparative study of PAS-phosphotungstic acid-Diamine Supra Blue FGL and immunological reactions for type I collagen.

Immunohistochemical techniques proved valuable in histological studies of various types of collagens. However drawbacks include non-specific reactions of antibodies, masking of antigens, and the high cost of antibodies. This study was undertaken to ascertain the specificity of the PAS-phosphotungstic acid-Diamine Supra Blue FGL (PAS-PTA-DSB-FGL) reaction for type I collagen, differentiating it from other collagens. Duplicate series of methacarn-fixed sections of various tissues were treated with the PAS-PTA-DSB FGL reaction and the peroxidase-antiperoxidase (PAP) technique for type I collagen and the staining patterns were compared. Fibers binding the blue dye were found only at sites reacting with antibodies against type I collagen. These observations indicate that the PAS-PTA-DSB FGL procedure is suitable for visualization of type I collagen, e.g. in screening of large series of sections and in the practice of surgical and autopsy pathology.

Bronchi↗
PubMed1982

Substructure of granules from serous cells of porcine tracheal submucosal glands.

The ultrastructure of serous cells from porcine tracheal submucosal glands was studied by conventional transmission electron microscopy (TEM), and by cytochemical methods to stain for complex carbohydrates. In tissue fixed and processed for TEM, and stained with uranyl acetate and lead citrate, the condensing granules of serous cells occasionally possessed a hexagonal and sometimes a lamellar substructure. Tissue fixed in paraformaldehyde-glutaraldehyde and stained with periodic acid-thiocarbohydrazide-silver proteinate (PTS) or with phosphotungstic acid (PTA) showed secretory granules stained for complex carbohydrates and revealed a substructure similar to that noted in the condensing granules. The dark staining substructure revealed by either the PTS or the PTA technique appeared to correspond to electron-lucent areas observed in the condensing granules by conventional TEM. The PTS staining probably demonstrated the presence of neutral glycoprotein, since the serous-cell granules did not react with a dialyzed iron stain for acidic glycoproteins. Treatment of periodic acid oxidized thin sections with pronase or pepsin prior to thiocarbohydrazide and silver proteinate treatment decreased the intensity of the PTS staining, but did not digest away any components of the granules. The substructure revealed by the carbohydrate stains may be a reflection of the mechanism of packaging or the macromolecular structure of the glycoproteins in the serous-cell granules.

Animals↗
PubMed1978

Purification of plasma membranes from roots of barley: specificity of the phosphotungstic Acid-chromic Acid stain.

Plasma membrane vesicles from roots of barley (Hordeum vulgare L., var. Arivat) had an equilibrium density in sucrose of about 1.16 grams per cubic centimeter, but could not be purified satisfactorily with the procedure developed for roots of other plant species. The reported procedure involving differential centrifugation to remove mitochondria (peak density of 1.18 grams per cubic centimeter) and subsequent density gradient centrifugation to purify plasma membrane vesicles was modified to include a narrower differential centrifugation fraction (13,000 to 40,000g instead of 13,000 to 80,000g) and a narrower density range in the sucrose gradient (1.15 to 1.18 grams per cubic centimeter instead of 1.15 to 1.20 grams per cubic centimeter). The fraction obtained by the modified procedure was between 60 and 70% pure as determined by staining with the phosphotungstic acid-chromic acid procedure, which was judged to be reliable for identifying plasma membrane vesicles in subcellular fractions from barley roots. The plasma membrane fraction was enriched in K(+)-stimulated ATPase activity at pH 6.5. The presence of nonspecific ATP-hydrolyzing activity in the plasma membrane fraction made it difficult to determine if the ATPase had properties in common with those reported for cation absorption in barley roots.

Journal Article↗
PubMed1982

The use of Mallory's phosphotungstic acid-hematoxilin (PTAH) stain in renal pathology.

High quality histology is needed in renal biopsy examination, especially when material for immunofluorescence and/or electron microscopy is not available. For these purposes Mallory PTAH stain was tried on 34 renal biopsies and the results were compared with the immunofluorescent and ultrastructural findings (used as a control of the reliability of the method). PTAH, besides showing extracellular structures, cytological details and rather subtle abnormalities, was able to detect the presence and the site of deposits. PTAH stains the material which appears as electron dense deposits in electron microscopy and as granular deposits in immunofluorescence, whereas it fails to stain linear deposits in immunofluorescence (which are not electron dense). Moreover, a good correspondence between PTAH and electron microscopic data was detected as far as the location of deposits is concerned.

Amyloidosis↗
PubMed1998

Localization of glucose-6-phosphatase activity and carbohydrates in boar caput epididymal principal cells.

Unidentified tubulovesicular profiles have been reported in the apical cytoplasm of boar caput epididymal principal cells in addition to vesicles considered to be involved in endocytosis and secretion. The main aim of the present study was to clarify the character of these organelles and to differentiate them from the endocytic apparatus. Glucose-6-phosphatase (G6Pase) activity was determined as the reporter enzyme of the endoplasmic reticulum (ER) and phosphotungstic acid was used to visualize carbohydrate moieties in both the proximal and distal caput. Phosphotungstic acid revealed the glycocalyx of the endocytic apparatus, which was similar in both regions studied, and also stained specific granules of the proximal caput. Glucose-6-phosphatase showed the tubulovesicular profiles to be sparsely granulated ER that was poorly developed in the proximal caput and very abundant in the apical cytoplasm of the distal caput principal cells. The function of such large amounts of sparsely granulated ER with corresponding G6Pase activity in caput epididymal principal cells is unknown.

Animals↗
PubMed2002

Comparison of a homogeneous assay with a precipitation method for the measurement of HDL cholesterol in diabetic patients.

OBJECTIVE: To compare direct-measured HDL cholesterol with HDL cholesterol measured by a precipitation method. RESEARCH DESIGN AND METHODS: We compared a homogeneous assay for direct HDL cholesterol analysis with the phosphotungstic acid magnesium chloride precipitation method in 55 type 1 diabetic patients, 70 type 2 diabetic patients, and 82 nondiabetic normal control subjects with plasma triglyceride levels <4.6 mmol/l. The cholesterol content of HDL determined by the direct assay was overall 0.1 mmol/l higher in all three groups than HDL cholesterol measured after precipitation, but the two methods were closely correlated (r(2) = 0.98, P < 0.001). RESULTS: HbA(1c), blood glucose, serum albumin, serum bilirubin, or triglyceride did not influence the differences of the two HDL cholesterol measurements. Because we have previously shown HDL cholesterol isolated by phosphotungstic acid precipitation to be lower than that by ultracentrifugation, the positive bias found in this study was expected. It seems that the direct HDL cholesterol assay reacts with apolipoprotein (apo) B-containing lipoproteins in the fraction with a density of >1.063; these apo B-containing lipoproteins are suggested to be coprecipitated with the phosphotungstic acid method. We also measured LDL cholesterol directly by a LDL cholesterol plus method and found no significant differences between this method and LDL cholesterol calculated from Friedewald's formula. CONCLUSIONS: Direct homogeneous assay for HDL cholesterol determination in diabetic patients seems not to exhibit a negative bias, in contrast to the precipitation method, when compared with the ultracentrifugation method. In addition, the direct assay saves time and is not influenced by type of diabetes or degree of metabolic control.

Adult↗
PubMed1983

Cytochemical studies of nuclear basic proteins in control and vitamin B12 starved Euglena.

In avitaminosis B12, Euglena gracilis Z is blocked in the cell cycle in the S/G2 phase. In these blocked cells, transcription and traduction go on and the amount of DNA is less than doubled and remains constant during the blockage. Chromatin clumps observed in situ with classical electron microscopic methods are always condensed in control cells but are not visualized in B12 starved cells. Two cytochemical reactions, ethanolic phosphotungstic acid and ammoniacal silver reaction, specific for lysine- or arginine-rich residues, are performed to reveal basic nuclear proteins of chromatin. With these two methods, control chromatin in situ always shows a condensed aspect, whereas the starved chromatin appears dispersed. These cytochemical differences might be considered to result from a different supramolecular organization of the two kinds of chromatin.

Animals↗
PubMed1979

CPC-PTA section staining of acid glycans.

A cetylpyridinium chloride-phosphotungstic acid procedure suggested by Kelényi and Kiss (1976) for the staining of mast cell granules in ultrathin sections was slightly modified with regard to pretreatment, pH of incubation, and rinsing of sections. The method also proved suitable for the demonstration of various mucosubstances and of cartilage proteoglycan aggrebates.

Animals↗
PubMed1982

Four methods for determining uric acid compared with a candidate reference method.

Uric acid as measured in serum by three different uricase (EC 1.7.3.3) methods (aca, Ektachem, and SMAC) and by the SMAC method with phosphotungstic acid was compared with a candidate Reference Method for uric acid. Serum specimens from 83 patients (uric acid concentrations, 19 to 141 mg/L) were analyzed by all five methods. Results were compared by using linear regression analysis, and the mean difference between results by the candidate Reference Method and the four other methods was calculated. Compared with the candidate Reference Method, the aca method gave the smallest deviation from zero for the intercept and the smallest mean difference, and the SMAC phosphotungstic acid method showed a slope closest to unity. The SMAC uricase method had the largest intercept and greatest deviation of the slope from unity.

Autoanalysis↗
PubMed1983

A new principle in polychrome staining: a system of automated staining, complementary to hematoxylin and eosin, and usable as a research tool.

A staining system is described in which each stage forms a separate module or unit. All reagents, concentrations of dye, ratios of phosphotungstic acid to dye, pH values, temperature and staining times are standardized and only aqueous solutions used. The technic uses equal strength solutions of orange G, acid fuchsin and methyl (or aniline) blue, in ascending order of molecular size, at pH 2.5 (range: 2.3 to 2.7). Phosphotungstic acid is incorporated in the dyebaths, not used separately, and the combination of this with ferric alum hematoxylin (Lillie's by preference) and either naphthol yellow S or picric acid as a primer, enables fibrin and cytoplasmic components to be demonstrated vividly, with other tissues shown in clear contrasting colors. Erythrocytes are yellow, fibrin red and collagen blue. The system permits substitution of dyes, lending itself to both manual and computer recording and analysis, helped by a notation system for identifying variants. Many of the factors are variable at will. The system aids research into the mechanism of polychrome staining, and, by extrapolation, into the mechanism of action of other stains. Two manually or machine usable progressive polychrome technics intended for routine use are described. They identify tissue components consistently, complementing the standard hematoxylin and eosin stain, and deserve equal attention during reporting. Variants may be used for one-minute one-stage staining of frozen sections, or to give strong colors with 2 millimicrons acrylic sections.

Coloring Agents↗