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PubMedDate not supplied

[Carboyydrates localization on ultrathin sections of "Brucella" and "Escherichia" cells in smooth (S) and rough (R) phase (author's transl)].

Carbohydrates localization on ultrathin sections of Brucella abortus, melitensis and Excherichia coli cells has been studied by the periodic acid thiocarbohydrazide-silver proteinate (PATAg) and phosphotungstic acid (PTA) methods. Carbohydrates are mainly localized on the cell envelope of Brucella and Escherichia but there are several differences between these two bacteria. The differences are discussed and a Brucella polysaccharide envelope model is proposed. The PATAg method gives the same silver grain deposits on Brucella S and R and Escherichia S and R. The phosphotungstic acid method differenciates Brucella S and R cells by lack of contrast on the outer leaflet of the outer membrane of the latter, but does not differenciate E. coli S or R cells. The Brucella outer membrane contains less polysaccharides than that of E. coli. There is a seemingly symetric distribution of polysaccharide in the Brucella outer membrane as compared to an asymetric distribution in E. coli. The peptidoglycan of Brucella reacts strongly as compared with that of E. coli. The polysaccharides present a dispersed pattern in Brucella cytoplasm, whereas in E. coli they appear as dense, strongly reactive clusters very close to the cytoplasmic membrane.

Brucella↗
PubMedDate not supplied

Manipulating the Papanicolaou staining method. Role of acidity in the EA counterstain.

The role of phosphotungstic acid as the main pH-controlling agent in Papanicolaou's EA stain was analyzed, including its effect on the competition between eosin Y and light green, the principal components of EA solutions, and this acid was compared with HCl as an acidifying agent. The amount of phosphotungstic acid in the solution determined the color of the cytoplasm. Our study led to a better understanding and a simplification of the staining procedure. With the acquired knowledge, the Papanicolaou counterstain can be standardized to suit the taste of the individual cytologist and can add to the predictability of the results.

Buffers↗
PubMed1986

Late preovulatory synthesis of proteoglycans by the human oocyte and cumulus cells and their secretion into the oocyte-cumulus-complex extracellular matrices.

Light- and electron-microscope autoradiography using 3H-glucosamine and 3H-fucose as precursors was employed to investigate proteoglycan synthesis and secretion by late preovulatory human oocytes and cumulus cells. Both the oocyte and cumulus cells were found to be important cellular sources supplying proteoglycans to the oocyte-cumulus-complex extracellular matrices, i.e., the zona pellucida and the cumulus intercellular matrix. Both the oocyte and cumulus cells were shown to secrete labelled proteoglycans into the zona pellucida. Labelled proteoglycans were also detected in the cumulus intercellular matrix. Chase experiments revealed the labelled molecules to be relatively closely associated with both the zona pellucida and the cumulus intercellular matrix. Staining with chromic acid and phosphotungstic acid showed proteoglycan material to penetrate from the cumulus intercellular matrix into pores of the zona pellucida. This material is thought to be a structural equivalent of the newly synthesized proteoglycans secreted by cumulus cells and migrating into the zona pellucida (as detected by autoradiography). It is concluded that newly synthesized proteoglycans secreted by the oocyte and cumulus cells in the late preovulatory period are a component of the microenvironment in which fertilization takes place.

Autoradiography↗
PubMed1975

[Immunochemical identification of carcinoembryonic antigen in ovarian adenocarcinoma and pseudomucinous cystoma extracts].

Carcinoembryonic antigen (CEA) was found in 3 of 17 individual extracts of adenocarcinoma and in 7 or 8 individual samples of pseudomucinous cystoma of the human ovaries. Rabbits were immunized for the purpose of preparation of specific antisera against CEA by individual fractions (ammonium sulfate, sulfosalicylic acid and phosphotungstic acid) of CEA-positive adenocarcinomas of the ovary.

Adenocarcinoma↗
PubMed1985

Isolation of alpha 1-acid glycoprotein from human plasma using high-performance liquid chromatography.

A method for the rapid isolation of purified alpha 1-acid glycoprotein (AGP) from small volumes of human plasma using HPLC has been developed. The method involves preparation of the seromucoid fraction of plasma by sequential perchloric acid and phosphotungstic acid precipitations, followed by chromatography on an HPLC TSKG-3000 column. The yield was high (0.75 mg AGP/ml plasma) and the procedure takes less than 1 day. The method lends itself to easy automation and is particularly suitable for isotopic turnover studies requiring multiple plasma samples.

Chromatography, High Pressure Liquid↗
PubMed2006

A quantum chemical study of the decomposition of Keggin-structured heteropolyacids.

Heterpolyacids (HPAs) demonstrate catalytic activity for oxidative and acid-catalyzed hydrocarbon conversion processes. Deactivation and thermal instability, however, have prevented their widespread use. Herein, ab initio density functional theory is used to study the thermal decomposition of the Keggin molecular HPA structure through the desorption of constitutional water molecules. The overall reaction energy and activation barrier are computed for the overall reaction HnXM12O40-->Hn-2XM12O39+H2O. and subsequently used to predict the effect of HPA composition on thermal stability. For example, the desorption of a constitutional water molecule is found to be increasingly endothermic in the order silicomolybdic acid (H4SiMo12O40)<phosphomolybdic acid (H3PMo12O40)<silicotungstic acid (H4SiW12O40)<phosphotungstic acid (H3PW12O40), in agreement with the experimental ordering of their thermal stability. The presence of an adjacent Keggin unit may stabilize the structural defect created by the water desorption, thus suggesting that constitutional water loss is an initial step toward the decomposition into a bulk mixed oxide. The equilibrium concentration of defective Keggin units is determined as a function of temperature and water partial pressure. It is concluded that the loss of constitutional water molecules is a plausible deactivation mechanism of the acid catalyst. The intermediate structures along the decomposition path are proposed as possible active sites for oxidation catalysis. The results presented herein provide molecular level insight into the dynamic nature of the heteropolyacid catalyst structure.

Journal Article↗
PubMed1972

Purification of an ion-stimulated adenosine triphosphatase from plant roots: association with plasma membranes.

A membrane-bound adenosine triphosphatase (EC 3.6.1.3) that requires Mg(++) and that is stimulated by monovalent ions has been purified 7- to 8-fold from homogenates of oat (Avena sativa L. Cult. Goodfield) roots by discontinuous sucrose-gradient centrifugation. The enzyme was substrate specific; adenosine triphosphate was hydrolyzed 25 times more rapidly than other nucleoside triphosphates. The membrane fraction containing adenosine triphosphatase was enriched in plasma membranes, which were identified by the presence of a glucan synthetase (EC 2.4.1.12), a high sterol to phospholipid ratio, and by a stain consisting of periodic acid, chromic acid, and phosphotungstic acid that is specific for plant plasma membranes. Oat-root plasma membranes and the associated adenosine triphosphatase were purified on either a 6-layer discontinuous sucrose gradient or on a simplified gradient consisting of only two sucrose layers.These results represent the first demonstration that plant plasma membranes contain an adenosine triphosphatase that is activated by monovalent ions, and this finding further implicates the enzyme in the absorption of inorganic ions by plant roots.

Journal Article↗
PubMed1983

Early and dose-dependent decrease of retrograde axonal transport in acrylamide-intoxicated rats.

The effect of retrograde axonal transport of doses of acrylamide ranging from 50 to 500 mg/kg was studied in sensory nerve of rats. Accumulation of trichloroacetic acid-phosphotungstic acid-insoluble label was measured in a collection segment distal to a double ligature placed on the sciatic nerve at intervals 9-15 h and 9-24 h following injection into the dorsal root ganglion of the fifth lumbar root of [35S]methionine and [3H]fucose. After a dose of 100 mg/kg of acrylamide no neurological signs of neuropathy had yet appeared, but retrograde buildup of protein label was significantly reduced for the long interval (2.20 +/- 0.49 arbitrary units (AU) (mean +/- SD) versus 2.81 +/- 0.57 AU in controls, 2p = 0.034). No abnormality of the short interval appeared before a dose of 500 mg/kg was reached. The retrograde transport abnormality was dose-related (r = -0.85, n = 28, and 2p = 1.2 x 10(-8)), as was the degree of neuropathy evaluated by "blind" neurological scoring (r = 0.88, n = 14, and 2p = 2.8 x 10(-5)). After a dose of 500 mg/kg, when the rats were severely disabled with almost total incoordination of the hindlegs, the retrograde accumulation of the long interval was profoundly depressed (1.08 +/- 0.28 AU versus 2.81 +/- 0.57 AU in controls, 2p = 1.2 x 10(-7)). Similar changes were seen in accumulation of glycoprotein label. After the rats had recovered for 4-10 weeks neurological signs of neuropathy had disappeared and the transport abnormality had improved. To test the specificity of acrylamide on the retrograde transport defect N-hydroxymethylacrylamide and methylene-bisacrylamide, which do not induce neuropathy, were studied. None of these related compounds influenced the transport. These observations imply that in acrylamide intoxication a defect in the amount of material carried by retrograde axonal transport rather than in "turnaround" time or in transport velocity is present, that the transport abnormality precedes the development of neuropathy, and that it is related to the degree of the neurological disability. We suggest that the retention of protein in the distal axons in the functional counterpart of the well-known accumulation of vesicular organelles in the preterminals.

Acrylamide↗
PubMed1997

Muscarinic-induced mucin secretion and intracellular signaling by hamster tracheal goblet cells.

Hamster tracheal epithelial cell cultures were used to investigate muscarinic regulation of high-molecular-weight glycoconjugate (HMWG) secretion by airway goblet cells. HMWG were radiolabeled with N-acetyl-D-[1-(3)H]glucosamine, precipitated with trichloroacetic acid and phosphotungstic acid, and counted by liquid scintillation. Carbachol (100 microM) increased HMWG secretion (166.6 +/- 18.7%, P < 0.001, n = 20), and this response was blocked by the muscarinic receptor antagonist atropine. Ca2+ may not be essential for carbachol response since 1) carbachol-activated secretion was not inhibited by chelating extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by reducing both extracellular and intracellular Ca2+ with BAPTA-acetoxymethyl ester in low-Ca2+ medium; 2) the carbachol response was only partially blocked in low-Ca2+ medium; and 3) calcium ionophore did not stimulate HMWG secretion. However, carbachol-stimulated secretion was abolished by pertussis toxin (PTX), indicating the involvement of a PTX-sensitive guanine nucleotide-binding regulatory protein (G protein), and by the protein kinase C (PKC) inhibitor chelerythrine chloride. Furthermore, carbachol-stimulated secretion was not inhibited by overnight incubation with phorbol 12-myristate 13-acetate. In conclusion, carbachol-stimulated secretion of HMWG appears to be coupled to a PTX-sensitive G protein and requires the activation of a phorbol ester-insensitive PKC isoform.

Alkaloids↗
PubMed1984

Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings.

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.

Animals↗
PubMed1993

Muramidase-mediated damage to Candida yeast cells. Histochemical and immunochemical characterization of accumulating wall-like material.

Hen egg-white lysozyme is known to be fungicidal to blastoconidia of Candida albicans under defined in vitro conditions. This lethal action leads to changes in the layering of cell wall and to plasmolysis, caused by unremitting accumulation of wall-like material between the yeast cell wall and cytoplasmic membrane. Here, several methods were applied on ultrathin sections to define the nature of wall-like material: histochemical staining with periodic acid-thiocarbohydrazide-silver proteinate, periodic acid-alkaline bismuth, and phosphotungstic acid at low pH; the localization of the carbohydrate residues with lectin-gold complex; immunocytochemical staining with monospecific antibodies, factor 1 and 6, which recognized major cell wall antigens. The wall-like material was almost uniformly highlighted with periodic acid-thiocarbohydrazide-silver proteinate, factor 1 antibody, concanavalin A-gold and wheat germ agglutinin-ovomucoid-gold, indicating the presence of mannoproteins and chitin. The serotype A-specific epitope recognized by factor 6 antibody was not detected in the wall-like material, although it was demonstrated in the outer cell wall layers after 2 h of exposure to lysozyme.

Antibodies, Monoclonal↗
PubMedDate not supplied

[Ultrastructure of Mycoplasma sp. demonstrated by phosphotungstic acid-chromic acid pH 0,4].

Ultrastructural observation of Mycoplasma sp. revealed by the phosphotungstic acid-cromic acid, pH 0,4. Arq. Inst. Biol., São Paulo, 45(3) :169-176, 1978. Cells of IB-RS-2 (clone C-26-3) swine line were examined under electron microscopy, employing the differential coloration with PTA-ACr 5 %, pH 0,4, during 30 minutes, at 37 degrees C. Through this method we could observe the intracitoplasmatic forms, electronically dense, rather pleomorfic, with sizes varying between 60nm and 640nm and a unitary membrane of 90A similar to the extracellular forms described as Mycoplasma sp.

Animals↗
PubMed1966

The fine structure of elastic fibers.

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.

Acetates↗
PubMedDate not supplied

Mucin-producing bronchioloalveolar-cell carcinoma. With special reference to a characteristic structure revealed by phosphotungstic acid-hematoxylin staining.

In four cases of bronchioloalveolar-cell carcinoma of the mucin-producing type, the cells were characterized electron microscopically by cored microvilli seen on the free surface of the tumor cells, structures that corresponded well to the phosphotungstic acid-hematoxylin (PTAH) stained structures seen by light microscopy in histologic specimens. The exfoliative cytology specimens contained corresponding PTAH-stained structures on the cell surfaces, namely, a prominent cell membrane with a "beaded" or "peg-like" configuration and, in places, a "feathery" nature, findings quite dissimilar to the surfaces of other well-differentiated adenocarcinoma cells. The cytologic specimens also exhibited characteristic nuclear indentations and tightly connected cell groupings. These results indicate the existence of a type of bronchioloalveolar-cell carcinoma not derived from type II alveolar epithelial cells or Clara cells.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1975

[Determination of urinary total proteins and some protein fractions].

In unconcentrated specimens measurements have been made of total proteins, by DOETSCH gel-filtration method, and of albumin, transferrin and of alpha-2-macroglobulin, by LAURELL electroimmunodiffusion technique. The results confirm that the gel-filtration method provides specific, reliable measurement of proteins in amount as little as 50 mg/1. The proteinuria of 21 samples of urine has been determined by the DOETSCH method and by the biuret method after protein precipitation by perchloric, trichloracetic and phosphotungstic acid. Only the use of phosphotungstic acid as precipitant provides a good correlation of results between the biuret method and the direct gel-filtration-biuret method. LAURELL electroimmunodiffusion technique was improved to allow the determination of 5 mg/1 of albumin, and transferrin, and of 1 mg/1 of alpha-2-macroglobulin. The AA. emphasize the advantages of using sensitive methods which do not require the preliminary concentration of the sample, for the analysis of urinary proteins.

Albuminuria↗