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Oncocytomas of the submandibular gland. A series of 22 cases and a review of the literature.

BACKGROUND: Oncocytomas are benign salivary gland neoplasms that represent approximately 1.5% of all salivary gland tumors. Oncocytomas of the submandibular gland, however, are decidedly uncommon. METHODS: Twenty-two cases of submandibular gland oncocytomas from the files of the Oral and Otolaryngic Tumor Registries of the Armed Forces Institute of Pathology were reviewed, and analysis of the histologic criteria, histochemical and immunohistochemical reactions, and ultrastructural and clinical follow-up data was performed. RESULTS: The patients included 11 females and 11 males, age 21-88 years, with a mean age at presentation of 58.7 years. Clinically, the tumors were generally asymptomatic masses in the submandibular gland that increased in size over a period ranging from several weeks to 20 years and were occasionally associated with pain (n = 9). The tumors ranged in greatest dimension from 0.7 to 7 cm and were circumscribed to encapsulated. Histologically, the tumors were characterized by large epithelial cells with eosinophilic, granular cytoplasm. The cytoplasm stained positively with stains used to demonstrate mitochondria (phosphotungstic acid-hematoxylin, Novelli, Cresylecht violet V, and Kluver-Barrera Luxol fast blue stains). Immunohistochemical reactions demonstrated an epithelial origin (keratin and epithelial membrane antigen), whereas markers for myoepithelial derivation (S-100 protein, actin, and glial fibrillary acidic protein) were not identified. At the time this study was conducted, all patients with submandibular oncocytomas were either alive without evidence of disease or had died without evidence of recurrent disease, with surgical resection the only treatment. CONCLUSIONS: Submandibular gland oncocytomas are rare, benign tumors. The tumor cells are filled with mitochondria, which are easily demonstrated by histochemical reactions. Complete surgical resection is adequate therapy.

Adenoma, Oxyphilic↗

Phosphotungstate as a useful eluent for hepatitis-B virus surface antigen purification by heparin-sepharose affinity chromatography.

On the basis of the heparin-like effect of phosphotungstate (PTA), we have shown that it is useful for the purification of hepatitis-B virus surface antigen (HBsAg) using heparin-Sepharose affinity chromatography. HBsAg was eluted with 0.2-0.6 M NaCl. HBsAg was also eluted with PTA at approximately 1 mM, and the HBsAg fraction thus obtained contained fewer impurities than the corresponding fraction eluted with NaCl. Moreover, PTA yielded HBsAg and hepatitis-B virus e-antigen simultaneously. PTA may specifically reduce the affinity of HBsAg for heparin as well as simply competing with heparin for an anion-binding site of HBsAg. Residual PTA in the eluate was easily decomposed by alkalization, which was useful for subsequent studies.

Chromatography, Affinity↗

Nucleus of the boar spermatozoon: structure and modifications in frozen, frozen-thawed, or sodium dodecyl sulfate-treated cells.

After cryosubstitution and Epon embedding, or after Nanoplast embedding and very thin sectioning, the chromatin of ejaculated or diluted boar spermatozoa appears to be formed of DNA fibers embedded in a quite homogeneous matrix. After sodium dodecyl sulfate (SDS) treatment, and to a lesser extent after freeze-thawing, the DNA fibers are present mostly between cords, probably proteinaceous in nature. The quantity of free sulfhydryl (SH) groups, as calculated from staining by DACM and flow fluorometry, is increased in thawed or SDS-treated cells. The quantity of NH2 groups, calculated from electron microscopy image analysis of alcoholic phosphotungstic acid-stained cells, is decreased in thawed nuclei. The DNA is more accessible to the fluorochrome ethidium bromide after freeze-thawing, and its sensitivity to HCl hydrolysis is modified, during the Feulgen-like staining procedure using acriflavine. The X-ray energy dispersive analysis of cryosections of nuclei indicates that the slight separation of DNA and nucleoproteins in freeze-thawed spermatozoa could result from a dramatic modification of the nuclear ionic environment during thawing.

Animals↗

Reconstruction of the centrosome cycle from cryoelectron micrographs.

The absence of detailed in vitro studies leaves the molecular events involved in the centrosome cycle poorly characterized. Most earlier studies have employed electron microscopy of thin or thick sections of cells. Here we have analyzed the structure of centrosomes isolated from nonsynchronized human lymphoblastic KE37 cells using cryoelectron microscopy of vitrified specimens. The centrosomes were classified into five categories depending on the number of centrioles (one or two), the respective orientation of the two centrioles in a pair (orthogonal or disoriented), and the presence or absence of appendages at the distal extremity of the centrioles (referred to as mature and immature, respectively). A detailed analysis of the centriole dimensions in these categories allowed us to reconstruct the centrosome cycle in KE37 cells. Our results suggest that centriole assembly is completed only when the mother centriole of an immature orthogonal pair separates from its daughter in preparation to centrosome duplication. Our study shows that an in vitro approach based on cryoelectron microscopy of vitrified specimens can be used to obtain detailed structural information on the centrosome cycle.

Cell Cycle↗

Single-molecule imaging of human insulin receptor ectodomain and its Fab complexes.

The insulin receptor (IR) is a four-chain, transmembrane dimer held together by disulfide bonds. To gain information about the molecular envelope and the organization of its domains, single-molecule images of the IR ectodomain and its complexes with three Fabs have been analyzed by electron microscopy. The data indicate that the IR ectodomain resembles a U-shaped prism of approximate dimensions 90 x 80 x 120 A. The width of the cleft (assumed membrane-distal) between the two side arms is sufficient to accommodate ligand. Fab 83-7, which recognizes the cys-rich region of IR, bound halfway up one end of each side arm in a diametrically opposite manner, indicating a twofold axis of symmetry normal to the membrane surface. Fabs 83-14 and 18-44, which have been mapped respectively to the first fibronectin type III domain (residues 469-592) and residues 765-770 in the insert domain, bound near the base of the prism at opposite corners. These images, together with the data from the recently determined 3D structure of the first three domains of the insulin-like growth factor type I receptor, suggest that the IR dimer is organized into two layers with the L1/cys-rich/L2 domains occupying the upper (membrane distal) region of the U-shaped prism and the fibronectin type III domains and the insert domains located predominantly in the membrane-proximal region.

Dimerization↗

Hepatocyte growth factor prevents lipopolysaccharide-induced hepatic sinusoidal endothelial cell injury and intrasinusoidal fibrin deposition in rats.

BACKGROUND: Acute endotoxemia is known to cause activation of Kupffer cells as well as serious injury in parenchymal and nonparenchymal cells in the liver. We have recently shown that a continuous recombinant hepatocyte growth factor (rHGF) supply prevents lipopolysaccharide (LPS)-induced liver injury in rats. As an attempt to elucidate the mechanism, here we investigate the cytoprotective effect of rHGF on sinusoidal endothelial cells (SECs) in LPS-induced liver injury in rats. MATERIALS AND METHODS: In order to supply rHGF continuously to the liver, syngenic rat fibroblasts genetically modified to secret rat rHGF were implanted in the spleen. Fourteen days after cell implantation, we injected LPS intravenously and evaluated SEC damage histologically and blood chemically. RESULTS: Phosphotungstic acid-hematoxylin staining revealed that rHGF treatment greatly attenuated intrasinusoidal LPS-induced fibrin deposition. The ultrastructural changes in SECs caused by LPS administration in control rats were barely detectable in rHGF-treated rats. Blood chemical analyses showed that rHGF potently suppressed the LPS-induced increase in serum hyaluronic acid and transaminase levels. CONCLUSIONS: Our results indicate an important role for HGF in SEC protection in vivo and would suggest a novel therapeutic strategy for liver diseases with SEC injury.

Alanine Transaminase↗

A critical evaluation of the relationship between the presynaptic network, synaptic vesicles and dense projections in central synapses.

Synapses of the oculomotor nucleus of Echidna have been examined ultrastructurally with the aim of integrating data obtained from osmicated and nonosmicated PTA stained material. Particular emphasis has been laid on the relationship between the synaptic vesicles of the osmicated material and the presynaptic network and vesicular grid of the PTA material. This relationship has been explored qualitatively by examining osmicated material of varying qualities of fixation. Such material contains dense projections in addition to synaptic vesicles, and various vesicular network appearances. A variety of measurement techniques have shown that the PTA network is characterised by reticular strands, spaces, and regular hexagonal units smaller than vesicles, these observations prompting the formulation of a "vesicle-network coincidence" model of the presynaptic terminal. This model has been tested by tracing the profiles of vesicles within the PTA network and comparing their size and shape frequency distributions with those of osmicated synaptic vesicles. The distributions have been found to be essentially similar, suggesting that vesicles can be located within the network, and that the hexagonal network units are formed only in the presence of an underlying vesicular matrix. Additionally, the following points have emerged: 1) the dense projections in the two types of material appear to be quivalent; 2) a loose correlation exists between dense projections and vesicles in osmicated terminals, increase in the area of the dense projections being associated with a decrease in the area of the vesicles; 3) network and dense projection units are similar. In view of the similarity between network and dense projection units, the demonstrated vesicular basis of the network raises the question of whether dense projections are entirely independent structures, or whether they depend in part for their existence on the nearby presence of synaptic vesicles.

Animals↗

An ultrastructural study of the microfilaments in rat brain by means of heavy meromyosin labeling. I. The perikaryon, the dendrites and the axon.

Nervous tissue pieces from the caudate nucleus and the substantia nigra of the rat were incubated in cold glycerol solutions of decreasing concentrations and then transferred into standard phosphate buffer (pH 7.0) or into tris-K+-Mg++-Ca++ buffer (pH 7.9) containing HMM, prepared from rabbit skeletal muscle by tryptic digestion. As controls, pieces were immersed for an identical period in the same buffers (1) without HMM or (2) with HMM to which had been added 2.5 mM Na+ pyrophosphate or 5 mM ATP. In control neurons smooth-surfaced microfilaments, about 50 A in diameter, were observed. After reaction with HMM, the microfilaments were increased in number and density and in width to 180-200 A. A meshwork was formed. Arrowheads pointing in the same direction were spaced at regular intervals (300-350 A) among short segments of the surfaces of the microfilaments, depending upon the plane of section. More often, however, typical arrowheads were not observed, and the surfaces of the microfilaments were seen coated with polarized side-arms cross-bridging the spaces between adjacent elements at more or less regular intervals. When cross-sectioned, the microfilaments appeared as dense dots from which a material of lesser electron density radiated. Following incubation in HMM solutions containing Na+ pyrophosphate or ATP, no arrowhead structures were seen. Of particular interest was the structural relation of the actin-like filaments with occasional, tapered myosin-like filaments, and with the plasma membrane, which served as anchor points. Mitochondria and smooth ER membranes were observed to be attached to the actin-like filaments or enmeshed in the network. The microtubules, as well as most of the neurofilaments, were disrupted by the glycerination procedure at 4 degrees, and thus no precision about the structural relationship of the actin-like filaments with the latter elements could be added. The role of the actin-like filaments in the transport of material, by a mechanism of chemomechanical transduction, throughout the neuron from sites of synthesis to functional locations, and between several functional locations, is discussed.

Actins↗

An ultrastructural study of the microfilaments in rat brain by means of E-PTA staining and heavy meromyosin labeling. II. The synapses.

To identify structures involved in the translocation of the synaptic vesicles towards the presynaptic membrane, an ultrastructural study has been undertaken by means of (1) the E-PTA stain and (2) the HMM-labeling procedure. Using serial sections of E-PTA stained nervous tissue, especially those made in transversal and tangential planes, the geometric order of the presynaptic grid and of its constituents has been described in detail. It consisted of dense projections having the shape of small truncated pyramids cut parallel to their hexagonal bases which rested on the electron-lucent presynaptic membrane. The dense projections were arranged at the points of equilateral triangles. Around each dense projection, six asymmetric hexagonal holes were seen to be arrayed in an hexagonal pattern, forming thus the presynaptic sieve. From the spiny tops of the dense projections, which appeared as specialized structures of the dense material coating the inner surface of the plasma membrane at the level of the synaptic cleft, fine filaments, 40--60 A in diameter, radiated and formed a three-dimensional meshwork pervading the presynaptic bag. The dense cytoplasmic coating delineating the plasma membrane served as anchor points for these microfilaments. Upon incubation with rabbit skeletal muscle HMM the microfilaments underwent specific structural changes, consisting of: (1) a striking increase in diameter; (2) the association of periodic and polarized substructures with their surfaces. The synaptic vesicles and mitochondria were seen to be attached to the numerous HMM-decorated filaments or enmeshed in the network formed by these filaments. The actin-like filaments were anchored to the plasma membrane at many points and to the presynaptic dense projections. Following incubation in the buffer alone or in buffer HMM solutions containing Na+ pyrophosphate or ATP, no arrowheaded structures were observed. Thus, a network consisting of actin-like filaments was demonstrated in the presynaptic bag. Of particular interest was the structural relation of the actin-like filaments with the occasional, tapered myosin-like filaments. The role of the presynaptic actin-like network in the transport of synaptic vesicles towards the presynaptic membrane by a mechanism of chemomechanical transduction is discussed. In the postsynaptic dendrite or dendritic spine, a filamentous network was observed to be attached to the subsynaptic web by means of the E-PTA stain and of the HMM-labeling procedure. The occurrence of an actin-like meshwork in the postsynaptic region is suggested to produce changes in the macromolecular configuration of the postsynaptic membrane by a "mechanoenzyme" system similar to that described in the mitochondrial membrane.

Actins↗

Interdigitating reticulum cells in the popliteal lymph node of the rat. An ultrastructural and cytochemical study.

Electronmicroscopic and cytochemical studies were performed to localize interdigitating reticulum cells (IDC) in the popliteal lymph node of the rat. The morphological features of the IDC of the rat correspond to those described for other species, but also show similarities to normal macrophages in the rat. This is considered to be an argument in favour of the common origin of IDC's and macrophages. Ultrahistochemical studies with horseradish peroxidase (HRP) reveal no phagocytotic capacity of IDC's. After perfusion fixation containing ruthenium red (RR) the surface coat stains heavily: RR is also found deep in the membrane invaginations of the IDC, indicating the presence of polyanionic sialoglycoproteins. The post-capillary-venules (PVC) are very permeable to both HRP and RR. The phosphotungstic acid-chromic acid stain (PTA-CrA) also reveals glycoproteins in the surface coat; these glycoproteins are susceptible to alpha-neuraminidase, whereas glycoproteins in the Golgi complexes, lysosomes and in the vesicular complexes of IDC are not. The glycoproteins of the latter are susceptible to 0.1 N NaOH. These findings indicate that IDC produce different kinds of glycoprotein, one of which may be secreted and act as a factor for stimulating peripheral T-lymphocytes. Intimate contact between IDC's and PCV's could be observed. It is therefore conceivable that IDC's play an important role in the homing of T-lymphocytes.

Animals↗