A study of staining for electron microscopy using collagen as a model system—IV. Phosphotungstate/tungstate negative staining patterns and their correlation with sequence data

The type I collagen fibril (for which the axial distribution of amino acid residues is known) has been used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils were compared with chemical data by a computer-aided correlation procedure. Stains used were: phosphotungstic acid, pH 3.2 and 7.0, lithium tungstate, pH 7.2, and methylamine tungstate, pH 6.6. In all cases, the results of the correlation analyses point to the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains as the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some preferred uptake of heavy metal ions on charged side chains (a positive staining contribution) can be demonstrated by partial correlation analysis but, under the staining conditions used here, the effect is largely masked by the much greater negative staining component.     

A study of staining for electron microscopy using collagen as a model system—VI. Uranyl nitrate negative staining patterns and their correlation with sequence data

Collagen is used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) were compared with chemical data by a computer-aided correlation procedure. The stain used was uranyl nitrate, pH 3.2 and 4.9. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some contribution of positive staining can also be demonstrated by the analysis described here.     

A study of staining for electron microscopy using collagen as a model system—VIII. Simulation of the negative staining pattern

Simulated negative staining patterns of collagen fibrils were prepared for visual display by a graphical procedure in which amino acid side-chains along the staggered molecules were weighted according to their stain-excluding capacity. The simulated patterns were then compared directly with electron-optical images of collagen fibrils negatively stained with sodium phosphotungstate or lithium tungstate. These visual comparisons confirm previous observations that satisfactory matching occurs when side-chains are weighted according to their ‘bulkiness’ (average cross-sectional area or ‘plumpness’). Optimal matching at the edges of the overlap zones occurred when a hairpin-like conformation was assumed for the N-terminal telopeptides and a condensed conformation for the hydrophobic part of the C-terminal telopeptides. The negative staining pattern is known to include some element of positive staining; visual matching suggests that this additional uptake of positive staining ions occurs predominantly in the more accessible gap zone in a fibril D-period. A slight mismatching between observed and simulated patterns can be understood if the gap zone suffers greater axial shrinkage than the overlap zone when specimens are prepared for electron microscopy.     

A study of staining for electron microscopy using collagen as a model system—V. The effect of suberimidate fixation on negative staining patterns

The effects of the fixative dimethylsuberimidate (DMS) on negative staining patterns were studied using reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) as a model system and comparing electron-optical data and chemical data by a computer-aided correlation procedure. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant factor in determining the stain-excluding property of a DMS-fixed negatively stained collagen fibril as it is in unfixed collagen. Some contribution of positive staining can also be demonstrated after DMS-fixation by partial correlation analysis. Other evidence suggests that (unlike glutaraldehyde) DMS does not produce any morphological alterations to the negative staining pattern.     

Ultrastructure, serogrouping and localization of surface antigens of Bacteroides intermedius

The surface ultrastructure of 21 strains of Bacteroides intermedius was investigated by electron microscopy. Rat monoclonal antibodies (mAbs) were used to define serogroups and to detect the location of surface antigens. All 21 isolates had capsules as demonstrated by the use of wet and dry Indian ink stains. Negative staining of whole cells with 1% (w/v) methylamine tungstate showed that all 21 isolates carried clumped peritrichous fibrils with strain dependent morphology, density and length (less than or equal to 0.75 micron). Fibrils on 11 of 13 fresh clinical isolates were more conspicuously clumped and easily visible, whereas those on 6 of 8 laboratory strains were indistinct and were at the limits of the resolution of the negative staining technique. Staining with ruthenium red (RR), followed by thin sectioning, revealed a dense, amorphous RR staining layer (RRL), up to 24.8 +/- 3.0 nm thick, adjacent to the outer membrane on all of 15 strains examined. All isolates had a less dense RR staining matrix (RRM) extending away from the RRL. The structure of the RRM varied between strains. Four rat mAbs (37BI6.1, 38BI1, 39BI1.1 and 40BI3.2) were used to serogroup the 21 strains of B. intermedius. Immunonegative staining revealed that the mAbs were not directed against fibrilis. Antigens recognized by mAb 37BI6.1 and mAb 39BI1.1 were located on the surfaces of cells, beneath fibrils, and on extracellular vesicles. mAb 38BI1 recognized an antigen which was most accessible on lysed cells, and non-specific binding of mAb 40BI3.2 to grids prevented its localization on the cell surface.     

Demonstration of sialic acid groups in the glomerular basement membrane of the rat with phosphotungstic acid at low pH

Summary This paper reports an unrecognized aspect of phosphotungstic acid staining at low pH. It provides an on-section staining method in which sialic acid-containing molecules can be demonstrated in the laminae rarae of the rat glomerular basement membrane. The staining in the basement membrane became negative after perfusion with the following cations: protamine sulphate, hexadimethrine, Alcian Blue, Ruthenium Red and Toluidine Blue. Blocking ws not achieved with Alcian Blue at about pH 1. The staining was also abolished after mild methylation and demethylation restored the contrast. This is suggestive of the involvement of carboxyl groups. Prior digestion with pronase, trypsin and neuraminidase rendered the laminae rarae negative, whereas hyaluronidase, chondroitinase ABC and crude heparinase were without effect. This indicates that sialic acid groups are detected by this method and that heparan sulphate does not interfere. The staining of the epithelial plasma membrane, also carrying sialic acid groups, remained positive after neuraminidase treatment. It is presumed that this method can be applied successfully for detecting changes in the sialic acid content of the laminae rarae in rat glomerular basement membranes under normal and pathological conditions.     

Interaction of rat liver glucocorticoid receptor with sodium tungstate.

Effects of sodium tungstate on various properties of rat liver glucocorticoid receptor were examined at pH7 and pH 8. At pH 7, [3H]triamcinolone acetonide binding in rat liver cytosol preparations was completely blocked in the presence of 10--20 mM-sodium tungstate at 4 degrees C, whereas at 37 degrees C a 30 min incubation of cytosol receptor preparation with 1 mM-sodium tungstate reduced the loss of unoccupied receptor by 50%. At pH 8.0, tungstate presence during the 37 degrees C incubation maintained the steroid-binding capacity of unoccupied glucocorticoid receptor at control (4 degrees C) levels. In addition, heat-activation of cytosolic glucocorticoid-receptor complex was blocked by 1 mM- and 10 mM-sodium tungstate at pH 7 and pH 8 respectively. The DNA-cellulose binding by activated receptor was also inhibited completely and irreversibly by 5 mM-tungstate at pH 7, whereas at pH 8 no significant effect was observed with up to 20 mM-tungstate. The entire DNA-cellulose-bound glucocorticoid-receptor complex from control samples could be extracted by incubation with 1 mM- and 20 mM-tungstate at pH 7 and pH 8 respectively, and appeared to sediment as a 4.3--4.6 S molecule, both in 0.01 M- and 0.3 M-KCl-containing sucrose gradients. Tungstate effects are, therefore, pH-dependent and appear to involve an interaction with both the non-activated and the activated forms of the glucocorticoid receptor.     

Electron-Microscopic Demonstration of a “Head and Stalk” Structure of the Leaf Vacuolar ATPase in Mesembryanthemum crystallinum L.

The structure of the vacuolar ATPase from mesophyll tonoplasts of Mesembryanthemum crystallinum has been studied by electron microscopy using negatively stained specimens of membrane-bound and detergent-solubilized ATPase molecules. We observed a high density of particles on the surface of tonoplast vesicles and “head and stalk” structures on the edge of the membrane, similar to the F₀F₁-ATPases of mitochondrial and chloroplast membranes. The staining conditions, which are often critical for such small objects, were improved by using methylamine tungstate as negative stain for the membrane-bound ATPase. Compared to other staining solutions generally applied, dissociation of the F₁-like enzyme complex from the membrane was best prevented and structural damage of the vesicles was least observed with methylamine tungstate. In freeze-fracture electron microscopy of tonoplast vesicles, where dissociation never occurs since no detergent is used, we also observed “head and stalk” structures on the edge of the membranes, beside many particles on the fracture faces. The detergent-solubilized ATPase forms string-like structures, caused by the aggregation of the hydrophobic membrane-embedded F₀-like part of the enzyme. After negative staining the F₁-like enzyme complex is arranged alternately along both sides of the string and connected by a narrow stalk.     

pH-dependent effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor

Effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor were examined and were found to be pH-dependent. When freshly prepared hen oviduct cytosol containing progesterone receptor was heated at 37°C for 20 min, its ability to bind [³H]progesterone decreased to 20% level of unheated samples. At pH 7, presence of 2–3 mM tungstate during the above incubation period reduced this loss of binding. At higher tungstate concentrations (>5 mM), this stabilizing effect was gradually abolished. Similar results were obtained with preparations that contained [³H]progesterone-receptor complexes; 70–80% of which remained after a 20 min incubation at 37°C in the presence of 2–3 mM tungstate at pH 7. At pH 8, presence of tungstate (1–10 mM) during the 37°C incubation stabilized both the steroid-bound and the unoccupied progesterone receptor in a concentration-dependent manner. The extent of steroid binding by the receptor at 4°C remained unchanged in the presence of up to 10 mM tungstate at both pH 7 and pH 8 assay conditions while presence of 20 mM tungstate lowered this binding capacity. These results indicate that tungstate effects may be mediated via its interaction with the progesterone receptor.     

Resolution of the isoenzymes of soybean lipoxygenase using isoelectric focusing and chromatofocusing

Isoelectric focusing in thin-layer polyacrylamide gels has been applied to the analysis of the enzymes involved in the formation and destruction of peroxides in soybeans [Glycine max (L.)], lipoxygenases and peroxidases, respectively. As a result of differences in pH optima for catalytic activity, lipoxygenases were selectively detected by adjusting the pH employed for activity-specific staining. Type-1 lipoxygenase was revealed not only by staining based on the conversion of linoleic acid to hydroperoxide but also by two stains based on the reduction of the hydroperoxide. These methods were found to be suitable for the analysis and characterization of isoenzyme patterns in different soybean cultivars. A substantial difference in the distribution of lipoxygenases maximally active near pH 7 was observed for cultivars Provar and Vickery. A similar degree of separation of the isoenzymes was achieved on a larger scale using chromato-focusing in the pH range 7.4-5.0.     

Modeling the effects of sodium chloride, acetic acid, and intracellular pH on survival of Escherichia coli O157:H7

Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primary antimicrobial compounds in these products are acetic acid and NaCl, which can alter the intracellular physiology of E. coli O157:H7, leading to cell death. For combinations of acetic acid and NaCl at pH 3.2 (a pH value typical for non-heat-processed acidified vegetables), survival curves were described by using a Weibull model. The data revealed a protective effect of NaCl concentration on cell survival for selected acetic acid concentrations. The intracellular pH of an E. coli O157:H7 strain exposed to acetic acid concentrations of up to 40 mM and NaCl concentrations between 2 and 4% was determined. A reduction in the intracellular pH was observed for increasing acetic acid concentrations with an external pH of 3.2. Comparing intracellular pH with Weibull model predictions showed that decreases in intracellular pH were significantly correlated with the corresponding times required to achieve a 5-log reduction in the number of bacteria.     

Thermodynamic studies on the interaction between sodium n-dodecyl sulphate and histone H2B

The thermodynamic parameters for the interaction of the anionic detergent sodium n-dodecyl sulphate (SDS) with H2B at pH 3.2, 6.4 and 10 have been measured at 27 degrees C and 37 degrees C by equilibrium dialysis to determine the Gibbs energies of detergent binding. The data have been used to obtain the enthalpy of interaction from the temperature dependence of the equilibrium constants from the Van't Hoff relation. The enthalpy of interaction between H2B and SDS is endothermic at pH 3.2, 6.4 and 10. The shapes of the enthalpy curves at pH 3.2 and 10 show some small exothermic contribution which probably indicates folding of H2B. The interactions of H2B-SDS are dominated by the increase in entropy on detergent binding. The larger negative free energy, enthalpy and entropy changes at pH 6.4 are consistent with greater denaturation relative to pH 3.2 and 10.     

Glutaraldehyde-induced changes in the axially projected fine structure of collagen fibrils

The fine structure of the collagen fibril, as seen in axial projection, is changed by treatment with glutaraldehyde. The changes are detectable in electron-optical staining patterns and in the intensities of the low-angle meridional X-ray diffraction maxima. Current knowledge of the amino acid sequence of collagen and of the axial arrangement of molecules in fibrils permits interpretation in terms of specific alterations to the axial distribution of electron density along the fibril. Analysis of fibril staining patterns from glutaraldehyde-treated calf skin collagen shows that uptake of staining ions in positive staining patterns is inhibited at residues known to interact with glutaraldehyde (lysyl, hydroxylysyl and probably histidyl side-chains) and on other charged residues in the immediate neighbourhood of the glutaraldehyde-reactive residues. This can be seen as a "stain-exclusion effect" due to the presence of bulky polymeric complexes of glutaraldehyde molecules at cross-linking sites. Such stain exclusion accounts for the drastic changes in the negative staining pattern following treatment with glutaraldehyde. The intensity changes observed in the low-angle meridional X-ray reflections from rat tail tendon, similarly treated, also can be explained by the presence of these bulky complexes. Existing data have been used to predict a model of the altered electron density profile indicating the axial distribution of glutaraldehyde along a D-period of moist tendon collagen.     

Integration of DIGE and bioinformatics analyses reveals a role of the antiobesity agent tungstate in redox and energy homeostasis pathways in brown adipose tissue

Our previous results demonstrated that tungstate decreased weight gain and adiposity in obese rats through increased thermogenesis and lipid oxidation, suggesting that brown adipose tissue was one of the targets of its antiobesity effect. To identify potential targets of tungstate, we used DIGE to compare brown adipose tissue protein extracts from the following experimental groups: untreated lean, tungstate-treated lean, untreated obese, and tungstate-treated obese rats. To distinguish direct targets of tungstate action from those that are secondary to body weight loss, we also included in the analysis an additional group consisting of obese rats that lose weight by caloric restriction. Hierarchical clustering of analysis of variance and t test contrasts clearly separated the different experimental groups. DIGE analysis identified 20 proteins as tungstate obesity direct targets involved in Krebs cycle, glycolysis, lipolysis and fatty acid oxidation, electron transport, and redox. Protein oxidation was decreased by tungstate treatment, confirming a role in redox processes; however, palmitate oxidation, as a measure of fatty acid beta-oxidation, was not altered by tungstate, thus questioning its putative function in fatty acid oxidation. Protein network analyses using Ingenuity Pathways Analysis highlighted peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) as a potential target. We confirmed by real time PCR that indeed tungstate up-regulates PGC-1alpha, and its major target, uncoupling protein 1, was also increased as shown by Western blot. These results illustrate the utility of proteomics and bioinformatics approaches to identify targets of obesity therapies and suggest that in brown adipose tissue tungstate modulates redox processes and increases energy dissipation through uncoupling and PGC-1alpha up-regulation, thus contributing to its overall antiobesity effect.     

The Determination of Apparent Isoelectric Points of Cell Structures by Staining at Controlled Reactions

The staining reactions at controlled pH-values of various dyes with the nucleus and cytoplasm of Trichonympha collaris under different conditions were investigated. When staining intensity was plotted against pH, it was found that with each dye a different curve was obtained. “Isoelectric points” obtained by superposition of acid and basic dye curves varied for the same material with the dyes employed. It was found that, with the same dye, the curves of staining intensity plotted against pH varied with the buffer system utilized. Moreover, the intensity of staining at any pH was found to vary directly with the concentration of dye and inversely with the concentration of buffer. Various factors modifying staining intensity were studied. In the staining of a protein in buffered solution, it was shown that staining intensity (the index of the concentration of the dye-protein compound) at a given pH-value is dependent upon the interaction of the dye-protein, buffer-protein and dye-buffer systems, and that as the dye or buffer or their concentrations were varied, the resultant “isoelectric points” which were obtained also varied. In view of these facts and of the present lack of knowledge of dyes and dye-protein combinations it would be impossible to determine a true isoelectric point by staining at controlled pH-values without further extensive work on the subject. It follows that no true isoelectric points have hitherto been obtained for nucleus, cytoplasm or other tissue elements by staining at controlled pH.     

Effect of NO3- transport and reduction on intracellular pH: an in vivo NMR study in maize roots

The effect of NO3- uptake on cellular pH was studied in maize roots by an in vivo 31P-NMR technique. In order to separate the effects on cytoplasmic pH due to NO3- uptake from those due to NO3- reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. 15N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither NO3- uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during NO3- uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that NO3- uptake is an acidifying process and suggest a possible involvement of NO3- reduction in pH homeostasis. In the presence of NO3-, a transient depolarization of transmembrane electric potential difference (Em) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolarization accompanied by a greater ability to recover Em was found. This was related to a higher activity of the plasma membrane (PM) H+-ATPase. When NO3- was administered as potassium salt, its uptake increased and a greater depolarization of Em took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K+ in the control of plasma membrane activities and cell pH homeostasis. A possible involvement of cytoplasmic pH in the control of PM H+-ATPase expression during nitrate exposure is suggested.     

Anti-diabetic and anti-obesity agent sodium tungstate enhances GCN pathway activation through Glc7p inhibition

Tungstate counteracts diabetes and obesity in animal models, but its molecular mechanisms remain elusive. Our Saccharomyces cerevisiae-based approach has found that tungstate alleviated the growth defect induced by nutrient stress and enhanced the activation of the GCN pathway. Tungstate relieved the sensitivity to starvation of a gcn2-507 yeast hypomorphic mutant, indicating that tungstate modulated the GCN pathway downstream of Gcn2p. Interestingly, tungstate inhibited Glc7p and PP1 phosphatase activity, both negative regulators of the GCN pathway in yeast and humans, respectively. Accordingly, overexpression of a dominant-negative Glc7p mutant in yeast mimicked tungstate effects. Therefore tungstate alleviates nutrient stress in yeast by in vivo inhibition of Glc7p. These data uncover a potential role for tungstate in the treatment of PP1 and GCN related diseases.     

Larval temperature experience determines sensitivity to cold-shock-induced wing color pattern changes in the blue pansy butterfly Junonia orithya

The butterfly wing color patterns are unique to a species but are modified in response to cold-shock and tungstate treatments at the pupal stage, producing characteristic temperature–shock (TS) phenotypes that are distinct from the color patterns of seasonal polyphenism. In this study, we examined the efficiency of cold-shock and tungstate treatments for color pattern modifications at the pupal stage in relation to larval rearing conditions for the fall or summer morph using the blue pansy butterfly Junonia orithya. We found that larvae reared under the low-temperature condition that induces the fall morph exhibited hardiness against the color pattern changes imposed by cold-shock or tungstate treatment at the pupal stage. When larvae were fed an artificial diet containing tungstate under the high-temperature condition that induces the summer morph, they were still vulnerable to color pattern changes imposed by cold-shock or tungstate treatment at the pupal stage. Furthermore, larvae reared under the high-temperature condition were subjected to cold-shock or tungstate treatments at the pupal stage. In addition to the expected TS-type changes, these individuals exhibited a reduced number of eyespots in adults, which is a feature of the fall morph. These results suggest that the temperature condition experienced by the larvae, but not their consumption of tungstate, determines the sensitivity of the wing imaginal discs to cold-shock and tungstate treatments at the pupal stage.     

Specific tumor markers in diagnostic cytology. Immunoperoxidase studies of carcinoembryonic antigen, lysozyme and other tissue antigens in effusions, washes and aspirates

The immunoperoxidase technique was used to identify specific tumor markers in exfoliated cells in fine needle aspirates and body fluids. Carcinoembryonic antigen (CEA) and lysozyme staining was evaluated in cytocentrifuge preparations from 42 malignant effusions and aspirates and 16 benign effusions. Reactive mesothelial cells were negative for CEA and lysozyme or showed faint peripheral cytoplasmic staining. Malignant cells from 50% of the adenocarcinomas studied were positive for CEA. All tumors studied were negative for lysozyme. These staining patterns are helpful in the differential diagnosis of reactive mesothelial and adenocarcinoma cells, a frequent diagnostic dilemma. Moreover, demonstration of specific tumor antigens (e.g., prostatic acid phosphatase, calcitonin and immunoglobulin) helped define the origin of metastatic malignancy in selected cases. Estrogen receptor activity was also identified in tumor cells using this technique. Immunoperoxidase was helpful in the evaluation of malignant cytologic specimens from patients with more than one tumor. Interpretation of staining patterns is discussed, with reference to the limitations of the technique. Immunoperoxidase methods maintain cytologic detail, are readily adaptable to diagnostic cytology and increase the specificity of cytologic diagnosis.     

The use of a basic dye (azure A or toluidine blue) plus a cationic surfactant for selective staining of RNA: a technical and mechanistic study.

Selective purple staining of RNA-rich structures such as basophilic cytoplasms of exocrine pancreas and plasma cells, Nissl substance, and nucleoli was achieved by treating tissue sections as follows. Stain dewaxed sections for 1/2 hour in a dyebath containing 0.1% w/v axure A or toluidine blue and 1% cationic surfactant (Hyamine 2389, a 50% w/v aqueous solution of diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride; or benzyldimethylammonium chloride, or cetylpyridinium bromide, or cetyltrimethylammonium bromide) buffered to pH 7 with phosphate. Rinse in water, blot, air dry and mount in synthetic resin. Intense purple staining of RNA-rich regions occurred after fixation in neutral formalin or in Carnoy's or Gendre's fluids, though satisfactory results were also found after fixation in acetone or alcohol. Chromatin generally stained a very pale azure after all fixations, though occasionally nuclei were unstained (Gendre's or Zenker's fluids). Subjecting tissue sections to acid hydrolysis or to digestion by RNAase eliminated or reduced the purple staining, but left the azure staining of nuclei unaffected. Satisfactory staining of RNA-rich structures was not critically dependent on the precise concentrations of dye, surfactant or inorganic salts in the dyebath, nor on pH, staining time or chemical nature of the surfactant. The staining patterns can be rationalized with a tissue model that considers both surface charge and permeability factors, since present in the dyebath are small dye cations and large cationic surfactant micelles. As micelles and dye will both quickly penetrate basophilic structures considered to be porous, such as chromatin, competition will then greatly reduce staining of such substrates. But the large micelles will only slowly penetrate regions considered to be more impermeable, such as basophilic cytoplasms, so consequently small fast moving dye ions may enter and stain without competition.