Sodium Silicotungstate Research Articles

Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals

Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.

Tungstate compounds as an alternative to uranyl acetate for enumerating viral-infected prokaryote cells in aquatic systems

We examined whether tungstate compounds (sodium phosphotungstate and sodium silicotungstate) can be used as a safe alternative to uranyl acetate (UA) for the purpose of detecting and counting viruses inside aquatic prokaryotic cells using transmission electron microscopy (TEM). Tungstate compounds used as negative stains at neutral to slightly acidic pH are less effective in the detection of viral particles inside the cells. However, the use of tungstate compounds at low pH (3 or 4) substantially improved viral images, being positively contrasted inside the cells. With this improvement, the estimates of the frequency of viral-infected cells derived by the tungstate method agreed well with those determined by the use of UA for both lake and marine waters. We also found that the average burst size of the lake sample differed little between tungstate and the UA approach; however, the average burst size in a marine sample was underestimated by the tungstate method because of the failure to resolve highly packed viral particles. The above results suggest that the use of tungstate compounds at low pH can be a useful alternative to the UA approach for estimating the frequency of viral-infected cells in aquatic samples, but results on the burst size need to be interpreted cautiously when the burst size is high.

An Archaeal Peptidase Assembles into Two Different Quaternary Structures

Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids.

Unconventional negative stains: Heavy metals are not required for negative staining

Salts of heavy metals (tungsten, uranium, and molybdenum) have long been used as negative stains for the transmission electron microscopy of protein molecules, supramolecular assemblies, and viruses, Negative staining still reveals details about protein structure only to around 25 Å (= 2.5 nm), most likely due to several major technical deficiencies in all traditional stain reagents. Experimental tests of unconventional compounds show that potassium aluminum sulfate, ammonium borate, and sodium tetraborate all dry into a glassy layer, and provide sufficient contrast to image the 86 Å lattice periods in thin crystals of catalase. Computer-generated power spectra of high-dose images indicate that sodium tetraborate can preserve periodic order in crystalline catalase to the same level (i.e., 28 Å) as the heavy metal negative stain, sodium silicotungstate. Sodium tetraborate also reveals the 40 Å wide central channel within tobacco mosaic virus, and the 25 Å thick protein shell of ferritin molecules. Images with these light atom compounds should be formed with a decreased electron beam exposure, in order to preclude the radiation-induced formation of voids within the dried stain. The results establish that heavy metal constituents are not necessary for a compound to function as a negative stain, and suggest that it should be possible to identify new reagents having the missing desired properties needed to obtain higher resolution imaging of protein structure with negative staining.

Towards the Molecular Architecture of the Asymmetric Unit Membrane of the Mammalian Urinary Bladder Epithelium: A Closed “Twisted Ribbon” Structure

The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm. Bovine AUM plaques negatively stained with anionic sodium silicotungstate revealed structural detail to 3.1 nm resolution. Correlation averaging resolved each particle into 12 stain-excluding domains arranged in two concentric rings (inner ring radius (rin)=3.7 nm, outer ring radius (rout)=6.6 nm), each with six domains which were rotated by roughly 30° relative to each other.Negative staining with cationic uranyl formate increased the resolution to 2.2 nm and unveiled distinct connections between adjacent AUM particles. These connections may provide a molecular basis for the observed insolubility of the plaques in many detergents. Examination of the luminal face of freeze-dried/unidirectionally metal-shadowed AUM plaques established a left-handed vorticity of the 16 nm protein particles, whereas the cytoplasmic face exhibited no significant surface corrugations. Three-dimensional reconstruction from sodium silicotungstate-stained specimens revealed the AUM particles to be built of six “V-shaped” subunits anchored upright in the membrane. The mass density distribution within uranyl formate-stained AUM particles was similar except that the inner tip of each V was bridged to the outer tip of an adjacent V, so that the 16 nm AUM particle appeared as a continuous, “twisted ribbon” embracing a central cavity. Finally, mass measurements of unstained/freeze-dried plaques by scanning transmission electron microscopy yielded a total mass of 1,120 kDa per membrane-bound AUM particle. By imposing constraints on the possible uroplakin stoichiometries within AUM plaques, these data provide a first glimpse of the molecular architecture of the 16 nm particles constituting the plaques.

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two‐phase partitioning

Plasma membrane vesicles were isolated from the roots of 7‐day‐old rice plants (Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two‐phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate‐sensitive K+, Mg2+‐ATPase activity that was 18 times higher in the upper (PEG‐rich) phase than in the lower (Dextran‐rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 mM KCl and 3 mM ATP with apparent Km (ATP) and Vmax of 0.75 mM and 79 μmol (mg protein)−1 h−1, respectively. With 50 mM KCl and 7 mM ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 mM and Vmax of 159 μmol (mg protein)−1 h−1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate.

Comparison of negatively stained and frozen hydrated samples of influenza viruses A and B and of vesicular stomatitis virus

Three negative strand viruses are visualized by negative staining using uranyl acetate (UAc) and sodium silicotungstate (SST) and by cryoelectron microscopy of frozen hydrated samples. We find that both stains induce changes in the shapes of viruses relative to the image in cryoelectron microscopy. Influenza viruses A and B are rather strongly deformed by SST whereas staining with UAc makes them more round than ice-embedded virus. SST stained vesicular stomatitis virus looks similar to the frozen hydrated virus but this virus is also made round by staining with UAc. SST was useful to visualize the spikes of the viruses and their internal structures, while UAc did not outline these details. The stain-induced deformations are discussed in relation to the specific viral structures.

Ultrastructure of Two Mutants in the H-Subunit of Recombinant Human Ferritin: Ordered Arrays Indicate Altered Surface Properties

Two major types of polypeptide subunits, H and L, coassemble (n = 24) as heteropolymers to form molecules of the iron-storage protein, ferritin [1]. The significance of the H-type subunits for molecular structure and function remains unclear. The present study has examined the ultrastructure of recombinant human ferritins which are homopolymers of wild-type or mutant H-subunits [2]. Both mutations change the aminoacid sequence along the outer surface of the molecule; 9Cd has a single substitution of glycine for the lysine at position 86, and Ml has a duplication of 18 aminoacids [2].Recombinant ferritins were overexpressed in E. coli. and purified as described by Levi et al [2]. Samples (0.1-0.5 mg/ml) were deposited onto a thin carbon film stripped from freshly cleaved mica and carried on 400 mesh grids; these specimen carriers had been made hydrophilic by glow discharge treatment. After negative staining with 2% sodium silicotungstate (pH 7.0), the specimens were dried and examined at lOOkV with a JEOL 100CX electron microscope.

A thermodynamic study of the preferential interaction of the polyoxometallate electrolyte Na4SiW12O40· 14H2O with ethers in aqueous solution

Free-energy and enthalpy changes associated with the interaction of a sodium silico-tungstate salt (Na4SiW12O40·14H2O) with diethylether and tetrahydrofuran in dilute aqueous solutions (< 1 mol kg–1 ether) have been investigated using precise vapour-pressure and microcalorimetry techniques. The standard partial molar volume of the oxometallate salt in pure water at a temperature of 298.15 K has been determined from density measurements in dilute solutions: text-decoration:overlineVo= 660.5 cm3 mol–1. It is shown that the solvation process is enthalpy driven in very dilute salt or ether solutions and entropy driven at higher ether concentrations; the enthalpy/entropy compensation leads to a decreasing standard function of transfer of the electrolyte from water to aqueous ether solutions. The available data are compatible with a two-step solvation process: first ether molecules solvate the oxometallate anion at given polarized sites and secondly, with increasing ether concentration, a favourable hydrophobic type of interaction takes place between the solvated anion and free ether molecules. An analysis of the salting constant of tetrahydrofuran in the aqueous sodium silico-tungstate solution using the scaled-particle theory is also presented.

Purification of Hydrogenase from Chlamydomonas reinhardtii

A method is described which results in a 2750-fold purification of hydrogenase from Chlamydomonas reinhardtii, yielding a preparation which is approximately 40% pure. With a saturating amount of ferredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H(2) produced per milligram protein per minute. The molecular weight was determined to be 4.5 x 10(4) by gel filtration and 4.75 x 10(4) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an abundance of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H(2) production; these properties are similar to those of bacterial hydrogenases. The enzyme is less thermally stable than most bacterial hydrogenases, however, losing 50% of its activity in 1 hour at 55 degrees C. The K(m) of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanced under slightly acidic conditions. Purified hydrogenase also accepts electrons from a variety of artificial electron mediators, including sodium metatungstate, sodium silicotungstate, and several viologen dyes. A lag period is frequently observed before maximal activity is expressed with these artificial electron mediators, although the addition of sodium thiosulfate at least partially overcomes this lag.

A method for the stabilization of toluidine blue vital staining for histological and ultrastructural studies

A method previously developed to stabilize methylene blue vital staining has been applied to toluidine blue, a basic dye with a closely related chemical structure commonly used as a vital stain for the screening of oral and esophageal lesions. The stain was made stable by adding 1% sodium silicotungstate to an aldehyde fixative (formaldehyde or glutaraldehyde). Sections were prepared for light and electron microscopy.

Electron microscopic study on the chromatin extracted from wheat germ and seedlings.

Extracted chromatin from wheat germ and 3 days-germinated seedlings is negatively stained with sodium silicotungstate, and examined by means of electron microscopy. The chromatin consists of fibrous elements, and is about 200A in diameter. The germ chromatin appears to be made up of a number of aggregates which have a mean diameter of 2, 200A. In contrast, no aggregates are present in the seedling chromatin.

Selection of Mutants with Disorganized Septation and Transformation to L forms of Nocardia Corynebacterioides Nom Nov and N. asteroides

Morphology of the cell wall and organization of the cytoplasm of Nocardia corynebacterioides nom nov and Nocardia asteroides ATCC 14597, were examined by electron microscopy after the induction of autolysis using 0.012M ammonium carbonate. Organisms were cultured in casaminic acidbroth modified according to Abrams (1955), medium PPLO (Difco) modified according to Sharp (1954), a modified mixture of electrolytes according to Dienes (1956) and a medium based on amino-acids in proportions of 0.05 mg %. In all of these media we used in an alternate or combined form 1.000 U/ml of penicillin or 100 mg/ml of cycloserine (Sigma). After treatment in ammonium carbonate and culture in the media mentioned above, incubated at 37°C, the growth of the colonies on Agar-peptone and Agar-amino-acid presented a smooth surface and alterations of pigmentation.Cultures in solid or liquid media were observed by phase contrast and with a JEM 7-A electron microscope. The cells were negatively stained with sodium silicotungstate pH 7.6 and fixed in 2% osmium according to Ryter and Kellenberger.

An electron microscope study of Rauscher leukemia virus

The effect of various stains on the morphology of Rauscher leukemia virus was examined. The virus appeared as a round, membrane-bound structure when prefixed with glutaraldehyde or formalin and stained with uranyl acetate (UA). Other stains, such as sodium phosphotungstate, sodium silicotungstate, or ammonium molybdate caused disruption of over 80% of the virus particles. They then appeared as smaller, tailed particles or swollen, heavily stained membranous structures. In the prefixed, UA-stained preparations, the particles were accurately measured using the catalase crystal internal marker technique. The intact virus had an average diameter of 1470 Å. Also present were smaller particles with an outer diameter of 1090 Å, which occurred at a level of 20–30%. The proportion of these smaller structures could be increased to 70% of the total particles by a brief treatment with 0.01% dodecyl sodium sulfate (SDS). This suggests that the outer envelope of the intact virus was selectively removed, thus exposing an internal nucleoid. This is consistent with the observation that isolated viral outer envelopes measured 200 Å across. The nucleoids obtained by SDS-treatment banded in cesium chloride (CsCl) at an average density of 1.226 g/ml, compared to the value of 1.18 g/ml for the fully enveloped virion, which also suggests the loss of a lipid envelope. Occasionally, nucleoid particles were fortuitously stained to reveal an internal single or double-stranded whorled substructure. The double-stranded form was 2.5 times more prevalent in virus harvested at 3 hr than at 24 hr. The contour length of both whorled forms was about the same and corresponds to a three-dimensional coil 1.1 – 1.5 μ long. Upon prolonged treatment of virus harvested at 3 hr with 0.01% SDS, the whorled structures could be liberated from the nucleoids. They appeared as strands about 1.18 μ long, and banded in CsCl at an average density of 1.343 g/ml. Their localization in the particle by UA staining, as well as their density suggest that these strands are nucleoprotein in nature. Models based on how the strand is packaged in the nucleoid are suggested to explain how the various internal forms could arise in the virus population.

The synthetic polytripeptides (Pro-Pro-Gly) 10 and (Pro-Pro-Gly) 20 form micro-crystalline structures similar to segmental structures formed by collagen

Previous studies demonstrated that (Pro-Pro-Gly) 10 and (Pro-Pro-Gly) 20 prepared by a specific modification of the Merrifield technique form triple-helical structures in aqueous solution similar to tropocollagen. In the present studies we used electron microscopy to demonstrate that at about 4 °C the same polytripeptides also form larger structures which we consider to be micro-crystalline segments. Examination of the micro-crystalline segments by negative staining with sodium silicotungstate demonstrated that one lateral dimension was relatively constant and was approximately equal to the theoretical length of the polytripeptides in a collagen-like triple helix. Also, when (Pro-Pro-Gly) 20 was negatively stained with uranyl acetate, smaller structures were seen which were of the same length as the “regular dimension” of the micro-crystalline segments and which probably were individual triple helices. Selective positive staining of either the amino-terminal or the carboxy-terminal groups indicated that the ends of the polytripeptide chains were packed in an anti-parallel fashion within the segments. The results obtained by both negative and positive staining are best explained by a scheme in which (a) the polytripeptide chains within triple helices are in register and parallel, and (b) the micro-crystalline segments are formed by the lateral aggregation of triple helices in an anti-parallel fashion.

Ultrastructure of exfoliation material (busacca deposits).

The peripheral exfoliation band was scraped from seventeen lenses that were removed because of senile cataract. A suspension of the exfoliation substance, negatively stained with sodium silicotungstate, was studied and compared with the sectioned exfoliation material. It is concluded that the exfoliation substance probably consists of a collagen material, that perhaps comes from basement membranes.

Ultrathin frozen sections. I. Methods and ultrastructural preservation.

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25-4% glutaraldehyde for 1-4 hr, are washed overnight in buffer at 3 degrees C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70 degrees C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35 degrees C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.

A modified method for the determination of total penicillin in procaine penicillin

AbstractA method is described for the chemical assay of total penicillin (salt) in procaine penicillin [penicillin (procaine salt)]. The method depends on the conversion of procaine penicillin to sodium penicillin [penicillin (sodium salt)] by precipitation with sodium silicotungstate, the sodium penicillin being assayed iodimetrically.

A method for the determination of procaine penicillin.

THE iodimetric estimation of procaine penicillin presents difficulties because of the interaction of procaine with iodine. Sodium penicillin to which an equivalent weight of procaine hydrochloride has been added gives results which may be from 3 to 5 per cent lower than for the untreated penicillin. Although the ferricyanide method of Hiscox1 is said to be free from the above objection, we could not obtain consistent results with it, a spread of ± 4 per cent being obtained in a series of determinations on a sample of sodium penicillin. The removal of the procaine ion by precipitation followed by iodimetric determination of the penicillin seemed the most promising line of attack, and sodium silicomolybdate and sodium silicotungstate were both found suitable for this purpose, although the latter possesses certain advantages over the former and is obtainable commercially. Both substances give with procaine salts heavy precipitates which are granular in the presence of sodium chloride (3 per cent) and can readily be centrifuged to a compact mass permitting decantation of the supernatant liquid. Precipitation is virtually complete, and neither precipitant has any action upon iodine or penicillin.