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PubMed1985

Colorimetric micromethod for the determination of protein in solutions with silver ions and dithizone.

A method of determination of protein based on the measurement of complexed Ag+ and AgCl absorbed on the macromolecules is described. This reaction is carried out in the presence of chloride ions and detergents (Triton X-100 or SDS) in alkaline solution. The silver ions are detected with dithizone. With this method it is possible to estimate 0.1 microgram bovine serum albumin in 0.5 ml sample with a coefficient of variation of 16%, and 1.5 micrograms/sample with a coefficient of variation of 3.6%. The method is simple, rapid, inexpensive, and very sensitive.

Azo Compounds↗
PubMed1978

Binding of mercurials to membrane suspensions and undenatured proteins.

Binding capacities of membrane suspensions and dissolved compounds for mercurials were titrated by a new potentiometric method. Critical steps included a silver electrode of new design, the use of L-cysteine as a thiol buffer, a nitrogen atmosphere, and pretreatment of samples with equimolar mercurial and cysteine. Titrations had a sharp endpoint, accurate +/- 26 nmole methylmercury or +/- 8 nmole mercuric salt. Measurements of binding capacity of bovine serum albumin averaged 93% of the titer predicted for one SH group per molecule; those of human hemoglobin yielded 86-91% of the titer predicted for two SH groups per molecule. Yields dropped with exposure of protein solutions or membrane suspensions to atmospheric oxygen. Brain microsomes had significantly higher binding capacities (per milligram of protein) than red blood cell ghosts. The ratio of endpoint titers of CH3HgCl to HgCl2 averaged 2:1 in assays of cysteine, proteins, and membranes, showing that the assay was free of denaturation artifacts and protein-protein interference. Solutions of EDTA showed measurable binding of Hg2+ but not of CH3Hg+. Satisfactory titrations were also obtained with N-ethylmaleimide.

Animals↗
PubMed1985

The carbohydrates of secretory granules and the glycocalyx in developing mucoid cells.

Complex carbohydrates in secretory granules and at the apical cell surface of mouse gastric mucoid cells were studied during embryogenesis and in the early postnatal period by various cytochemical methods; the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) and tannic acid-uranyl acetate (TA-UA) procedures made neutral mucosubstances (NMS) visible, whereas the hexose residues of glycoconjugates were identified using WGA-, RCA II- and ConA-ferritin. The glycocalyx was stained with ruthenium red (RR). During differentiation of the embryonic mucoid cells the number of secretory granules increased in parallel to the increase in their carbohydrate component. NMS-stainable parts in secretory granules also had binding sites for the conjugates RCA II- and WGA-ferritin, but the binding of ConA could not be identified. The increasing quantity of NMS in secretory granules was correlated with the increased amount of PA-TCH-SP and TA-UA positive substances in the apical glycocalyx only in 14- and 18-day-old embryos. The observed uniform affinity for RR and lectin conjugates in all analysed developmental stages remains to be explained.

Animals↗
PubMed2000

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.

Animals↗
PubMed1990

Application on Timm sulphide silver method for electron microscope localization of lead ions in blood cells.

The application of the Timm sulphide silver method for the demonstration of lead ions in peripheral blood cells is investigated, using male white adult Wistar rats treated with a single dose of lead acetate, range 150 mg/kg b.w. To improve the Timm reaction for electron microscopy, fixation of whole cells with glutaraldehyde fixative solution saturated with H2S, an agarose embedding and physical development of thick sections without prior cryostat sectioning are presented. At 6.0 hours after injection both erythrocytes as well as white cells reveal the positive Timm reaction. All types of blood cells contain numerous cytoplasmic precipitates illustrating the intracellular lead accumulation. It is shown that the invaginations of white cell nuclear membrane possess a storing function as areas of lead depots. As a rule, the neutrophils display a highest amount of cytoplasmic precipitates and exclusively a low amount of reaction products in basophils is observed. At 14 days after injection, precipitates are present only in erythrocytes and monocytes. A suggestion of a possible functional significance of changes in the Timm staining pattern in blood cell types is discussed in this paper.

Animals↗
PubMed2005

Detection of adsorption of Ru(II) and Os(II) polypyridyl complexes on gold and silver nanoparticles by single-photon counting emission measurements.

The present study describes a new application of ruthenium(II) tris(bipyridine) (Ru(bpy)3(2+)) and osmium(II) tris(bipyridine) (Os(bpy)3(2+)) as phosphorescent labels for the quantification of surface binding of molecules to gold and silver nanoparticles. The fraction of Ru(bpy)3(2+) and Os(bpy)3(2+) that is in solution can be distinguished from the surface-bound fraction by the relative lifetimes and integrated emission yields as determined by time-correlated single-photon counting (TCSPC) spectroscopy. Complementary steady-state measurements were carried out to confirm surface attachment of the phosphorescent label molecules. Although the emission of solutions of Ru(bpy)3(2+) and Os(bpy)3(2+) is quenched proportional to the concentration of 10 nm Au or 20 nm Ag nanoparticles, the quenching is static and not diffusional quenching observed in Stern-Volmer plots. The results demonstrate that time-resolved spectroscopy provides a rapid method for the measurement of surface binding of labeled molecules on metallic nanoparticles. While steady-state measurements require the preparation of a series of samples with varying quencher concentrations and a reference, the method described herein requires a single sample plus reference. The mechanism for phosphorescence quenching on Au and Ag nanoparticles is discussed in terms of energy and electron transfer theories.

Adsorption↗
PubMed2004

Surface-enhanced-Raman-scattering-inducing nanoprobe for spectrochemical analysis.

This paper describes a new nanoprobe that induces the surface-enhanced Raman scattering (SERS) effect when brought into contact with chemicals on any type of surface. The SERS-inducing probe was fabricated from an optical fiber that was tapered to a tip 100 nm in diameter. A thin layer of silver islands was applied to the tip of the tapered fiber via thermal evaporation to induce the SERS effect. The small scale of the tip may be amenable to localized, nondestructive SERS-based analyses of surfaces with high spatial selectivity. Because the contact probe itself induces the SERS effect, no modification of the sample is required. Direct analysis at submicrometer spatial selectivity is therefore possible for analyte compounds on any type of surface. Various optimization studies and preliminary evaluations were performed. A 10-nm silver thickness was determined to yield the optimum SERS effect. A 25% relative standard deviation in SERS signal was observed for five different probe tips. As a demonstration of the SERS-inducing capability of the probe, Raman spectra were recorded for glass surfaces coated with brilliant cresyl blue and p-aminobenzoic acid before and during contact with the SERS-inducing nanoprobe.

4-Aminobenzoic Acid↗
PubMed1990

Recovery of the pyrrolic nucleus of pyrrolizidine alkaloid metabolites from sulphur conjugates in tissues and body fluids.

Reactive pyrrolic metabolites formed when pyrrolizidine alkaloids are given to rats can alkylate soluble and tissue-bound thiol groups. Pyrrolic thioethers thus formed are relatively stable, and may persist in tissues for long periods. A simple procedure has been developed for recovering the nucleus of the pyrrolic metabolite from such S-binding, whether in solution or attached to solid tissues, in an easily identifiable form. The thioether bond was broken by silver nitrate and the pyrrolic moiety allowed to react with ethanol or methanol to form an ethoxy or methoxy derivative. The chemical basis of the procedure was established by model experiments on a preparative scale, but for small scale recovery from tissues, pyrrolic ethers were extracted and identified by TLC, HPLC, capillary GC and mass spectrometry. Because the pyrrolic derivatives thus formed were easily recognised and unrelated to any physiological compound, the recovery method described, especially when applied to blood samples, provided a way to monitor animals for previous exposure to toxic pyrrolizidine alkaloids.

Animals↗
PubMed2006

Neurotrophic effects of polyhydroxylated steroids and steroid glycosides in cultured neuroblastoma cells.

The effects of steroid compounds from Pacific Ocean starfishes were studied using cultured neuroblastoma C-1300 cells. Vital observations and examination of silver-impregnated preparations showed that the test substances in a concentration of 2-10 microM stimulate differentiation and improves survival of neuroblastoma cells under adverse conditions (similarly to neurotrophins). These substances in high concentrations (20-40 microM) had no effect or exhibited cytotoxic activity. The screening test allowed us to select several compounds for further studies of neurotrophic and neuroprotective properties.

Animals↗
PubMed1990

Degradation of metal-labeled collagen implants: ultrastructural and X-ray microanalysis.

Three different metal salts, silver nitrate, uranyl acetate and lead citrate, are mixed with a collagen gel to produce 3 metal/collagen sponges. These sponges were implanted subcutaneously in the rat and samples harvested after 5 days of implantation. TEM observation shows that sponges are degraded and digested by macrophages, polymorphonuclear cells (PMN) and fibroblasts. We have observed that the location of the precipitates differs according to the metal added to the collagen. Lead precipitates stay longer on the collagen mesh while silver precipitates, after 5 days, are soon digested and are found in phagosomes of macrophages. Uranium precipitates are digested with the collagen and uranium/collagen associated pictures are seen in phagolysosomes. Metal precipitates accumulated in phagolysosomes of macrophagic cells are recognized by X-ray microanalysis. The degradation process of implanted collagen is discussed.

Animals↗
PubMedDate not supplied

Skimmed-milk blocking improves silver post-intensification of peroxidase-diaminobenzidine staining on nitrocellulose membrane in immunoblotting.

A silver-staining procedure for enhancing the sensitivity of protein detection on nitrocellulose membranes in immunoblotting is described. After completing any peroxidase-Ni-diaminobenzidine immunostaining, nitrocellulose sheets are placed in a physical developer, containing sodium tungstate and ascorbic acid, until the desired grade of silver-intensification has been reached. In this way a 16- to 64-fold amplification of intensity of the initial immunostaining can be achieved. False positive silver staining of protein bands and of background are suppressed by replacing bovine serum albumin, the blocking agent most frequently used in immunoblotting, with skimmed milk.

3,3'-Diaminobenzidine↗
PubMed1989

Intra-operative spinal cord evoked potentials during cervical and lumbo-sacral microsurgical DREZ-tomy (MDT) for chronic pain and spasticity (preliminary data).

We have undertaken the intra-operative study of spinal cord surface evoked potentials in patients operated upon for pain and/or spasticity using the microsurgical DREZ-tomy (MDT) procedure. The goals of this work were 1) to collect data on spinal cord evoked potential components and 2) to analyze the effects of MDT on spinal cord physiology. The MDT consists of a therapeutic lesion in the ventro-lateral aspect of the dorsal root entry zone, directed to the activatory circuitry, and aiming at returning the dorsal horn physiology towards inhibition. Averaged evoked potentials to peripheral nerve electrical stimulations were obtained from various loci on the surface of the dorsal columns of the cervical and lumbo-sacral spinal cord in 19 patients, using a small uninsulated silver ball electrode. An initial far-field positivity was found, corresponding to a compound action potential in the proximal part of the brachial (or lumbo-sacral) plexus. Pre-synaptic compound action potentials were identified, most often composed of multiple successive sharp peaks. A post-synaptic field potential generated in the dorsal horn was recognized. The MDT caused an immediate and irreversible decrease of amplitude down to a disappearance of the dorsal horn potential. This decrement was proportional to the amount of operated cord segments. In contrast, there has been a relative post-MDT sparing of the pre-synaptic action potentials originating from the operated cord segments, and the scalp contralateral parietal N 20 has been only reversibly affected by the therapeutic lesion. We thus argue for a specific involvement of dorsal horn physiology by the MDT, with a relative sparing of the dorsal column system.

Electric Stimulation↗
PubMed1995

Distribution of neurons of origin of zinc-containing projections in the amygdala of the rat.

The present study describes the distribution of neurons of origin of zinc-containing pathways in the amygdaloid complex of the rat, using the selenium method for simultaneous retrograde labeling of all zinc-containing neurons. With this method, vesicular ionic zinc is precipitated intravitally with selenium compounds and transported retrogradely to the parent neurons, where it can be visualized by silver amplification. Neurons labeled retrogradely with silver-amplified precipitate were observed in all amygdaloid nuclei except for the lateral olfactory tract nucleus, the accessory olfactory tract nucleus and the central nucleus. Very few labeled cell bodies were seen in the anterior amygdaloid area and the medial nucleus. The amygdalo-hippocampal area and the amygdalo-piriform transition area both showed a substantial number of labeled somata throughout their rostrocaudal extent. In the anterior cortical nucleus, very few labeled cell bodies were found in the rostral pole, whereas they were abundant in the caudal quarter of the nucleus. In the posterolateral cortical nucleus, the number of labeled cell bodies increased gradually; there were none in the rostral pole, but most of the neurons in the caudal part were labeled. The posteromedial cortical nucleus contained a great number of labeled somata, but with some variation in the rostrocaudal extent of the nucleus. Considerable numbers of labeled neurons were observed throughout the lateral nucleus. In the basolateral nucleus, a small number of labeled cell bodies was present in the rostral half, but a gradual increase was observed in the caudal direction. Finally, in the basomedial nucleus, very few labeled cell bodies were present in the rostral two-thirds, whilst a considerable number was encountered in the caudal one-third. Possible functional implications of neuronal zinc are considered. The distribution of neurons of origin of zinc-containing projections has been compared with previously described intrinsic connections of the rat amygdala, and tracts that may possibly be zinc-containing are outlined and discussed. It is concluded that in all probability a substantial proportion of the intrinsic connectivity of the rat amygdaloid complex is zinc-containing.

Amygdala↗
PubMed1984

Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide.

A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.

Animals↗