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PubMed1983

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement.

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.

Aldehydes↗
PubMed1983

Ultrastructural localization of complex carbohydrates present in the sublingual gland of rabbits.

Stainings with dialized iron (DI), high-iron diamine-thiocarbohydrazide-silver proteinate HID-TCH-SP), tannic acid-uranylacetate (TA-U), tannic acid-ferric chloride (TA-F), and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) were applied to samples of sublingual glands of rabbits. Comparing the results obtained with what had been previously demonstrated biochemically, it is possible to assert that the faintly electron-opaque, granules of the cells of the preterminal tracts contain sulphated glycoconjugates because of the presence of sulphated hexosamines and hexoses, acid glycoconjugates because of the presence of sialic acid and uronic acids, and neutral glycoconjugates because of the presence of hexoses.

Animals↗
PubMed1983

Periodate-labile structures at the normal human cutaneous basement membrane zone.

The distribution of carbohydrates containing 1,2-glycol groups at the basement membrane zones of the dermo-epidermal junction and dermal blood vessels was investigated using the periodic acid-thiosemicarbazide-silver proteinate technique. Controls included pre-incubation with dimedone, dimedone treatment both before and after periodate oxidation, and dimedone treatment followed by thiosemicarbazide and silver proteinate. At the dermo-epidermal junction the anchoring fibrils were stained selectively after both short (45 min) and long (71 h) thiosemicarbazide incubation periods, there being no staining of the lamina densa; however, the lamina densa surrounding blood vessels was labelled. After prolonged treatment with thiosemicarbazide, fibrils were observed traversing the lamina lucida at the dermo-epidermal junction, and collagen fibres were stained faintly. Pre-incubation with dimedone to block aldehyde groups did not affect the staining. However, when periodic acid treatment was omitted, or when sections were incubated with dimedone both before and after periodate oxidation, only melanin granules and the cytoplasm of fibroblasts were stained. Thus, anchoring fibrils are rich in carbohydrates containing 1,2-glycol groups susceptible to periodate oxidation, and correspond to the periodic acid Schiff-positive layer seen at the dermo-epidermal junction. Furthermore, the basement membrane zones of dermal blood vessels and the dermo-epidermal junction differ in their carbohydrate distribution.

Adult↗
PubMed1979

Ultrastructural localization of complex carbohydrates in rat monocytes and peritoneal and alveolar macrophages.

Complex carbohydrates are demonstrated in lysosomes and other organelles of rat monocytes and macrophages by means of ultrastructural morphochemical techniques for localizing acidic groups and vicinal glycols that are characteristic of glycoconjugates. The presence of glycoconjugates in various cell organelles differ in monocytes and macrophages from different sites. Bone marrow and blood monocytes contained glycoproteins with periodate-reactive vicinal glycols, as shown by the periodate-thiocarbohydrazide-silver proteinate sequence in lysosomes, a population of small vesicular structures, and Golgi lamellae. However, these cells contained no dialyzed iron or high iron diamine reactivity indicative of acidic groups. Alveolar macrophages were similarly periodate-thiocarbohydrazide-silver proteinate-reactive, and contained a variable degree of dialyzed iron and high iron diamine reactivity demonstrative of acid mucosubstances in lysosomes. Peritoneal macrophages, on the other hand, exhibited highly reactive lysosomes with all three techniques, and revealed a population of periodate-thiocarbohydrazide-silver proteinate-positive vesicles not previously recognized and not readily discernable by other means. These cells also displayed periodate-thiocarbohydrazide-silver proteinate staining in large cisternal elements, possibly related to Golgi-endoplasmic reticulum-lysosome (GERL) and in other Golgi complex-related elements, as well as in cisternae of rough endoplasmic reticulum.

Animals↗
PubMed1995

Modulation of melanogenic protein expression during the switch from eu- to pheomelanogenesis.

Mammalian melanocytes can produce two basic types of melanin, eumelanin and pheomelanin, within discrete organelles termed melanosomes. The physiological signals that regulate this switch are extrinsic to the melanocyte, and include alpha-melanocyte stimulating hormone and the agouti protein. Tyrosinase, encoded at the albino locus, is the enzyme essential for the synthesis of both types of melanin, but other tyrosinase-related proteins (e.g. TRP1 encoded at the brown locus and TRP2 encoded at the slaty locus) regulate eumelanogenesis catalytically at steps distal to tyrosinase (as 5,6-dihydroxyindole-2-carboxylic acid oxidase and DOPAchrome tautomerase, respectively). The silver protein is another melanosomal protein, and although it has some limited homology to the tyrosinase-related proteins, it does not have any known enzymatic function and probably serves as a structural matrix protein. The role of each of those melanosomal proteins in pheomelanogenesis, however, is still unclear. In this study, we have compared the expression and catalytic functions of those proteins in pheomelanic and eumelanic hair bulb melanocytes. There was no detectable expression of TRP1 or TRP2, or either of their enzymatic activities, in hair bulbs of lethal yellow (Ay/a) newborn mice, and tyrosinase activity was present at a reduced level compared to that found in hair bulbs of black (a/a) newborn mice. Similar results were observed in regenerating hair bulbs of adult lethal yellow mice and in hair bulbs of 5- to 7-day-old agouti mice (A/A), an age where pheomelanin is produced predominantly. Expression of the silver protein was similarly not observed in hair bulbs of the pheomelanic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

[Silver staining of proteins separated by polyacrylamide gel electrophoresis: mathematical model].

Silver staining of proteins separated by electrophoresis in the thin layer of polyacrylamide gel can be quantitatively measured when the results are statistically treated. In the mathematical model, the reduction of protein-bound silver ions (crucial silver staining step) is considered to be autocatalytic. According to the model, the reaction rate strongly depends on the stability constant of silver complexes with functional groups of protein amino acids; and the shape of the calibration curve is determined by the ratio of concentration of silver-binding side chain groups to the reciprocal value of the stability constant. The complex shape of the curves can be explained the combination of autocatalysis and saturation. High sensitivity of this reaction to experimental conditions, contribution of complexes with different stability constants to the intensity of protein band staining, and optical effects are analyzed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Interaction of radiolabelled Na,K-ATPase-liposomes with human peripheral blood mononuclear cells.

Artificial phospholipid vesicles (liposomes) containing in their membrane about eight Na,K-ATPase (sodium pump) molecules per vesicle were incubated in the presence of [110mAg]silver nitrate to label the membrane protein; silver binds specifically to the Na,K-ATPase protein. When such silver-labelled liposomes were incubated with freshly isolated human peripheral blood mononuclear cells, a large number of liposomes was found in cells as evidenced by their 110mAg content after washing them with powerful silver chelators. Thus, liposomes containing an integral membrane protein can be transferred to human peripheral blood mononuclear cells rapidly and without toxicity.

Cell Separation↗
PubMed2000

Improvement of silver impregnation technique (protargol) to obtain morphological features of protists ciliates, flagellates and opalinates.

The research on ciliates, flagelates and opalinates have been widespread by the utilization of techniques employing silver impregnation (protargol), modified by several authors. However, these are time consuming and its results are variable. The present work is a variant of the technique described by Tuffrau (1964, 1967) showing some adaptations made in our laboratory. The organisms can be preserved by different fixatives (alcoholic Bouin, Stieve's fluid, 2.5% glutaraldehyde and others) and then rinsed in destilled water followed by a fast clarification by 3% sodium hypochloride. If the organism is very sensitive to hypochloride, 4% sodium lauryl sulfate may be used and then washed 3 times in distilled water. The protista can be adhered to the glass slides with Mayer's glycerinated-albumin (1 glycerin vol. to 1 or 2 albumin vol.), diluted in water at a proportion of 1:10 Cv/v., or with 1% polylysine followed by fast washes with distilled water. After the slide preparation, they were covered with a layer of 0,8% Silver proteinate. Right after that, the slide has to be placed in a glass tray lined with moist tissue and covered to prevent the proteinate to dry. The tray was placed in a incubator at 40 degrees - 50 degrees C for 30 minutes. The slides are rinsed for 1 minute. with warm (35 degrees C) distilled water. The development of the material should be done with 0.4% hydroquinone with a maximum incubation time of 1 minute. It should be developed gradually, controlling the silver impregnation intensity by observation under optical microscope. Next, rinse in distilled water for 1 minute, and then, fix in 2,5% Sodium thiosulfate. Rinse the slide for two minutes before dehydrating it in an alcoholic serial 50-100 degrees. Finally rinse the slides in xylene. Mount the slides with Entellan MerckTM or Canada balsam.

Animals↗
PubMed1983

A sensitive silver stain for proteins in agarose gels.

A silver stain for proteins in agarose gels which is at least 10 times as sensitive as Coomassie blue is described. The method is simple to use and is particularly useful for the study of protein bands in the gamma region on electrophoresis of fluids such as cerebrospinal fluid in which the protein concentration is low. It readily detects bands of IgG containing 20 to 40 ng/band (approx 3 to 6 ng of IgG/mm2 of gel).

Electrophoresis, Agar Gel↗
PubMed1982

Quantitative autoradiography, electron microscopy, and granulometry of endocrine cells in the rabbit colon.

A high-resolution autoradiographic study was conducted on 268 thin sections of endocrine cells in the rabbit colon following injection of (3H) L-dopa. Using quantitative autoradiography, silver-proteinate impregnation, granulometry, and statistical analysis of cell populations by the Falck procedure, three cell types were identified--EC, L; and H--as well as a single cell with distinct ultrastructural characteristics. For the first time, a fine quantitative estimation of the handling ability of amine precursor related to the cross-sectional areas of each cell type was obtained, using a Kontron Digiplan image analyzer. All cells studied showed the ability to take up the precursor. Labeling indices were 6.63, 2.15, and 2.14 for EC, L, and H, respectively. These data and silver-proteinate impregnations provide good criteria for differentiating EC from H cells despite morphological similarities. After critical analysis of granule diameters, L cells were considered to be pluripotential in secretory activity.

Animals↗
PubMed1981

Electron microscopic studies of silver-strained proteins in nucleolar organizer regions: location in nucleoli of rat sympathetic neurons during light and dark periods.

Ag-AS staining of nucleolar organizer regions was carried out on interphasic superior cervical ganglia neurons of rats sacrificed during light and dark periods. While the Ag-AS technique has mostly been used monolayer cell lines or cell suspensions, the present study showed that in electron microscopy this technique is also applicable to small pieces of tissues. The finest pictures are obtained when (I) all solutions used for the staining procedure are at pH 4.5-4.7 and (2) the second step of the reaction involving ammoniacal silver and formalin developing solutions does not exceed 3 min. The results indicate that in the 2 time periods studied, a positive reaction took place exclusively in nucleolar fibrillar centres and in the fibrillar centres and in the fibrillar ribonucleoprotein (RNP) component (dense fibrillar component). The other nucleolar components, i.e. granular and vacuolar, were devoid of silver deposits. As previously shown in sympathetic neurons, the fibrillar centres of the nucleoli show a 10-fold increase in volume during the dark period. In this period, silver grains were located on both "giant" and small-sized fibrillar centres. The fibrillar RNP component seen either at the periphery of fibrillar centres or in the form of a well-delimited network showed the strongest reaction. The same distribution of silver grains was observed in the sympathetic neurons of rats sacrificed during the light period. Here again, silver accumulation occurred exclusively in the fibrillar centres and the fibrillar RNP component. The same difference in reactivity was observed as for the dark period, the fibrillar RNP component being the main site of the reaction.

Animals↗
PubMed1980

Ultrastructural cytochemistry and radioautography of complex carbohydrates in heterophil granulocytes from rabbit bone marrow.

The subcellular route of incorporation of complex carbohydrates into rabbit heterophil primary granules and their subsequent intragranular distribution during granule maturation were studied with ultrastructural, cytochemical, and radioautographic methods. High iron diamine (HID) staining of sulfated glycoconjugates in primary granules was partially diminished after treatment with chondroitinase ABC or after removal of N-sulfate groups with nitrous acid, but was not altered by exposure to hyaluronidase, trypsin, or HCl. Subsequent thiocarbohydrazide-silver proteinate (TCH-SP) straining of thin sections increased the density of the HID reaction product. Golgi-derived spherules and very immature morular granules stained weakly with HID-TCH-SP and labeled intensely after a 10 min incubation with 35SO4. After a 60 min 35SO4 pulse and a 60 min chase, an increase in radiolabeling was observed in granules with HID stained, fused morular material, and some labeling was present in more mature rim stained granules. Fully mature granules lacked HID or HID-TCH-SP staining, but contained most of the 35SO4 labels after a 60 min pulse and 18 hr chase in vitro. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing complex carbohydrates in Golgi-associated small vesicles. These vesicles lacked HID-TCH-SP staining and apparently contained neutral glycoprotein. They frequently bordered, in a rosette arrangement, the immature morular granules, but not the more mature primary granules. The PA-TCH-SP method localized complex carbohydrates in the rim of granules precursors and enclosed a spherule or morula, but failed to stain the sulfate-containing material in the morulas or spherules. PA-TCH-SP reactivity was diffusely distributed in moderately mature granules and was decreased in fully mature granules. These results indicate that heterophil primary granule contain several complex carbohydrates including O-sulfated and N-sulfated glycosaminoglycans, as well as vicinal glycol-containing glycoproteins. These complex carbohydrates are transported to immature primary granules by different Golgi-derived organelles. The complex carbohydrates are subsequently distributed differently within primary granules and become masked to staining as the granule matures.

Animals↗
PubMed1980

Application of the thiocarbohydrazide method for vicinal glycol group detection to the study of gastric mucosa endocrine cells.

The thiocarbohydrazide-silver proteinate (TCH SP) method was applied to the study of cat, rabbit and mouse gastric mucosa endocrine cells. After 24-h treatment with thiocarbohydrazide (TCH), glycogen was seen in the hyaloplasm of X, D, P, A and O cells but not in EC, EC-like or D1 cells. With flotation times as short as 30 to 40 min glycogen was readily detected in X cells. Secretory granules of EC cells were constantly stained, while those of D1 cells failed to react. In most experiments granules of X, A and O cells showed peripheral "staining", while in others staining of variable intensity affected the entire granular cross-section in X, D and P cells. With 72-h exposure to TCH, EC and EC-like cells showed particles resembling glycogen, even staining or only peripheral staining of certain EC cell granules. From the results of this and previous studies, EC cell staining is believed to be due wholly or partly, according to exposure times, to the action of silver proteinate, while that of certain non-EC cells is probably a specific indicator of complexed carbohydrates.

Animals↗
PubMed2003

Fluorescence properties of labeled proteins near silver colloid surfaces.

The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to several hundredfold or larger with two-photon excitation. The probes were moderately more photostable in the presence of silver islands. There was also a dramatic decrease in the lifetimes with the amplitude-weighted values decreasing from 7- to 35-fold. The data suggest that these spectral changes are due to both increased rates of excitation near the metallic particles and increases in the rates of radiative decay. Because these silver island surfaces are very heterogeneous, we are hopeful that larger increases in intensity and photostability can be obtained for probes situated at an optimal distance from the ideal island surfaces.

Avidin↗
PubMed2001

Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor.

H(2)O(2) and other reactive oxygen species are key regulators of many intracellular pathways. Within mammalian skin, H(2)O(2) is formed as a byproduct of melanin synthesis, and following u.v. irradiation. We therefore analyzed its effects on melanin synthesis. The activity of the rate-limiting melanogenic enzyme, tyrosinase, decreased in H(2)O(2)-treated mouse and human melanoma cells. This inhibition was concentration- and time-dependent in the B16 melanoma model. Maximal inhibition (50-75%) occurred 8-16 hours after a 20 minute exposure to 0.5 mM H(2)O(2). B16 cells withstand this treatment adequately, as shown by a small effect on glutathione levels and a rapid recovery of basal lipid peroxidation levels. Enzyme activities also recovered, beginning to increase 16-20 hours after the treatment. Inhibition of enzyme activities reflected decreased protein levels. mRNAs for tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, silver protein and melanocortin 1 receptor also decreased after H(2)O(2) treatment, and recovered at different rates. Downregulation of melanocyte differentiation markers mRNAs was preceded by a decrease in microphthalmia transcription factor (Mitf) gene expression, which was quantitatively similar to the decrease achieved using 12-O-tetradecanoylphorbol-13-acetate. Recovery of basal Mitf mRNA levels was also observed clearly before that of tyrosinase. Therefore, oxidative stress may lead to hypopigmentation by mechanisms that include a microphthalmia-dependent downregulation of the melanogenic enzymes.

Animals↗