Autometallography: tissue metals demonstrated by a silver enhancement kit

In biological tissue, minute accumulations of gold, silver, mercury and zinc can be visualized by a technique whereby metallic silver is precipitated on tiny accumulations of the two noble metals, or on selenites or sulphides of all four metals. In the present study a silver enhancement kit, primarily intended for the amplification of colloidal gold particles, has been used to demonstrate these catalytic tissue metals. Sections from animals exposed intravitally to aurothiomalatate, silver lactate, mercury chloride, sodium selenite or perfused with sodium sulphide were subjected to a commercial silver enhancement kit (IntenSE, Janssen Pharmaceutica). It was found that the kit performs adequately to the silver lactate gum arabic developer and to the photographic emulsion technique. The kit can be used as a silver enhancement medium for the demonstration of zinc by the Neo-Timm and selenium methods and for demonstration of gold, silver, and mercury in tissues from animals intravitally exposed to these metals. It can also be used for counterstaining silver treated osmium fixed tissues embedded in plastic.     

几种植物组织及其原生质体过氧化物酶同工酶的比较研究

对马尾松幼苗子叶和胚轴、甘蔗、小麦、烟草、黄花菜等幼叶及其原生质体的过氧化物酶同工酶(POI)分别作比较研究。观察到凡经纤维素酶处理的各植物组织POI酶带数均多于未经纤维素酶处理的组织的酶带数;除个别例外,后者一般又比无壁原生质体的酶带数多。此种差异随植株生长年龄而增大,表明植物组织内大部分POI主要存于质外体中。     

A silver impregnation technique for the study of steroid receptors in central nervous system tissue prepared for autoradiography

The commonly used silver stains were found to be unsatisfactory for nervous tissue processed for autoradiography. A silver impregnation procedure for central nervous system tissues prepared for the autoradiographic study of steroid receptors is described. The procedure is a combination of several silver and reticular strains made up in solutions containing dimethylsulfoxide. The technique clearly distinguishes perikarya of neurons, brain nuclei and fiber tracts without substantial loss of silver grains, and thus greatly facilitates the identification of steroid receptor nuclei at all levels of the central nervous system.     

A Silver Impregnation Technique for the Study of Steroid Receptors in Central Nervous System Tissue Prepared for Autoradiography

The commonly used silver stains were found to be unsatisfactory for nervous tissue processed for autoradiography. A silver impregnation procedure for central nervous system tissues prepared for the autoradiographic study of steroid receptors is described. The procedure is a combination of several silver and reticular stains made up in solutions containing dimethylsulfoxide. The technique clearly distinguishes perikarya of neurons, brain nuclei and fiber tracts without substantial loss of silver grains, and thus greatly facilitates the identification of steroid receptor nuclei at all levels of the central nervous system.     

The Action of Polygalacturonase and Cellulase Enzymes of Botryodiplodia theobromae Pat. on Yam (Dioscorea spp.) and Sweet Potato (Ipomoea batatas) Tissues

The action of an endo-polygalacturonase PG3 and cellulase of Botryodiplodia theobromae, in the crude and pure forms, on sweet potato and yam slices was investigated. The purified isoenzyme PG3 and the crude pectic enzyme preparations macerated and killed the cells of both tissues although the former did so at a faster rate. Purified cellulase neither macerated nor killed cells from either plant. Although the action was delayed, the crude preparation of cellulase slightly macerated and killed the cells of sweet potato. Yam tissue was, however, not macerated by crude cellulase. Plasmolysis of the cells of both tissues delayed the toxicity and macerating action of the pure PG3 on the tissues. This delay in cell death was, however, not observed when the plasmolysed cells were treated with the crude cellulase preparation.     

Immunofluorescent localization of the gastrin-secreting G cells in the pyloric antrum of the pig

Summary An indirect immunofluorescence technique, using anti-human gastrin serum, was applied to formalin-fixed, paraffin-embedded material from antral tissues of porcine stomach. After photographing the result, post-fixation in formalin was followed by the Grimelius silver technique. The argyrophil G cells, situated predominantly in the middle zone of the gastric glands, all showed positive gastrin immunofluorescence. Some weakly fluorescent cells failed to stain by the silver technique, suggesting that the latter is less sensitive than immunofluorescence in that it depends on the presence of the granular storage product.     

Occurrence and Localization of 9.5 Cellulase in Abscising and Nonabscising Tissues

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.     

Silver Staining for Nucleolar Organizing Regions of Vertebrate Chromosomes

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.     

Silver staining for nucleolar organizing regions of vertebrate chromosomes

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Digestive enzyme pattern of two stomachless filter feeders, silver carp, Hypophthalmichthys molitrix Val., and bighead carp, Aristichthys nobilis Rich.

Activities of digestive enzymes (trypsin and amylase) of microplanktophagous silver carp, Hypophthalmichthys molitrix , and macroplanktophagous bighead carp, Aristichthys nobilis , were investigated in relation to pH in 10 different segments of the gut. In addition, the possible existence of 'classical' lysozyme in both species and cellulase in silver carp were scrutinized, but no activities were detected. Tryptic and amylolytic activities decreased sharply from fore-gut to hind-gut indicating an efficient reabsorption mechanism. Trypsin and amylase of both species had a pH optimum at 8.3 and 7.0 respectively. The relatively low pH of 6.4 in the fore-gut of silver carp did not support an efficient operation of these enzymes, which was compensated by a higher enzyme concentration as compared to bighead carp. The latter, however, showed a higher pH of 7.3 in the fore-gut and thereby maintained similar activities as silver carp. The distinction of herbivorous and omnivorous fish in terms of digestion mechanisms is discussed.     

Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.     

Fungal melanin detection by the use of copper sulfide-silver

Silver-staining procedures were investigated for their effectiveness in identifying cell wall-based fungal melanins in live and fixed plastic embedded samples, particularly 1,8-dihydroxynaphthalene (DHN) based polyketide melanins. We developed a simple and reliable melanin-staining technique based on a silver accumulation method originally published for histological demonstration of heavy metal sulfides in mammalian tissues. Copper is bound to fungal melanin followed by formation of the copper sulfide at melanin sites in fungal cell walls, which then are amplified into vivid black stains using a silver enhancement step. The method demonstrates patterns of melanization in a range of fungal hyphae and is suitable for light and electron microscopy. Albino mutant fungi and normally nonmelanized fungi do not stain with the sulfide-silver technique. Mammalian melanocytes also were labeled by the technique, indicating its universality as a melanin probe.     

The Effects of pH, Ionic Strength, and Ethylene on the Extraction of Cellulase from Abscission Zones of Citrus Leaf Explants

The solubility of cellulase extracted from the abscission zones of citrus leaf explants (Citrus sinensis L. Osbeck) in sodium phosphate buffer depends on the pH of the extracting solution and, to a lesser extent, on the ionic strength. By increasing molarity from 0.01 to 0.16, the solubility of cellulase increased from 51% to 89% at pH 6.1 and from 70% to 98% at pH 7. In all cases, residual cellulase was further extracted from the pellet by buffer containing 1 m NaCl. Most of the enzymic activity was found in tissues proximal to the separation line, and activity of the cellulase which was soluble in phosphate buffer was closely correlated with abscission at both pH values. When extraction of cellulase at pH 6.1 with phosphate buffer was followed by a reextraction of the pellet with buffer containing 1 m NaCl, the activity of the cellulase soluble in the fortified buffer was also correlated with abscission. Pretreatment of explants with ethylene increased the solubility of cellulase in the phosphate buffer regardless of the pH used at the first extraction.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level

Summary The argyrophilic proteins of the nucleolar organizer region (Ag-NOR proteins) were specifically localized at the optical level with a modified one-step silver technique performed at 20° C.This method was applied to various materials including cells in smears, chromosomes, semi-thin sections of plastic-embedded cells and sections of paraffin-embedded human pathological tissues.In order to improve the visualization of the silver deposits we tested various modes of imaging, including bright-field, Nomarski contrast, reflected light and combined Nomarski contrast with reflected light. The use of Nomarski contrast is useful to define precisely the phases of mitosis. The use of reflected light, which is based on the ability of silver to reflect incident light specifically, gives images with an improved resolution compared to bright-field.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Identification of enzymes that are effective for isolating protoplasts from grass leaves

Cellulase C₁, cellulase Cx, and xylanase were isolated and purified from a cellulase preparation of Trichoderma viride as enzymes effective in the isolation of protoplasts from oat leaves. Pectin lyase which is specific for methyl-galacturonide linkages was also found to be a useful enzyme for the isolation of protoplasts from the tissues. This suggested that pectic polysaccharides with a high degree of esterification may play an important role in cell walls of Gramineae. It was necessary to use the mixture of cellulase C₁, cellulase Cx, xylanase, and pectin lyase for the rapid isolation of protoplasts, while a small amount of protoplasts could be isolated from oat leaves by cellulase C₁ plus xylanase or cellulase C₁ plus pectin lyase. The mixture of four enzymes also was effective in the isolation of protoplasts from the leaves of wheat, barley, and corn.     

Modification of cellulase and its application to extraction of diosgenin from Dioscorea zingiberensis C.H. Wright

In order to enhance the thermostability and efficiency of cellulase in the extraction of diosgenin from Dioscorea zingiberensis C.H. Wright, we applied polyethylene glycol (PEG) (400, 1000, 2000, and 4000) to modify cellulase. The modified cellulase, α-amylase and β-glycosidase were used to hydrolyze the material. The results show that the thermostability of modified cellulase is better than that of natural cellulase, the optimum pH value and temperature of modified cellulase are wider than that of natural cellulase, the activity of cellulase modified by activated PEG2000 is higher than that of cellulase modified by other modifiers, and its remaining activity is 58% of its initial value. With this technique, the purity of the product reaches 96%, the melting point is 201–204 °C, the yield rate and the extraction rate of the diosgenin reaches 2.80% and 96.6%, respectively. IR spectra and ¹H NMR spectroscopy were used to confirm the structure of the product.