Protargol Research Articles

Studies of the uptake and binding of trace metals in fungi. Part I: Accumulation and characterization of mercury and silver in the cultivated mushroom, Agaricus bisporus

AbstractThe champignon mushroom Agaricus bisporus was cultivated on compost labelled with the radioactive traers 110mAg and 203Hg and the uptake of these two metals studied. The radiotracer then served as an aid in elucidation and characterization of the fungal metal–protein species using gel chromatography. In the case of the silver–protein complex, but not of mercury, the majority of the metal was contained in the fractions of intermediate (ca 8000–10000 Da) molecular weight possibly corresponding to metallothioneins (MT) or MT‐like species.

Ultrastructure and cytochemistry of extramural substances associated with intercellular hyphae of several rust fungi

Several types of extramural substance(s) associated with rust fungal intercellular hyphae were identified using a variety of tissue processing techniques. With conventional glutaraldehyde–OsO4 fixing and uranyl acetate – lead citrate staining, little material could be discerned on the hyphal surfaces in nonsporulating areas except at locations of cell–cell contact, where a lightly staining fibrous or darker staining amorphous material was apparent. Freeze-substitution or freeze-fracturing preserved greater amounts of coating material, which could be distinguished from the outer fungal wall layers. In freeze-substituted samples the extramural material was amorphous in nonsporulating areas, whereas near sporulating zones it had a fibrous consistency, with the fibrils oriented perpendicularly to the fungal wall. At locations of cell–cell contact there was additional extramural material that was composed of randomly oriented fibrils or was amorphous and densely staining. All types of extramural material stained positively with the periodate – thiocarbahydrazide – silver proteinate or periodate – chromate–phosphotungstate stains, but Concanavalin A bound only to some of the dense amorphous material. Sodium ethoxide etching and platinum–carbon shadowing also revealed the extramural material. When rust-infected wheat leaves were flooded with suspensions of colloidal gold, adhesion of gold particles occurred outside the hyphal walls and coincided with the location of extramural material.

Effect of tunicamycin on glycogen accumulation in the stratum intermedium and odontoblasts of rat incisor.

Repeated injection of rats with tunicamycin over two days induced a 1- to 5-fold increase in glycogen. This accumulation occurred in the stratum intermedium of the enamel organ and in young secretory odontoblasts. In rats injected over 3 days, the number of glycogen particles was at least 10 times larger than in control rats, and large glycogen accumulations were observed in the cytosol of these two groups of cells. These results were obtained by staining with periodic acid-thiocarbohydrazide and silver proteinate, a specific method for the detection of glycoconjugates containing vic-glycol groups. The existence of a relationship between these local cytosolic accumulations of glycogen and the developmental stage of certain groups of cells was shown by the changes that occurred in glycogen distribution. The present results suggest that the stratum intermedium supplies energy for precursor transport.

Histochemical localization at the electron microscopic level of sulfated glycosaminoglycans in the rat gingiva

Using the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique, we investigated ultrastructural localization of sulfated glycosaminoglycans (GAGs) in the rat gingiva shortly after eruption, especially those associated with internal and external basal laminae. In the apical portion of the internal basal lamina, HID-TCH-SP stain deposits were distributed mainly in the region of the lamina lucida located between the lamina densa and the distal surface membrane of the junctional epithelium and inside the depression of the distal surface membrane adjacent to the basal lamina. Stain deposits were also detected on the surface membrane of the cytoplasmic protrusion. Interestingly, the density of HID-TCH-SP stain deposits in the internal basal lamina was highest in the apical portion of the junctional epithelium and decreased in the coronal direction, finally tending to disappear completely. On the other hand, in the external basal lamina the deposits were localized in the whole region of the basal lamina or at both sites of the lamina densa. HID-TCH-SP stain deposits were also detected external to the lamina densa in the basement membrane associated with capillaries and in the connective tissue where they were distributed in close relation to collagen fibrils. Testicular hyaluronidase digested most HID-TCH-SP stain deposits in the connective tissue, whereas those in the region of basement membranes resisted this enzymatic digestion.

Ultrastructural cytochemistry of hepatic lysosomes and their protein components is selectively revealed by the ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate reaction.

Proteins in lysosomal membranes, lysosomes and within the transtubular network are readily accessible for electron microscopic analysis by a new three-step method. Oxidative deamination of tissue-bound amino acids by ninhydrin in aqueous dimethyl sulfoxide and the concomitant formation of corresponding carbonyl groups comprise the first step. The addition reaction of thiocarbohydrazide to tissue-bound carbonyl groups comprises the second step, while the reduction of silver proteinate by tissue-bound thiocarbohydrazones is the final step of this sequential method. Glutaraldehyde-fixed and osmified ultrathin sections of rat liver embedded in LR White were oxidatively deaminated for 24 h by 1% w/v ninhydrin in aqueous 75% v/v dimethyl sulfoxide (DMSO). They were then incubated for 40 min in aqueous 1% w/v thiocarbohydrazide (TCH) and stained for 30 min at 50 degrees C with silver proteinate (SP). The ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate (N-DMSO-TCH-SP) reaction proved to be chemically specific and highly selective for ultrastructural resolution of the internal structure of lysosomes and their protein components. We conclude that the N-DMSO-TCH-SP reaction is the method of choice for cytochemical elucidation of the protein ultrastructure of lysosomes and their enzymatic aggregates.

Histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus

I investigated the ultrastructural localization and histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus, by use of the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. HID-TCH-SP stain deposits were localized in the dental basal lamina and in the whole thickness of developing enameloid matrix, particularly closely associated with enameloid collagen fibrils. Most HID-TCH-SP stain deposits in the enameloid were susceptible to testicular hyaluronidase but some stain deposits survived. HID-TCH-SP stain deposits in the basal lamina resisted the enzymatic digestion, and were regularly localized to the internal and external sites of lamina densa at an early stage of development, subsequently tending to be randomly arranged with the increase in thickness of enameloid matrix layer. Furthermore, enzymatic digestion with heparitinase preferentially removed HID-TCH-SP stain deposits in the region of the basal lamina. Thus, it was confirmed that most HID-TCH-SP stain deposits in developing enameloid matrix are chondroitin 4-sulfate and/or 6-sulfate and that the stain deposits in the basal lamina represent heparan sulfate. The chondroitin sulfates tended to disappear with the advancement of enameloid mineralization.

Host cell responses in susceptible hard pine tissue infected with Endocronartium harknessii

Compatible interactions between susceptible hypocotyl tissue of Pinus banksiana Lamb, and Endocronartium harknessii (J. P. Moore) Y. Hirat. were studied using light and transmission electron microscopy. Host endoplasmic reticulum was observed to be closely associated with the haustorial body, although staining with silver proteinate failed to show any similarity between the contents of the endoplasmic reticulum and extrahaustorial matrix. The haustorium was also commonly observed to be closely associated with the host nucleus, often indenting the latter, though never in direct contact. Chloroplasts in recently infected cells appeared similar to those in uninfected cells, but in more advanced infections large starch grains were observed in the chloroplasts of the outer cortex; such chloroplasts normally contained little starch. Collars were another common feature of infected cells. Collars were continuous with the host cell wall and reacted to silver proteinate in a similar manner to the cell wall; callose was not evident. Collars were associated with portions of the cell wall that were inwardly displaced by the fungus; however, cytoplasmic vesicles were also observed in association with the collar and possibly contributed to their development.

An ultrastructural study, including cytochemistry and quantitative analyses, of the interactions between pseudomonads and leaves of Phaseolus vulgaris L

Infection of Phaseolus vulgaris cultivars Red Mexican and Tendergreen with Pseudomonas fluorescens, P. syringae pv. coronafaciens, P.s. pv. phaseolicola races 1 and 3, and a mutant of race 3 (race 3 M1) with altered cultivar specific virulence was examined. In addition to qualitative observations of the development of colonies of bacteria and the responses in adjacent plant cells, quantitative analyses were made of the numbers of bacteria within sections of colonies, contact between bacteria and the plant cell wall, the accumulation of fibrillar acidic polysaccharides (which stained with ruthenium red) around bacteria, convolution of the plant cell membrane adjacent to bacteria, deposition of paramural papillae, rupture of the tonoplast and the occurrence of cytoplasmic disorganization. The presence at infection sites of material staining with the periodic acid, thiocarbohydrazide silver proteinate (PATAg) procedure to localize vicinal glycols was also quantified. Cells of P. fluorescens failed to multiply in the plant and seemed tightly bound at junctions between mesophyll cells. The pathovars of P. syringae all multiplied at similar rates during the first 12 h after inoculation and were not closely attached to the plant cell wall. Fibrillar, ruthenium red staining material, considered to be bacterial extracellular polysaccharides, accumulated around cells of the pathovars of P. syringae irrespective of the compatibility of their interaction with cultivars. Amorphous PATAg positive deposits formed around cells of the saprophyte and as condensed aggregates in colonies of P. syringae pathovars in tissue undergoing the hypersensitive reaction (HR) but not during compatible interactions. The PATAg positive material may be involved in the elicitation of responses by plants during the HR. The first response of plant cells to adjacent bacteria was the localized convolution of the plasma membrane; this response was neither race nor species specific. Deposition of paramural papillae was also found to be a non-specific response occurring during compatible and incompatible interactions. Some components of papillae were PATAg positive. Plasmolytic studies demonstrated that, during the HR, irreversible damage to the plasma membrane first occurred 5 and 8 h after initial convolution of the membrane following inoculation of cv. Tendergreen with P.s. pv. coronafaciens and P.s. pv. phaseolicola race 3 respectively. Tonoplast dysfunction appeared to follow irreversible damage to the plasma membrane and preceded loss of compartmentation and cytoplasmic collapse. The ultrastructural study showed that many plant responses were non-specific and therefore could be separated from irreversible damage to the plasma membrane which was the irrevocable event during the HR.

Argyria or cyanosis

A case of argyria secondary to the prolonged use of nasal drops that contained mild silver protein is presented. The patient was thought to be cyanosed immediately after a total knee replacement. Argyria, its differentiation from cyanosis and its prevention are discussed. The need for a careful drug history is noted.

Cytological studies of the M-haustorium of Endocronartium harknessii: morphology and ontogeny

M-haustoria of the endocyclic rust Endocronartium harknessii (J. P. Moore) Y. Hirat. were examined with light and transmission electron microscopy in infected seedlings of Pinus banksiana Lamb. The haustoria developed from unspecialized cells of the intercellular hyphae, each of which appeared capable of producing several haustoria. The haustoria were distinct from the intercellular hyphae in possessing a narrow septate neck region which terminated in a globose haustorial body. Periodic acid – thiocarbohydrazide – silver proteinate and periodic acid – chromic acid – phosphotungstic acid staining provided evidence of an additional wall layer in the haustorial neck not evident in the intercellular hyphae and suggested that the extra-haustorial matrix contained polysaccharides of mixed linkage as well as lipids. However, cellulase extraction and the use of gold-bound wheat-germ lectin showed that neither cellulose nor chitin, respectively, was a component of the matrix. Both the haustoria and the matrix were separated from the host cytoplasm by the extrahaustorial membrane. This membrane stained positively with periodic acid – chromic acid – phosphotungstic acid, while the noninvaginated portion of the host plasmalemma with which it was continuous usually did not. The matrix fluoresced strongly when stained with aniline blue in an apparently compatible reaction. Other stains such as analinonapthalenesulphonic acid and Calcofluor white showed evidence of protein and polysaccharide in the fungal walls. Light and transmission electron microscope observations showed that penetration pegs formed as narrow tubular evaginations of the haustorial mother cell which caused inward displacement of the host cell wall. They retained their peg-like appearance as they entered the cell lumen, but eventually their distal ends enlarged to form typical globose haustorial bodies.

Cortical Morphogenesis during Excystment in Histriculus similis (Hypotrichida: Oxytrichidae)1

ABSTRACT Cortical morphogenesis during excystment in Histriculus similis has been studied by light microscopy of preparations impregnated with silver proteinate (protargol). This morphogenesis shows clear differences from cortical morphogenesis during division. The oral, paroral, and fronto‐ventro‐transverse primordia originate from an extensive field of kinetosomes. The marginal cirral primordia and the dorsal bristle primordia appear at an early stage of morphogenesis. The left marginal cirral primordium originates from the oral primordium.

Enhancement of the periodic acid--Schiff (PAS) and periodic acid--thiocarbohydrazide--silver proteinate (PA-TCH-SP) reaction in LR white sections.

LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination: 1) The PAS reaction in semithin sections turns out stronger after partial (70% ethanol) than complete (100% ethanol) dehydration of the tissue before its transfer to 100% LR White. 2) Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end. 3) The use of hot silver proteinate (50 degrees C) plus strong silver enhancement (15-20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.--Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.

Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells.

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.

Ultrastructural observations on Helvellaceae (Pezizales). Ascosporogenesis of selected species of Helvetia

The ultrastructure of ascosporogenesis including observations on spore delimitation, primary wall deposition, and formation of the secondary wall is examined in three species of the genus Helvella. The double membranes delimiting the spores and the ascus plasmalemma stain identically with silver proteinate poststain. Primary wall formation is similar in all species examined, although very early development was observed only for H. macropus. Secondary wall formation, which results in the characteristic spore ornamentation, appears to be quite similar for the three species. In addition, observations are made on the structure and origin of the epispore and on cytological details of the epiplasm and sporoplasm relating to spore ontogeny.

Peroxidase-containing microgranules in human neutrophils: physical, morphological, cytochemical, and secretory properties

A microgranule fraction, isolated from human neutrophils by using a novel high-resolution Percoll density gradient system contained granules with the lowest density and diameter when compared with 12 other isopycnic granule fractions. Ultrastructurally, from 34% to 50% of the microgranules showed homogeneous diaminobenzidine (DAB) staining under conditions for localizing peroxidase reactivity. The presence of myeloperoxidase (MPO) was further confirmed by biochemical and spectral analysis and immunodiffusion methods. Periodate-thiocarbohydrazide- silver proteinate (PA-TCH-SP) intensely stained vicinal glycols in the matrix of greater than 97% microgranules in contrast to the weak or absent staining seen in larger primary granules. Directly sampled segmented neutrophils contained small DAB- and PA-TCH-SP-positive granules, which often appeared in clusters. These DAB-positive microgranules selectively remained within the cells after stimulation of exocytosis with the calcium ionophore A23187. The enriched DAB- positive microgranule fraction recovered from A23187-treated cells also contained lysozyme and beta-glucuronidase but lacked vitamin B12 binding protein activity. A similar small, DAB- and PA-TCH-SP-positive granule type was also identified in normal promyelocytes and was the predominant or only granule type observed in leukemic or preleukemic myeloid cells from four patients. This study demonstrates a unique subpopulation of MPO-containing microgranules in normal and leukemic human myeloid cells that are distinguished from (other) primary granules by their extremely low density, small size, content of complex carbohydrates, and resistance to secretion.

Histogenesis of alveolar cell carcinoma

Alveolar cell carcinoma (ACC) is a lung neoplasm characterized by the presence of lamellar bodies specific for normal type 2 alveolar cells. Tumor histogenesis is uncertain. The present study indicates that ACC originates from dedifferentiation of hyperplastic type 2 alveolar cells rather than migration of bronchial stem cells into alveoli as suggested earlier.An aliquot of human lung biopsy diagnosed as ACC was fixed in glutaraldehyde and osmium, treated with 1% aqueous uranyl acetate, and embedded in epoxy resin. Sections were stained for glycogen by periodic acid - thiosemicarbazide - silver proteinate, and post-stained by uranyl acetate and lead citrate.The tumor contained morphologically distinct cell clusters. Each cluster consisted of identical cells. Differences between clusters resulted from synchronous alterations in lamellar bodies, mitochondria, glycosomes, and ribosomes. These alterations revealed distinct stages in cell differentiation classified here as follows: stage 1-hyperplastic cells (Fig.1), stage 2-dedifferentiating cells (Fig.2), stage 3-undifferentiated cells (Fig.3), stage 4-differentiating cells (Fig.4).

Changements cytochimiques et ultrastructuraux des parois cellulaires de la pellicule du raisin, Vitis vinifera, durant la croissance et la maturation de la baie

Cytochemical and ultrastructural analysis of cell wall characteristics was performed in the dermal system of the grape berry (Vitis vinifera L. cv. Pinot noir), using a test for polysaccharides (periodic acid – thiosemicarbazide – silver proteinate, associated with dimethylsulfoxide extraction). Samples were examined at successive stages of fruit development. The main events of the first growth phase, i.e., thickening of hypodermal cell walls and their tangential extension, continued during the second growth phase. At all the stages of grape development, tangentially expanding walls were typical of actively growing plant cell walls, with an ordered texture, new layer deposition, and progressive rotation of microfibrillar subunits (bow-shaped patterns). At maturity, hydration, swelling of the walls, and an increase in the amount of soluble pectic substances were noticed. No redifferentiation of the cell walls occurred. The cuticle, already differentiated into two layers at the first growth phase, was clearly lamellar during the second. At maturity a number of layers, distinguishable by their cytochemical reactivity, were detected. Cuticle thickness increased during "véraison" and was maintained throughout the second growth phase. A considerable increase in the content of epicuticular waxes was noticed during ripening.

Ultrastructural distribution of sulfated glycosaminoglycans in epithelial-mesenchymal interface of developing rat tooth germs.

We investigated the ultrastructural distribution of sulfated glycosaminoglycans in the epithelial-mesenchymal interface of tooth germs by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion method. At an early stage in odontoblast differentiation, HID-TCH-SP stain deposits were sparsely distributed in the basement membrane and in the intercellular spaces. Subsequently, as formation of the initial predentin matrix began, HID-TCH-SP stain deposits were densely distributed in the interfibrillar spaces and the basement membrane. Testicular hyaluronidase digested most of those in the progenitor pre-dentin, whereas those in the region of basal lamina resisted enzymatic digestion. Testicular hyaluronidase-resistant HID-TCH-SP stain deposits were susceptible to heparitinase, indicating that the sulfated glycosaminoglycan in the basal lamina is heparan sulfate. Furthermore, the heparan sulfate tended to be regularly arranged at the sites of internal and external lamina densa. However, as progenitor pre-dentin matrix formation proceeded, the numbers of stain deposits temporarily increased and their distribution pattern became irregular, finally tending to disappear with the disruption of basal lamina.

The interface between haustoria of Puccinia poarum (monokaryon) and Tussilago farfara

The haustoria of the monokaryon of Puccinia poarum and associated structures in infected cells of Tussilago farfara leaves were examined by electron microscopy. Specimens were fixed with glutaraldehyde and osmium tetroxide and sections stained with uranyl and lead salts and by methods for the specific localization of carbohydrate (periodic acid, thiocarbohydrazide and silver proteinate (PATCHSP), plasma membranes (periodic acid, chromic acid and phosphotungstate (PACP)), or ATPase activity, all with appropriate controls. Haustoria occurred in mesophyll cells but were more abundant in vascular tissues where all cell types except sieve tubes were infected. They were filamentous and each was enclosed in an extension of the host plasma membrane (the extrahaustorial membrane). Between the haustoria and their extrahaustorial membranes, host wall-like material extended to varying distances from the penetration sites, sometimes covering the haustoria. Where this layer was incomplete, the proximal and distal matrices lay beyond it. The amorphous proximal matrix was distinguished mainly by strong reactions with uranyl acetate, PATCHSP and PACP and the distal matrix by radiating fibrils stained with PACP but unstained by PATCHSP. The plasma membrane lining the host cell wall and the extrahaustorial membrane over cell wall-like material gave positive reactions with PACP and for ATPase. Over proximal and distal matrices, the extrahaustorial membrane usually stained weakly with PACP and always lacked ATPase activity. The transitions in staining and ATPase reactions were usually gradual, extending over a distance of ~1·5 μm but staining indicating the presence of a neckband was never observed. The observations are discussed with reference to the development and physiology of the haustoria and infected cells. It is suggested that the extrahaustorial membrane is less fluid than a normal plasma membrane, that solute transport is less efficient and the nutritional relationship of individual haustoria less permanent than in infections by the dikaryotic phase of the fungus.

A modified periodic acid-thiocarbohydrazide-silver proteinate staining sequence for enhanced contrast and resolution of glycogen depositions by transmission electron microscopy.

Oxidation of araldite-embedded liver sections by 1% w/v aqueous H5IO6 for 15 min and a 5-min reaction of carbonyls with 1% w/v thiocarbohydrazide in 10% v/v acetic acid was employed for subsequent staining of glycogen with silver-proteinate (S-P). The network of branching intracellular glycogen aggregates was revealed by 15-min staining with S-P, whereas 24 hr incubation in S-P was necessary to enhance the contrast of glycogen inclusions. We conclude that the proposed modification of glycogen staining readily affords the means for its localization at a desired level of contrast and resolution.