Ultrastructure of intercellular hypha and haustorium of the rust fungus,Uromyces euphorbiae

The ultrastructure of intercellular hyphae and dikaryotic haustoria of Uromyces euphorbiae, and the host response to haustorial invasion was investigated. The intercellular hyphae share common characteristics with those of other uredinial stages of rust fungi. Three types of septa were recognized inside the intercellular hypha. This study showed that the extrahaustorial membrane was possibly formed before the development of the haustorium. The periodic acid-thiocharbohydrazide-silver proteinate technique showed that the haustorial mother cell wall at the penetration site, and the haustorial wall contained more carbohydrates than other fungal structures. In addition, the neckband, present around the haustorial neck, contains different material from those of the rest of the haustorial neck wall. The close associations of host organelles, such as the nucleus, chloroplasts, mitochondria, endoplasmic reticulum and microtubules, with the haustorium, is described.     

Haustoria inUrocystis (Tilletiales)

Haustoria of severalUrocystis spp. have been investigated by transmission electron microscopy. The haustoria are botryose and have an extrahaustorial matrix with vesiclelike bodies. The extrahaustorial membrane shows high ATPase activity in contrast to the haustorial plasmalemma. In walled off haustoria the haustorial plasmalemma stains more intensely than the extrahaustorial membrane. The vesicle-like bodies are ATPase negative. The role of the vesicle-like bodies is discussed.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday. Part 55 of a series Studies inHeterobasidiomycetes.     

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

Summary 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.     

The development of the placenta in the anthocerote Phaeoceros laevis (L.) Prosk

The development of the placenta in the anthocerote Phaeoceros laevis (L.) Prosk. was studied by transmission electron microscopy. By the time the sporophyte emerges from the involucre, a conspicuous placental region is formed by the intrusive growth of sporophyte foot haustorial cells into the adjacent gametophyte vaginula tissue. The separation of gametophyte cells by haustorial cells and their incorporation into the placenta are preceded by the loosening and swelling of their walls and the formation of a periplasmic space. This process causes the disruption of the plasmodesmata, and may eventually result in the complete isolation and consequent degeneration of the cells. Crystals are commonly observed in the vacuoles of gametophyte placental cells. Crystals become more abundant during cytoplasmic degeneration, and are released in the placental lacunae that result from the complete dissolution of gametophyte cells. During the subsequent phase of capsule elongation, the gametophyte placental cells that retain the symplastic connection with the adjoining gametophyte parenchyma develop a wall labyrinth typical of transfer cells. Obliteration of the wall labyrinth by deposition of lightly staining wall material is observed later in sporophyte development, in concomitance with capsule dehiscence. Crystals are negative to the periodic acid/thiocarbohydrazide/silver proteinate test for carbohydrates whilst they are completely digested by pepsin or protease, denoting protein composition.Abbreviation PATAg periodic acid/thiocarbohydrazide/silver proteinate     

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.     

Cytochemical and biochemical observations on the cell wall of the spore ofGlomus epigaeum

Summary The cell wall of the spore ofGlomus epigaeum Daniels and Trappe, which has fibrillar subunits regularly arranged in arcs, was studied ultrastructurally and biochemically.The periodic acid/thiocarbohydrazide/silver proteinate (PATAg) reaction for polysaccharide location (Thiéry 1967) and the silver methenamine reaction for protein location (Swift 1968) were performed on whole spores, progressively alkaline-extracted and autoclaved spores, and untreated and alkaline-extracted cell wall fractions. The cytochemical results and those obtained from frozen sections indicated that the fibrils forming the main structure of the outer and inner wall consist of chitin. Quantitative determinations showed that chitin is the most important component (47%) of the alkali-insoluble residue and represents 27.2% of the whole cell wall fraction. It occurs predominantly as the acetylated form. Cytochemical and biochemical observations showed that the matrix surrounding the fibrils is made of alkali-soluble, PATAg positive polysaccharides (4.98% of the whole cell wall fraction). Monomers were identified by gas liquid chromatography as being -lactone of glucuronic acid, and glucose, rhamnose and mannose. Alkali-soluble proteins are an important part of the matrix, being spread mostly throughout the inner wall and constituting a large portion (55.1 %) of the alkali-soluble fraction.From the results we derive a model in which the chemical components are interconnected to build up a macromolecular network, in agreement with electron-microscopic observations.     

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.     

Modification of the Silver Proteinate Impregnation Technique for Protozoa and Cultured Nerve Cells

Silver impregnation with silver-protein compounds is widely used for staining tissue sections and cell cultures. Some authors report that the results obtained with these methods have not always been reproducible because the reagent's composition varies according to the manufacturer. To avoid this problem in the method described in this paper, a silver proteinate, produced in our own laboratory is used. Although our method is based on Bodian's, the modifications we have made allows its use for both free-living cells (protozoa) and cells grown in culture (nerve cells). The significant modifications are 1) different fixation, 2) postfixation with Cajal's for-mol-bromide, 3) changes in the duration of the impregnation steps technique and 4) elimination of metallic copper. The method reported here enables us to use silver proteinate whenever we require it and to control the composition of the silver proteinate. This technique can be used for cells cultured in either plastic or glass.     

An Improved Silver Stain for Developing Nervous Tissue

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.l% formol as developed agent, 25% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

An improved silver stain for developing nervous tissue.

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

Extracellular beta-1,3-Glucanases in Stem Rust-Affected and Abiotically Stressed Wheat Leaves : Immunocytochemical Localization of the Enzyme and Detection of Multiple Forms in Gels by Activity Staining with Dye-Labeled Laminarin

Endo-β-1,3-glucanase activity in intercellular washing fluid (IWF) from leaves of wheat (Triticum aestivum) increased 10-fold 4 days after leaves were infected with the wheat stem rust fungus (Puccinia graminis f.sp. tritici), while exo-β-1,3-glucanase activity remained unchanged at a low level. Heat and ethylene stress had no effect, whereas mercury treatment resulted in a 2-fold increase in endo-β-1,3-glucanase activity. With a new method of activity staining using laminarin-Remazol brilliant blue as substrate in overlay gels, 18 electrophoretic forms of endo-β-1,3-glucanase were detected in IWF from unstressed leaves and up to 24 forms in IWF from stem rust-infected leaves. Most of the increase in β-1,3-glucanase activity and in the number of β-1,3-glucanases after rust infection was due to a nonspecific, stress-related effect on the plant, but two major forms of the enzyme probably originated from the fungus. β-1,3-Glucanase was localized cytochemically with anti-barley-β-1,3-glucanase antibodies. With preembedding labeling, the enzyme was demonstrated on the outside of host and fungal cell walls. Postembedding labeling localized the enzyme in the host plasmalemma and in the domain of host cell walls adjoining the plasmalemma, throughout walls of intercellular hyphal cells and haustoria, in the fungal cytoplasm, and in the extrahaustorial matrix. Cross-reactivity of β-1,3-glucanases from wheat and germinated uredospores of the rust fungus with the anti-barley-β-1,3-glucanase antibodies was confirmed in dot blot assays and on Western blots.     

小麦条锈菌吸器超微结构和细胞化学的研究

本文应用电镜技术和细胞化学方法,对小麦条锈菌吸器和入侵点的超微结构进行了研究。小麦条锈菌吸器由呈管状的颈部和顶端膨大的吸器体组成,颈部壁和吸器体壁相互连贯,均为两层,并且含有多糖物质。在颈部中段存在有一染色较深的颈环结构。观察发现吸器中的多核现象极为普遍。细胞化学染色结果表明:在吸器外间质内分布有多糖物质;经蛋白消酶解处理后,吸器外间质中可观察到染色较深的纤丝状物质。在入侵点部位,吸器母细胞壁因局部增厚而呈凸镜状,入侵栓壁由内、外两层构成,这两层分别与吸器母细胞壁的第六层和第五层相连接。本研究还观察到同一入侵点产生两个入侵栓的现象。     

Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities

Summary Rust haustoria isolated from infected leaf tissue strongly bind to ConA. This property was exploited to purify them by affinity chromatography on a ConA-Sepharose macrobead column. Haustoria were obtained with more than 90% purity and yields of up to 50%. Binding of haustoria to the column was partially inhibited by a ConA-specific sugar, methyl -D-mannopyranoside. Compared to ConA,Lens culinaris agglutinin and wheat germ agglutinin were less efficient affinity ligands. Using ConA-Sepharose, rust haustoria from a variety of sources could be isolated with equal efficiency, indicating that they have similar carbohydrate surface properties. The haustoria maintained their typical shape after the isolation procedure, which suggests a rather rigid wall structure. The morphology of haustoria was characteristic both for a given species and the nuclear condition of the rust mycelium. Electron microscopy of isolated haustoria revealed an intact haustorial wall surrounded by a fibrillar layer presumably derived from the extrahaustorial matrix. The matrix thus appears to represent a layer with gel-like properties which is rich in ConA-binding carbohydrates and connected to the haustorial wall but not to the host-derived extrahaustorial membrane.Abbreviations ConA Concanavalin A - LCA Lens culinaris agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - DAPI 4,6-diamidinophenylindol×2 HCl     

An association of carbohydrates with particles of arabis mosaic virus retained within Xiphinema diversicaudatum

Sections through the odontophore of Xiphinema diversicaudatum showed two types of staining for carbohydrates using the periodic acid - thiosemicarbazide -silver proteinate (PA-TSC-SP) reaction. The first consisted of thin, localised, intensely-stained patches on the lining of the food canal of all the specimens examined. The second type, found only in nematodes exposed to AMV-infected plants, revealed cloud-like areas of carbohydrate - containing material associated with the stained patches on the lining of the food canal. By staining alternate sections with uranyl acetate/lead citrate, these carbohydrate clouds were shown to contain virus particles. Although the cloud material could have originated from either the plant or the nematode, sections through a pellet of partially purified virus particles prepared from plants did not stain for carbohydrate. The possible role of carbohydrate in virus retention and transmission is discussed.     

Ultrastructural localization of carbohydrates on thin sections of Staphylococcus aureus with silver methenamine and wheat germ agglutinin-gold complex.

Postembedding staining of intracellular carbohydrates on thin sections of Staphylococcus aureus was studied by the silver methenamine and the wheat germ agglutinin-gold techniques. Staining of silver grains was observed on both the cell wall and the cross wall. The staining was interpreted to be due to teichoic acid. Labeling by wheat germ agglutinin-gold particles was observed on both the cell wall and the cross wall, and the staining pattern resembled that of silver methenamine staining. Therefore, the labeling was considered to be due to N-acetylglucosamine of teichoic acid. The combination of two types of cytochemical techniques was useful to localize and characterize the carbohydrates of the bacterial cell.     

Gold-conjugated arabinogalactan-protein and other lectins as ultrastructural probes for the wheat/stem rust complex

Summary Arabinogalactan-protein (AGP, -lectin) was isolated from leek seeds, tested for specificity, conjugated with gold colloids, and used as a cytochemical probe to detect -linked bound sugars in ultrathin sections of wheat leaves infected with a compatible race of stem rust fungus. Similar sections were probed with other gold-labeled lectins to detect specific sugars. AGP-gold detected -glycosyl in all fungal walls and in the extrahaustorial matrix. Other lectin gold conjugates localized galactose in all fungal walls except in walls of the haustorial body. Limulus polyphemus lectin bound only to the outermost layer of intercellular hyphal walls of the fungus. Binding of these lectins was inhibited by their appropriate haptens and was diminished or abolished in specimens pretreated with protease, indicating that the target substances in the tissue were proteinaceous or that polysaccharides possessing affinity to the lectin probes had been removed by the enzyme from a proteinaceous matrix by passive escape. Bindig of Lotus tetragonolobus lectin was limited to the two outermost fungal wall layers but was not hapten-inhibitable. Limax flavus lectin, specific for sialic acids, had no affinity to any structure in the sections. In the fungus, the most complex structure was the outermost wall layer of intercellular hyphal cells; it had affinity to all lectins tried so far, except to Limax flavus lectin and to wheat germ lectin included in an earlier study. In the host, AGP and the galactose-specific lectins bound to the inner domain of the wall in areas not in contact with the fungus. At host cell penetration sites, affinity to these lectins often extended througout the host wall, confirming that it is modified at these sites. Pre-treatment with protease had no effect on lectin binding to the host wall. After protease treatment, host starch granules retained affinity to galactose-specific lectins, but lost affinity for AGP.This paper is listed as Contribution No. 1330, Agriculture Canada Research Station Winnipeg     

Fibrin-enhanced endothelial cell organization

We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron-dense material that progressively became multilayered. Biosynthetic studies indicated that 2-4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15-20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.     

Effects of Oxidation on Neurofibrillar Argyrophilia

The effect of oxidation on neurofibrillar argyrophilia was studied by subjecting nervous tissues containing both normal and degenerating fibers to the action of potassium permanganate, periodic acid, chromic acid, lead tetraacetate, and sodium bismuthate prior to silver impregnation. The argyrophilic response of normal fibers to such treatment was studied with the Nonidez silver nitrate block technic, the double impregnation method of Bielschowsky on both blocks and sections, and a silver proteinate procedure. The response of degenerating fibers was studied by the Cajal formula 6 block technic and the modified Bielschowsky procedure of Nauta and Ryan for sections. The experimental data indicated that such oxidation did not produce any differential staining effects between normal or degenerating fibers.     

Comparative ultrastructural study on tris 1-aziridinyl phosphine oxide prefixed cuticles of some oligochaetes

The cuticle of five species of Oligochaeta, chosen to represent differences in size and a variety of biotopes, was studied electron microscopically after fixation with the acrolein-TAPO-osmium tetroxide method. Five distinct layers in the cuticle of all studied species were found. Staining with lead and uranyl ions or with silver proteinate visualized basically the same structural components of the cuticle, but the degree of electron opacity and the distribution of the electron-opaque stain in these components differed according to the staining method used. Since the acrolein-TAPO-osmium tetroxide method visualized the cuticular zones preferentially stained by Thiéry's silver proteinate method, it was concluded that the TAPO method may be considered suitable for the visualization of polysaccharides. Staining with phosphotungstic acid provided some information on the composition of the cuticle of Oligochaeta not obtained by staining ultrathin sections with lead and uranyl ions nor with silver proteinate. The conclusion is that phosphotungstic acid binds to polysaccharides which do not contain vicglycol groups nor active sites responsible for the positive reaction with lead and uranyl salts. Structural components in the cuticle of the oligochaetes studied were characteristic for each species. The taxonomic value of such components, however, must be confirmed by examination of a larger number of species of oligochaetes.     

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

Summary 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 afterpartial (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 ofhot silver proteinate (50° 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.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday