Morphological changes in the central vacuole ofBlastocystis hominis during in vitro culture

Summary Morphological changes in the central vacuole during the growth in in vitro culture ofBlastocystis hominis were investigated by light and electron microscopy. Most cells in log phase and an early stationary phase showed a positive staining reaction in the central vacuole with PAS or Sudan black B stain, whereas cells in late stationary phase showed few positive reactions. Electron microscopic observations revealed that 95% ofB. hominis cells in log phase and 50% of cells in early stationary phase, had a substantial accumulation of electron-dense material in the central vacuole. In contrast, only 25% of the organisms in late stationary phase had an electron-dense central vacuole, while more than 50% of cells had an electron-lucent central vacuole. These results indicate thatB. hominis accumulated carbohydrates and lipids in the central vacuole during cell growth and that the organism probably consumed these metabolic substances during stationary growth. Therefore, it is strongly suggested that the central vacuole is an important organelle for storage of metabolic substances, such as carbohydrates and lipids, required for cell growth.Abbreviations PBS phosphate-buffered saline - PAS periodic acid-Schiff     

Ultrastructural localization of basic proteins in Trypanosoma cruzi.

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) techniques were applied in epimastigote and trypomastigote forms of the pathogenic protozoa Trypanosoma cruzi to detect basic proteins at the ultrastructural level. With both techniques, reaction was observed in the nucleus and in some cytoplasmic vacuoles. In the kinetoplast of epimastigotes, reaction was observed only at its periphery. In trypomastigotes, however, an intense reaction was observed in the spherical kinetoplast. With the ethanolic phosphotungstic acid technique, reaction was also observed in ribosomes and at the peripheral doublet microtubules of the flagellum. The filaments which form the paraflagellar structure did not react.     

Herpetomonas samuelpessoai: electron microscopy and cytochemistry of electron-dense granules.

Herpetomonas samuelpessoai has membrane-bound electrondense granules in its cytoplasm. The electron density independs on postfixation with osmium tetroxide and is enhanced by uranyl acetate staining. The granules contain iron, have basic proteins cytochemically detected by the silver ammoniacal method, and have a peroxidase activity as detected by the diaminobenzidine method. Some of the granules also have acid phosphatase activity. It is suggested that the granules may represent either lysosomes or a storage form of tetrapyrrole derivatives which are essential for the growth and metabolism of most Trypanosomatidae.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

A Modified Nauta-Gygax Method for Human Brain and Spinal Cord

Relatively thick frozen sections of formalin-fixed human brains are treated subsequently with an equal-parts mixture of 1% oxalic acid and 1% hydroquinone for 30-60 min, 0.005% chromic acid for 10 min, 4% hydrobromic acid for 6 min, 1% phosphotungstic acid for 15 min, 0.05% potassium permanganate for 3-10 min, equal parts of 1% oxalic acid and 1% hydroquinone for 2-5 min. After thorough washing in distilled water, the sections are then soaked in 1.5% silver nitrate for 15-30 min, Laidlaw's ammoniacal silver carbonate for 2.5 min, and then reduced in the Nauta-Gygax reducer. The sections are washed and then passed through 1% sodium thiosulfate for 1-2 min; again washed, dehydrated, cleared and covered with synthetic resin.     

Localization of the synaptonemal complex under the light microscope

The use of osmium tetroxide fixation followed by postreatment with p-phenylenediamine gives an opportunity of locating the synaptonemal complex (SC) under the light microscope in mouse testes and Allium cepa anthers. When semi-thin sections from these materials were observed under phase contrast optics or dark field microscopy, fine threads in the pachytene nuclei were clearly visible. Post-staining of semi-thin sections with ammoniacal silver increased the contrast of the SC and allowed for observations using a bright field illumination. Ultrathin sections of osmium tetroxide/ p-phenylenediamine treated material showed that, under the electron microscope, this technique stains preferentially elements of the synaptonemal complex, while the surrounding chromatin remains unstained.     

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO₄ and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO₄ fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes.     

On the infrastructural localization of basic proteins in the nucleus and nucleolus of cells in pea axillary meristems submitted to or released from apical dominance

Summary In pea axillary meristems submitted to or released from apical dominance, basic nuclear proteins and their fractions (lysine or arginine-rich) were localized at the infrastructural level using convergent methods. In the inhibited nuclei, the condensed chromatin and the nucleoli are the most reactive regions to alcoholic solution of phosphotungstic acid and to ammoniacal silver nitrate. It is the same in the reactivated nuclei after the release from dominance, but the increase in diameter of the nucleoli is accompanied by the creation of a granular component which is observed around the nucleoli during the G₁ S or G₂ phases. This structure is built up essentially by a lysin-rich ribonucleoprotein complex characteristic of active nuclei.     

Vacuole proteins in parenchyma cells of secondary phloem and xylem of Dalbergia odorifera

Summary Light- and electron-microscopic observations were made on the stem parenchyma cells of Dalbergia odorifera T. Chen (Papilionaceae), a tropical deciduous tree. In the secondary phloem of branchlet and trunk, all of the parenchyma cells except companion cells contain vacuole proteins. Only the outer secondary xylem of branchlets, but not trunk secondary xylem, has proteins in the ray parenchyma and the vasicentric parenchyma. The xylem vacuole proteins begin to accumulate at the end of the growing period and they disappear after the first flush of growth in spring. The vacuole proteins in phloem cells, particularly in the cells near the cambium, also show seasonal fluctuations. Under the electron microscope, the vacuole proteins appear as fibrous materials in aggregation or in more or less even dispersion, and they occur in the large central vacuoles during both the growth and dormant periods. According to the published studies, the stem storage proteins in the temperate trees appear as small protein-storage vacuoles or protein bodies, and the proteins in the tropical trees occur in large central vacuoles. This distinction is assumed to be related to the differences in the nature of dormancy between temperate and tropical trees.     

Reappearance of hydrolytic activities and tonoplast proteins in the regenerated vacuole of evacuolated protoplasts

Mesophyll protoplasts of tobacco (Nicotiana tabacum L. cv. Xanthi) were evacuolated by centrifugation in a density gradient. Evacuolation resulted in the quantitative loss of vacuolar hydrolytic activities. The evacuolated miniprotoplasts were cultivated under different conditions, and the regeneration of the central vacuole was investigated by light and electron microscopy as well as by the determination of activities of vacuolar marker enzymes. Vacuoles and hydrolytic activities, as well as cell wall material reappeared faster when the cells were cultivated at low osmotic strength. A newly synthesized tonoplast polypeptide could be detected using a polyspecific serum raised against tonoplast proteins of barley (Hordeum vulgare L.). Both vacuolar proton pumps, the ATPase as well as the pyrophosphatase appear to be newly synthesized during the regeneration of the vacuole.Abbreviations GAP-DH NADP-dependent glyceraldehyde 3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PPi pyrophosphate - PPase pyrophosphatase We thank Dr. Ernst Wehrli, Labor für Elektronenmikroskopie I, ETH Zürich, for taking micrographs. Esther Vogt assisted in the determination of the hydrolases. Bafilomycin was kindly provided by Professor Altendorf, Osnabrück FRG. This work was supported by the Swiss National Foundation grant No. 31-25196.88.     

Ultrastructural and histochemical studies on guard cells

Serial thick sections of guard cells from Vicia faba L., Nicotiana tabacum L., Allium cepa L., Zea mays L. and Beta vulgaris L. were obtained systematically (600–800 nm) and viewed with the transmission electron microscope in an effort to demonstrate the presence or absence of a symplastic transport pathway within the stomatal complex. Eight to ten stomata from each species were examined, and no continuous plasmodesmata were found connecting guard cells to sister guard cells or to adjacent epidermal or subsidiary cells. Continuous plasmodesmata were observed in immature guard cells, but were sealed (truncated) during the development of the mature cell wall. Histochemical stains, phosphotungstic acid and silver methenamine, were used to demonstrate differentiation within the mature guard-cell wall. The structural differentiation of the stomatal apoplastic region is discussed in relation to fanctional specialization. Plasma-membrane elaborations or plasmalemmasomes were identified in the guard cells of Zea, and it is suggested that these structures may function in ion transport.Abbreviations PTA-HCl phosphotungstic acid and hydrochloric acid - SM silver methenamine - UA-LC uranyl acetate and lead citrate     

Osmoregulatory mutants that affect the function of the contractile vacuole inChlamydomonas reinhardtii

Summary Four independent osmoregulatory mutants,osml, osm3,osm4, and osm7, were isolated on the basis of their requirement for growth medium of high osmotic strength. In normal low-osmoticstrength medium, in contrast to wild-type cells, the mutants grow poorly or not at all; in distilled water mutant cells are immobilized and eventually swell and burst. The mutants were examined by ordinary brightfield and phase-contrast microscopy, videomicroscopy, and electron microscopy. The four mutants showed different defects in the contractile vacuole (CV) cycle. Timing of various stages of the CV cycle showed thatosm1 was affected primarily in the early stage of the cycle when the CV begins to grow,osm3 primarily in midcycle when vacuoles fuse to form the CV proper,osm7 at a late stage of the cycle at docking and fusion of the CV with the plasma membrane, andosm4 during contraction of the CV. At the electron microscopic level, in dilute medium, mutant cells by comparison with wild-type cells had large autophagosomes, swollen mitochondria, and dilated ER cisternae. Although electron microscopy showed general abnormalities of the contractile vacuoles consistent with the videomicroscopic observations of living cells, no obvious vacuole membrane abnormalities were seen which would explain the mutational defects. The mutations help define the separate processes that contribute to the coordinated CV cycle inChlamydomonas, and open the way to eventual isolation of some of the genes responsible for CV function.Abbreviations CV contractile vacuole - TAP Tris-acetate-phosphate medium - TAP+L medium supplemented with lactose - TAP+S medium supplemented with sucrose or other sugar     

Ultrahistochemical studies on the moderately electron dense bodies in teleostean endocardial cells

Summary The staining properties of phosphotungstic acid (PTA) on moderately electron dense bodies (MDB), are studied in the endocardium of Gadus morrhua. In MDB fixed in aldehydes only, and stained with PTA at a low pH (0–1), intensely electron dense material occurs on and beneath the limiting membrane. This latter area displays a declining electron density when stained with PTA solutions in which the pH is raised from 1 to 4. At pH>5 the peripheral matrix appears nearly unstained. Collagen fibres fixed as above, and then stained with PTA at a low pH, appear electron dense. These results suggest that the peripheral matrix of the MDB consists mainly of basic proteins.     

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella‐containing vacuole

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high‐throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.     

HISTOCHEMISTRY OF MYELIN—XII ANIONIC STAINING OF MYELIN BASIC PROTEINS FOR HISTOLOGY, ELECTROPHORESIS AND ELECTRON MICROSCOPY

Abstract— Phosphotungstic acid haematoxylin, trypan blue and amidoblack techniques have been developed as anionic dye methods for staining myelin basic proteins. All methods displayed central and peripheral nervous system myelin in histochemical prepa rations and stained brain basic proteins in electrophoretic polyacrylamide gels: phosphotungstic acid haematoxylin appeared to be the most selective of these techniques. Electron photomicrographs of peripheral nerve stained by phosphotungstic acid haematoxylin showed that the major part of myelin basic protein is located in the period dense line. The basic proteins stained by phosphotungstic acid haematoxylin showed an early loss in rat sciatic nerve undergoing Wallerian degeneration and had completely disappeared from the centre of 20 plaques of multiple sclerosis.     

葡萄果实发育过程中果肉细胞超微结构的观察

用透射电镜观察了“巨峰”葡萄(Vitis vinifera×V.labrusca)果实3个发育时期中果肉细胞超微结构的变化。果实第一次快速生长期的果肉细胞超微结构表现出物质和能量代谢旺盛的特点。缓慢生长期的果实虽外部形态平静少变,但果肉细胞超微结构表现出深刻的变化:细胞核形状特化为裂瓣状是最显著的特点;线粒体数目丰富;粗面内质网槽库膨大形成的囊泡富集,出现向液泡汇融和向质膜靠近的现象;质膜内陷;液泡膜完整。另外,原生质也出现一些降解的现象。但总体结构特点表明果肉细胞在此期处于十分活跃的物质周转代谢和信息交换过程中。果实第二次快速生长期果肉细胞超微结构表现出衰老降解的特点,但线粒体结构依然完整,数量仍然丰富,原生质膜也保持了很好的完整性,这似乎与维持第二次快速生长或成熟有关。     

Differentiation of Myofibrillae, Reticular and Collagenous Fibrils in Vertebrates

A procedure for the differentiation of the mesenchymal derivatives, myofibrillae, reticular and collagenous fibers is presented. Formol-Zenker fixation (5-12 hours) is followed by the washing, iodinization, dehydration and paraffin embedding steps routine for that fixative with the following modifications. Zirkle's butyl alcohol series is used for dehydration and infiltration with paraffin as well as in the alcohol slide series. Embedding paraffin used is Parawax plus 8-10% bayberry wax. Tissue-exposed surface of paraffin block is soaked in water overnight before cutting serial sections at 3-5μ. Sections are mounted using the dilute albumen method, and the slides, thoroughly dried at 37^oC. overnight, are left at 60^o for 10 minutes to melt the paraffin of the sections. Before staining, the sections are given a preliminary treatment with potassium permanganate and oxalic acid. For reticular staining a 10% silver nitrate bath is succeeded by an ammoniacal silver carbonate solution followed by reduction in 1% neutral formalin, toning in gold chloride and fixing in sodium thiosulphate. Myofibrillae, the sacroplasmic limiting membrane and other sarcous elements are stained by Heidenhain's azocarmine solution, adult tissues at room temperature and fetal tissues at 50 ^oC. Differentiation in phosphotungstic acid is followed by the staining of collagenous fibers. For adult tissue, light green SF (C.C.) is used and for fetal tissue, fast green FCF (C.C). A discussion of the preparation of ammoniacal silver solutions is included. Both stock and used solutions of ammoniacal silver have been in use by the author for over a period of two years.     

Transition of basic protein during spermatogenesis of <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck, 1765)

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.     

Structure and function in the grass ligule: The membranous ligule ofLolium temulentum L. as a secretory organ

Summary Using phosphotungstic acid and periodic acid/thiocarbohydrazide/silver proteinate (Thiéry test) aspects of polysaccharide ultracytochemistry were studied in the membranous ligule ofLolium temulentum L. Staining results are presented for all three tissues-abaxial and adaxial epidermes and mesophyll-but discussed only for the epidermes. PTA- and PATAg-staining of the adaxial epidermis suggested synthesis of a conjugated polysaccharide material in this tissue, its accumulation in the periplasmic space and its subsequent secretion to the outside of the ligule via gaps in the cuticle. The ligule of this grass is considered to be a secretory organ.     

Examination of the contribution of vacuolar proteases to intracellular protein degradation in Chara corallina.

The contribution of proteases in the central vacuole of Chara corallina internodal cells to overall cellular protein degradation was examined. I measured the decrease in the trichloroacetic acid (TCA)-precipitable radioactivity in the cell for a 6-d chase period after labeling cellular proteins with [3H]leucine. The kinetics of [3H]leucine-labeled protein disappearance showed that the half-life of the cellular soluble proteins was 4 to 5 d. This value did not change when cells were treated with (2S,3S)-trans-epoxysuccinyl-L-leucylamido- 3-methyl-butane ethyl ester, a permeant inhibitor of cysteine proteases. This inhibitor mostly inhibited bovine serum albumin-degrading activity in the vacuole. I also measured the release of TCA-soluble radioactivity from the TCA-insoluble fraction in the cell. This experiment showed that 13% of [3H]leucine-labeled cellular proteins were degraded in 1 d. This value agreed well with the half-life obtained for soluble proteins in the above experiment. This value did not change even when both trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, a cysteine protease inhibitor, and pepstatin A, an aspartic protease inhibitor, were introduced into the vacuole. With this operation, bovine serum albumin-degrading activity in the vacuole was almost completely inhibited. These data suggest that the cytoplasmic but not the vacuolar proteases contribute to cellular protein turnover in Chara internodal cells.