Axenic Paramecium caudatum. I. Mass culture and structure

To establish and grow Paramecium caudatum in mass axenic culture the culture medium of Soldo, Godoy & van Wagtendonk was modified by substituting phosphatidylethanolamine (PE) for TEM-4T and by a 10-fold increase in folic acid. Population densities of 4000 to 6000 cells/ml and a generation time of 20-26 h are regularly obtained. Optimal growth is obtained with PE-stigmasterol ratios between 40:1 to 400:1. Cells from 1-day-old axenic cultures have many lipid bodies aggregated in clumps (which disappear in 2 to 3 days) as well as foci of rough endoplasmic reticulum bordered by dictyosomes. The latter suggests a very active metabolism. Crystalline sheets found in both food vacuoles and lysosomes presumably play a role in digestion. Axenically grown cells also have abundant Golgi bodies (dictyosomes) and by late log phase become filled with lysosomes.     

Ultrastructural investigations of Arbutus unedo-Laccaria amethystea mycorrhiza synthesized in vitro

Summary Anatomy and ultrastructure of the arbutoid mycorrhiza of Arbutus unedo-Laccaria amethystea from axenic culture are described. In comparison to non-inoculated roots, the rhizodermal cells of mycorrhizas are of greater volume, their nuclei are enlarged and show an irregular shape, plasmalemma and cytoplasm with mitochondria, plastids, endoplasmic reticulum and dictyosomes are increased. Several ontogenetical states are documented. The arbutoid mycorrhiza as a connecting link between ectomycorrhiza and ericoid mycorrhiza is discussed.     

ULTRASTRUCTURAL CYTOLOGY OF CALLUS CULTURES OF STREPTANTHUS TORTUOSUS AS AFFECTED BY TEMPERATURE

Tissue culture cells of Streptanthus tortuosus var. orbiculatus (Cruciferae) which have acquired a spherical viruslike particle located in their nucleoli, designated cell line STV, developed supergranal chloroplasts and lost the ability to differentiate vascular tissues. The effect of temperature on the ultrastructural cytology of one line of the STV tissue, STV-I, was compared with the effect of temperature on the ultrastructural cytology of tissue culture cells lacking the viruslike particles (control cell lines). At 4 C, the cellular and ultrastructural appearance of control tissue culture cells differed from that of tissue grown at 22 C by producing increased amounts of endoplasmic reticulum and dictyosomes and by reduction of chloroplast thylakoids. STV-I cells were generally moribund as a result of 4 C treatment. Chloroplast thylakoids were also reduced in control tissue following growth at 10 C and the apparent quantities of endoplasmic reticulum and dictyosomes were similar to those observed in control cells grown at the control temperature (22 C), but less than those observed in tissue subjected to 4 C. STV-I tissue grown at 10 C demonstrated increased endoplasmic reticulum and dictyosomes and reduction of polysomal configurations. The mitochondrial morphology was variable and the cells contained supergranal chloroplasts and proplastids. At the control temperature (22 C), the fine structural appearance of control tissue culture cells was typical of parenchyma cells, but STV-I cells contained mitochondria of variable morphology and two types of chloroplasts— normal and supergranal. Control tissue grown at 30 C also contained proplastids, but these proplastids contained starch in contrast to the proplastids in control tissue grown at low temperatures. The ultrastructural cytology of STV-I cells grown at elevated temperature (30 C) was characterized by enlarged mitochondria containing massive lipid bodies and the presence of protoplastids with starch and supergranal chloroplasts.     

Effects of induced pinocytotic activity and extreme temperatures on the morphology of golgi bodies in Amoeba proteus

Summary The morphology of the Golgi apparatus of Amoeba proteus can be influenced by substances inducing pinocytotic activity as well as by extreme temperatures. During the ingestion of a solution of 0.5% egg white the number of Golgi bodies decreases from 100% measured in control cells to 82% measured in cells showing induced pinocytosis. Simultaneously the ratio of the surface area of the cisternae at the proximal face to that of the vesicles at the distal face of single dictyosomes remains constant (1.74–1.72).The decrease and increase of the temperature of the culture medium to 4° C and 30° C respectively, causes the disappearance of most of the dictyosomes. After keeping the cells for 3–10 h at these temperatures the number of Golgi bodies was only 5–10% of the controls. A continued treatment with cold or warm culture medium leads to a partial reorganization of dictyosomes. After 15 h the number of Golgi bodies counted per cell returned to 57% at 4° C and 38% at 30° C. The ratio of the surface area of the Golgi cisternae to the surface area of the Golgi vesicles also alters under the influence of extreme temperatures. The values measured after treating the cells for 3 h, 4 h 10 h and 15 h at 4° C and 30° C amounted to 0.75, 0.85, 1.14 1.53 and 0.93, 0.38, 0.88, 1.60, respectively, compared to 1.72 of control amoebae.The different values of the ratio of the surface area of cisternae to that of vesicles indicate that there are strong morphological changes of single dictyosomes.     

Structure and cytochemistry of the procambium in Salix buds during dormancy and dormancy breaking

This report presents a combined investigation of ultrastructural and enzymatic changes in the procambium from late winter to early spring. In January the procambial cells of dormant Salix buds have a convoluted plasma membrane with many plasmalemmasomes, numerous lipid bodies, large stacks of rough ER and plastids surrounded by smooth ER profiles. Several small lysosomes show activity of ATPase and acid phosphatases. In addition ER, nuclear envelopes, dictyosomes, and thylakoids have ATPase activity, and ER and plasmalemma, and nuclei also show acid phosphatase activity. In February metabolism seems to increase as indicated by lysosomes with membranous formations, dilated ER, nuclear envelopes, spiny vesicles, and polysomes. ATPase activity occurs in plasmalemma and vacuoles, and acid phosphatases in the middle lamella region of walls, in plasmalemma, vacuoles, ER, and nuclei. At the end of March, when growth starts inside the buds, but before they break, the stacks of rough ER disappear, and the vacuoles coalesce. Most of the lipid bodies have disappeared and the plastids have accumulated starch. Cell division and differentiation of procambial cells to protophloem and protoxylem have started. The distribution of ATPase increases; activity is found in walls and plasmalemma, and only a few small vacuoles still have ATPase and acid phosphatase activity. Notable is the appearance of ATPase in mitochondrial cristae and nucleoli and the occurrence of rather high levels also in endomembranes and dictyosomes.     

COMPARATIVE ANALYSIS OF THE FINE STRUCTURE OF YOUNG AND ADULT INDIVIDUALS OF DUNALIELLA SALINA (POLYBLEPHARIDACEAE,CHLOROPHYCEAE) WITH EMPHASIS ON THE FLAGELLAR APPARATUS1

Individuals of Dunaliella salina (Dunal.) Teod. change their shape during ontogenesis. Here we describe the fine structure of this species with emphasis on distinctions between young and adult individuals. The cell coat is present at early stages of cell development and may be synthesized by vesicles of nuclear membrane-associated endoplasmic reticulum. Scanning electron microscopical observations show differences in the surface pattern of the cell coat in young and adult cells. The nucleus of young cells is more or less spherical, whereas that of adult cells is pyriform. The Golgi apparatus is positioned immediately under the basal bodies and consists of three dictyosomes in young cells and six to eight dictyosomes in adult cells. The flagellar apparatuses of young and adult cells have a 1/7 o'clock (i.e. clockwise) displacement of basal bodies and are grossly similar, but there are subtle differences between specific components. Two non-axonemic basal bodies (1′, 2′) appear in a plane perpendicular to that determined by the flagella-bearing basal bodies (1, 2). The cruciate microtubular rootlet system has a 4–2–4–2 alternation pattern. In adult cells, rhizoplasts emerge from each terminal body and run parallel to the four rootlets.     

Cytochemical localization of peroxidase in soybean suspension culture cells and protoplasts: Intracellular vacuole differentiation and presence of peroxidase in coated vesicles and multivesicular bodies

Summary The ultrastructural localization of peroxidase in soybean (Glycine max L.) suspension culture cells and protoplasts is reported. In cells peroxidase is found primarily in the cell wall and at the tonoplast. Protoplasts and cells contain a vacuolar system which is differentiated with respect to peroxidase content since some vacuoles are found which do not contain peroxidase reaction product. The Golgi dictyosomes, coated and smooth vesicles contain peroxidase. Some of the multivesicular bodies have the reaction product as well. The results are discussed in terms of the pathways of sorting of peroxidase between the cell wall and vacuoles of cultured cells.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

Intranuclear Virus-like Bodies in the Amoeboflagellate Naegleria gruberi*

SYNOPSIS. Intranuclear virus-like bodies were seen in cultures of the EG strain of Naegleria gruberi from soil. Introduction of these virus-like particles into cultures seemed to coincide with use of chicken embryo extract as a supplement in culture media used to maintain axenic amoebae in the laboratory; the appearance of the virus-like units is triggered by transfer of axenic lines of N. gruberi EG into monobacterial culture medium. The particles, ～ 100 nm in diameter, are mainly restricted to the nucleus of the cell. Passage of particles from the nucleoplasm into the cytoplasm is suggested by theit association with tubular projections from the nuclear membrane, and particles have been seen in the cytoplasm of the amoebae. The virus-like bodies resemble reovirus.     

Ultrastructural localization of acid phosphatase in cultured cells ofDaucus carota

Summary Acid phosphatase localization has been studied, using the lead salt method, in suspension-cultured cells of the wild carrot. Enzyme activity in most of the cells was restricted to the walls and vacuoles. However, in some senescent cells activity was also seen in the nucleus, at one face of the dictyosomes, and in nearby dictyosome-derived vesciles.The activity in the walls was closely associated with the central portion of the wall which ultimately disintegrates in auxin-containing media. However, the large vesicles which accumulate in this portion of the wall as it breaks down never showed acid phosphatase activity, nor did the multivesicular bodies which appear to transfer vesicular material into the wall space. Although multivesicular bodies in plant cells resemble the multivesicular lysosomes of animal cells, no evidence could be obtained in this study for the presence in such bodies of hydrolytic enzymes.     

Spore germination and sporeling developlnent in Jungermannia sphaerocarpa Hook.

Abstract

The globular protonema of Jungermannia sphaerocarpa developed outside the spore wall in axenic culture. Up to six gametophytes were produced by each protonema, and sporeling development to the attainment of sexual maturity is described. Significantly fewer and larger oil bodies occurred in the rounded mature leaves compared with bifid immature leaves and with cells of the protonema. Within the numerous rhizoids, an intense purple pigmentation was developed and maintained.     

CYTOCHALASIN B INFLUENCES DICTYOSOMAL VESICLE PRODUCTION AND MORPHOGENESIS IN THE DESMID EUASTRUM1

Cytochalasin B (CB) applied to young developing cells of the desmid Euastrum oblongum Ralfs ex Ralfs, at concentrations that do not entirely inhibit cytoplasmic streaming, retarded cell growth and caused malformations of cell shape. While the basic symmetry of the cell was maintained, only the first indentations were formed and the cell body appeared to be swollen. Electron microscopic investigations revealed that vesicle production at the dictyosomes was disturbed by cytochalasin. In contrast to untreated control cells, where vesicles with electron-dense contents (“dark vesicles”) were formed during primary wall formation, vesicles pinched off by the dictyosomes during CB treatment exhibited an “empty” appearance. These vesicles, which correspond to the “dark vesicles” in size, were accumulated around the dictyosomes without being transported to the plasma membrane and were frequently connected to the trans-cisternae of the Golgi bodies. We speculate that CB may influence the transfer of products from the endoplasmic reticulum (ER) to the dictyosomes via transition vesicles, which results in a disturbed vesicle production at the Golgi bodies. CB also causes a shift in ER and dictyosome distribution. Moreover, a cortical actin system appears to be involved in the cell shaping of Euastrum. The arrangement of microtubules around the nucleus is not affected by the drug.     

Brefeldin A induces reversible dissociation of the Golgi apparatus in the green algaMicrasterias

Summary The fungal metabolite brefeldin A (BFA) causes inhibition of cell growth inMicrasterias denticulata after 2 h incubation, combined with slight malformation of the cell shape. The BFA effects on cell development are accompanied by a gradual decrease in the number of Golgi cisternae and severe structural and morphological changes of the dictyosomes which are already visible after only 10 min exposure. When the treatment is prolonged the number of dictyosomes is markedly reduced, leading to almost complete loss of Golgi bodies, particularly in the young semicell. Groups of primary wall material-containing vesicles accumulated in areas of former dictyosomes, and previously unknown vesicular bodies are found. Restitution of almost normal dictyosomes occurs within 5 h when the cells are allowed to recover from BFA treatment.Micrasterias cells incubated in BFA at concentrations below 15 M maintain their ability to divide over several generations. Our results indicate that, of the various inhibitors of the secretory pathway tested against growingMicrasterias cells, BFA is the only drug which induces complete and reversible dissociation of dictyosomes in the growing semicell. This allows deductions about the function of the processes targeted by BFA during cell development inMicrasterias.Abbreviations BFA brefeldin A - CPA cyclopiazonic acid - ER endoplasmic reticulum - TM tunicamycin     

Elektronenmikroskopische Untersuchungen zur Genese des Golgi-Apparates (Dictyosomen) und ihrer Kernabhängigkeit beiAcetabularia

Summary The genesis of dictyosomes has been studied on nucleate and enucleateAcetabularia cells.Dictyosomes differentiatede novo from prestages, the perinuclear bodies, which are synthesized under the actions of nuclear ribonucleoprotein. Consequently the formation of dictyosomes is nucleus (DNA) dependent.This was shown by experiments using actinomycin and puromycin and by experiments on darkened and enucleate cells.There is a relationship between dictyosome structure and function and the morphogenetic activities of a cell.

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Ultrastructure of the polygerm ofAgeniaspis fuscicollis Dalm. (Chalcidoidea,Hymenoptera)

Summary The extraembryonic food supply of developingAgeniaspis fuscicollis polygerm from the host haemolymph is mediated by the trophamnion. The nutritive materials are absorbed by micropinocytosis. Besides microvilli and micropinocytotic vesicles, the following organelles appear in the trophamnion: multivesicular bodies, dictyosomes, mitochondria, and microtubules. The multivesicular bodies may be considered as a form of yolk. The storage material of the trophamnion is lipid. Numerous polyribosomes appear in the inner part of the trophamnion indicating intense protein synthesis. Food supply from the trophamnion to the embryo cells also proceeds by micropinocytosis. The polyembryo is surrounded by a cyst made up of host cells. Large numbers of tracheoles supplying the developing parasitic embryos with oxygen are invaginated into the cyst cells. The ultrastructure of theAgeniaspis fuscicollis polygerm shows a close trophic relationship between the polyembryonally developing parasite and its host.This research was supported in part by Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw     

THE PAS/ACCUMULATION BODIES IN PROROCENTRUM LIMA AND PROROCENTRUM MACULOSUM (DINOPHYCEAE) ARE DINOFLAGELLATE LYSOSOMES1

Numerous spherical bodies containing electron-dense material, fibrous material, and membranous material are present in the cytoplasm of two dinoflagellate species, Prorocentrum lima (Ehr.) Dodge and Prorocentrum maculosum Faust. Similar bodies have been observed in other dinoflagellates and have been termed accumulation bodies or PAS bodies. In both Prorocentrum species, these bodies autofluoresce under blue light excitation and increase in size with cell culture age. They possess acid phosphatase activity, react positively with the periodic acid/Schiff reagent, and stain with acridine orange. All these properties are characteristic of eukaryotic lysosomes; thus, we propose that dinoflagellate accumulation bodies and PAS bodies are identical organelles and are, in fact, dinoflagellate lysosomes.     

Significance of the cytoskeleton for cytoplasmic organization and cell organelle dynamics in epithelial cells of fresh-water sponges

Summary The cytoskeleton in epithelial cells ofSpongilla lacustris is constructed of microtubules radiating from the nuclear region and terminating at the cell periphery as well as microfilaments forming a cortical layer beneath the plasma membrane and distinct fibers in the cytoplasmic matrix. Single frame analysis and in vivo labeling of mitochondria with Rh 123, endosomes or lysosomes with TRITC-BSA, endoplasmic reticulum (ER) with DiOC₆ (3) and dictyosomes with C₆-NBD-ceramide points to the microtubular system as a candidate for controlled cytoplasmic organization and active transport of these cell organelles. In epithelial cells with an intact microtubular system, mitochondria and endosomes or lysosomes show a regular shuttle movement between the nucleus and the cell periphery with a velocity of 1.3–1.4 m/s; the ER forms a more or less dynamic two-dimensional network in the entire cytoplasmic matrix, and dictyosomes are arranged in a ring-like manner around the nucleus. In epithelial cells treated with colchicine or colcemid, mitochondria and endosomes or lysosomes gather in the perinuclear region and become immobile; the ER accumulates near the cell center, whereas most dictyosomes distribute randomly over the whole cytoplasm. Transformation of the microfilament system with cytochalasin D has no influence on cell organelle distribution and dynamics but impedes cell locomotion and cell surface activities.Abbreviations BSA bovine serum albumin - C₆-NBD-ceramide 6-[(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)caproyl]sphingosine - DiOC₆(3) 3,3-dihydroxyloxacarbocyanine jodide - DMSO dimethylsulfoxide - EGTA ethylenediaminetetraacetic acid - GTX glycerol-Triton-X-100 - PBS phosphate buffered saline - PEG polyethylene glycol - PIPES 1,4-piperazine-N,N-bis-(2-ethanesulfomc) acid - Rh 123 rhodamine 123 - TRITC tetramethylrhodamine isothiocyanate     

A COMPARISON OF ROOT CAP CELLS OF EPIPHYTIC,TERRESTRIAL AND AQUATIC PLANTS

Outer cells from the root cap of Cattleya orchids are characterized by their secretory activity. They are arranged in layers intercalated with layers of secretory product and form a protective mantle over the root tip. The ultrastructure of these cells is similar to those of terrestrial roots (for example Zea mays) in that they are characterized by copious quantities of endoplasmic reticulum and numerous dense-staining prevacuolar bodies. In contrast, most root cap cells of water hyacinth and duckweed are highly vacuolate with no dense-staining prevacuolar bodies. The endoplasmic reticulum is sparse and dictyosomes are small and without secretory activity.     

ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN AXENIC CULTURE AND ITS RESPONSE TO THE ENDOPHYTIC FUNGUS MYCOSPHAERELLA ASCOPHYLLI AND EPIPHYTIC BACTERIA1

Ascophyllum nodosum (L.) Le Jol. was freed from bacteria and the endophytic fungus Mycosphaerella ascophylli Cotton by repeated treatment with chlorine solutions and grown in artificial seawater. Two types of axenic culture of different origin were obtained. Type 1 was developed from apices of A. nodosum collected in the sea. Type 2 was from plants which developed from adventitious embryos on rhizoids formed by type 1. This is the first time A. nodosum has been cultivated axenically. Growth of the axenic alga was increased by IAA, 21P and zeatin. Without external growth regulators some strains of the axenic alga deteriorated within a year; others developed a filamentous habit. Sulfur in a reduced state also stimulated growth. Addition of either glucose, mannose or mannitol to the medium caused the formation of calluslike layers of loosely packed colorless cells under the epidermis of the thalli and the epidermis was sloughed off. No increase in thallus length was noticed. Mycosphaerella ascophylli in axenic culture did not excude any substances stimulating growth of the alga, but that does not exclude an influence of the fungus on the alga in vivo. The fungus, when growing within the alga, seemed to have little influence on algal morphology. A bacterized but fungus-free A. nodosum was cultivated in an artificial seawater for 8 years. In the bacteria-free alga, the fungus protruded from the epidermis and evidently utilized the alga as a carbon source. The bacteria thus seem much more important than the fungus for normal growth of the Ascophyllum plant.     

THE BIOLOGY OF HARVEYELLA MIRABILIS (CRYPTONEMIALES,RHODOPHYCEAE). VII. STRUCTURE AND PROPOSED FUNCTION OF HOST-PENETRATING CELLS1

The parasitic red alga Harveyella mirabilis (Reinsch) Schmitz & Reinke was examined by light and electron microscopy to determine the structural mechanism involved in nutrient transfer. The host-penetrating rhizoidal cells are unique in possessing an extensive and apparently dynamic endomembrane system as well as other unique cytoplasmic inclusions. The membrane system consists of the plasmalemma, pinocytotic vesicles, multivesicular and concentric bodies, endoplasmic reticulum, dictyosomes, micro-body-like structures and an extensive vacuolar system. It is proposed that this system is active in the uptake and processing of host-derived nutrients. Plasmalemmal extensions (plasmalemmavilli) of Harveyella medullary cells may also function in nutrient uptake.