Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Lateral hydrodynamic effects of rotating filaments

On the basis of the filament rotation model that was elaborated for interpretations in cell motility, the lateral hydrodynamic effects of rotating filaments have been investigated by large-scale model experiments. Helices were rotated by small electric motors in a medium of high viscosity (honey or polyethyleneglycol). The observed effects, hitherto not investigated in detail by hydrodynamics, show some features that were attributed to the indefinable formative power or vital force of the past. The main effects generated by the rotating filaments are (1) flows and flow patterns with impact zones where flows collide, (2) regions of excessive pressure and negative pressure (corner effect) along a wall, (3) grooves and smoothly shaped ridges on a free fluid surface, and (4) rolling motions of freely hanging filaments. All effects and flow patterns depend on the appropriate distribution of rotating and counterrotating filaments. Each change of the rotational direction means a dramatic alteration. The application of the observed effects explains largely the function of the microtubule/microfilament hoops or helices during the cytokinesis of a plant cell. Interpretations or simulations are described for events as the formation of secondary wall thickenings, the orientation of their microfibrils, the motion of the preprophase band microtubules, the formation of the phragmosome, the migration, stationary position and shape of the preprophase nucleus, the girdle-, septum- and H-piece formation of cell walls in algae and some events of morphogenesis inMicrasterias. Further interpretations are related to the lateral flows and to invaginations of free cell membranes, to lateral filament motions, to the right-left problem, to the selfintertwining of filaments, to the rotation of a cell body by its flagellum, to the repulsion of chromatids during meiosis and to the tetragonal and hexagonal arrangement of filaments.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

Antiinsectan properties of a novel Fusarium-derived sterol sulfate

The antiinsectan properties of the Fusarium graminearum-derived novel sterol sulfate, 4,4,24-trimethylcholesta-8,14,24(28)-trien-2,3,11,12-tetrol 12 acetate, 3-sulfate were tested on the corn earworm (Heliothis zea (Boddie), the fall armyworm (Spodoptera frugiperda (J. E. Smith), and the driedfruit beetle (Carpophilus hemipterus L.). The sterol sulfate could not be used as a sterol source by any of the insects. The sterol sulfate inhibited growth of S. frugiperda and C. hemipterus larvae at 2500 and 4000 ppm, respectively. Defensive implications of this compound are discussed.     

Different regeneration potential of mesophyll protoplasts from cultivated and a wild species of tomato

Mesophyll protoplasts were isolated from leaves of three cultivars of Lycopersicon esculentum (L.) Mill., namely Hilda 72, Rutgers and Rentita, and from the wild tomato species Lycopersicon peruvianum (L.) Mill. Protoplasts from L. peruvianum divided and grew actively in a liquid medium according to Zapata et al. (1977), whereas protoplasts from the tomato cultivars Hilda 72 and Rutgers showed comparable rates for cell division only, when the content of major elements in this medium was reduced to one half of the original concentration and when mannitol as osmoticum was replaced by glucose. In Rentita protoplasts no cell division could be observed in about 15 different modifications of the five basic culture media tested. The morphogenetic potential of these tomato cells was assessed by comparing the root and shoot formation of protoplasts and of leaf explants. L. peruvianum exhibited the highest potential. Calli derived from protoplasts regenerated roots on Murastrige-Skoog agar containing 1 M benzylaminopurine (BAP) plus 10 M indole-3-acetic acid (IAA) and 0.1 M BAP plus 1 M IAA. Shoot formation occurred in the combinations of 10 M BAP with 0.1, 1.0, and 10 M IAA. Plantlets regenerated from the L. peruvianum calli could be grown in soil. No shoots or roots were regenerated from calli of Hilda 72 and Rutgers protoplasts in all combinations of BAP and IAA tested in the range from 0.1 M to 100 M, thus indicating the rather low morphogenetic potential of these protoplasts as compared to protoplasts from L. peruvianum leaves.Abbreviations BAP benzylaminopurine - IAA indole-acetic acid - TMV tobacco mosaic virus     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Cell multiplication and ultrastructure ofMicrasterias denticulata (Desmidiaceae) grown under salt stress

Cells ofMicrasterias denticulata Bréb. were kept in nutrient solution of high osmolality (salt stress) for four weeks. In a special cell multiplication test it was established that cell division is gradually inhibited at increasing salt concentrations and totally arrested at the highest concentration (26 mosm/kg). Recovery studies proved that even cells from the highest concentration range start dividing immediately after being placed in aqua bidest. thus indicating the full reversibility of the inhibiting effect. — Cells of the highest concentration range show marked ultrastructural changes. Besides an enormous accumulation of starch and oil bodies and a condensed appearance of the ground plasma, a reduction of mitochondria, ER and the Golgi-system is found. The most striking effect occurs on the vacuolar system which appears extremely reduced and condensed. The cell wall is thickened by the formation of an additional cell wall layer with a spongy electron microscopical appearance. Through the cell wall many droplets of a probably fat-like substance are excreted. — In summary, salt stress induces growth-inhibited akinete cells in the sense ofFritsch; these can be reactivated by decreasing the salt concentration. The salt-induced akinete state seems to be an ecological adaption to unfavourable conditions rather than a degeneration of the cells.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.23. 12. 1988     

Ultrastructure and early development of the pore plate sensilla ofGymnomerus laevipes (Shuckard) (Vespoidea,Eumenidae)

Summary The oval pore plates (approx. 17 m long) are separated from the antennal cuticle by a furrow, the inner wall of which is flexible. The thin perforated plates are strengthened by an encircling and a middle ledge, the latter of which branches into about 100 almost parallel rims. Each pore plate is innervated by about 20 sense cells. The dendrites fork into numerous branches occupying the outer receptor lymph cavity below the perforated plate. Each pore plate is associated with one thecogen cell, two trichogen cells, one tormogen cell and one envelope cell 4. A so-called additional cell surrounds the sensillum in the imaginal stage. The envelope cells in the later of the two pupal stages examined, have reached an arrangement which immediately precedes the secretion of the cuticulin layer. The surface of the duplicate trichogen cells is almost equal in area to the completed perforated plate. A dendritic sheath, entirely reduced in the imago, protrudes into the exuvial space, where it encloses a single dendrite.In the younger pupal stage the Sensillenanlage forms a crater, whereby envelope cell 4 overtops the other envelope cells. The distal ends of the trichogen cells are divided into several appendages that form the bottom of the crater.     

Immuno-gold localization of α-L-arabinofuranosyl residues in pollen tubes of Nicotiana alata Link et otto

Immuno-gold labelling using a monoclonal antibody (PCBC3) with a primary specificity for -L-arabinofuranosyl residues was used to locate these residues in pollen tubes of Nicotiana alata grown in vivo. The antibody bound to the outer fibrillar layer of the pollen-tube wall: the inner, non-fibrillar wall layer was not labelled. Cytoplasmic vesicles (0.2 m diameter) were also labelled. The antibody may bind to an arabinan in the pollen-tube wall.     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

Calcium control of differentiation in Phytophthora palmivora

A method has been developed for the preparation of zoospores from Phytophthora palmivora which allows the ionic composition of the suspension medium to be closely controlled. Sub-micromolar concentrations of calcium ions have been shown to play a key role in maintaining the zoospore state and in the transition to the cyst stage. Restriction of free Ca²⁺ to between 0.2 and 1 M resulted in zoospores which could be maintained for several hours before they finally encysted and germinated. When exposed to citrus-pectin, or 3 mM SrCl₂, or to vigorous shaking, these zoospores underwent rapid synchronous encystment. At free Ca²⁺ concentrations below 0.1 M, zoospores lysed slowly. If exposed to inducers of encystment before lysis had occurred, the zoospores failed to respond to pectin or to vigorous shaking. However, they did differentiate in response to SrCl₂ addition. Provided the free Ca²⁺ was maintained between 0.02 and 0.2 M, zoospores survived gentle centrifugation, a procedure which previously had resulted in encystment.Abbreviations IM (ion-mix) release medium containing 100 M KCl, 10 M CaCl₂, and 10 M MgCl₂     

The rotifer trap of Zoophagus

The predacious watermold Zoophagus insidians traps loricate rotifers on short, lateral branches of the main, hyphal axis. These branches or traps are packed at their distal ends with a number of vesicles filled with an electron-dense matrix. Electron micrographs of the mycelium disclose a two-layered wall; the outer layer is electron dense and the inner, electron transparent. The outer dense layer on the tip of the trap is organized into a number of fine ridges and occasional discontinuities. Thin sections through recently trapped rotifers indicate that the cilia of the animals are stuck to the trap by a glue. This adhesive is derived from secretion of the matrix of the vesicles aggregated in the tip of the trap. The secretion mechanism is triggered by the animal and is accompanied by: 1. The separation of the two layers of the wall, 2. fusion of the vesicles with the cell membrane and 3. extrusion of the glue through pits in the tip of the inner wall of the trap. After snaring a rotifer, the previously arrested branch grows as a haustorium into the body cavity of the animal. The host tissues disintegrate within a few hours and appear to be the main nutrient source of the fungus.     

Isolation of a β-glucosidase binding and activating polysaccharide from cell walls of Trichoderma reesei

The extracellular -glucosidase from the filamentous fungus Trichoderma reesei QM 9414 is mainly bound to the cell wall of the fungus and only partially released into the medium. Isolation of the cell walls and its hydrolysis by enzymatic treatment with Aspergillus niger cellulase released -glucosidase, which appeared tightly associated with a cell wall polysaccharide. This polysaccharide was purified by gel filtration and ion exchange chromatography and was shown to consist of mannose, galactose, glucose, galacturonic acid and glucuronic acid. It was devoid of protein and phosphate. It reassociated both with extracellular -glucosidase as well as -glucosidase released from the fungus' cell wall. Addition of the polysaccharide to the -glucosidase in vitro increased the enzyme's activity against 4-nitrophenyl--glucoside twofold. These findings suggest, that the isolated polysaccharide functions as an anchor glycan for the -glucosidase in Trichoderma reesei.     

Changes in glucan synthetase activity and plasma membrane proteins during encystment of the cellular slime mold Polysphondylium pallidum

The activity of glucan synthetase increased dramatically during encystment of Polysphondylium pallidum cells. The majority of activity was present in purified plasma membranes. Activity, measured as glucose incorporation from UDPG into NaOH-insoluble glucan, increased 30–40 fold in the membranes. Increases in activity within the cells preceded plasma membrane increases and the enzyme appeared to be rapidly transported to the plasma membrane. Intracellular activity was relatively low. When cells were incubated with UDPG and when phloretin was included to inhibit glucose uptake, no NaOH-insoluble glucan was synthesized. Hence, the UDPG-binding site was not exposed at the cell-surface. When the NaOH-insoluble glucan was digested with endo--1,4-glucanase the products were cellobiose and glucose. The glucan could also be precipitated from Schweizer's reagent with acetic acid. These results suggest that the glucan contained predominantly -1,4-linkages and may be cellulose. Experiments with cycloheximide confirmed that protein synthesis was required for encystment. Labeling of cells with [1-¹⁴C]-acetate showed that the synthesis of certain plasma membrane proteins was developmentally regulated. A number of proteins (e.g., myosin heavy chains and actin) were synthesized during the lag phase and their synthesis was subsequently reduced or ceased altogether. Immediately prior to the commencement of cyst wall formation seven new plasma membrane proteins were synthesized. These proteins were not detected intracellularly, indicating rapid transfer to the plasma membrane. The possible relationship between the seven developmentally regulated proteins and a postulated multi-enzyme-complex involved in cellulose synthesis is discussed. Their synthesis may be related to the increase in particles in the outer leaflet of the plasma membrane observed during encystment with freeze-etching (G.W. Erdos and H.R. Hohl, 1980, Cytobios, 29, 7–16).     

Model of neural visual system with self-organizing cells

This paper describes a model of a neural visual system of a higher animal, in which the capability of pattern recognition develops adaptively. To produce the adaptability, we adopted self-organizing cells, and with them modeled feature-detecting cells which were discovered by Hubel and Wiesel and whose plasticity was found by Blakemore and Cooper. Combining the self-organizing cells and the learning principle of a Perceptron-type system, we constructed a model of the whole visual system. The model is also equipped with an eye movement control mechanism for gazing, which reduces the number of selforganizing cells required for pattern recognition, thus contributing to their quick self-organization. Computer simulation and an experiment using a hardware simulator showed that self-organizing cells quickly become sensitive to the features often seen and that the resulted system can classify patterns with a rather small number of feature-detecting cells.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Differentiation and redifferentiation of a transfer cell: Development of septal nectaries ofAloe andGasteria

Summary The epithelial cells of the septal nectaries ofGasteria andAloe have a second wall layer consisting of numerous irregular wall protuberances along the outer wall. They, thus, are typical transfer cells. The wall protuberances develop a short time before the nectar secretion begins. At this time and during the secretory phase the amount of microtubules is at a minimum. After the secretion phase the protuberances disappear gradually while often callose is formed between them. Simultaneously a third wall layer is deposited which covers the remnants of the second layer. During the first developmental stages the cells contain amyloplasts with changing amounts of starch. In the redifferentiation phase they become chloroplasts with a crystalline prolamellar body and small stacks of thylakoids.     

Localization and function of an FMRFamide-like substance in the aorta of Helix aspersa

Summary With an antiserum (aFM) against the molluscan cardio-active FMRFamide (Phe-Met-Arg-Phe-NH₂) numerous immunoreactive axons were found in the outer, longitudinal, muscle layer of the anterior aorta of Helix aspersa. Immunoreactive axons were rare in the inner, circular, muscle layer. At the ultrastructural level four types of axons could be distinguished. The granules containing the immunoreactive substance (mean diameter ca. 100 nm) are present in type-2 axons. The effect of synthetic FMRF-amide was tested in vitro on preparations of ring- and tubule-shaped pieces of the anterior aorta. Physiological doses (3 × 10^-7 M) provoked contractions of the circular muscle fibres, but had no effect on the longitudinal muscle cells. Apparently in vivo the FMRF-like substance diffuses from the richly innervated longitudinal muscle layer to the circular muscle layer, where it exerts its effect. This conclusion is sustained by the observation that the contents of the aFM-immunoreactive granules in type-2 axons are released by exocytosis in a non-synaptic fashion.     

Cell types in the adenohypophysis of the hagfish,Myxine glutinosa (Cyclostomata)

Summary The anterior pituitary of the Atlantic hagfish, Myxine glutinosa, was investigated by transmission electron microscopy. The cells of the adenohypophysis are arranged in follicles surrounded by connective tissue. Five cell types containing granules and one agranular cell type were identified. At the present state of the study the function of these cells remains open to discussion.This study was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (Grant no. 4070) and the Stiftungs- und Förderungsgesellschaft der Paris Lodron-Universität in Salzburg. The authors are grateful to the Director of the Biological Station in Drøbak (Norway), Amanuensis Finn Walvig, for procuring the material     

Comparative particle-induced cytotoxicity toward macrophages and fibroblasts

The cytotoxicity caused by the debris resulting from wear of prostheses can produce major damage to tissues around the implant. We have compared particle internalization by macrophages and fibroblastsin vitro and analyzed cell death. J774.2 macrophages and L929 fibroblasts were incubated with 0.43 and 2.81 m alumina particles or 0.45 and 3.53 m polystyrene (PS) beads. Incubation of J774.2 cells with alumina particles of both sizes and 0.5 and 1.0 mg/ml PS beads significantly decreased cell numbers in a particle concentration-dependent manner. L929 cells were not affected by lower concentrations of 0.43 m alumina particles (which aggregate at high concentrations) and they internalized 0.45 m PS beads without any decrease in cell numbers. Particles were more cytotoxic for macrophages than for fibroblasts. Particles caused the size of both types of cells to increase in correlation with cytotoxicity. Trypan blue exclusion and lactate dehydrogenase release showed cell membrane leakage for both types of cells incubated with PS beads for 24 h. Apoptosis was assessed by annexin V–FITC, propidium iodide staining and assay of caspase 3 activity. Macrophage death appeared to depend on both necrosis, caused mainly by 3.53 m PS beads, and apoptosis, mainly due to 0.45 m PS beads. The release of the inflammatory cytokine IL-6 appears to be nonlinearly correlated with cytotoxicity. Thus, the size of the internalized particles affects macrophages and fibroblasts differently, and the increase in cell size can be used as a preliminary criterion of particle cytotoxicityin vitro.     

Comparison of [35S]GTPγS binding to plasma membranes and endomembranes prepared from soybean and rat

Summary Plasma membranes were prepared from soybean hypocotyls and roots by aqueous two-phase partitioning and subsequent free-flow electrophoresis. The highly purified plasma membranes bound [³⁵S]GTPS with a relatively high affinity (K_d10nM). The binding was saturable and specific as it was indicated by the displacement of bound [³⁵S]GTPS by unlabeled GTPS and GTP, but not by ATPS, ATP, UTP or CTP. ITP was intermediate in its ability to displace [³⁵S]GTPS. When soybean plasma membrane proteins were separated by SDS-PAGE and displayed by autoradiography, two major [³⁵S]GTPS binding proteins were revealed with apparent molecular weights of 24 and 28 kDa. Results with plasma membranes from soybean hypocotyls and roots were similar but differed from those with plasma membranes prepared from rat liver and adipocytes where only a single major [³⁵S]GTPS binding activity with a molecular weight of 28 kDa was observed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - G protein hetero-trimeric GTP binding protein with , , subunits - G_n protein GTP binding protein detected on nitrocellulose blots - GTPS guanosine 5-[-thio]triphosphate - IAA 3-indoleacetic acid - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis