Transport of rosettes from the golgi apparatus to the plasma membrane in isolated mesophyll cells ofZinnia elegans during differentiation to tracheary elements in suspension culture

Summary Rosettes of six particles have been visualized by freeze-fracture in the protoplasmic fracture (PF) faces of: a) the plasma membrane, b) Golgi cisternae, and c) Golgi-derived vesicles in mesophyll cells ofZinnia elegans that had been induced to differentiate synchronously into tracheary elements in suspension culture. These rosettes have been observed previously in the PF face of the plasma membranes of a variety of cellulose-synthesizing cells and are thought to be important in cellulose synthesis. InZinnia tracheary elements, the rosettes are localized in the membrane over regions of secondary wall thickening and are absent between thickenings. The observation of rosettes in the Golgi cisternae and vesicles suggests that the Golgi apparatus is responsible for the selective transport and exocytosis of rosettes in higher plants, as has been previously indicated in the algaMicrasterias (Giddings et al. 1980). The data presented indicate that the Golgi apparatus has a critical role in the control of cell wall deposition because it is involved not only in the synthesis and export of matrix components but also in the export of an important component of the cellulose synthesizing apparatus. The rosettes are present in the plasma membrane and Golgi vesicles throughout the enlargement of the secondary thickening, suggesting that new rosettes must be continually inserted into the membrane to achieve complete cell wall thickening.Abbreviations EF Golgi vesicles, exoplasmic fracture; the plasma membrane, extracellular fracture - PF protoplasmic fracture     

Investigations of the turnover of the putative Cellulose-synthesizing particle “rosettes” within the plasma membrane ofFunaria hygrometrica protonema cells

Summary YoungFunaria protonemata were treated with Monensin (10^–6 M) and Cytochalasin (CB) (2×10^–5 M). The influence of the inhibitors on a) elongation growth, b) cell fine structure and c) particle rosettes within the plasma membrane after freeze fracture was observed. Monensin stopped cell growth, caused swelling of the mitochondria and plastids and inhibited the secretory activity of the Golgi apparatus within about 15 minutes. The number of rosettes in the PF of the plasma membrane was distinctly reduced after 4–5 minutes and decreased further to only very few after 30 minutes. The tip to base gradient in distribution was maintained for a long time. The effects were reversible, regeneration occurred within 3 hours. CB treatment showed no effect on elongation growth and cell fine structure. The number of rosettes, however, was strongly reduced within 3 minutes exposure time and their distribution was nearly uniform then. Number and tip to base gradient increased again after 6 minutes intoxication. The results are discussed in regard to the turn over of the rosettes.Abbreviations CB Cytochalasin B - PF protoplasmic fracture face - F-vesicle flat vesicle - F-Actin filamentous actin - G-Ac-tin globular actin     

Comparative physiology of nitrogenase activity and vesicle development for Frankia strains CpI1, ACN1 ag,EAN1pec and EUN1f

The relationship between nitrogen fixation and development of a specialized cell structure, called the vesicle, was studied using four Frankia isolates. Nitrogenase activity was repressed in all four strains during growth with ammonia. Strain CpI1 formed no vesicles during NH₄ growth. Strains ACN1 ^ag , EAN1_pec and EUN1_f produced low numbers of vesicles in the presence of ammonia. Following transfer to nitrogen-free media, a parallel increase in nitrogenase activity and vesicle numbers occurred with all four isolates. Appearance of nitrogenase activity was more rapid in those strains that possessed some vesicles at the time of shift to N₂ as a nitrogen source. The ratio of vesicle numbers to level of nitrogenase activity varied widely among the four strains and in response to different growth conditions and culture age of the individual strains. Optimum conditions of temperature, carbon and energy source, nitrogen source and availability of iron and molybdenum were different for each of the four strains. Those conditions that significantly reduced nitrogenase activity were always associated with decreased numbers of vesicles.     

The plasma membrane of the Funaria caulonema tip cell: morphology and distribution of particle rosettes,and the kinetics of cellulose synthesis

Freeze-fracturing of Funaria hygrometrica caulonema cells leads to a cleavage within the plasma membrane. The extraplasmatic and the plasmatic fracture faces differ in their particle density. The plasmatic fracture face in caulonema tip cells or in tip cells of side branches, but never in other caulonema cells, is further characterized by the occurrence of particle rosettes. The highest density of rosettes is found at the cell apex but decreases steeply toward the cell base. The shape of the rosettes varies remarkably; 20% of them are found in an incomplete, presumably disintegrating or aggregating state. The complete rosette has a diameter of about 25 nm and consists of five to six particles. The size of the single particles varies between 4 nm to 10 nm. The rosettes are thought to posses cellulose-synthase activity. It is assumed that one rosette produces one elementary fibril; rough calculations, considering the number of rosettes and the estimated amount of cellulose produced in the tip region, indicate that an elementary fibrillar length of 900 nm is formed in 1 min by one rosette. The consequence of the kinetics on the life-time of the rosettes and the cellulose-synthase activity are discussed.Abbreviations EF extraplasmatic fracture face - PF plasmatic fracture face     

Investigations of the turnover of the putative cellulose-synthesizing particle “rosettes” within the plasma membrane ofFunaria hygrometrica protonema cells

Summary In youngFunaria protonemata the influence of various inhibitors and treatments on cell elongation, fine-structure, and particle rosettes within the plasma membrane, putative parts of cellulose synthase complexes, was investigated. Cycloheximide (3×10^–5M) inhibited growth, reduced the number of rosettes and evened the gradient of rosette distribution at the beginning of treatment. The cell fine-structure was unaffected. Actinomycin D (10^–5M and 10^–4) caused an initial but transient decrease in rosette number. Alterations in cell elongation and fine-structure have not been observed. Application of 2.6-dichlorobenzonitrile (10^–5 M) for some minutes reduced the number of rosettes remarkably, while cell elongation seemed to be normal after the filaments had been transferred back to normal medium. An incubation of 2 h or longer stopped growth and caused cells to burst. The number of rosettes then rose to about 50% of the control values. When applied for 7 h biofluor (5×10^–4 M) promoted growth slightly, but generally it retarded it when used for a longer time. It did not markedly affect the number of rosettes. A short heat stock stopped elongation, caused the disappearance of rosettes and affected the structure of the mitochondria and of the Golgi apparatus. Plasmolysed cells did not grow and, initially, did not have rosettes. At reduced turgor, wider cells are formed. Freeze fracturing under UHV conditions and shadowing at very low specimen temperature revealed a small, central depression in the 8 nm rosette particles, suggesting that they are composed of subunits. Our results provide further evidence that the rosettes are parts of the cellulose synthase complexes. Their existence clearly depends on protein synthesis and on the constitution of the plasma membrane, but not on cellulose crystallization.     

The role of actin in root hair morphogenesis: studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug

Root hairs develop from bulges on root epidermal cells and elongate by tip growth, in which Golgi vesicles are targeted, released and inserted into the plasma membrane on one side of the cell. We studied the role of actin in vesicle delivery and retention by comparing the actin filament configuration during bulge formation, root hair initiation, sustained tip growth, growth termination, and in full-grown hairs. Lipochito-oligosaccharides (LCOs) were used to interfere with growth ( De Ruijter et al . 1998 , Plant J. 13, 341–350), and cytochalasin D (CD) was used to interfere with actin function. Actin filament bundles lie net-axially in cytoplasmic strands in the root hair tube. In the subapex of growing hairs, these bundles flare out into fine bundles. The apex is devoid of actin filament bundles. This subapical actin filament configuration is not present in full-grown hairs; instead, actin filament bundles loop through the tip. After LCO application, the tips of hairs that are terminating growth swell, and a new outgrowth appears from a site in the swelling. At the start of this outgrowth, net-axial fine bundles of actin filaments reappear, and the tip region of the outgrowth is devoid of actin filament bundles. CD at 1.0 μ m , which does not affect cytoplasmic streaming, does not inhibit bulge formation and LCO-induced swelling, but inhibits initiation of polar growth from bulges, elongation of root hairs and LCO-induced outgrowth from swellings. We conclude that elongating net-axial fine bundles of actin filaments, which we call FB-actin, function in polar growth by targeting and releasing Golgi vesicles to the vesicle-rich region, while actin filament bundles looping through the tip impede vesicle retention.     

Clathrin localization and dynamics in Aspergillus nidulans

Cell growth necessitates extensive membrane remodeling events including vesicle fusion or fission, processes that are regulated by coat proteins. The hyphal cells of filamentous fungi concentrate both exocytosis and endocytosis at the apex. This investigation focuses on clathrin in Aspergillus nidulans, with the aim of understanding its role in membrane remodeling in growing hyphae. We examined clathrin heavy chain (ClaH‐GFP) which localized to three distinct subcellular structures: late Golgi (trans‐Golgi equivalents of filamentous fungi), which are concentrated just behind the hyphal tip but are intermittently present throughout all hyphal cells; the region of concentrated endocytosis just behind the hyphal apex (the “endocytic collar”); and small, rapidly moving puncta that were seen trafficking long distances in nearly all hyphal compartments. ClaH localized to distinct domains on late Golgi, and these clathrin “hubs” dispersed in synchrony after the late Golgi marker PH^OSBP. Although clathrin was essential for growth, ClaH did not colocalize well with the endocytic patch marker fimbrin. Tests of FM4‐64 internalization and repression of ClaH corroborated the observation that clathrin does not play an important role in endocytosis in A. nidulans. A minor portion of ClaH puncta exhibited bidirectional movement, likely along microtubules, but were generally distinct from early endosomes.     

An Emulsion of Sulfoquinovosylacylglycerol with Long-Chain Alkanes Increases Its Permeability to Tumor Cells

The α-anomer form of sulfoquinovosyl-monoacylglycerol with a saturated C18 fatty acid (α-SQMG-C_18:0) is a natural sulfolipid that is a clinically promising antitumor agent. It forms vesicles, micelles or an emulsion in water, depending on several physicochemical conditions. The type of aggregate formed appears to strongly influence the bioactivity level. Thus, we investigated the nature of the aggregates in relation to their bioactivities. The structure of the α-SQMG-C_18:0 assembly was greatly affected by the type of additive used in the preparation. Emulsification with ethanol and n-decane might be more effective at inhibiting tumor cell growth than the micelle or vesicle preparations. α-SQMG-C_18:0 formed an “emulsion-like-aggregate” in ethanol containing an n-decane concentration in the range of 1.03–103 mM. These ethanol/n-alkane/α-SQMG-C_18:0 aggregates inhibited cell growth in a dose-dependent manner, under optimum conditions (i.e., ethanol containing 103 mM of n-decane or n-dodecane dispersed in phosphate-buffered saline or culture medium). Based on these data, we discuss the relationship between the molecular action of and antitumor activity by α-SQMG-C_18:0.     

Critical evaluation of the VSC model for tip growth

The vesicle supply centre (VSC) model (Bartnicki-Garcia et al., 1989) for hyphal tip growth is powerful because it can model diverse developmental morphologies and predicts cellular organization based in current cell biology. It predicts that tip growth results from the random distribution of cell surface synthesizing vesicles from a point in the tip, the VSC, which determines their pattern of impact and fusion at the plasma membrane. We derive equations for tip-high gradients of vesicle fusions, generated by mechanisms not related to a supply centre, which create typical hyphal morphologies. These equations direct the conceptual basis for tip growth to vesicle fusion gradients, presumably mediated by a putative membrane skeleton associated with the plasma membrane. We also show that the organization and behaviour of motile organelles in growing hyphal tips of the oomycete,Saprolegnia ferax, argue against the presence of an apparatus capable of generating the distribution of vesicles postulated by the VSC model. We conclude that the VSC model is unlikely to describe the mechanistic basis of tip growth inS. ferax, and therefore, at best, it is not universally applicable.     

The effects of ruthenium red,lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle transport,vesicle fusion and tip extension in pollen tubes

The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine, all antagonists of Ca²⁺ function in cells, have been studied in growing pollen tubes of Tradescantia virginiana. All four drugs inhibit pollen-tube growth but bring about different ultrastructural changes at the growing tips and within the cytoplasm. The results strongly support the hypothesis that Ca²⁺ plays a vital role in the mechanism of pollen-tube tip growth. The effect of ruthenium red provides evidence that sequestration of Ca²⁺ by mitochondria critically adjusts the concentration of these ions at tube tips. Fluorescein isothiocyanate appears to be a potent inhibitor of vesicle fusion at the plasma membrane, with vesicles accumulating in the tip at rates equivalent to those determined previously for their production. Both vesicle fusion and tip extension are regulated by Ca²⁺ but appear to be independently controlled processes.     

Symplast domains in extrastelar tissues of Egeria densa Planch.

A set of hydrophilic fluorescent dyes of known molecular weight has been used to determine the molecular exclusion limit and the extent of apical, epidermal and cortical symplasts in the root, stem and leaf of Egeria densa. These dyes are unable to pass the plasmalemma, so that any cell-to-cell movement of injected dye must occur via the symplast. The shoot-apex symplast has a high molecular exclusion limit, excluding dyes with a molecular weight of 749 dalton (fluorescein hexaglycine) and greater but allowing dyes of up to 665 dalton (fluorescein diglutamic acid) to pass. The leaf epidermal symplast is similar to that in the apex: fluorescein pentaglycine (674 dalton) moves to a limited extent, but fluorescein hexaglycine is immobile. Stem and root epidermal cells have a lower molecular exclusion limit, only the dye 6-carboxyfluorescein (376 dalton) is able to move from cell-to-cell. Cortical and epidermal tissues in both the stem and the root have similar symplast permeabilities. However, a barrier to dye (6-carboxyfluorescein) movement is found between the epidermis and the cortex in both organs. Barriers are also found at the nodes between expanded internodes. The stem barriers are not found in the unexpanded nodes near the shoot tip; apparently they are formed early during internode expansion. In the root tip, a barrier to the movement of dye is found between the root cap and the remainder of the root. Plasmodesmata are found linking all cell types studied, even cells where barriers to dye movement occur. Thus, the plant, far from being one uniform symplast, consists of a large number of symplast domains, which may or may not differ in molecular exclusion limit.Abbreviations F fluorescein isothiocyanate isomer I - Glu l-glutamic acid - (Glu)₂ l-glutamylglutamic acid - (Gly)₅ l-pentaglycine - (Gly)₆ l-hexaglycine     

Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution

Summary The hyphal tip ofSclerotium rolfsii was examined after fixation by freeze substitution. The Spitzenkörper consisted of a dense mass of apical vesicles and microvesicles surrounding a vesicle-free zone. Linear arrangements of microvesicles were occasionally observed within the Spitzenkörper. Abundant microfilaments were seen within the Spitzenkörper region, often in close association with apical vesicles and microvesicles. Microtubules passed through the Spitzenkörper and terminated at the plasmalemma at the extreme hyphal apex. Filasomes were mostly observed within the apical region and were in close proximity to the plasmalemma. Rough ER, mitochondria, microtubules, and vacuoles were abundant in the subapical region and were usually oriented parallel to the long axis of the hypha. Ribosomes were aligned on the outer surfaces of mitochondria. Golgi body equivalents were observed throughout the subapical region and appeared as inflated cisternae of varying shapes and electron opacities. Relationships to other basidiomycetous hyphal tip cells are discussed.Abbreviations AV apical vesicle - C Celsius - diam diameter - f filasome - G Golgi body equivalent - h hour - nm nanometer - M mitochondria - ME membranous elements; min minute - MV microvesicle - MVB multivesicular body - N nucleus - OsO₄ osmium tetroxide - R ribosome - ER endoplasmic reticulum - S Spitzenkörper - Va vacuole - m micrometer     

The role of microtubules during the cytokinetic events of cell wall ontogenesis and papilla development inCarteria crucifera

Summary An ultrastructural study of cytokinesis, cell wall ontogenesis, and papilla development/form inCarteria crucifera Korsh. andChloromonas rosae Ettl was undertaken. After typical phycoplast-mediated cytokinesis, wall ontogenesis begins at the level of Golgi apparatus activation and secretion to the outside of the daughter cells of fibrillar wall precursors which self assemble into the typical chlamydomonad wall (sensuRoberts 1974). As wall ontogenesis approaches the flagellar region of the cell, several precisely timed events occur: flagellar apparatus formation, flagellar emergence, protoplasmic extension in the future papilla area underlined by series of parallel aligned microtubules, wall formation (at least the W₂–W₆ layers), retraction of the protoplasmic extension and loss of underlying microtubules, and final wall modification (gap filling by W₁ material) to yield the characteristic wall papilla. The transient cytoplasmic extensions mimic the shape of the future wall papilla and are maintained, at least inCarteria, by underlying microtubules. Structural and developmental properties of the papilla are characterized and phylogenetic implications are discussed.This research was supported by National Science Foundation Grant DEB 78-0554.     

Association of a 19- and a 21-kDa GTP-binding protein to pancreatic microsomal vesicles is regulated by the intravesicular pH established by a vacuolar-type H+-ATPase

Summary Evidence suggests that certain ras-related small molecular weight GTP-binding proteins (smg-proteins) are involved in intracellular membrane trafficking and vesicle fusion. We have previously shown that intravesicular acidification due to a vacuolar-type H⁺-ATPase, which is Cl^– dependent and highly sensitive to the specific inhibitor bafilomycin, enhances GTP-induced fusion of pancreatic microsomal vesicles (Hampe, W., Zimmermann, P., Schulz, I. 1990. FEBS Lett. 271:62–66). This process may involve function of smg-proteins. The present study shows that MgATP (2 mm), but neither MgATPS nor ATP in the absence of Mg²⁺, increases association of 19- and 21-kDa smg-proteins to the vesicle membrane as monitored by their [-³²P]GTP binding. The affinity of smg-proteins for [-³²P]GTP was not altered by MgATP. Bafilomycin B₁ (10^–8 m), the protonophore CCCP (10^–5 m), and replacement of Cl^– in the incubation buffer by CH₃COO^– or NO ₃ ^– resulted in an almost complete inhibition of the MgATP-dependent association of the 19- and 21-kDa smg-proteins to the vesicle membranes. Furthermore, the MgATP effect on both smg-proteins was found to be due to the intravesicular pH and not to the H⁺ gradient over the vesicle membrane. We conclude that association of a 19-kDa (immunologically identified as the ADP-ribosylation factor, arf) and a yet unidentified 21-kDa GTP-binding protein to vesicle membranes is regulated by the intravesicular pH established by a vacuolar-type H⁺-ATPase.The arf-antibodies were kindly supplied by Dr. R.A. Kahn. We thank Prof. Dr. D. Gallwitz, Dr. R. Jahn, and Dr. E.G. Lapetina for kindly providing the ypt 1-, rab 3-, and rap 1-antibodies, respectively. ADP-ribosyltransferase C₃ from Clostridium botulinum was kindly supplied by Prof. Dr. K. Aktories. This work was supported by the Jung-Stiftung für Wissenschaft und Forschung. S.Z. was supported by a grant of the Deutsche Forschungsgemeinschaft (Ze 237/3-1).     

Chloride Channel Function in the Yeast TRK-Potassium Transporters

The TRK proteins—Trk1p and Trk2p— are the main agents responsible for “active” accumulation of potassium by the yeast Saccharomyces cerevisiae. In previous studies, inward currents measured through those proteins by whole-cell patch-clamping proved very unresponsive to changes of extracellular potassium concentration, although they did increase with extracellular proton concentration—qualitatively as expected for H⁺ coupling to K⁺ uptake. These puzzling observations have now been explored in greater detail, with the following major findings: a) the large inward TRK currents are not carried by influx of either K⁺ or H⁺, but rather by an efflux of chloride ions; b) with normal expression levels for Trk1p and Trk2p in potassium-replete cells, the inward TRK currents are contributed approximately half by Trk1p and half by Trk2p; but c) strain background strongly influences the absolute magnitude of these currents, which are nearly twice as large in W303-derived spheroplasts as in S288c-derived cells (same cell-size and identical recording conditions); d) incorporation of mutations that increase cell size (deletion of the Golgi calcium pump, Pmr1p) or that upregulate the TRK2 promoter, can further substantially increase the TRK currents; e) removal of intracellular chloride (e.g., replacement by sulfate or gluconate) reveals small inward currents that are K⁺-dependent and can be enhanced by K⁺ starvation; and f) finally, the latter currents display two saturating kinetic components, with preliminary estimates of K_0.5 at 46 μM [K⁺]_out and 6.8 mM [K⁺]_out, and saturating fluxes of ∼5 mM/min and ∼10 mM/min (referred to intracellular water). These numbers are compatible with the normal K⁺-transport properties of Trk1p and Trk2p, respectively.This revised version was published online in August 2005 with a corrected cover date.     

Ultrastructure of spermatogenesis in Cerastoderma glaucum (Cardiacea) and Spisula subtruncata (Mactracea)

Summary

In Cerastoderma glaucum, Sertoli cells are rich in lipids, glycogen and lysosomes, and premeiotic cells exhibited nuage, a prominent Golgi complex and endoplasmic reticulum cisternae encircling the nucleus. The Golgi complex gives rise to proacrosomal vesicles during mid-spermiogenesis, and the round acrosomal vesicle, with a dense fibrillar core, migrates laterally while linked to the plasma membrane as it develops the subacrosomal material. In its final position, the vesicle becomes cap-shaped (0.6 μm) and differentiates into apical light and basal dense regions. The elongated and helicoidal nucleus (8–9.9 μm) has a thin tip (0.3 μm) that invades the subacrosomal space, and in the midpiece (0.8 μm) two of the four mitochondria extend laterally to the nucleus (1.5–2.1 μm). In Spisula subtruncata, Sertoli cells are rich in lipids, glycogen and phagocytosed sperm. Premeiotic cells exhibit nuage, a prominent Golgi complex that gives rise to proacrosomal vesicles from the leptotene stage and a flagellimi that is extruded at zygotene. The acrosomal vesicle forms during the round spermatid stage and differentiates into a large and dense basal region and an apical light region. It then migrates while linked to the plasma membrane by its apical pole. Development of the subacrosomal perforatorium is associated with nuage materials and endoplasmic reticulum vesicles. The mature cap-shaped (0.6 μm) acrosomal vesicle exhibits a large apical and irregular region with floccular contents and a basal dense region. The round nucleus becomes barrel-shaped (1.5 μm) and the midpiece (0.8 μm), with four mitochondria, contains a few glycogen particles.     

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (β-D-glucosyl)₃ Yariv phenylglycoside [(β-D-Glc)₃] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(β-D-Glc)₃ complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall. Received: 14 August 1997 / Accepted: 9 September 1997     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

Electrophysiological investigation of the amino acid carrier selectivity in epithelial cells fromXenopus embryo

Summary The electrical responses induced by external applications of neutral amino acids were used to determine whether different carriers are expressed in the membrane of embryonic epithelial cells ofXenopus laevis. Competition experiments were performed under voltage-clamp conditions at constant membrane potential.Gly,l-Ala,l-Pro,l-Ser,l-Asn andl-Gln generate electrical responses with similar apparent kinetic constants and compete for the same carrier. They are [Na] _o and voltage-dependent, insensitive to variations in [Cl] _o and [HCO₃] _o , inhibited by pH _o changes, by amiloride and, for a large fraction of the current, by MeAIB. The increase in [K] _o at constant and negative membrane potential reduces the response, whereas lowering [K] _o augments it. l-Leu,l-Phe andl-Pro appear to compete for another carrier. They generate electrogenic responses insensitive to amiloride and MeAIB, as well as to alterations of membrane potential, [Na] _o and [K] _o . Lowering [Cl] _o decreases their size, whereas increasing [HCO₃] _o at neutral pH _o increases it.It is concluded that at least two and possibly three transport systems (A, ASC and L) are expressed in the membrane of the embryonic cells studied. An unexpected electrogenic character of the L system is revealed by the present study and seems to be indirectly linked to the transport function. l-Pro seems to be transported by system A or ASC in the presence of Na and by system L in the absence of Na. MeAIB induces an inward current.     

Germination and parasitation of the resting sporangia ofSynchytrium endobioticum

Summary The cytoplasmic organization of the long-lived, thick walled resting stage of the sporangium ofSynchytrium endobioticum (Schilb.) Perc. is described. The cytoplasm of the resting sporangium contains a large number of closely packed lipid bodies and irregular electron dense bodies, which are interspaced with fine channels of cytoplasm. These ultrastructural observations are discussed in relation to the hypothesis ofBally (1912) andCurtis (1921) that zoospore primordia are already present during the resting stage. It is shown that the zoospore primordium is actually a lipid body and an osmiophilic body and the strands postulated to connect the individual zoospore primordia are actually the fine channels of cytoplasm.A new inner wall layer is laid down prior to the start of the germination. It is this wall layer which will protrude to form the vesicle in which sporogenesis takes place. The germination process observed, protrusion of a vesicle through a crack in the sporangial wall, the migration of the sporangial content into the vesicle, and the formation of a single, membrane-bound sporangium within this vesicle, is in full agreement with the recent light microscopic studies ofSharma andCammack (1976). These observations support the transfer ofS. endobioticum from the subgenusMesochytrium to the subgenusMicrosynchytrium (bothsensu Karling 1964).A major objective of the study, to obtain ultrastructural evidence for the location of the meiotic divisions in the life cycle, was not fulfilled.Three different fungi were observed to parasitize the resting sporangium ofS. endobioticum. These infections are discussed in relation to other mycoparasites of plant pathogenic fungi. The possibility of using a mycoparasite for the biological control of potato wart disease is considered to be without practical relevance.