Subcellular site of the biosynthesis ofO-acetylated sialic acids in bovine submandibular gland

Bovine submandibular glands were homogenized and fractionated under conditions which yielded subcellular fragments from mainly one cell type, the mucous acinar cell, as judged by morphological analysis of the glands before and after homogenization. The majorN-acetylneuraminate-9(7)-O-acetyltransferase activity was detected in the cytosolic fraction, a result supported by the high specific radioactivity of free sialic acids isolated after [¹⁴C]acetate-labelling experiments. Separation of membranes on a Ficoll density gradient gave six fractions which were analyzed biochemically and morphologically. The particulate activities of acetyltransferase and sialyltransferase were found in fractions containing smooth and mitochondrial membranes. MembraneO-acetyl sialic acids were present at the highest levels in these fractions and also had the highest specific radioactivity after [¹⁴C]acetate-labelling experiments. Significant amounts of theO-acetyltransferase activity also occur in the cytosol and are consistent with a model ofO-acetyl sialic acid biosynthesis involving both cytosolic and smooth membrane sites ofO-acetylation.     

Variation in polar-group content in lipids of cowpea (Vigna unguiculata) Cell cultures as a mechanism of haloadaptation

Callus and cell suspension cultures of cowpea (Vigna unguiculata) were induced with 2,4-dichlorophenoxyacetic acid and grown at different NaCl concentrations. The cell biomass yield and its total lipid content decreased with increasing salinity. However, while the hexose content in lipids was higher, the amount of lipid phosphorus was significantly lower in both agar and cell suspension cultures. Ion-transport rates with artificial membranes prepared with different lipid fractions showed that lipids from cells grown in a saline medium were less permeable to Na⁺ and to Cl^- than those grown in a non-saline medium. Also the permeability of membranes prepared with glycolipids was lower than those prepared with phospholipids and whole lipids. Apparently, the increase of hexose/phosphorus ratio in membrane lipids is induced in response to the halo-adaptation process.     

Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod ⁺fix^- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod⁺ fix^- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.     

A study of dynamic membrane phenomena during the gastric secretory cycle: Fusion,retrieval and recycling of membranes

Summary The possibility of recycling, fusion and retrieval of membranes during the gastric secretory process was studied in isolated gastric mucosae of the toadBufo marinus. Incorporation and efflux of¹⁴C-inulin and horseradish peroxidase (HRP) into the tissue as well as transmission and freeze-fracture electron microscopic studies during the secretory cycle were done. HRP and¹⁴C-inulin were incorporated into the tubulovesicular membrane system during the secreting-resting transition. Upon restimulation, markers were released towards the lumen. Marker efflux preceded onset of H⁺ secretion. Morphological transformations in the oxyntic cell as evidenced from transmission and freeze-fracture electron microscopy preceded acid secretion coinciding with marker efflux. At this time, images that have been associated with membrane fusion were found in the apical membranes of oxyntic cells. The results are consistent with a model where membrane area increases by a fusion mechanism at the expense of the tubulovesicular system. This transformation precedes the onset of H⁺ secretion. Upon cessation of the stimulus or inhibition, membranes are retrieved and the tubulovesicular system reformed. Retrieved membranes could be then reutilized in the next secretory cycle.     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Calcium carbonate deposition in a cell wall sac formed in mulberry idioblasts

Summary. Although calcium carbonate is known to be a common biomineral in plants, very little attention has been given to the biological control of calcium carbonate deposition. In mulberry leaves, a subcellular structure is involved in mineral deposition and is described here by a variety of cytological techniques. Calcium carbonate was deposited in large, rounded idioblast cells located in the upper epidermal layer of mulberry leaves. Next to the outmost region (“cap”) of young idioblasts, we found that the inner cell wall layer expanded to form a peculiar outgrowth, named cell wall sac in this report. This sac grew and eventually occupied the entire apoplastic space of the idioblast. Inside the mature cell wall sac, various cellulosic membranes developed and became the major site of Ca carbonate deposition. Concentrated Ca²⁺ was pooled in the peripheral zone, where small Ca carbonate globules were present in large numbers. Large globules were tightly packed among multiple membranes in the central zone, especially in compartments formed by cellulosic membranes and in their neighboring membranes. The maximum Ca sink capacity of a single cell wall sac was quantified using enzymatically isolated idioblasts as approximately 48 ng. The newly formed outgrowth in idioblasts is not a pure calcareous body but a complex cell wall structure filled with substantial amounts of Ca carbonate crystals. Correspondence and reprints: Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

A 31P NMR study of the interaction of amphibian antimicrobialpeptides with the membranes of live bacteria

Summary Amphibian skin is a rich source of peptides that are specific to pathogens and act by disrupting bacterial membranes. Three antimicrobial peptides were isolated from the skin glands of Australian tree frogs,Litoria caerulea andLitoria genimaculata. NMR spectroscopy was used to observe changes induced by these peptides in the³¹P resonances of bacterial membranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics disrupted the membranes ofBacillus cereus andStaphylococcus epidermidis (Gram-positive), leading to an increase in the isotropic³¹P NMR signal. Caerin 4.1, a narrow-spectrum antibiotic, however, did not affect the³¹P spectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents on the membranes of live bacteria.     

Electron microscopy of the K2 killer effect of Saccharomyces cerevisiae T206 on a mesophilic wine yeast

A mesophilic wine yeast, Saccharomyces cerevisiae CSIR Y217 K ^– R ^– was subjected to the K₂ killer effect of Saccharomyces cerevisiae T206 K ⁺ R ⁺ in a liquid grape medium. The lethal effect of the K₂ mycoviral toxin was confirmed by methylene blue staining. Scanning electron microscopy of cells from challenge experiments revealed rippled cell surfaces, accompanied by cracks and pores, while those unaffected by the toxin, as in the control experiments, showed a smooth surface. Transmission electron microscopy revealed that the toxin damaged the cell wall structure and perturbed cytoplasmic membranes to a limited extent.     

A31P NMR study of the interaction of amphibian antimicrobial peptides with the membranes of live bacteria

Amphibian skin is a rich source of peptides that are specificto pathogens and act by disrupting bacterial membranes. Threeantimicrobial peptides were isolated from the skin glands ofAustralian tree frogs, Litoria caerulea and Litoriagenimaculata. NMR spectroscopy was used to observe changesinduced by these peptides in the ³¹P resonances of bacterialmembranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics, disrupted the membranes ofBacillus cereus and Staphylococcus epidermidis (Gram-positive), leadingto an increase in the isotropic ³¹P NMR signal. Caerin 4.1, anarrow-spectrum antibiotic, however, did not affect the ³¹Pspectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents onthe membranes of live bacteria.     

Cytotoxicity of equinatoxin II from the sea anemoneActinia equina involves ion channel formation and an increase in intracellular calcium activity

Summary Equinatoxin Il is a 20-kDa basic protein isolated from the sea anemoneActinia equina. The aim of our work was to investigate the primary molecular basis for the cytotoxic effects of equinatoxin II in two model systems: single bovine lactotrophs and planar lipid bilayers. Previous work has shown that equinatoxin II produces rapid changes in cell morphology, which are dependent on external calcium. It has also been reported that addition of equinatoxin II increases membrane electrical conductance, which suggests that the cytotoxic action of equinatoxin II involves an increase in the permeability of membranes to Ca²⁺. Extensive changes in cytosolic Ca²⁺ activity are thought to invoke irreversible changes in cell physiology and morphology. In this paper, we show that morphological changes brought about by equinatoxin II in bovine lactotrophs are associated with a rapid rise in cytosolic Ca²⁺ activity, monitored with a fura-2 video imaging apparatus. Moreover, incorporation of equinatoxin II into planar lipid bilayers produces Ca²⁺ permeable ion channels. This suggests that the mode of equinatoxin II cytotoxicity involves the formation of cation (Ca²⁺) permeable channels in cell membranes.     

Effect of mitotic inhibitors on the ultrastructure of root meristem cells

After 5 h in 10^-3M vinblastine the most obvious effects upon Vicia faba L. cells are seen in the spindle apparatus. These include the microtubules themselves as indicated by c-type metaphases and the pole regions of otherwise intact spindles, leading to multipolar anaphases and to telophases with more than two daughter nuclei. Vesicle transport may be undisturbed and new cell walls can be formed, although cases of disturbed cell plate and cell wall formation were noted occasionally, accompanied by myelinizations in phragmosomes. After 24 h in the same concentration of vinblastine, divisions are no longer observed and the plasma membranes are severely affected. They show extensive myelinizations, accumulations of lipids and dehiscence from the cell walls which are frequently thickened and irregularly formed. Of the other cellular membranes, the nuclear envelope and, more frequently, the tonoplast may be affected. Electron-dense deposits appear in the vacuoles. Comparable, though less severe, changes including multipolar anaphases and myelinizations result from treatment with lumicolchicine, but not with colchicine. Vinblastine leads to the appearance of filament bundles both in cytoplasm and karyoplasm, lumicolchicine to morphologically identical filaments in the cytoplasm alone. The nature of these filaments is unknown.Abbreviation VLB vincaleukoblastine     

Structure and possible functions of the calcospherite-rich cells (R* cells) in the digestive gland of the shore crab CArcinus maenas

Summary R^*-cells of the digestive gland of Carcinus maenas have been investigated functionally and morphologically. A comparison of the capacity of separated cell suspensions to synthesize glycogen gave support to the hypothesis that R and R^* cells belong to the same cell line. The unexpected observation of R^* cells in gastric juice suggests that their release could represent a mode of redistribution of carbohydrate stores when the feeding activity of the crab is lower. Under electron microscopy, the calcospherites of R^* cells appeared to be surrounded by multiple membranous layers, and displayed tubular and vesicular structures in their core. High glucose-6-phosphatase (G6Pase) activity in the subcellular fraction that is enriched in calcospherites suggests that these membranes are derived from the endoplasmic reticulum, via a process in which the enzyme plays a key role. We propose that this is the way by which the R cell differentiates into R^* cell.     

β-1-4-and β-1-3-glucan synthases are associated with the plasma membrane of the fungus Saprolegnia

Cytoplasmic membranes from mycelium or protoplasts of Saprolegnia monoica (a cellulosic cell-wall fungus) were separated by continuous sucrose-density-gradient centrifugation. Glucan synthases assayed at low (micromolar uridine 5-diphosphate (UDP) glucose for -1-4-glucan synthase) and high (millimolar UDP glucose for -1-3-glucan synthase) substrate concentrations were associated with membranes exhibiting vanadate-sensitive, oligomycin-insensitive ATPase and equilibrating at density 1.16 g cm^-3. Synthase activities were also bound to membranes of lower density (1.10 and 1.145 g cm^-3). Plasma membranes were stabilized by coating protoplasts with concanavalin A. After lysis of the protoplasts, plasma membranes recovered by low centrifugal forces were isolated in continuous isopycinic gradients. Both synthase activities peaked with [³H]concanavalin A and Na-vanadate ATPase indicating that the synthetases are located at the plasma membrane. Treatments of intact protoplasts with cold glutaraldehyde or proteases before disruption lead to a diminution of glucan-synthase activities indicating that at least part of the enzymes of plasma membrane face the outside of the cell.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - GSI -1,4-glucan synthase - GSH -1,3-glucan synthase - UDP uridine 5-diphosphate     

Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber

Embryogenic cell suspensions of rubber derived from immature inflorescences and inner integuments of immature fruits released 3.1 ± 0.2 × 10⁷ protoplasts g^-1 f. wt. (mean ± s.e.m, n = 10) and 3.2 ± 0.2 × 10⁷ protoplasts g^-1 f. wt., with mean viabilities of 83 ± 2% and 77 ± 8%, respectively. Sustained mitotic division was observed only when protoplasts were cultured in KPR liquid medium on nitrocellulose membranes overlying the same semi-solid medium containing Lolium multiflorum nurse cells. Protoplast-derived cell colonies were produced within 2 months of culture. Protoplast-derived cell colonies proliferated, upon subculture to MS-based regeneration medium, with 40% of the protoplast-derived calli developing somatic embryos. The latter germinated into plants on the same medium after 3 months of culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermoluminescence properties of the S2-state in chloride-depleted water oxidizing complexes after reconstituting treatments with various monovalent anions

Under conditions that assured rebinding of the extrinsic 17 and 23 kDa polypeptides, Cl^--depleted Photosystem II membranes isolated from spinach chloroplasts were subjected to reconstituting treatments in media containing NaF, NaCl, NaBr, NaI or NaNO₃, or they were kept in a medium without any added salt other than the buffer. After removing most of the unbound reconstituting anions by washing, the O₂-evolution activities and thermoluminescence properties of the membranes were compared. While the temperature of maximal thermoluminescence emission was lowest for membranes treated with Cl^-, no uniform correlation was evident between the temperature profile of the thermoluminescence emission and the apparent activating effectiveness of the anions in the membranes' water oxidizing machinery. However, the differences between the thermoluminescence features did conform to a trend according to which the emission temperatures were upshifted as the size of the activating anion increased, and its hydration energy decreased, i.e. Cl^-<Br^-<NO₃ ^-<I^-. The inactive F^- anions were not well retained by the membranes. To explain the experimental data it is suggested that the structural environment of the charge accumulating Mn-center is influenced by the ionic conditions encountered by the Photosystem II membranes after Cl^- removal, further enforced by the binding of compatible anions, and then stabilized by the 17 and 23 kDa extrinsic polypeptides. If, as some concepts imply, the anion binding sites are located at or near the functional Mn, only very exceptional characteristics of the water-oxidizing mechanism may account for the observation that the potentially electron-donating I^- anion can serve as activator and that it stabilizes rather than destabilizes the S₂-state.Abbreviations Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - Mes 2-(N-morpholino)ethane sulfonic acid - Pheo the pheophytin a of the Photosystem II reaction center - PS photosystem     

Absence of plasma membrane H+-ATPase in plasmodesmata located in pit-fields of the young reactive pulvinus ofMimosa pudica L.

Summary Immunocytochemical techniques were employed to study the spatial distribution of the plasma membrane H⁺-ATPase within various cell types of the young reactive primary pulvinus ofMimosa pudica L. These cells were interconnected by large numbers of plasmodesmata, being concentrated within pit-fields. Although we could routinely detect evidence of the H⁺-ATPase along the plasma membrane, immunolabelling was rarely, if ever, observed along the plasma membranes of the plasmodesmata. This finding is discussed with respect to the likely specialized supramolecular structure of the plasmodesma.Abbreviations SEL size exclusion limit of plasmodesmata     

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil

Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [³H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [³H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [³H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTPγS and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [³H]alkylacyl-GP and [³H]alkylacyl-GPethanol, phospholipase D breakdown products from [³H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [³H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes. © 1996 Wiley-Liss, Inc.     

Identification and mitotic partitioning strategies of vacuoles in the unicellular red alga <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis>

Cyanidioschyzon merolae is considered as a suitable model system for studies of organelle differentiation, proliferation and partitioning. Here, we have identified and characterized vacuoles in this organism and examined the partitioning of vacuoles using fluorescence and electron microscopy. Vacuoles were stained with the fluorescent aminopeptidase substrate 7-amino-4-chloromethylcoumarin l-arginine amide, acidotrophic dyes quinacrine and LysoTracker, and 4′,6-diamidino-2-phenyl indole, which, at a high concentration, stains polyphosphate. Vacuoles have been shown to be approximately 500 nm in diameter with a mean of around five per interphase cell. The vacuolar H⁺-ATPase inhibitor concanamycin A blocked the accumulation of quinacrine in the vacuoles, suggesting the presence of the enzyme on these membranes. Electron microscopy revealed that the vacuoles were single membrane-bound organelles with an electron-dense substance, often containing a thick layer surrounding the membrane. Immunoelectron microscopy using an anti-vacuolar-H⁺-pyrophosphatase antibody revealed the presence of the enzyme on these membranes. In interphase cells, vacuoles were distributed in the cytoplasm, while in mitotic cells they were localized adjacent to the mitochondria. Filamentous structures were observed between vacuoles and mitochondria. Vacuoles were distributed almost evenly to daughter cells and redistributed in the cytoplasm after cytokinesis. The change in localization of vacuoles also happened in microtubule-disrupted cells. Since no actin protein or filaments have been detected in C. merolae, this result suggests an intrinsic mechanism for the movement of vacuoles that differs from commonly known mechanisms mediated by microtubules and actin filaments.     

Involvement of a lipid-linked intermediate in the transfer of galactose from UDP [14C]galactose to exogenous protein in castor bean endosperm homogenates

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of galactose from UDP[¹⁴C]galactose into chloroform/methanol (2:1)-soluble glactolipids. At least two galactolipids were formed. Most of the [¹⁴C]galactose was present in a galactolipid synthesized by the microsomal membranes, the remainder was present in a second galactolipid synthesized by other cellular membranes, possibly Golgi-derived. The addition of asialo-agalacto-fetuin reduced incorporation of [¹⁴C]galactose into the microsomal galactolipid with a concomitant increase in microsomal [¹⁴C]galactoprotein. Asialo-agalacto-fetuin did not affect galactolipid or galactoprotein synthesis by nonmicrosomal fractions. The results suggest that the endoplasmic reticulum is a major site of protein galactosylation in castor bean endosperm cells, and that galactose transfer from UDP-galactose to protein occurs via a lipid-linked intermediate.Abbreviations ER endoplasmic reticulum - ASGF asialoagalacto-fetuin - IDPase inosine diphosphatase - TCA trichloroacetic acid