The influence of fluoro-phenyl-alanine on the synthesis of simian virus 40 DNA and T antigens.

Treatment of Simian Virus 40 (SV40) infected monkey cells with fluorophenylalanine (FPA) resulted in increased uptake of thymidine by the cells, and progressive inhibition of both viral and cellular DNA synthesis. Viral DNA synthesis was more sensitive to inhibition by FPA than cell DNA synthesis. Synthesis of SV40 T antigens was however unaffected by FPA, as judged from immunofluorescence assays. The M.W. of the major polypetides immunoprecipitated from cell extracts by antibodies from tumor bearing hamster sera was similarly unaffected. It is suggested that T antigen synthesized in the presence of FPA is non functional.     

Comparative evaluation of the glycosaminoglycan composition in plasma membranes of normal and transformed mouse liver cells

Using electrophoresis in agarose gel, a comparative study of composition of membrane-bound glycosaminoglycans (GAG) from normal mouse liver cells and from o-aminoazatoluene-induced mouse hepatoma cells was carried out. Differences in the composition and localization of GAG were revealed. The plasma membrane fraction of hepatoma cells contained no GAG; the bulk of GAG (approximately 98%) was localized in the plasma membrane free fraction. Within this fraction GAG contained no heparan sulfates with a high electrophoretic mobility that were detected in plasma membranes of normal liver cells. The possible involvement of proteoglycans bound to cell surface in transmembrane signal transfer is discussed.     

The isolation of endosome-derived vesicles from rat hepatocytes.

Intracellular 5'-nucleotidase involved in membrane circulation in rat hepatocytes is latent, and is protected from inhibition when whole cells are incubated with inhibiting antiserum at 2 degrees C [Stanley, Edwards & Luzio (1980) Biochem. J. 186, 59-69]. These two criteria were used to identify intracellular membrane vesicles containing 5'-nucleotidase on Ficoll density gradients. A sharply defined turbid band containing intracellular 5'-nucleotidase isolated on density gradients was further fractionated by immunoadsorption of plasma-membrane fragments derived from the cell surface of surface-inhibited cells on to an anti-(immunoglobulin G) immunoadsorbent. The resulting non-adsorbed membrane fraction consisted of vesicles of uniform size (approx. 65 nm diam.), but was not identifiable as any known organelle. This fraction could account for approx. 5% of the total cell 5'-nucleotidase activity, and the enzyme activity measured was 55% latent. The fraction had a restricted polypeptide composition but similar phospholipid composition compared with plasma membrane. We suggest that the vesicles observed in this fraction were derived from the endocytic pathway.     

Integrin-like Protein Is Involved in the Osmotic Stress-induced Abscisic Acid Biosynthesis in Arabidopsis thaliana

We studied the perception of plant cells to osmotic stress that leads to the accumulation of abscisic acid （ABA） in stressed Arabidopsis thaliana L. cells. A significant difference was found between protoplasts and cells in terms of their responses to osmotic stress and ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction are essential in identifying osmotic stress. Western blotting and immunofluorescence localization experiments, using polyclonal antibody against human integrin β1, revealed the existence of a protein similar to the integrin protein of animals in the suspension-cultured cells located in the plasma membrane fraction. Treatment with a synthetic pentapeptide, Gly-Arg-Gly-Asp-Ser （GRGDS）, which contains an RGD domain and interacts specifically with integrin protein and thus blocks the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, but not in protoplasts. These results demonstrate that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-Iike proteins played important roles in osmotic stress-induced ABA biosynthesis in Arabidopsis thaliana.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Characterization of the cell wall and outer membrane of Rhodopseudomonas capsulata.

Sucrose density gradient centrifugation of cell envelopes of chemotrophically grown cells of Rhodopseudomonas capsulata St. Louis (= ATCC 23782) resulted in the separation of a cytoplasmic membrane from a cell wall fraction (buoyant densities, 1.139 and 1.215 g/cm3, respectively). The cell wall fractions (untreated or Triton extracted) contained peptidoglycan- and lipopolysaccharide-specific components. Their neutral sugar content, mainly rhamnose and galactose, was high (250 and 100 micrograms/mg [dry weight] of material) due to a non-lipopolysaccharide polymer. The fatty acid content was low (less than or equal to 60 micrograms/mg [dry weight] of material), and half of it was contributed by lipopolysaccharide (3-OH-C10:0, C12:1, and 3-oxo-C14:0). The predominant other fatty acid was C18:1. An outer membrane fraction, obtained by lysozyme treatment of the Triton-extracted cell wall, showed essentially the same chemical composition except for almost complete removal of peptidoglycan. Saline extraction (0.9% NaCl, 37 degrees C, 2 h) removed a lipopolysaccharide-protein(-phospholipid?) complex from whole cells of R. capsulata St. Louis. The polypeptide patterns of the cell wall and outer membrane as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comprised 20 to 25 different polypeptides (most of them very faint) and were dominated by a single, heat-modifiable major protein (Mr 69,000 after solubilization below 60 degrees C; Mr 33,000 at temperatures above 70 degrees C).     

Protein Composition of the Cell Wall and Cytoplasmic Membrane of Escherichia coli

Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions. The fraction with the higher density was enriched in fragments derived from the cell wall, as indicated by the high content of lipopolysaccharide, the low content of cytochromes, and the similar morphology of the fragments and intact cell walls. The less-dense fraction was enriched in vesicles derived from the cytoplasmic membrane, as indicated by the enrichment of cytochromes, the enzymes lactic and succinic dehydrogenase and nitrate reductase, and the morphological similarity of the vesicles to intact cytoplasmic membrane. Both fractions were rich in phospholipid. The protein composition was compared by mixing the cytoplasmic membrane-enriched fraction from a (3)H-labeled culture with the cell wall-enriched fraction from a (14)C-labeled culture and examining the resulting mixture by gel electrophoresis. Thirty-four bands of radioactive protein were resolved; of these, 27 were increased two- to fourfold in the cytoplasmic membrane-enriched fraction, whereas 6 were similarly increased in the cell wall-enriched fraction. One of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope. This protein accounted for 70% of the total protein of the cell wall, and its occurrence in the envelope from spheroplasts suggests that it is a structural protein of the outer membranous component of the cell wall.     

Yeast 1,3-beta-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic reticulum

1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.     

Effect of osmotic stress on the growth of epicotyls of Cicer arietinum in relation to changes in cell wall composition

The inhibition of growth by polyethlene glycol (PEG)-induced osmotic stress led to modifications in the changes taking place in cell wall composition during normal growth of epicotyls of Cicer arietinum L. cv. Castellana. Epicotyls growing under normal conditions showed a decrease in the amount of pectic fractions and an increase in the hemicellulosic fractions and α-cellulose that led to an increase in the rigidity and a loss in growth capacity. Among the hemicellulosic fractions, the KI-2 fraction (insoluble fraction of 10% KOH-extracted hemicelluloses) seemed to be the only one related to the elongation process and subsequent rigidity. During normal growth a decrease was observed in the total amount of galactose, mainly from the pectic fractions. The inhibition of elongation led to an increase in the amount of the cell walls, due to inhibition of cellular elongation. PEG prevented the increase in the hemicelluloses and the α-cellulose, indicating that these changes were related to elongation. Thus, during the inhibition of elongation there is probably an inhibition of new synthesis that prevents cell wall rigidity and maintains cell wall growth capacity. Changes in the pectic fractions during growth were not affected by the inhibition of elongation, showing that these fractions are related to cell wall loosening rather than to elongation. Study of the cell wall composition confirms the idea that the autolytic process is regulated by changes in the cell wall structure during epicotyl growth     

Transport and processing of staphylococcal enterotoxin B.

A larger, membrane-bound form of staphylococcal enterotoxin B was shown by in vivo pulse-chase analysis to be the kinetic precursor to extracellular enterotoxin B. Processing of the enterotoxin B precursor molecules can apparently occur either cotranslationally or posttranslationally. Subcellular fractionation of cells revealed that all of the precursor toxin was associated with the membrane fraction. Once processed and released from the membrane, it was transiently associated with the cell wall before being released into the extracellular environment. The cell-wall-associated enterotoxin B was completely resistant to protease treatment and to extraction by high- or low-salt solutions at 0 to 2 degrees C, although it could be easily released from the cell by removal of the cell wall with lysostaphin. These data imply that newly formed enterotoxin B may be temporarily sequestered in specialized regions that require cell wall integrity before being released into the extracellular environment.     

Transforming Growth Factor β1Induces Squamous Carcinoma Cell Variants with Increased Metastatic Abilities and a Disorganized Cytoskeleton

Previous studies indicated that mouse transformed keratinocytes undergo an epithelial–fibroblastic conversion when cultured in the presence of TGF-β₁. This conversion is associatedin vivowith a squamous-spindle carcinoma transition. We derived epithelioid (A6, FPA6) and spindle (B5) clonal cell variants from a squamous carcinoma cell line (PDV) after treatment with TGF-β₁. FPA6 cells were isolated from the ascites fluid of an A6-tumor-bearing mouse. FPA6 and A6 cell lines produced in nude mice mixed carcinomas with a squamous and poorly differentiated component. Both cell lines coexpressed keratins and vimentin and synthesized E-cadherin protein, although FPA6 cells cultured at early passages (FPA6-ep) had reduced levels of E-cadherin mRNA and increased synthesis of keratin K8, a marker of malignant progression. Immunofluorescence analysis revealed that FPA6-ep cells exhibited a disorganized cytoskeleton with keratins forming focal juxtanuclear aggregates and loss of F-actin stress fibers and cortical bundles, and E-cadherin was localized in the cytoplasm out of cell–cell contact areas. Sporadic cells in A6 and PDV cultures also presented those anomalous keratin structures, suggesting that FPA6 cells originated from a subpopulation of A6 tumor cells that metastasized into the peritoneal cavity. The analysis of the spontaneous and experimental metastatic potentials of the cell lines showed that epithelioid and fibroblastic cell variants had acquired metastatic abilities compared to PDV which was nonmetastatic. The FPA6-ep cell line exhibited a highly aggressive behavior, killing the animals at about 17 days after intravenous injection of the cells into athymic mice. The phenotype of FPA6-ep cells was unstable and reverted at later passages in which the normal organization of keratin and F-actin in filaments and the localization of E-cadherin at cell–cell contacts were restored. This phenotypic reversion occurred concomitantly with a reduction of the experimental metastatic potential of FPA6 cells.     

The Arabidopsis Rab GTPase RabA4b localizes to the tips of growing root hair cells

Spatial and temporal control of cell wall deposition plays a unique and critical role during growth and development in plants. To characterize membrane trafficking pathways involved in these processes, we have examined the function of a plant Rab GTPase, RabA4b, during polarized expansion in developing root hair cells. Whereas a small fraction of RabA4b cofractionated with Golgi membrane marker proteins, the majority of this protein labeled a unique membrane compartment that did not cofractionate with the previously characterized trans-Golgi network syntaxin proteins SYP41 and SYP51. An enhanced yellow fluorescent protein (EYFP)-RabA4b fusion protein specifically localizes to the tips of growing root hair cells in Arabidopsis thaliana. Tip-localized EYFP-RabA4b disappears in mature root hair cells that have stopped expanding, and polar localization of the EYFP-RabA4b is disrupted by latrunculin B treatment. Loss of tip localization of EYFP-RabA4b was correlated with inhibition of expansion; upon washout of the inhibitor, root hair expansion recovered only after tip localization of the EYFP-RabA4b compartments was reestablished. Furthermore, in mutants with defective root hair morphology, EYFP-RabA4b was improperly localized or was absent from the tips of root hair cells. We propose that RabA4b regulates membrane trafficking through a compartment involved in the polarized secretion of cell wall components in plant cells.     

Immunogold localization of callose and other cell wall components in pea nodule transfer cells

Summary Transfer cells are located adjacent to xylem and phloem elements in pea nodule vascular tissues. The composition of the labyrinthine wall intrusions was investigated by immunogold labeling using specific antibody probes. Callose antigen was found at the base of newly formed cell wall intrusions and also in adjacent plasmodesmata. Sections through developed labyrinthine intrusions revealed that wall ingrowths had an internal structure with small domains of callose suggesting the presence of channels or vents. Xyloglucan and pectin antigens were uniformly distributed within the wall, but the distribution of extensin antigens was variable, with different antigens being detected in different regions of the wall ingrowth. A lectinlike glycoprotein, PsNLEC-1, was localized in intercellular spaces associated with nodule transfer cells. Previously, expression of this component was observed in other types of cells showing complex involution of the plasma membrane, namely root cortical cells harboring arbuscular mycorrhizae and nodule cells harboring nitrogen-fixing rhizobia.     

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.     

Distribution of Lipids in the Wall and Cytoplasmic Membrane Subfractions of the Cell Envelope of Escherichia coli

Cell wall and membrane subfractions of the cell envelope of Escherichia coli have been isolated by a procedure involving particle electrophoresis and sucrose gradient density centrifugation. The lipid content of each fraction has been investigated. The individual phospholipids of both fractions are quantitatively similar except that the proportion of lysophosphatidylethanolamine is greater in the wall than in the membrane. Fatty acid analysis of the phospholipids of each fraction revealed that the wall phospholipids contain a greater proportion of palmitic acid. Coenzyme Q is almost exclusively localized in the cell membrane.     

The effect of ascorbic acid on arachidonic acid and prostaglandin E2 metabolism in B16 murine melanoma cells

Ascorbic acid (Asc), arachidonic acid (AA) and prostaglandin E2 (PGE2) are reported to be important in maintaining the stability of the cell matrix. Asc has also been shown to influence fatty acid (FA) and PGE2 synthesis, with the result that effects of Asc on cell growth are suggested to be mediated through the metabolism of these two compounds. This study examined the effect of Asc, supplemented over the concentration range of 0-100 micrograms/ml, on the in vitro cell growth of non-malignant LLCMK (monkey kidney) cells and malignant B16 murine melanoma cells. The effects of Asc supplementation on AA and PGE2 levels in the cell stroma and membrane fractions of the two cell types was also determined. Asc had no significant inhibitory or stimulatory effect on the growth of either the B16 or LLCMK cells. The total percentage AA composition determined in the B16 control cells (combined stroma and membrane fractions), was similar to that determined in the LLCMK control cells. Asc supplementation of the B16 cells, resulted in an inverse relationship between B16 cell growth and total percentage AA composition. PGE2 concentration in the control B16 cells (combined stroma and membrane fractions) was significantly higher than that detected in the control LLCMK cells. No PGE2 was detected in the B16 stroma fraction, with all appearing to be located in the membrane fraction. However, upon the supplementation of the B16 cells with increasing Asc concentrations, PGE2 appeared to be mobilized from the membrane fraction, resulting in increasing PGE2 levels in the stroma fraction relative to the membrane fraction. This was accompanied by a significant decrease in PGE2 concentration, in the membrane fraction. B16 cell growth and total (stroma and membrane fractions) PGE2 concentration in these cells was inversely related, when cultures were supplemented with increasing levels of Asc. Asc supplementation of the LLCMK cells did not appear to have any significant effect on AA or PGE2 metabolism in these cells.     

Cell wall changes in nisin-resistant variants of Listeria innocua grown in the presence of high nisin concentrations

Abstract Two nisin-resistant variants of a strain of Listeria innocua were isolated after growth in the presence of 500 and 4000 IU ml⁻¹ of nisin A showed increased cell wall hydrophobicity, resistance to phage attack and three different cell wall-acting antibiotics, as well as to the peptidoglycan hydrolytic enzymes lysozyme and mutanolysin, as compared to the parental strain. Transmission electron microscopy revealed marked thickening of the wall of nisin-resistant cells with an irregular surface. Differences in thickness were lost after cell wall purification and no significant difference in gross wall composition was observed between the parental and resistant variants. Cell wall changes in nisin-resistant listeriae are attributed to abnormal cell wall synthesis and autolysin inhibition, the latter possibly associated with subtle changes in cell wall structures and function.     

Cell-associated pheromone peptide (cCF10) production and pheromone inhibition in Enterococcus faecalis

In Enterococcus faecalis, the peptide cCF10 acts as a pheromone, inducing transfer of the conjugative plasmid pCF10 from plasmid-containing donor cells to plasmid-free recipient cells. In these studies, it was found that a substantial amount of cCF10 associates with the envelope of the producing cell. Pheromone activity was detected in both wall and membrane fractions, with the highest activity associated with the wall. Experiments examining the effects of protease inhibitor treatments either prior to or following cell fractionation suggested the presence of a cell envelope-associated pro-cCF10 that can be processed to mature cCF10 by a maturase or protease. A pCF10-encoded membrane protein, PrgY, was shown to prevent self-induction of donor cells by reducing the level of pheromone activity in the cell wall fraction.     

An improved procedure for the isolation of plasmodesmata embedded in clean maize cell walls

A cell wall preparation of high purity was obtained using a procedure which involved repeated grindings of etiolated maize mesocotyl tissue and filtration through 200 mesh nylon cloth, followed by cell disruption via a nitrogen disruption bomb, and recovery of the cell walls via filtration. The cell wall fraction was free of particulate contaminants as determined both by phase-contrast and electron microscopy. The only membrane components found associated with the wall fraction as determined by electron microscopy were pladmodesmata embedded in the cell wall. The specific concentration of PAP26, a plasmodesmatal-associated polypeptide, was greatly increased in the cleanest cell wall fraction. A second plasmodesmatal-associated protein, PAP27, which was previously shown to be associated with the neck region of the plasmodesmata, was diminished as a result of passage through the nitrogen disruption bomb suggesting a partial fragmentation of the plasmodesmata. In addition to PAP26, the specific concentrations of at least three other cell wall-associated polypeptides with molecular weights of 80, 21 and 18 kDa, as revealed by SDS-PAGE, were also increased greatly in the cleanest cell wall fraction.     

Effect of restrictive temperature on cell wall synthesis in a temperature-sensitive mutant of Bacillus stearothermophilus.

A temperature-sensitive mutant of Bacillus stearothermophilus, TS-13, was unable to grow above 58 degrees C, compared to 72 degrees C for the wild type. Actively growing TS-13 cells lysed within 2 h when exposed to a restrictive temperature of 65 degrees C. Peptidoglycan synthesis stopped within 10 to 15 min postshift before a shut down of other macromolecular syntheses. Composition of preexisting peptidoglycan was not altered, nor was new peptidoglycan of aberrant composition formed. No significant difference in autolysin activity was observed between the mutant and the wild type at 65 degrees C. Protoplasts of TS-13 cells were able to synthesize cell wall material at 52 degress C, but not at 65 degrees C. This wall material remained closely associated with the cell membrane at the outer surface of the protoplasts, forming small, globular, membrane-bound structures which could be visualized by electron microscopy. These structures reacted with fluorescent antibody prepared against purified cell walls. Production of this membrane-associated wall material could be blocked by bacitracin, which inhibited cell wall synthesis at the level of transport through the membrane. The data were in agreement with previous studies showing that at the restrictive temperature this mutant is unable to alter its membrane fatty acid and phospholipid composition with temperature such that it is not able to maintain a membrane lipid composition which permits normal membrane function at the restrictive temperature.