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PubMed1997

Polysaccharide hydrolases in germinated barley and their role in the depolymerization of plant and fungal cell walls.

Cell wall degradation is an important event during endosperm mobilization in the germinated barley grain. A battery of polysaccharide and oligosaccharide hydrolases is required for the complete depolymerization of the arabinoxylans and (1 --> 3,1 --> 4)-beta-glucans which comprise in excess of 90% by weight of these walls. The (1 --> 3,1 --> 4)-beta-glucan endohydrolases release oligosaccharides from their substrate and are probably of central importance for the initial solubilization of the (1 --> 3,1 --> 4)-beta-glucans, but beta-glucan exohydrolases and beta-glucosidases may be important additional enzymes for the conversion of released oligosaccharides to glucose. The latter enzymes have recently been purified from germinated barley and characterized. There is an increasing body of evidence to support the notion that the (1 --> 3,1 --> 4)-beta-glucan endohydrolases from germinated barley evolved from the pathogenesis-related (1 --> 3)-beta-glucanases which are widely distributed in plants and which hydrolyse polysaccharides that are abundant in fungal cell walls. Arabinoxylan depolymerization is also mediated by a family of enzymes, but these are less well characterized. (1 --> 4)-beta-Xylan endohydrolases have been purified and the corresponding cDNAs and genes isolated. While the presence of (1 --> 4)-beta-xylan exohydrolases and alpha-L-arabinofuranosidases has been reported many times, the enzymes have not yet been studied in detail. Here, recent advances in the enzymology and physiology of cell wall degradation in the germinated barley grain are briefly reviewed.

Carbohydrate Sequence↗
PubMed1987

Identification of polysaccharide hydrolases involved in autolytic degradation of zea cell walls.

Cell walls of Zea mays (cv L.G.11) seedlings labeled with (14)C were treated with alpha-amylase from Bacillus subtilis to remove starch and mixed linkage glucans. These walls released arabinose, xylose, galactose, and galacturonic acid in addition to glucose when they were allowed to autolyze. Methylation analysis was performed on samples of wall which had been incubated autolytically and the results indicated that degradation of the major polymer of the wall, the glucoarabinoxylan, had occurred. A number of glycanases could be dissociated from the wall by use of 3 m LiCL. The proteins which were released were found to contain a number of exoglycosidase activities in addition to being effective in degrading the polysaccharide substrates, araban, xylan, galactan, laminarin, mannan, and polygalacturonic acid. The effects of these enzymes on the wall during autolysis appear to result from endo-activity in addition to exo-activity. The structural changes that occurred in the cell walls during autolysis were found to be related to the changes previously found to occur in cell walls during auxin induced extension.

Journal Article↗
PubMed1984

In vitro characterization of tomato fruit softening : the use of enzymically active cell walls.

Cell wall isolated from pericarp of normal tomato (Lycopersicon esculentum Mill. cv ;Rutgers') fruit released pectic polymers in a reaction apparently mediated by wall-bound polygalacturonase that appears with the onset of ripening. Release was negligible in wall preparations from normal green and the ripening mutant rin fruit. Pectin solubilization was most extensive at pH 2.5 with a less significant peak at 5.5. Brief exposure to low (1.5) or high (7.5) pH resulted in reduction of autolytic activity, which was also inhibited by high temperature, Ca(2+), and treatments employed to dissociate protein from cell wall. Uronic acid solubilization was significantly enhanced by 150 millimolar NaCl and by increasing temperature within the physiological range. These data indicate that the release of polyuronide from isolated cell walls is enzymic and may provide a convenient and reliable system for the study of softening metabolism.

Journal Article↗
PubMed1972

Effect of temperature on the integrity of Bacillus psychrophilus cell walls.

Cell walls isolated from Bacillus psychrophilus autolyse at temperatures which support growth. At temperatures above the maximum growth temperature (28 C), a nonenzymatic lysis occurs. Removal of autolytic enzyme activity with 10 m LiCl had little effect on the rate or extent of lysis at elevated temperatures (37 and 45 C). Nonenzymatic lysis was characterized chemically by a decrease in the liberation of N-terminal groups, and the effects of pH, Ca(2+), and ethylenediaminetetraacetic acid suggest that ionic linkages are involved in much of the integrity of the cell wall of this psychrophile. The nonenzymatic absorbance decrease at 45 C can be reversed to the extent of 70 to 100% at 0 C. Centrifugation of a heat-lysed wall suspension separated a soluble protein component which is required for low-temperature reaggregation. Preliminary evidence indicates the insoluble residue which remains after temperature-mediated lysis is primarily peptidoglycan.

Autolysis↗
PubMed1991

Protective efficacy of a 62-kilodalton antigen, HIS-62, from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

We reported previously that a detergent extract of the cell wall and cell membrane of Histoplasma capsulatum yeast cells contains antigens recognized by T cells. In T-cell immunoblot analysis, a region encompassing 62 kDa was stimulatory for an H. capsulatum-reactive T-cell line and T-cell clones derived from C57BL/6 mice. In this study, we isolated a 62-kDa band, termed HIS-62, from electrophoresed cell wall and cell membrane of H. capsulatum yeast cells and examined its antigenicity and immunogenicity. C57BL/6, BALB/c, and CBA/J mice that were immunized with viable H. capsulatum yeast cells mounted a delayed-type hypersensitivity response to HIS-62 that was stronger than that of normal controls. Spleen cells from each strain of mouse immunized with viable yeast cells proliferated vigorously in response to HIS-62; conversely, splenocytes from control animals did not recognize this antigen. A T-cell line and 5 of 5 T-cell clones from C57BL/6 mice, 10 of 15 BALB/c T-cell hybridomas, and 8 of 12 CBA/J T-cell hybridomas recognized HIS-62. A cutaneous delayed-type hypersensitivity response to the antigen was apparent in each strain of mouse that was injected with 80 micrograms of HIS-62 mixed with Freund adjuvant. In addition, spleen cells from HIS-62-immunized mice proliferated in vitro in response to this antigen. Vaccination of each strain of mouse with 80 micrograms of HIS-62 conferred protection against a lethal intravenous challenge with H. capsulatum yeast cells. Thus, HIS-62 appears to be an important target of the cellular immune response to H. capsulatum and induces a protective immune response in mice.

Animals↗
PubMed1974

Regulation of bacterial cell walls: turnover of cell wall in Staphylococcus aureus.

The cell wall of Staphylococcus aureus was shown to undergo turnover during exponential growth. The rate of turnover, about 15% per generation, was identical for both cell wall polymers, peptidoglycan and teichoic acid. Both the old and newly synthesized wall material appeared to undergo turnover at similar rates. The rate of turnover followed first-order kinetics until more than 90% of the original wall was lost. Cell wall turnover was completely blocked under conditions of unbalanced synthesis known to inhibit cellular autolysis, e.g., addition of chloramphenicol. Cell wall turnover was shown to occur in a number of different strains of S. aureus and appears to be widely distributed in this species.

Bacterial Proteins↗
PubMed1991

Auxin-enhanced glucan autohydrolysis in maize coleoptile cell walls.

Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve the sensitivity of the measurements and to facilitate kinetic analysis, isolated cell walls were consolidated within a small column, and the autolysis rate was directly determined from the sugar content of the effluent. This protocol revealed that the maximum rate of autohydrolysis of walls prepared from segments occurs within the first 2 hours and a steady decline commences almost immediately. Walls from indoleacetic acid pretreated segments (0.5-4 hours) released sugar at a higher rate initially (110-125% of controls) and the enhanced rate of autolysis continued for 6 to 8 hours, but then it became equivalent to that of the controls. Pretreatment of the segments at acidic pH had no effect on the measurable rates of autolysis. The (1-->3), (1-->4)-beta-d-glucan content of the walls and the extractable glucanase activities support the hypothesis that temporal enhancement of autohydrolysis is a function of auxin on enzyme activity. The progressive decline in autolysis during prolonged incubations is consistent with the decrease in the quantity of the beta-d-glucan in the wall. The relationship between glucan content and autolysis rate is supported by the observation that while glucose pretreatment of segments had only a small effect on initial autolysis rates, the presence of the sugar during pretreatment served to extend the interval over which higher rates of autolysis could be sustained. The results demonstrate that autolysis is related to auxin-induced wall metabolism in maize coleoptiles.

Journal Article↗
PubMed2005

Mating-induced shedding of cell walls, removal of walls from vegetative cells, and osmotic stress induce presumed cell wall genes in Chlamydomonas.

The first step in sexual differentiation of the unicellular green alga Chlamydomonas reinhardtii is the formation of gametes. Three genes, GAS28, GAS30, and GAS31, encoding Hyp-rich glycoproteins that presumably are cell wall constituents, are expressed in the late phase of gametogenesis. These genes, in addition, are activated by zygote formation and cell wall removal and by the application of osmotic stress. The induction by zygote formation could be traced to cell wall shedding prior to gamete fusion since it was seen in mutants defective in cell fusion. However, it was absent in mutants defective in the initial steps of mating, i.e. in flagellar agglutination and in accumulation of adenosine 3',5'-cyclic monophosphate in response to this agglutination. Induction of the three GAS genes was also observed when cultures were exposed to hypoosmotic or hyperosmotic stress. To address the question whether the induction seen upon cell wall removal from both gametes and vegetative cells was elicited by osmotic stress, cell wall removal was performed under isosmotic conditions. Also under such conditions an activation of the genes was observed, suggesting that the signaling pathway(s) is (are) activated by wall removal itself.

Amino Acid Sequence↗
PubMed1988

Application of chromatographic methods to the analysis of macrophage factors induced by Nocardia opaca cell walls.

Cell walls from Nocardia opaca induce the production of mitogenic factors by mouse peritoneal macrophages in vitro. These factors stimulate thymocytes from C3H/HeJ mice. Supernatants of peritoneal cell culture exhibiting this activity were fractionated by chromatographic procedures such as gel filtration and metal chelate affinity chromatography and the biological activities assayed. These fractionation studies indicate that several biologically active products occur in the supernatant. Four factors monokines (M) with different apparent molecular masses M1 (100,000), M2 (50,000), M3 (16,000) and M4 (7000) were obtained, one of which (M3) was identical to interleukin 1 (IL1). Several of the biochemical parameters of one of these factors, M2, were analyzed. It was found that this monokine had many properties in common with IL1: stimulation of proliferation of thymocytes from C3H/HeJ mice, similar amino acid composition and mobility during isoelectric focusing.

Amino Acids↗
PubMed1980

Preparation of zymosan from yeast cell walls.

Cell walls of the yeast Saccharomyces cerevisiae after disintegration and protoplasm removal by centrifugation and repeated washing were suspended in 0.5 M Na2HPO4, pH 7.8-8.0, as a 5% or 10% suspension, depending on the mode of heating. The suspension was boiled for 3 h, purified by repeated washing with water and ethanol and dried. The yield was approximately 1.8% of the starting amount of pressed commercial baker's yeast.

Cell Wall↗
PubMed1990

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls.

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.

Carbohydrate Sequence↗
PubMed2006

Se-enriched mycelia of Pleurotus ostreatus: distribution of selenium in cell walls and cell membranes/cytosol.

The incorporation of Se to fungi has been studied, focusing on element distribution among different cellular compartments and, in particular, polysaccharide structures contained in cell walls. Se-enriched mycelia of Pleurotus ostreatus were obtained in submerged cultures. The incorporation of selenium from the growth medium to mycelia was observed with the relative distribution between cytosol plus cell membranes fraction (CCM) and cell walls fraction (CW) of about 44 and 56%, respectively. CCM fractions were analyzed by size exclusion chromatography with on-line UV (280 nm) and ICP-MS detection (80Se). The results obtained showed selenium binding to components of different molecular masses (about 24% of total selenium coeluted with the compounds of molecular mass > 10 kDa). A polysaccharide-containing fraction of mycelia was treated alternatively with Tris-HCl at pH 7.5 or with chitinase. Better solubility and increased contribution of low molecular mass compounds were observed in chitinase extracts (UV detection), confirming the degradation of polysacharides by the enzyme. The total area under the ICP-MS chromatogram of chitinase extract was 2 times higher with respect to the area for Tris-HCl extract. Furthermore, the relative contribution of selenium in the low molecular mass fraction (molecular mass < 1 kDa) in chitinase extract was 72% as compared to 45% in Tris-HCl extract (based on peak area measurements with respect to total area under the chromatogram). The results obtained suggest selenium binding to chitin-containing polysaccharide structures in fungi cell walls.

Cell Membrane↗
PubMed1979

Thermal activation of peroxidase from tobacco leaf mesophyll cell walls.

Cell wall-bound peroxidase isolated from tobacco leaf mesophyll cell walls was found to consist of two isoenzymes (P1 and P2). Each exists in the form of a single subunit with the same molecular weight (35 000) as determined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Both isoenzymes exhibited maximum activities at 70 degrees. P1 increased to 265% and P2 to 140% of the activities assayed at 27 degrees; below this temperature the two isoenzymes had the same specific activity. On hydrolysis, P1 showed a carbohydrate content of 26.22% when the monosaccharides were analyzed by gas liquid chromatography; P2 gave 21.45%.

Carbohydrates↗
PubMed1992

Role of Kupffer cells in developing streptococcal cell wall granulomas. Streptococcal cell wall induction of inflammatory cytokines and mediators.

Hepatic granulomas are induced by intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats. Kupffer cells rapidly clear SCW from the blood, and the authors examined Kupffer cells further for a role in SCW-hepatic inflammation. Isolated Kupffer cells cultured with SCW secreted high levels of tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), transforming growth factor beta (TGF beta), and prostaglandin E2 (PGE2). SCW transiently induced increased steady-state levels of IL-1 beta and TNF alpha mRNA; in contrast, constitutive expression of TGF beta 1 mRNA in Kupffer cells was not affected by SCW. Low concentrations of SCW induced the accumulation of intracellular IL-1 and TGF beta bioactivity, with intracellular IL-1 bioactivity remaining high through at least 72 hours of culture. Kupffer cells isolated 1, 7, and 21 days after SCW injection did not express IL-1 beta or TNF alpha mRNA greater than control levels and exhibited marked hyporesponsiveness to secondary in vitro stimulation with SCW or LPS. SCW transiently induces Kupffer cells to secrete a variety of soluble mediators that contribute to hepatic inflammation by inducing leukocyte recruitment and activation and fibroproliferation. The transient nature of the Kupffer cell response and the hyporesponsiveness to secondary stimulation may be a mechanism by which the hepatic inflammation is negatively regulated.

Animals↗
PubMed1985

Interaction of the staphylococcin-like peptide Pep 5 with cell walls and isolated cell wall components of Gram-positive bacteria.

Unlike bacteriocins of Gram-negative bacteria, the strongly basic staphylococcin-like peptide Pep 5 lacked specific receptor mediated binding to sensitive Gram-positive bacteria. Studies with whole cells, purified cell walls, teichoic acids, and lipoteichoic acids strongly suggested that it binds reversibly via electrostatic interaction to negatively charged groups. Thus, Pep 5 binding could be reversed by sufficiently high concentrations of monovalent (K+, 150-250 mM) and divalent (Ca2+, 15-30 mM) cations (Fig. 1, 2) and by low pH (pH 2), where Pep 5 binding groups are protonated. Cells of Staphylococcus cohnii 22 with a reduced teichoic acid content showed a reduced Pep 5 binding capacity (Fig. 3). The results indicate that teichoic, teichuronic, and lipoteichoic acids are the unspecific cell wall binding sites for Pep 5.

Adsorption↗
PubMed1965

CHARACTERIZATION OF CONJUGATION FACTORS IN ESCHERICHIA COLI CELL WALLS. I. INHIBITION OF RECOMBINATION BY CELL WALLS AND CELL EXTRACTS.

Lancaster, John H. (University of Texas, Austin), E. P. Goldschmidt, and Orville Wyss. Characterization of conjugation factors in Escherichia coli cell walls. I. Inhibition of recombination by cell walls and cell extracts. J. Bacteriol. 89:1478-1481. 1965.-An assay procedure was devised to determine quantitatively the interference with conjugation of cell-wall fragments from both male and female strains of Escherichia coli. The fertility of the donor is reflected in the assay. Phenol extracts from the cell walls were active, and chemical analysis suggested that the activity resided in the lipopolysaccharide fraction.

Cell Extracts↗
PubMed2001

The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes.

We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes.

Amino Acid Sequence↗
PubMed1998

The making of the architecture of the plant cell wall: how cells exploit geometry.

Cell wall deposition is a key process in the formation, growth, and differentiation of plant cells. The most important structural components of the wall are long cellulose microfibrils, which are synthesized by synthases embedded in the plasma membrane. A fundamental question is how the microfibrils become oriented during deposition at the plasma membrane. The current textbook explanation for the orientation mechanism is a guidance system mediated by cortical microtubules. However, too many contraindications are known in secondary cell walls for this to be a universal mechanism, particularly in the case of helicoidal arrangements, which occur in many situations. An additional construction mechanism involves liquid crystalline self-assembly [A. C. Neville (1993) Biology of Fibrous Composites: Development Beyond the Cell Membrane (Cambridge Univ. Press, Cambridge, U.K.)], but the required amount of bulk material that is able to equilibrate thermally is not normally present at any stage of the wall deposition process. Therefore, we have asked whether the complex ordered texture of helicoidal cell walls can be formed in the absence of direct cellular guidance mechanisms. We propose that they can be formed by a mechanism that is based on geometrical considerations. It explains the genesis of the complicated helicoidal texture and shows that the cell has intrinsic, versatile tools for creating a variety of textures. A compelling feature of the model is that local rules generate global order, a typical phenomenon of life.

Journal Article↗