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Quantitative analysis of Actinomyces cell walls.

Quantitative data on the amino acid composition of cell walls of five species of Actinomyces were obtained by using a Beckman-Spinco amino acid analyzer. The major amino acids in A. israelii, A. naeslundii, A. eriksonii, and A. bovis species included alanine, glutamic acid, lysine, aspartic acid, and ornithine, as reported by previous workers, whereas A. propionicus contained diaminopimelic acid. Other amino acids, including glycine, valine, leucine, proline, isoleucine, and threonine, were present in at least some of the walls in quantities equal to or slightly less than that of lysine. This raised the question of whether these may represent cross-links in the peptidoglycan or other cell wall structural components or whether the wall preparations contained nonpeptidoglycan material despite the use of electron microscopy as a standard of purity; further work is required to supply the answer. The quantitative data furnish relative molar concentrations of amino acids, which can provide definitive identification of some of the species and differentiation of Actinomyces from other members of the Actinomycetales and from morphologically similar genera such as Corynebacterium and Propionibacterium.

Actinomyces↗

[Age and changes in the cell wall of Cunninghamella japonica].

Changes in the composition of cell walls were studied in hyphae of Cunninghamella japonica in the course of aging. The content of neutral sugars, uronic acids and lipids remained virtually the same while the content of protein, aminosugars and chitin changed in the course of the cultural growth. The presence of chitosan in the cell wall of Cun. japonica is discussed.

Amino Sugars↗

Mechanical effects of plant cell wall enzymes on cellulose/xyloglucan composites.

Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

Base Sequence↗

Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth.

Hydroxyl radicals (OH) are capable of unspecifically cleaving cell-wall polysaccharides in a site-specific reaction. I investigated the hypothesis that cell-wall loosening underlying the elongation growth of plant organs is controlled by apoplastically produced OH attacking load-bearing cell-wall matrix polymers. Isolated cell walls (operationally, frozen/thawed, abraded segments from coleoptiles or hypocotyls, respectively) from maize, cucumber, soybean, sunflower or Scots pine seedlings were pre-loaded with catalytic Cu or Fe ions and then incubated in a mixture of ascorbate + H2O2 for generating OH in the walls. This treatment induced irreversible wall extension (creep) in walls stretched in an extensiometer. The reaction could be promoted by acid pH and inhibited by several OH scavengers. Generation of OH by the same reaction in living coleoptile or hypocotyl segments caused elongation growth. Auxin-induced elongation growth of maize coleoptiles could be inhibited by OH scavengers. Auxin promoted the production of superoxide radicals (O2(-)), an OH precursor, in the growth-controlling outer epidermis of maize coleoptiles. It is concluded that OH fulfils basic criteria for a wall-loosening factor acting in auxin-mediated elongation growth of plant species with widely differing cell-wall polysaccharide compositions.

Cell Wall↗

Structure and chemical composition of the cell walls from the haploid yeast and mycelial forms of Ustilago maydis.

Isolated or in vivo cell walls from the yeast and mycelial forms of haploid Ustilago maydis were not stained by the normal osmium procedure for electron microscopy. KMnO4 stained mycelial walls, revealing a layered structure with a loose electron-dense layer at the cell surface, but stained only the outer surface layer of yeast walls. Walls were purified from extracts obtained by ballistic and ultrasonic disruption. Chemical analysis showed that composition of yeast and mycelial walls was similar. Yeast walls contained higher amounts of neutral sugars and protein, whereas mycelial walls contained more chitin and phosphate. No chitosan or uronic acids were detected. Higher proportions of xylose and mannose were present in yeast walls, whereas the amounts of glucose and galactose were higher in mycelial walls. Fucose, arabinose, and ribose were detected in yeast walls only. Electrophoretic patterns of proteins extracted with SDS, beta 1, 3-glucanase, or chitinase were similar in walls of both morphologies, although some differential bands were identified. Most antigenic proteins appeared in the covalently bound fraction of the wall. Some were common to both morphologies, but others were stage specific.

Amino Acids↗

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.

Bacterial Proteins↗

Relatedness and classification of Streptococcus mutans and "mutans-like" streptococci.

The "mutans-like" streptococci can be separated into five species (Streptococcus mutans, S. rattus, S. sobrinus, S. cricetus, and S. ferus) that belong to the same rRNA homology cluster. New valuable chemical characters for differentiation of the five species--such as peptidoglycan type, presence of cell wall teichoic acid, and cell wall sugar composition--are described. The peptidoglycan type and the cell wall sugar composition can be determined by rapid procedures.

Animals↗

Elastic properties of the composite outer hair cell wall.

We propose a mathematical model for analyses of the elastic properties of the wall of the outer hair cell (OHC) in the inner ear. The model reflects the properties of the major components of the OHC wall: the subsurface cisternae, the cortical lattice, the plasma membrane, and the radial pillars. The wall is treated as a composite consisting of three elastic cylindrical shells. Two inner shells, isotropic and orthotropic/ are adjacent to each other, and the outermost isotropic shell is connected to the combined inner shell by elastic springs. We derive Flugge-type equations for the composite wall and apply the model to the interpretation of the experiments with axial loading and with inflation of the OHC. We derive expressions for the axial stiffness and the wall strains measured in these experiments in terms of the elastic properties of the wall components. We also consider a conceivable experiment with torsion of the OHC and obtain relations between the torque (the axial reaction) and the angle of torsion. These solutions provide necessary information for the future determination of the OHC elastic properties.

Biomechanical Phenomena↗

Olive fruit cell wall: degradation of pectic polysaccharides during ripening.

Olive fruits at three stages of ripening (green, cherry, and black) have been studied. After cell wall isolation, the compositions of the cell wall and that of the phosphate-soluble polysaccharides were determined. In cell walls, decreases in arabinose, xylose, glucose, and uronic acid levels were observed, together with a slight increase in mannose on ripening. At the beginning of ripening, fragments of pectic polymers were the major constituents of the phosphate-soluble fraction, with the hemicellulosic ones increasing toward the end of the process. The molecular weight of the fragments solubilized was approximately 6 kDa. After cell wall fractionation, the pectic polysaccharides soluble in imidazole and sodium carbonate were also studied. In both fractions, between the green and cherry stages of ripening, a significant loss of homogalacturonans took place. Between the cherry and black stages of ripening, rhamnogalacturonan side chains were also released in addition to homogalacturonans. In any of the pectic fractions, changes in apparent molecular weight were quantified.

Arabinose↗

Downregulation of the Petunia hybrida alpha-expansin gene PhEXP1 reduces the amount of crystalline cellulose in cell walls and leads to phenotypic changes in petal limbs.

The expansins comprise a family of proteins that appear to be involved in the disruption of the noncovalent bonds between cellulose microfibrils and cross-linking glycans, thereby promoting wall creep. To understand better the expansion process in Petunia hybrida (petunia) flowers, we isolated a cDNA corresponding to the PhEXP1 alpha-expansin gene of P. hybrida. Evaluation of the tissue specificity and temporal expression pattern demonstrated that PhEXP1 is preferentially expressed in petal limbs during development. To determine the function of PhEXP1, we used a transgenic antisense approach, which was found to cause a decrease in petal limb size, a reduction in the epidermal cell area, and alterations in cell wall morphology and composition. The diminished cell wall thickness accompanied by a reduction in crystalline cellulose indicates that the activity of PhEXP1 is associated with cellulose metabolism. Our results suggest that expansins play a role in the assembly of the cell wall by affecting either cellulose synthesis or deposition.

Cell Size↗

Cell-wall architecture and lignin composition of wheat developed in a microgravity environment.

The microgravity environment encountered during space-flight has long been considered to affect plant growth and developmental processes, including cell wall biopolymer composition and content. As a prelude to studying how microgravity is perceived - and acted upon - by plants, it was first instructive to investigate what gross effects on plant growth and development occurred in microgravity. Thus, wheat seedlings were exposed to microgravity on board the space shuttle Discovery (STS-51) for a 10 day duration, and these specimens were compared with their counterparts grown on Earth under the same conditions (e.g. controls). First, the primary roots of the wheat that developed under both microgravity and 1 g on Earth were examined to assess the role of gravity on cellulose microfibril (CMF) organization and secondary wall thickening patterns. Using a quick freeze/deep etch technique, this revealed that the cell wall CMFs of the space-grown wheat maintained the same organization as their 1 g-grown counterparts. That is, in all instances, CMFs were randomly interwoven with each other in the outermost layers (farthest removed from the plasma membrane), and parallel to each other within the individual strata immediately adjacent to the plasma membranes. The CMF angle in the innermost stratum relative to the immediately adjacent stratum was ca 80 degrees in both the space and Earth-grown plants. Second, all plants grown in microgravity had roots that grew downwards into the agar; they did not display "wandering" and upward growth as previously reported by others. Third, the space-grown wheat also developed normal protoxylem and metaxylem vessel elements with secondary thickening patterns ranging from spiral to regular pit to reticulate thickenings. Fourthly, both the space- and Earth-grown plants were essentially of the same size and height, and their lignin analyses revealed no substantial differences in their amounts and composition regardless of the gravitational field experienced, i.e. for the purposes of this study, all plants were essentially identical. These results suggest that the microgravity environment itself at best only slightly affected either cell wall biopolymer synthesis or the deposition of CMFs, in contrast to previous assertions.

Cell Membrane↗

The chemical composition of the cell wall of Chlamydomonas gymnogama and the concept of a plant cell wall protein.

Cell walls of Chlamydomonas gymnogama, shed during sexual mating, were collected and analyzed. Ultrastructural examination indicates that the walls are free of cytoplasmic contamination and that they exhibit a regular lamellate structure. The walls are composed of glycoprotein rich in hydroxyproline. The hydroxyproline is linked glycosidically to a mixture of heterooligosaccharides composed of arabinose and galactose. Altogether, the glycoprotein complex accounts for at least 32% of the wall. The amino acid composition of the walls is extraordinarily similar in widely different plant species. The implications of these similarities as well as the widespread occurrence of these glycoproteins are discussed.

Amino Acids↗

Biochemical changes in Bifidobacterium bifidum var. pennsylvanicus after cell wall inhibition. VIII. Composition and metabolism of phospholipids at different stages and conditions of growth.

1. The phospholipid content and composition of Bifidobacterium bifidum var. pennsylvanicus is markedly influenced by the growth phase, the pH and the presence of human milk in the culture medium. 2. The lipid-phosphorus content of the cells increases during the first period of active growth, but decreases later. The lipid-phosphorus content of the cells in the stationary phase is at constant pH 5.5 about 45 percent of that at constant pH 6.8 and final pH 5.2. This difference is caused by a general reduction of all types of phospholipids. 3. Phosphatidylglycerol content decreases during growth, diphosphatidylglycerol increases in the first period, but decreases later and especially in the stationary phase by an increase of its lysoderivatives. The phosphogalactolipids rise during growth in the non-controlled pH-culture to 70 percent of the phospholipids in the stationary phase. When pH is constant at 6.8 and 5.5 glycerolphosphorylgalactosyldiglyceride remains at a constant level of about 20 percent during growth. At pH 6.8 glycerophosphorylmonogalactosylmonoglyceride increases to 24 percent during cultivation; at pH 5.5 this lipid contributes only a few percent. 4. Under all pH conditions, lack of human milk in the culture medium causes a marked increase of the lipid-phosphorus content of the cell, together with a high increase of the relative amounts of diphosphatidylglycerol and phosphatidylglycerol and a decline of the phosphogalactolipids. The same changes are observed after inhibition of cell wall synthesis by various antibiotics, but not after inhibition of protein synthesis.

Bacitracin↗

Effects of growth medium selection on plasmid DNA production and initial processing steps.

Cultures of recombinant Escherichia coli containing the plasmid pSVbeta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium.

Biotechnology↗

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy.

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.

Aspergillus nidulans↗

Chemical composition of the cell wall of lactic acid bacteria and related species.

In order to examine the relationship between biological activities and the cell wall content, the murein type and the teichoic acid of the cell wall from five strains of bacteria were studied. Two of these Lactobacillus casei CRL 431 and L. acidophilus CRL 730, are used in a commercial fermented milk (BIO MILK), which is believed to be beneficial for health. The other strains, Lactococcus lactis CRL 526, Pediococcus pentosaceus CRL 923 and Propionibacterium acidipropionici CRL 1198 were included in order to compare the cell wall structures of active and inactive strains. A method was designed to confirm the amino acids of the peptidoglycan in impure substrates. Four of the studied strains, L. casei, L. acidophilus, L. lactis and P. acidipropionici, contained glycerol teichoic acids. L. casei, L. acidophilus, P. pentosaceus and L. lactis contained A4 alpha type murein, while P. acidipropionici contained A3 gamma type. The capacity of orally administered peptidoglycans of the studied strains to stimulate phagocytosis by mouse peritoneal macrophages was analyzed. Only the PG of L. casei showed this activity. No differences were observed between active and inactive strains with respect to the chemical composition of the peptidoglycan. Therefore the biological activity is unlikely to be due to the peptidoglycan structure.

Animals↗

Halococcus morrhuae: a sulfated heteropolysaccharide as the structural component of the bacterial cell wall.

The qualitative and quantitative composition of purifed cell wall of Halococcus morrhuae CCM 859 was determined. Glucose, mannose, galactose; glucuronic and galacturonic acids; glucosamine, galactosamine, gulosaminuronic acid; acetate, glycine and sulfate are found as major constituents. The amino sugars are N-acetylated. It was not possible to fractionate the cell wall in chemically different polymers. Evidence is presented that the major cell wall polymer of this strain is a complex heterolgycan which seems, like the peptidoglycan of most bacteria, to be responsible for the rigidity and stability of the cell wall. In addition it could be proved that this heteroglycan is sulfated and therefore differs considerably from previously described bacterial cell wall polymers.

Amino Sugars↗