Residual Cell Wall Research Articles

Effect of Delignification on In Vitro Digestion of Alfalfa Cellulose

Alfalfa forages containing low or high content of cell walls were ground in a Wiley mill through 1- or 8-mm screens. Portions of the forages were delignified with permanganate. Delignified and intact alfalfas were digested in vitro to ascertain rate and extent of digestion of cell wall and cellulose. Initial and residual cell wall and cellulose were greater for intact high cell wall and 8-mm alfalfa than for low cell wall and 1-mm alfalfa whereas potentially digestible (initial minus undigestible) cell wall and cellulose did not differ. Cellulose content of delignified forages was considerably greater than that of intact forages. Cell wall of low cell wall digested slightly faster (.0550/h) than high cell wall (.0485/h), and 8-mm forage had a slightly higher rate of digestion (.0505/h) of cell wall than 1-mm (.0485/h). Within intact or delignified alfalfas, cellulose digested similarly for either cell wall amount or particle size. Cellulose of delignified forages had a longer digestion lag (23h) than intact forages (4.5 to 6.0h), but once cellulose digestion began, it was much more rapid for the delignified forages (.161/h) than for intact (.048/h). Delignification greatly increased both rate and extent of digestion of cellulose in alfalfa.

Clq BINDING AND COMPLEMENT ACTIVATION BY CAPSULAR AND CELL WALL COMPONENTS OF S. PNEUMONIAE TYPE XIX

Cell wall components (purified cell walls, teichoic acid and residual cell walls) from S. pneumoniae type XIX showed antibody independent C1q binding capacity, as assessed by C1q deviation test, with teichoic acid being the most efficient. Specific capsular substance did not bind C1q. All substances tested produced C1 activation in normal human serum, but not in hypo-gamma-globulinemic serum. Thus, teichoic acid showed high C1q binding capacity but did not activate C1 in the absence of antibodies. Teichoic acid was an effective activator of alternative pathway. Specific capsular substance did not activate the alternative pathway in C1q deficient serum or in Mg2+ -EGTA chelated normal serum.

Effect of delignification upon in vitro digestion of forage cellulose.

Orchardgrass forages harvested at two maturities (early and late) were ground through two screens (1 and 8 mm) and digested in vitro as intact forage and forage delignified by permanganate oxidation. Initial and residual cell wall, initial and residual cellulose and potentially digestible cellulose were greater in late intact forage than in the early. In the delignified forage, late cut forage had less residual cellulose than did the early, but initial and potentially digestible cellulose were similar. Particle size had less consistent and smaller effects upon cell wall and cellulose than did maturity. Cellulose of intact orchardgrass was 64% digested at 72 h vs 94% for cellulose of delignified orchardgrass. Digestion rate of cellulose was .0197 and .0220 logn units/hr for early and late cut intact forage and .0554 and .0719 logn units/hr for early and late cut delignified forage. Removal of the inhibitory effects of lignin increased the amount of digestible cellulose, increased the rate at which cellulose degraded and decreased the indigestible cellulose residue. Reduction in lignin could greatly improve forage intake and utilization at moderate levels of animal production.

Quantitative determination of endosperm modification and its relationship to the content of 1,3∶1,4-β-glucans during malting of barley

The modification of barley endosperm during malting was quantitatively determined using the fluorochrome Calcofluor. The technique involved image analyser measurement of the area of residual cell wall material in longitudinally cut half seeds. The average modification was found to be highly correlated to both 1,3–1,4-β-glucan content and endo-β-glucanase activity, while a low correlation coefficient was obtained between modification and acrospire length of individual seeds.

On the sequential determination of α-amylase transport and cell wall breakdown in germinating seeds of Hordeum vulgare

By means of immunohistochemical α-amylase determination techniques and Calcofluor white M2R New staining of residual endosperm cell walls it was possible to observe transport of α-amylase and breakdown of cell wall material in germinating seeds of Hordeum vulgare cv. Nordal.When barley seeds of which the distal tip had been removed, were treated with gibberellic acid, the importance of the distal aleurone cells in the production of both α-amylase and cell breakdown hydrolases was clearly seen.

Activation of the Alternative Pathway by Pneumococcal Cell Wall Teichoic Acid

Abstract Previous studies on the interaction of pneumococci and the alternative pathway have shown that the cell wall, rather than the cell membrane or capsule, plays a major role in the activation of the alternative pathway. The present study was performed in order to identify the biochemical component of the pneumococcal cell wall responsible for activating the alternative pathway. Teichoic acids were extracted from purified pneumococcal cell walls by three different procedures (0.05 M periodate at 37°C; 100% formamide at 170°C; 5% TCA at 4°C). In each case the teichoic acids were able to consume C3 in C4-deficient guinea pig serum (C4D GPS) at concentrations as low as 3 to 10 µg/ml. In contrast, the residual cell walls, which contained the peptidoglycan, lost their activity in proportion to the amount of teichoic acid that had been extracted. Notably, cell walls extracted with periodate lost over 98% of their teichoic acid and the residual cell wall peptidoglycan was unable to consume C3 in C4D GPS at concentrations as high as 1 mg/ml.

Activation of the Alternative Complement Pathway by Pneumococcal Cell Wall Teichoic Acid

Abstract Previous studies have shown that the cell wall plays a major role in the activation of the alternative pathway by the pneumococcus. The present studies were performed in order to identify the biochemical component of the pneumococcal cell wall responsible for activating the alternative pathway. Teichoic acids were extracted from purified pneumococcal cell walls by three different procedures, and in each case they were able to activate the alternative pathway at concentrations as low as 3 to 10 µg/ml. In contrast, the residual cell walls, which contained the peptidoglycan, lost their activity in proportion to the amount of teichoic acid that had been extracted. Notably, cell walls extracted with periodate lost over 98% of their teichoic acid and the residual peptidoglycan was unable to activate the alternative pathway at concentrations as high as 1 mg/ml. When an autolysin defective pneumococcal strain is grown in the presence of penicillin, cell wall polymers are secreted into the medium. Crude preparations of secreted cell wall polymers were found to be able to activate the alternative pathway. The activity was removed by absorption with TEPC-15 myeloma with anti-choline specificity. Since both cell wall teichoic acid and lipoteichoic acid contain choline and are removed by TEPC-15 absorption, the activity in the crude preparations of cell wall polymers could have been due to one or both of these compounds. However, purified lipoteichoic acid was unable to activate the alternative pathway. Thus, the ability of crude preparations of cell wall polymers to activate the alternative pathway was due to the presence of cell wall teichoic acid. The results of these studies demonstrate that cell wall teichoic acid, rather than peptidoglycan, is responsible for pneumococcal activation of the alternative pathway.

Carbon Dioxide Fixation in Helminthosporium cynodontis

A mycelial suspension of Helminthosporium cynodontis (ATCC24938), grown on glucose-peptone-yeast extract broth and exposed to NaH14CO3 for 5 h, fixed significant quantities of 14C into the following fractions (%): small molecular weight components, 7-4; lipid and lipoproteins, 3-9; nucleic acids, 59; the residual protein and cell wall fragments, 29-2. The labelled protein components were (%): aspartate, 39; glutamate, 18; cystine, 15; threonine, 9. Radioactive nucleic acid components were (%): adenine, 18; guanine, 18; cytidylate, 34; uridylate, 30. When the mycelium was grown in Czapek-Dox glucose medium and incubated in this medium plus NaH14CO3, the nucleic acid fraction contained 29-9% and the residual protein 49-5% of the cellular radioactivity. The removal of CO2 from the atmosphere did not reduce growth. Pyruvate carboxylase (PC) and phosphoenolypyruvate carboxykinase (PEPCK) activities were demonstrated in extracts of H. cynodontis. Synthesis of PEPCK was stimulated under conditions promoting gluconeogenesis and was reduced under conditions promoting glycolysis, while PC synthesis was similar under both conditions.

Variations in properties of L-forms of Pseudomonas aeruginosa.

In a study of the pathogenic potentials of Pseudomonas L-forms, three unstable L-forms were derived by carbenicillin inductionfrom a mouse virulent strain of Pseudomonas aeruginosa, Rosenthal 180. One L-form, induced on a sucrose-stabilized medium, grew more slowly and differed in a number of properties from two other L-forms induced on a medium supported with polyvinylpyrilidone. After adaptation to a common liquid medium, the three L-forms differed with respect to colonial shape on solid medium, growth rate, certain biochemical properties, antibiotic sensitivities and antigenic surface, and virulence for mice. The L-form may revert in vitro to a serotype different from that of the parent culture. The revertant may acquire new antibiotic resistances and sensitivities in the absence of previous exposure to the drugs and enhanced resistance to the L-inducing agent. The three L-forms showed a characteristically lower, but wide, range of virulence than did the parental form. Though death of mice was accompanied by reversion of the L-forms in vivo to the bacterial form, reversion in vivo was not necessary for virulence of L-forms. Modification of residual cell wall antigens accompanied the induction of each L-form as determined by type-specific antisera.

Deuterium isotope effects in enzymatic transamination

The ionic fraction from the hydrolysate of residual cell walls of Scenedesmus obliquus grown in a medium containing 99.6% 2H 2O was subjected to ion-exchange chromatography for the fractionation of fully deuterated l-glutamic acid. l-[2- 2H]-Glutamic acid and l-[3,3,4,4- 2H 4]glutamic acid were synthesized from l-glutamic acid and l-[2,3,4,4- 2H 5]glutamic acid, respectively. A kinetic study of enzymatic transamination was conducted using the four l-glutamic acid analogs ( l-glutamic acid and three deuterated analogs) as the substrates. The rate of transamination was followd by photometric measurements at 280 mμ. Maximum velocity determinations, based on the binary mechanism of the enzymatic transamination, were performed on each of the l-glutamic acid analogs. The deuterium isotope effect reflected in the rate of transmission was estimated through maximum velocity analysis. The compound with deuterium in the α-position showed an isotope effect of 1.85. A similar isotope effect was observed for the compound with deuterium at the α-, β- and γ-carbons. The data indicate that the bond to the α-carbon and not to the β-carbon is ruptured during transamination. It is clearly demonstrated, therefore, that an α-elimination mechanism is involved in the enzymatic transamination.

Lysostaphin-induced, osmotically fragile Staphylococcus aureus cells.

Staphylococcus aureus FDA 209P cells when suspended in 24% (w/v) NaCl were rendered osmotically fragile by exposure to lysostaphin for time intervals ranging from 2 to 60 min. Such cells were analyzed chemically and serologically for evidence of residual cell wall material, were plated in hypertonic sucrose medium to determine revertibility to normal, and were subjected to manometric studies to determine metabolic capabilities. Most of the cells (95%) which were exposed to lysostaphin (0.5 or 1.0 unit/ml) for 2 min, although osmotically fragile, retained their cell wall hexosamine and were capable of reverting to osmotically normal cells when plated in hypertonic medium. Cells exposed to lysostaphin for 5 and 10 min also retained much of their cell wall hexosamine, but lost their ability to revert to normal staphylococci. Cells exposed to lysostaphin for 2 to 10 min continued to react with staphylococcus anti-k antiserum. Complete removal of cell wall hexosamine was attained only after exposure to lysostaphin for 20 min or more; these cells failed to react with k antiserum. Lysostaphin-induced L-type colonies were extremely rare in our experiments, even if incubation times and media were optimal for their detection. Lysostaphin-induced staphylococcal protoplasts were as active metabolically in manometric studies as were untreated staphylococci.

Polysaccharide-degrading Enzymes are Unable to Attack Plant Cell Walls without Prior Action by a “Wall-modifying Enzyme”

A study of the degradation of plant cell walls by the mixture of enzymes present in Pectinol R-10 is described. A "wall-modifying enzyme" has been purified from this mixture by a combination of diethylaminoethyl cellulose, Bio Gel P-100, and carboxymethyl cellulose chromatography. Treatment of cell walls with the "wall-modifying enzyme" is shown to be a necessary prerequisite to wall degradation catalyzed by a mixture of polysaccharide-degrading enzymes prepared from Pectinol R-10 or by an alpha-galactosidase secreted by the pathogenic fungus Colletotrichum lindemuthianum. The action of the "wall-modifying enzyme" on cell walls is shown to result in both a release of water-soluble, 70% ethanol-insoluble polymers and an alteration of the residual cell wall. A purified preparation of the "wall-modifying enzyme" is unable to degrade a wide variety of polysaccharide, glycoside, and peptide substrates. However, the purified preparation of wall-modifying enzyme has a limited ability to degrade polygalacturonic acid. The fact that polygalacturonic acid inhibits the ability of the "wall-modifying enzyme" to affect cell walls suggests that the "wall-modifying enzyme" may be responsible for the limited polygalacturonic acid-degrading activity present in the purified preparation. The importance of a wall-modifying enzyme in developmental processes and in pathogenesis is discussed.

Stability and comparative transport capacity of cells, mureinoplasts, and true protoplasts of a gram-negative bacterium.

The outer layers of the cell envelope of a pseudomonad of marine origin were removed by washing the cells in 0.5 m NaCl followed by suspension in 0.5 m sucrose. The term mureinoplast has been suggested for the rod-shaped forms which resulted from this treatment. As previously established, these forms lacked the outer cell wall layers but still retained a rigid peptidoglycan structure. Mureinoplasts remained stable if suspended in a balanced salt solution containing 0.3 m NaCl, 0.05 m MgSO(4), and 0.01 m KCl but, unlike whole cells, lost ultraviolet (UV)-absorbing material if suspended in 0.5 m NaCl or 0.05 m MgCl(2). Sucrose added to the balanced salt solution also enhanced the loss of UV-absorbing material. Addition of lysozyme to suspensions of mureinoplasts in the balanced salt solution produced spherical forms which, by electron microscopy and the analysis of residual cell wall material, appeared to be true protoplasts. Only undamaged mureinoplasts, as judged by their capacity to fully retain alpha-aminoisobutyric acid, were capable of being converted to protoplasts. Protoplasts and undamaged mureinoplasts retained 100% transport capacity when compared to an equal number of whole cells. The Na(+) requirement for transport of alpha-aminoisobutyric acid and the sparing action of Li(+) on this Na(+) requirement were the same for both protoplasts and whole cells. These observations indicate that, in this gram-negative bacterium, the cell wall does not participate in the transport process though it does stabilize the cytoplasmic membrane against changes in porosity produced by unbalanced salt solutions. The results also indicate that the requirements for Na(+) for transport and for the retention of intracellular solutes are manifested at the level of the cytoplasmic membrane.

Electron microscopic studies on streptococci. I. M antigen.

The presence of M antigens on group A streptococci is associated with hairlike fimbriae that cover the surface of the streptococcal cell wall and are demonstrable by electron microscopy. These fimbriae also may be associated with R antigen. Like M protein, the surface fimbriae are destroyed by trypsin treatment and reappear when "trypsinized" streptococci are reincubated in fresh, trypsin-free broth. Ferritin-conjugated, type-specific antibodies localize on homologous M+ cells in a pattern suggestive of several M antigenic sites along the length of individual surface fimbria. The M-associated fimbriae remain on the residual cell wall after removal of the bulk of group-specific polysaccharide through nitrous acid extraction. This suggests attachment of the fimbriae to the mucopeptide and minor polysaccharide components remaining in the nitrous acid-extracted wall. The pattern of localization of ferritin-conjugated antibodies on homologous streptococci before and after trypsin exposure and upon reincubation of the trypsinized cells in fresh medium suggests the following hypothesis: M antigen is secreted by the cell, is partially excreted through the otherwise intact cell wall, and is bound by the wall so that M protein occupies a peripheral, exposed position on the surfaces of the streptococcal cell wall.

The nature of trichloroacetic acid-soluble derivatives of d-alanine in Streptococcus faecalis

In the absence of vitamin B 6, Streptococcus faecalis requires d-alanine for growth and assimilates this from the medium into the cells, where it occurs largely in combined form. Of the d-alanine in the cells, a majority is present in the cell walls; substantial amounts, however, are extracted by treatment with cold or hot aqueous trichloroacetic acid (4). Fractionation of the cold trichloroacetic extract from cells grown with d-alanine-1-C 14 showed that 74% of the radioactivity of this extract was due to free d-alanine while approximately 15% was due to a component identified as d-alanyl- d-alanine. Other minor radioactive components of this extract were not identified. Dialysis of the hot trichloroacetic acid extract separates it into a dialyzable fraction that contains most of the radioactive d-alanine in uncharacterized but nonnucleotide form and a nondialyzable fraction that contains 39% of the radioactivity of the extract and 12% of the weight of the original dry cells. This component contains the same amino acids and carbohydrates as the cell wall and arises through partial degradation of the cell wall fraction by treatment with hot trichloroacetic acid. It sediments as a single substance in the ultracentrifuge, with molecular weight of 10,000–20,000, and may thus represent a material suitable for further structural investigations. The residual cell wall material is more resistant to degradation by trichloroacetic acid; its composition has not been determined.