Hydrolysis of plant polysaccharides and GLC analysis of their constituent neutral sugars

The release and degradation of sugars from onion cell walls and potato cell wall polysaccharides were followed during hydrolysis with trifluoroacetic acid so that the optimum hydrolysis conditions could be determined. After 6 hr hydrolysis in 2 M acid at 100°, calculated recovery factors of different monosaccharides from potato pectic fractions ranged from 61 to 96%. Lower yields of monosaccharides were obtained from intact onion cell walls, while the yield from cellulose was less than 0.2%. A new GLC column for the separation of alditol acetates derived from cell wall sugars is described.     

Biosynthesis of liver catalase in rats treated with allylisopropylacetylcarbamide. I. Immunochemical assay of catalase in liver cell fractions.

Rats were injected twice intraperitoneally with 20 mg of allylisopropylacetylcarbamide (Sedormid) per 100 g of body weight at an interval of 12 hr. The level of catalase [EC 1.11.1.6] in various liver cell fractions was determined both enzymatically and immunochemically 12 hr after the second injection. 1. The decrease in catalase protein assayed by the immunochemical method directly confirmed the inhibition of biosynthesis of the enzyme by this porphyrinogenic drug. 2. The occurrence of a considerable amount of catalase protein with no enzymatic activity was demonstrated both in the peroxisomes and in the supernatant fraction. 3. The amount of catalase-synthesizing polysomes in hepatic cell was reduced in Sedormid-treated rats by the extent comparable to the decrease in the concentration of liver catalase.     

Effect of Salicylic Acid and Phenylserine on the Hypersensitive Reaction of Tobacco to Tobacco Mosaic Virus

Injection of leaves of tobacco (Nicotiana tahacum cv. ‘Xanthi’ nc) with salicylic acid (SA) or phenylsene (PS) had an effect on the local lesion development caused by tobacco mosaic virus (TMV), depending upon the concentration used and the time interval between injection and challenge inoculation. Maximum reduction in lesion size was obtained with 0.75 mM SA or with 8 mM PS. Concentrations higher than 1 mM SA or 25 mM PS damaged the leaf tissue, PS being far less toxic than SA. The leaves responded rapidly to injection with SA or PS. A time interval of only 1 h between injection and TMV inoculation reduced the lesion size significantly. Isolated tobacco cell walls incubated with SA yielded carbohydrate fractions capable of reducing lesion size significantly after injection. Cell walls incubated without SA or with PS did not yield active carbohydrate fractions.     

Degradation of Streptococcal Cell Wall Antigens In Vivo

Specific chemical modification of group A polysaccharide antigen to the A-variant structure was demonstrated in the lymphoid organs of mice by autoradiography by use of radioantibodies specific for these structures. Both antigenic moieties persisted and were still discerned 10 weeks after injection of the group A cell wall. In rabbit skin, the group A specificity was altered after a prolonged period. Unlike the situation for the mouse, polysaccharide A was not converted to A-variant structure, but another specificity common to both polysaccharides persisted at the site of injection. Mucopeptide, separated from the polysaccharide of group A cell walls, was eliminated from the site of injection in rabbit skin between 4 and 8 hr after injection. Group D streptococcal cell walls were also rapidly eliminated from tissue, and were no longer detectable 8 hr after injection into rabbit skin or 24 hr after injection into mice. The rapid degradation of these structures was correlated with their susceptibility to lysozyme in vitro and was in contrast to the prolonged persistence of group A cell walls, which were completely resistant to egg white lysozyme. This persistence in tissue correlated with the capacity of group A cell wall fragments to induce a chronic inflammatory process, whereas the isolated mucopeptide or group D cell walls produced only an acute necrotoxic reaction.     

Immunization of Mice with Components of Pasteurella multocida

Log-phase cells of Pasteurella multocida strain P-1059 were used to prepare isolated culture filtrate, cell wall, and cytoplasmic components. Culture filtrate was further separated by column chromatography. A portion of cytoplasm and culture filtrate was conjugated to ferritin by means of metaxylylene diisocyanate. Cell walls induced more protection in mice than the conjugated or unconjugated cytoplasm or culture filtrate. The cell walls caused edema and erythema when given intradermally in rabbits, whereas cytoplasm and culture filtrate produced dermal necrosis. The first of four chromatographically separated fractions of culture filtrate was possibly more immunogenic in mice than cell walls. This fraction was less reactive intradermally in rabbits than cell walls but more reactive than the other fractions.     

Biological properties of streptococcal cell-wall particles. 3. Dermonecrotic reaction to cell-wall mucopeptides

Abdulla, Essa M. (University of North Carolina School of Medicine, Chapel Hill), and John H. Schwab. Biological properties of streptococcal cell-wall particles. III. Dermonecrotic reaction to cell-wall mucopeptides. J. Bacteriol. 91:374-383. 1966.-Intradermal injection of rabbits and guinea pigs with mucopeptide suspensions produced an acute necrotic lesion which reached maximal severity within 24 hr and gradually subsided with scar formation. Necrosis was evident within 4 hr after injection of 100 mug, and an indurated area (10 x 10 mm) was produced with as little as 5.0 mug. Mucopeptides from six bacterial strains were studied. Comparison of cell walls and derived mucopeptides showed that the acute necrotic lesion tended to be more severe as the residual polysaccharide was decreased. Hyperimmunization with mucopeptide reduced the acute reaction, with evidence of immunological specificity. Incubation with lysozyme also modified the reaction in relation to extent of digestion. Toxicity was related to particle size, since extended sonic vibration decreased activity. Histological sections showed intense accumulations of polymorphonuclear leukocytes, along with altered collagen. A chronic nodular lesion appeared about 7 days after injection of the intact cell-wall fragments. In contrast to the acute necrotic reaction, this lesion was rarely produced by the mucopeptide separated from polysaccharide.     

Biochemical studies on the cell wall degradation of Fusarium oxysporum f. sp. lycopersici race 2 by its own lytic enzymes for its biocontrol

An exhaustive cell wall fractionation of Fusarium oxysporum f. sp. lycopersici race 2 ( Fol 2) with alkali in a sequential procedure yields only three polysaccharide fractions: F1_s (alkali and water soluble), F1₁ (alkali soluble and water insoluble) and F4 (alkali-insoluble residue). These fractions amounted respectively to 15, 1.3 and 52% of the cell wall and have been characterized by infra-red spectroscopy and gas liquid chromatography-mass spectrometry (GLC-MS). F1_s is a β-gluco-galacto-mannan, F1₁ is mainly composed of a β-1, 3-glucan and F4 is a β-1,3-glucan-chitin complex. The F1_s is a very complex polysaccharide and its hydrolysis requires the action of different enzymes. The lysis of the cell wall and its three fractions with lytic enzymes from Fol 2 has been studied and a correlation between the lysis of the cell wall and the lysis of these fractions was found. The amount of glucose, galactose and mannose in F1_s and cell wall hydrolysates were quantified by GLC and they indicated the hydrolysis of the gluco-galacto-mannan polysaccharide. In the hydrolysis of F4 and cell walls N -acetylglucosamine was also found and quantified. When chlamydospores of this fungus were treated with Fol 2 lytic enzymes, the sugars liberated were principally mannose and N -acetylglucosamine. These results indicate that Fol 2 produces during its autolysis the necessary enzymes to hydrolyse its own cell walls. This fact suggests that a biological control of Fol 2 with its own lytic enzymes, conveniently immobilized, could be developed.     

Acquired Resistance in Hypersensitive Tobacco against Tobacco Mosaic Virus, Induced by Plant Cell Wall Components

Isolated cell walls from Nicotiana tabacum L. cv. Xanthi-nc were partially digested with enzymes present in the fungal preparation cellulase Onozuka R-10. Released carbohydrate material was separated from the enzymes by gel filtration and fractions were injected into the intercellular spaces of one half of each of several Xanthi-nc leaves. Two to three days after injection the whole leaves were inoculated with TMV. A large reduction in mean lesion diameter was observed in the injected areas compared with those in untreated tissue.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

A Paracoccidioides brasiliensis polysaccharide having granuloma-inducing, toxic and macrophage-stimulating activity

The occurrence of a polysaccharide fraction of Paracoccidioides brasiliensis cell wall with toxic, granuloma-inducing and macrophage-stimulating activities was demonstrated. After fractionation of the lipid-extracted wall with 1 M-NaOH, three fractions were obtained: (1) an alkali-insoluble fraction; (2) an alkali-soluble, acid-insoluble fraction and (3) an alkali-soluble, acid-soluble fraction. When the three fractions were injected into mice only fraction (1) was able to induce chronic lung inflammation, causing a marked loss in body weight and death at a dose of 6 mg per animal. Analysis of the stimulation of peritoneal macrophages of mice (measured by cell spreading on glass) after intraperitoneal injection of fraction 1 showed that 75% of the cells were able to spread even 20 d after inoculation.     

Inhibiting Materials for γ Phage Adsorption to the Cell Wall of Bacillus anthracis,Strain Pasteur No. 2-H

Cell wall preparations of Bacillus anthracis, strain Pasteur No. 2-H, were treated with heat or with acetone and ether. Both of the treated cell walls preparations inactivated γ phage. The centrifuged supernatant of the heat-treated cell walls was fractionated on Sephadex G-200, and four fractions containing reducing sugars were obtained. The first fraction had the phage-inactivating activity. On the other hand, the fourth fraction had no phage-inactivating activity, but strongly inhibited phage adsorption to the cell walls. In the fourth fraction, glutamic acid, alanine, 2, 6-diaminopimelic acid and glucosamine were detected by paper chromatography after acid hydrolysis. Authentic D,L -2, 6-diaminopimelic acid and D -glucosamine markedly inhibited phage adsorption to the cell walls. D -Galactosamine, D -mannosamine and L -lysine also showed similar activities. Results suggest the possibility that one or a combination of these substances defines the characteristics of phage adsorption to the cell walls of B. anthracis, strain Pasteur No. 2-H.     

Comparative Study of the Cell Walls of the Yeastlike and Mycelial Phases of Histoplasma capsulatum

Cell walls were prepared from the yeastlike and mycelial phases (YP and MP) of Histoplasma capsulatum and from Saccharomyces cerevisiae by mechanical disruption and washing. Lipids were extracted with methanol-ether, chloroform, and acidified methanol:ether; a final extraction was made with ethylenediamine. The lipid contents of H. capsulatum YP and MP walls were about the same. Qualitative and quantitative analyses were made of the products obtained from treatment of the cell walls, or fractions from them, with weak acid or with enzymatic preparations containing glucanase and chitinase activities. YP walls contained much larger quantities of chitin and smaller quantities of mannose and amino acids than the MP walls. H. capsulatum MP was shown to resemble S. cerevisiae by low chitin content and by the presence of a mannose polymer, soluble in ethylenediamine and water. H. capsulatum MP chitin appeared to be intimately associated with glucose in the wall, since enzymatic hydrolysis of the residue after mild acid hydrolysis of cell walls or fractions from them resulted in the release of glucose and acetylglucosamine; only acetylglucosamine was released from YP walls with such treatment. By electron microscopic observations, the unextracted MP cell walls were much thinner than the YP, and neither wall appeared laminated.     

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: SEM imaging (part 2)

In the first part of our work, we combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis experiments coupled with chemical analysis of the different fractions of corn stover showed significant differences in cell wall structure before and after liquid hot water pretreatment. The extent of enzyme hydrolysis followed the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Since similar lignin contents remained after liquid hot water pretreatment of leaves, rind, and pith, our results indicated that the amount of lignin alone is not sufficient to explain the different enzymatic hydrolysis characteristics of the fractions. While the role of structural characteristics on enzyme hydrolysis of cell walls is measured as described in part I, the SEM images presented in this part II of our work show that sugar yields from enzymatic hydrolysis of corn fractions correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment.     

Alterations in the Morphology of Bacillus subtilis After Exposure to β-Lysin and Ultraviolet Light

Viable counts, turbidities, and electron micrographs of Bacillus subtilis exposed to beta-lysin and ultraviolet light (UV), singly or in combination, were compared in an attempt to relate death with changes in morphology. The decreases in survival of both the beta-lysin- and UV-treated cells were rapid and preceded decreases in turbidity, as well as the changes in morphology. No significant differences were observed in turbidity reduction or morphological alterations of control cells from those of cells exposed to UV light. These cells developed prominent subcell wall spaces during incubation in the hypertonic stabilizing medium. No observable damage in either the cell wall or the cell membrane had taken place during 4 hr, but by 20 hr extensive damage of these two structures was apparent. The control and UV-treated cells exposed to beta-lysin did not develop prominent subcell wall spaces. Within 2 hr, lesions were observable in their cell walls, and the cytoplasmic membranes were permeable to phosphotungstic acid. The damage to these structures became more extensive with time. Although the visible changes of control and UV-treated cells were evident much later than those induced by beta-lysin, the morphological alterations in all cells were similar. It appeared that beta-lysin caused an accelerated release of an autolytic enzyme which digested the cell walls.     

Subpopulations of physiological and ovarian follicular fluid peptide induced apoptotic cells

Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries enhances apoptotic changes in ovarian granulosa cells of mice. To get an insight into the cell subpopulations responding to OFFP, the heterogeneity of granulosa cells was resolved. Subpopulations of granulosa cells were obtained from ovaries of immature mice treated with PMSG alone and autopsied 48 hr (control) and 72 hr after injection (atretic) and from animals injected OFFP 24 hr after PMSG injection and autopsied 24 hr later (OFFP treated) by separation on discontinuous Percoll gradient. Four fractions were collected and studied for their relative distributions and percent apoptotic cells measured by acridine orange staining. FSH binding to granulosa cell (sedimenting as a major) fraction was studied by radio receptor assay. There is a difference in densities in subpopulations of apoptotic cells induced by OFFP and those generated during the physiological process of atresia. This difference may be a reflection of different granulosa cell subpopulations involved in peptide response or differences in phases as the cells transit from normal to apoptotic phenotype. FSH binding to granulosa cells from OFFP treated animals was significantly less than those from control and atretic group.     

Resistance of Zygorhynchus Species to Lysis

Zygorhynchus vuilleminii, a nonmelanin-containing fungus, was not lysed by mycolytic actinomycetes. Several enzymes and Streptomyces enzyme preparations digesting walls of other fungi were without appreciable activity on walls of Zygorhynchus species. A bacterium able to solubilize a portion of the Zygorhynchus wall released little or no reducing sugars from these structures. Fractions of Z. vuilleminii walls were resistant to glucanase hydrolysis, but certain fractions were digested by chitinase and microbial enzyme preparations. The walls and several wall fractions were not readily susceptible to degradation by a soil community. Walls of lysis-resistant Zygorhynchus species contained glucosamine, fucose, glucuronic acid, and galactose but little or no glucose. Resistant wall fractions were rich in uronic acid and fucose, whereas the readily degradable fractions contained abundant glucosamine. Cultural conditions affected the extent of digestion and composition of the walls. Possible reasons for the resistance of Zygorhynchus to lysis in nature are discussed.     

The production of toxins by Botrytis fabae in relation to growth of lesions on field bean leaves at different humidities

Filtrate extracts from liquid cultures of B. fabae and extracts from spreading chocolate spot lesions contained at least two heat-stable, light-labile phytotoxic compounds. Lesions similar in appearance to those of chocolate spot developed after injection of fractions containing these compounds into healthy bean leaves. Of 15 plant species injected with an extract from lesions, Vicia faba appeared to be the most susceptible to damage. Evidence is presented to support the hypothesis that under conditions of low humidity the concentrations of toxic compounds in an infected leaf become high enough to kill healthy cells surrounding infected tissue. The dead tissue then dries out preventing further fungal growth and lesion spread. In saturated air, however, the toxic compounds diffuse throughout the lamina and become too dilute to kill uninfected tissue. Tissue does not become desiccated and the fungus continues to spread.     

Study of the composition of cell wall of group A Streptococcus after hydrolysis using muramidase from Streptomyces levoris

The aim of the experiment was to study the lysis products of cell walls of group A streptococci resulting from exposure to N-acetylmuramidase. It was shown that for isolating surface proteins free of polysaccharide and peptidoglycan fragments it was necessary to treat the streptococcal cell walls with endo-beta-N-acetylmuramidase for no more than 30 minutes. Prolonged hydrolysis with muramidase led to the presence of polysaccharide and the peptidoglycan fragments in the protein fractions, intracellular wall proteins covalently bound to the peptidoglycan fragments and polysaccharide being also released.     

Chemical and enzymatic variation in the cell walls of pathogenic Candida species

Cell walls, isolated from seven pathogenic species of Candida, were lipid extracted and fractionated by treatment with ethylenediamine or enzymatically hydrolyzed using chitinase and laminarinase. Two different chitinase preparations were used, one from Streptomyces sp. which had some beta-1,3-glucanase activity, and another from Serratia marcescens which did not have glucanase activity. Laminarinase was a commercial preparation. The monosaccharide constituents of whole cell walls and the fractions derived from them were determined qualitatively and quantitatively by gas-liquid chromatography of the products of a mild acid hydrolysis and by the phenol - sulfuric acid assay of the products of a stronger acid hydrolysis. The monomeric constituents of the enzymatic hydrolyses were analyzed using gas-liquid chromatography. Approximately 50% of all walls was soluble in ethylenediamine. Glucose and mannose were the only monosaccharides found in all of the fractions derived from ethylenediamine extraction examined. Similarities among the strains, based upon relative amounts of glucose and mannose, were more apparent than differences, but statistical analyses of the data revealed a general trend of decreasing similarity in the following order, C. albicans and C. stellatoidea, C. tropicalis and C. parapsilosis, and C. pseudotropicalis, C. guilliermondii, and C. krusei. In the enzymatic assays, mannose and glucose were released by laminarinase, whereas glucose and N-acetyl-D-glucosamine or N-acetyl-D-glucosamine alone were released by the chitinases. These assays supported the trend in relationships cited above, with the data being somewhat more definitive.     

Localization of a mosquito-larval toxin of Bacillus sphaericus 1593.

Cell-free wall, membrane, and cytoplasmic fractions were prepared from Bacillus sphaericus 1593, which exhibited toxic activity against larvae of the mosquito Culex pipiens var. quinquefasciatus. Breakage of 12- to 14-h cells by sonication or French pressure cell yielded toxic material which could be assayed in a standard mosquito larva bioassay. When sporulating cells of strain 1593 were fractionated, the majority of the toxic activity was localized in the cell wall rather than in the plasma membrane or cytoplasm. The toxin located in the bacterial cell wall was relatively stable, in that activity was unaffected by treatment with trypsin, pronase, CHCl3-CH3OH-water, Triton X-100, 8 M urea (30 min), heat (80 degrees C, 12 min), sonication, refrigeration, lyophilization, or freezing. Activity was destroyed by boiling for 10 min or by 0.01 N NaOH. Only about 1.0% of the activity present in purified cell walls could be recovered by a 2-h extraction with 8 M urea or 3 M guanidine hydrochloride. A comparison of the toxicity of a cell-free cell wall fraction with that of a sample consisting entirely of heat-stable spores indicated that the spore preparation was about 10 times more active.