Insoluble Cell Wall Fraction Research Articles

Phenolics and Antioxidant Properties of Fruit Pulp and Cell Wall Fractions of Postharvest Banana (Musa acuminata Juss.) Cultivars

Banana fruits are important foods, but there have been very few studies evaluating the phenolics associated with their cell walls. In the present study, (+) catechin, gallocatechin, and (-) epicatechin, as well as condensed tannins, were detected in the soluble extract of the fruit pulp; neither soluble anthocyanidins nor anthocyanins were present. In the soluble cell wall fraction, two hydroxycinnamic acid derivatives were predominant, whereas in the insoluble cell wall fraction, the anthocyanidin delphinidin, which is reported in banana cell walls for the first time, was predominant. Cell wall fractions showed remarkable antioxidant capacity, especially after acid and enzymatic hydrolysis, which was correlated with the total phenolic content released after the hydrolysis of the water-insoluble polymer, but not for the posthydrolysis water-soluble polymer. The acid hydrolysis released various monosaccharides, whereas enzymatic hydrolysis released one peak of oligosaccharides. These results indicate that banana cell walls could be a suitable source of natural antioxidants and that they could be bioaccessible in the human gut.

The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of theChlamydomonascell wall

Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.

The Chaotrope-soluble Glycoprotein GP2 Is a Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

Organization of cell walls in Sandersonia aurantiaca floral tissue.

Visual symptoms of the onset of senescence in Sandersonia aurantiaca flowers begin with fading of flower colour and wilting of the tissue. When fully senescent, the flowers form a papery shell that remains attached to the plant. The cell walls of these flowers have been examined to determine whether there are wall modifications associated with the late stages of expansion and subsequent senescence-related wilting. Changes in the average molecular size of pectin were limited through flower opening and senescence, although there was a loss of neutral sugar-containing side-branches from pectins in opening flowers, and the total amounts of pectin and cellulose continued to rise in cell walls of fully senescent sandersonia flowers. Xyloglucan endotransglycosylase activity increased in opening and mature flowers, but declined sharply as flowers wilted. Concomitantly, the proportion of hemicellulose polymers of increasing molecular weight increased from flower expansion up to the point at which wilting occurred. Approximately 50% of the non-cellulosic neutral sugar in mature flower cell walls was galactose, primarily located in an insoluble cell wall fraction. Total galactose in this fraction increased per flower with maturity, then declined at the onset of wilting. Beta-galactosidase activity was low in expanding tepals, but increased as flowers matured and wilted.

Larvicidal Effect of a Cell-Wall Fraction Isolated from Alder Decaying Leaves

Decaying alder leaves in water from Alpine Aedes breeding sites, particularly their toxicity to larval Culicidae, were investigated and characterized with comparative toxicological and chemical methods. Bioassays that used third-instar Aedes aegypti as a reference species indicated that the larvicidal effect of crude leaf litter varied with decaying age of the litter, while no toxicity was detected from leaching water of the mosquito breeding sites. Ten-month-old leaf litter was the most toxic. Comparison of the different soluble and insoluble fractions obtained after sequential extraction of decomposed litter allowed us to localize the toxicity factor to an insoluble cell-wall fraction. The toxicity seems to be linked to phenolic activity. It is higher than that found for tannic acid solutions used as a reference to mimic the larvicidal effects of the molecules naturally occurring in decaying litter. The pattern of establishing the larvicidal effect of alder leaf litter in water of Alpine Aedes breeding sites is discussed.

Chloroacetamide Mode of Action, I: Inhibition of Very Long Chain Fatty Acid Synthesis in Scenedesmus acutus

Abstract Herbicidal chloroacetamides cause a very sensitive inhibition of fatty acid incorporation into an insoluble cell wall fraction of Scenedesmus acutus. The molecular basis was investigated in more detail. After incubation of the algae with [14C]oleic acid and saponification, the remaining pellet was solubilized and fractionated consecutively with chloroform / methanol, phosphate buffer, amylase, pronase, and finally with dioxane/HCl. By acid hydrolysis in dioxane a part of the cell wall residue was solubilized showing inhibition of exogenously applied oleic acid and other labelled precursors such as stearic acid, palmitic acid, and acetate. After extraction of this dioxane-soluble subfraction with hexane, HPLC could separate labelled metabolites less polar than oleic acid. T heir formation was completely inhibited by chloroacetam ides, e.g. 1 μᴍ metazachlor. This effect was also observed with the herbicidally active 5-enantiomer of metolachlor while the inactive R-enantiomer had no influence. These strongly inhibited metabolites could be characterized by radio-HPLC /MS as very long chain fatty acids (VLCFAs) with a carbon chain between 20 and 26. Incubating am etazachlor-resistant cell line of S. acutus (Mz-1) with [14C]oleic acid, V LCFA s could not be detected in the dioxane/ HCl-subfraction. Furthermore, comparing the presence of endogenous fatty acids in wildtype and mutant Mz-1 the VLCFA content of the mutant is very low, while the content of long chain fatty acids (C16 -18) is increased, particularly oleic acid. Obviously, the phytotoxicity of chloroacetam ides in S. acutus is due to inhibition of VLCFA synthesis. The resistance of the mutant to metazachlor has a bearing on the higher amount of long chain fatty acids replacing the missing VLCFAs in essential membranes or cell wall components.

A Cell Wall-associated, Receptor-like Protein Kinase

Physical connections between higher plant cell walls and the plasma membrane have been identified visually, but the molecules involved in the contact are unknown. We describe here an Arabidopsis thaliana protein kinase, designated Wak1 for wall-associated kinase, whose predicted extracytoplasmic domain contains several epidermal growth factor repeats and identity with a viral movement protein. Wak1 fractionates with insoluble material when plant tissue is ground in a variety of buffers and detergents, suggesting a tight association with the plant extracellular matrix. Immunocytochemistry confirms that Wak1 is associated with the cell wall. Enzymatic digestion of the cell wall allows the release of Wak1 from the insoluble cell wall fraction, and protease experiments indicate that Wak1 likely has a cytoplasmic kinase domain, and the EGF containing domain is extracellular. Wak1 is found in all vegetative tissues of Arabidopsis, and has relatives in other angiosperms, but not Chlamydomonas. We suggest that Wak1 is a good candidate for a physical continuum between the cell wall and the cytoplasm, and since the kinase is cytoplasmic, it also has the potential to mediate signals to the cytoplasm from the cell wall.

Immunological Identification of a Putative Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

To identify precursors of the insoluble glycoprotein framework of the Chlamydomonas cell wall, a polyclonal antibody was raised against the mixture of polypeptides released from the insoluble wall fraction by chemical deglycosylation. This antibody preferentially cross-reacted with a '150 kDa' salt-soluble cell wall glycoprotein. The conclusion that this '150 kDa' glycoprotein is a putative precursor of the insoluble cell wall fraction was corroborated by the results of pulse-chase experiments and by experiments with antibodies raised against the '150 kDa' salt-soluble glycoprotein and against its 100 kDa deglycosylation product, respectively. Whereas the antibody against the '150 kDa' glycoprotein preferentially recognized carbohydrate side chains, the antibody against its 100 kDa deglycosylation product was found to have essentially the same specificity towards glycosylated and deglycosylated cell wall components as the antibody against the deglycosylation products of the insoluble wall fraction. Furthermore, the antibody against the deglycosylated, insoluble wall fraction recognized almost the same set of peptide fragments derived by V8 protease treatment from the '150 kDa' salt-soluble cell wall glycoprotein and its 100 kDa deglycosylation product, respectively, as the antibody against the 100 kDa deglycosylated cell wall polypeptide.

Rapid metabolism of isoflavonoids in elicitor-treated cell suspension cultures of Pueraria lobata

Upon treatment with yeast extract as an elicitor to cell suspension cultures of Pueraria lobata, the constitutive isoflavonoid conjugates (7- O-glucoside-6″- O-malonyl esters) showed a rapid decline in their contents within 4 hr, followed by both reaccumulation of the conjugates and formation of isoflavones. Simultaneous treatment of a potent translation inhibitor, cycloheximide (200 μM), could not suppress the elicitor-inducible decrease of the pre-existing isoflavonoids, although it resulted in almost complete inhibition of both reaccumulation of the conjugates and formation of isoflavones. Exogenously applied 14C-labelled isoflavones were readily converted to their respective malonyl glucosides in the cells. When yeast extract was applied to the cell cultures in which [ 14C]-daidzein had been incorporated into its malonyl glucoside, the methanol-soluble radioactivity decreased rapidly within 4 hr and it was not recovered for 48 hr, indicating that most of the conjugate was incorporated into an insoluble cell wall fraction. The rapid response upon elicitation was selective for isoflavonoids and not for flavonoids.

Morphogen-Based Chemical Flypaper for Agaricia humilis Coral Larvae.

Larvae of the scleractinian coral Agaricia humilis settle and metamorphose in response to chemosensory recognition of a morphogen on the surfaces of Hydrolithon boergesenii and certain other crustose coralline red algae. The requirement of the larva for this inducer apparently helps to determine the spatial pattern of recruitment in the natural environment. Previous research showed that the inducer is associated with the insoluble cell wall fraction of the recruiting algae or their microbial epibionts, and that a soluble but unstable fragment of the inducing molecule can be liberated by limited hydrolysis, either with alkali or with enzymes specific for cell wall polysaccharides. We now show that the parent morphogen can be solubilized by gentle decalcification of the algal cell walls with the chelators EGTA or EDTA, suggesting that the morphogen may be a component of the calcified recruiting alga itself, rather than a product of any noncalcified microbial epibionts. The solubilized inducer is subsequently purified by hydrophobic-interaction and DEAE chromatography. The purified, amphipathic morphogen retains activity when tightly bound to beads of a hydrophobic-interaction chromatography resin, and this activity (tested with laboratory-reared larvae) is identical in the ocean and the laboratory. We have attached the purified, resin-bound inducer to surfaces coated with a silicone adhesive and thus produced a potent artificial recruiting substratum--i.e., a morphogen-based chemical "flypaper" for A. humilis larvae. This material should prove useful in resolving the role of chemosensory recognition of morphogens in the control of substratum-specific settlement, metamorphosis, and recruitment and in the maintenance of species isolation mechanisms in the natural environment.

Solubilization of limulus test reactive material(s) from Candida cells by murine phagocytes.

Solubilization of limulus test reactive materials from Candida was examined in the presence or absence of phagocytic cells. Solubilized limulus test reactive materials (LTRM) were detected in culture supernatant, and hot water and sodium hydroxide extracts of the acetone dried cells of Candida parapsilosis. Suspensions of Candida cells also reacted with limulus test, and LTRM were released from the acetone dried cells by serum treatment. After treatment of the acetone dried cells with polymorphonuclear leucocytes (PMN) or macrophages (M phi), a significant amount of LTRM was solubilized. Significant amounts of LTRM were also released by PMN during treatment of live and growing C. parapsilosis. The reactivity of LTRM was completely inhibited by the addition of excess amount of purified (1----3)-beta-D-glucan, suggesting LTRM from Candida cells as described above would contain (1----3)-beta-D-glucan. These results suggested that LTRM during fungal infection would come from the extracellular water soluble polysaccharide fraction as well as the insoluble cell wall fraction solubilized by the action of phagocytes.

Isoxaben Inhibits the Synthesis of Acid Insoluble Cell Wall Materials In Arabidopsis thaliana

The effect of the herbicide isoxaben on the incorporation of radiolabeled glucose, leucine, uracil, and acetate into acid insoluble cell wall material, protein, nucleic acids, and fatty acids, respectively, was measured. Dichlobenil, cycloheximide, actinomycin D, and cerulenin, inhibitors of the incorporation of these precursors into these macromolecular components, functioned as expected, providing positive controls. The incorporation of radiolabeled glucose into an acid insoluble cell wall fraction was severely inhibited by isoxaben at nanomolar concentrations. Amitrole, fluridone, ethalfluralin, and chlorsulfuron, as well as cycloheximide, actinomycin D, and cerulenin did not inhibit incorporation of glucose into this fraction, ruling out a general nonspecific effect of herbicides on glucose incorporation. The evidence thus suggests that isoxaben is an extremely powerful and specific inhibitor of cell wall biosynthesis.

Orinithine as a constituent of the peptidoglycan of Chloroflexus aurantiacus, diaminopimelic acid in that of Chlorobium vibrioforme f. thiosulfatophilum

L-Ornithine is the only diamino acid of the peptidoglycan of the gliding phototrophic Chloroflexus aurantiacus. The other constituents are L- and D-alanine, D-glutamic acid, N-acetyl-glucosamine and N-acetyl-muramic acid (in part as muramic acid-6-phosphate), all in approximate equimolar ratios to L-ornithine, aside from small amounts of glycine and histidine. Furthermore unlike typical Gram-negative bacteria, protein is not bound to this peptidoglycan. Instead, the rigid layer (sodium dodecyl sulfate insoluble cell wall fraction) contained large amounts of a complex polysaccharide consisting of sugar O-methyl ethers, hexoses and pentoses. Its binding site is presumably muramic acid-6-phosphate of the peptidoglycan.

The structure of a mannitol teichoic acid from Bifidobacterium bifidum ssp. Pennsylvanicum

The isolation and analysis of the cell wall and cell wall fractions of Bifidobacterium bifidum ssp. pennsylvanicum are presented. With lysozyme a solubilized cell wall fraction is obtained which contains muramic acid, glucosamine, rhamnose, glucose, mannitol, phosphate and all peptidoglycan amino acids. Its composition did not change with culture age. A glycogen-like glucose polymer which is of cytoplasmic origin is identified in the insoluble cell wall fraction. The solubilized cell wall fraction contains a glucosylated rhamnose polymer which is linked by glycosidic bonds to the peptidoglycan fragments. This polymer is a 1,2-linked or an alternating, 1,2/1,3-linked α-rhamnose chain substituted on average at every second rhamnose residue with an α-linked glucose molecule. Various experiments gave evidence that mannitol and phosphate are present in 4,6-linked mannitol phosphate oligomers which are linked by phosphodiester bonds to the glucosylated rhamnose polymer. These oligomers may fulfill the functions of the more common wall teichoic acids.

An Extracellular Macromolecular Complex from the Surface of Soybean Suspension Cultures

A cell-free, insoluble cell wall fraction is described which floats on the surface of suspension cultures of Glycine max L. Merrill var. Acme cells. Its accumulation is governed by both the shaking speed and the medium volume, a shaking speed of 110 to 120 revolutions per minute with a medium volume of about 100 to 120 milliliters in a 250-milliliter flask being optimal. Various factors which could control the accumulation of the complex were tested and are discussed, and scanning electron micrographs of the complex being released from the cell surface are presented.The composition of the complex by weight is 46% galacturonic acid, 36% protein, 11% lignin (apparent), 4.4% arabinose, 2% ash, and 0.5% methyl ester. Evidence for an intimate relationship between the uronic acid and protein fractions is presented. The protein contains hydroxyproline, and the bulk of it is tightly bound to the complex, although a portion can be removed with high salt treatments.