Coniferyl alcohol oxidase activity of a cell-wall-located class III peroxidase

1999 ◽  
Vol 26 (5) ◽  
pp. 411 ◽  
Author(s):  
A. Ros Barceló ◽  
G. J. Aznar-Asensio
Keyword(s):  
Cell Wall ◽  
Oxidase Activity ◽  
Cell Walls ◽  
Alcohol Oxidase ◽  
Visible Spectrum ◽  
Coniferyl Alcohol ◽  
Class Iii ◽  
Apparent Homogeneity

Coniferyl alcohol oxidase activity was determined in cell walls from hypocotyls of the following species belonging to the family Asteraceae: Calendula officinalis, Callistephus sinensis, Cosmos bipinnanthus, Helianthus annuus, Helianthus debilis and Zinnia elegans. In all the cases studied, coniferyl alcohol oxidase activity was partially located ionically-bound to cell walls and resided in a basic peroxidase, the activity of which was stimulated by H 2 O 2 . This enzymatic activity was insensitive to freezing and was inactivated by high H 2 O 2 concentrations, as tested both in vitro and in situ by using purified cell wall fractions. The peroxidase with coniferyl alcohol oxidase activity was purified from Z. elegans hypocotyls until apparent homogeneity, as checked by SDS-PAGE. It showed a visible spectrum typical of a haem-containing high-spin ferric secretory (class III) plant peroxidase. Coniferyl alcohol oxidase activity of this basic peroxidase constitutes about 0.25% of the activity shown in the presence of H 2 O 2 . The significance of the coniferyl alcohol oxidase activity in vivo was studied in Z. elegans hypocotyls by means of histochemical tests, which revealed that it was located in the H 2 O 2 -producing lignifying xylem cells. The results obtained from the histochemical probes suggest that the coniferyl alcohol oxidase activity of this basic peroxidase is physiologically irrelevant in tissues that accumulate H 2 O 2 , as is the case of the lignifying xylem, where the peroxidase activity of the enzyme favorably competes with the oxidase activity of the enzyme.

scholarly journals Putative Role of β-1,3 Glucans in Candida albicans Biofilm Resistance

2006 ◽  
Vol 51 (2) ◽  
pp. 510-520 ◽  
Author(s):  
Jeniel Nett ◽  
Leslie Lincoln ◽  
Karen Marchillo ◽  
Randall Massey ◽  
Kathleen Holoyda ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Cell Viability ◽  
Amphotericin B ◽  
Cell Walls ◽  
Positive Impact ◽  
Indirect Evidence ◽  
Phenotypic Properties

ABSTRACT Biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface. Nearly all device-associated infections involve growth in the biofilm life style. Biofilm communities have characteristic architecture and distinct phenotypic properties. The most clinically important phenotype involves extraordinary resistance to antimicrobial therapy, making biofilm infections very difficulty to cure without device removal. The current studies examine drug resistance in Candida albicans biofilms. Similar to previous reports, we observed marked fluconazole and amphotericin B resistance in a C. albicans biofilm both in vitro and in vivo. We identified biofilm-associated cell wall architectural changes and increased β-1,3 glucan content in C. albicans cell walls from a biofilm compared to planktonic organisms. Elevated β-1,3 glucan levels were also found in the surrounding biofilm milieu and as part of the matrix both from in vitro and in vivo biofilm models. We thus investigated the possible contribution of β-glucans to antimicrobial resistance in Candida albicans biofilms. Initial studies examined the ability of cell wall and cell supernatant from biofilm and planktonic C. albicans to bind fluconazole. The cell walls from both environmental conditions bound fluconazole; however, four- to fivefold more compound was bound to the biofilm cell walls. Culture supernatant from the biofilm, but not planktonic cells, bound a measurable amount of this antifungal agent. We next investigated the effect of enzymatic modification of β-1,3 glucans on biofilm cell viability and the susceptibility of biofilm cells to fluconazole and amphotericin B. We observed a dose-dependent killing of in vitro biofilm cells in the presence of three different β-glucanase preparations. These same concentrations had no impact on planktonic cell viability. β-1,3 Glucanase markedly enhanced the activity of both fluconazole and amphotericin B. These observations were corroborated with an in vivo biofilm model. Exogenous biofilm matrix and commercial β-1,3 glucan reduced the activity of fluconazole against planktonic C. albicans in vitro. In sum, the current investigation identified glucan changes associated with C. albicans biofilm cells, demonstrated preferential binding of these biofilm cell components to antifungals, and showed a positive impact of the modification of biofilm β-1,3 glucans on drug susceptibility. These results provide indirect evidence suggesting a role for glucans in biofilm resistance and present a strong rationale for further molecular dissection of this resistance mechanism to identify new drug targets to treat biofilm infections.

scholarly journals Confirmation of Cage Effect and Prebiotic Production Potential of a β-Mannanase, with SBM as Substrate Using Microscopy and Wet Chemistry

2021 ◽  
Vol 13 (2) ◽  
pp. 23
Author(s):  
L. M. Gomez-Osorio ◽  
Hwa Gyun Oh ◽  
Jung Jin Lee
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Degree Of Polymerization ◽  
In Vitro Assays ◽  
Wall Structure ◽  
Specific Substrate ◽  
Mannan Oligosaccharides

In vitro assays were carried out to investigate the solubilization of cell walls and generation of mannan oligosaccharides of a b-mannanase-containing commercial product on SBM. Using commercial dosages of the b-mannanase (500 g per ton of feed) cell wall degradation of mannan in SBM cell walls was visualized and an increase in reducing ends (0.12±0.02 mg/mL) and the generation of mannan oligosaccharides of degree of polymerization 2 and 4 (22.9±3.2 mg/L and 398.8±25.4 mg/L) were also measured using HPLC. Mannan, which is H-bonded to cellulose and xyloglucan, was solubilized using a single monocomponent enzyme, allowing for visualization of the disintegration of the entire SBM cell wall structure. This work is the first of its kind using strictly commercial dosage levels of enzyme for evaluating efficacy of the same microscopically. These data confirm the hypothesis that there most likely is a need for only a single relevant NSP enzyme targeting its specific substrate, independent of the concentration of the latter within the complex polysaccharide matrix in the plant cell wall to experience the beneficial effects of the enzyme both in vitro and in vivo. An analogy to compare our data would be destruction of the foundation (mannan) of a building or a bridge (soybean cell wall) which would inevitably lead to dismantling or demolition the entire building or bridge.

scholarly journals Sulfation of fucoidin in focus embryos: III. Required for localization in the rhizoid wall

1978 ◽  
Vol 78 (3) ◽  
pp. 866-873 ◽  
Author(s):  
WE Hogsett ◽  
RS Quatrano
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Alginic Acid ◽  
Fluorescein Isothiocyanate ◽  
Sulfated Polysaccharide ◽  
Specific Region ◽  
Fucus Distichus ◽  
Rhizoid Formation

Zygotes of the brown alga Fucus distichus L. Powell accumulate a sulfated polysaccharide (fucoidin) in the cell wall at the site of rhizoid formation. Previous work indicated that zygotes grown in seawater minus sulfate do not sulfate the preformed fucan (an unsulfated fucoidin) but form rhizoids. Under these conditions, we determined whether sulfation of the fucan is required for its localization in the rhizoid wall. This was accomplished by developing a specific stain for both the fucan and fucoidin. Using a precipitin assay, we demonstrated in vitro that the lectin ricin (RCA(I)) specifically complexes with both the sulfated and desulfated polysaccharide. No precipitate is observed when either is incubated in 0.1 M D-galactose or when RCA(I) is mixed with laminarin or alginic acid, the other major polysaccharides in Fucus. RCA(I) conjugated with fluorescein isothiocyanate (FITC) is also shown to bind specifically to fucoidin using a filter paper (DE81) assay. When added to zygotes, RCA(I)-FITC binds only to the site of fucoidin localization, i.e., the rhizoid cell wall. However, RCA(I)-FITC is not observed in the rhizoid wall of zygotes grown in the absence of sulfate. This observation is not due to inability of RCA(I)-FITC to bind to the fucan in vivo. Chemically desulfated cell walls that contained fucoidin in the rhizoid wall bind RCA(I)-FITC only in the rhizoid region. Also, the concentration of fucose-containing polymers and polysaccharides that form precipitates with RCA(I) is the same in embryos grown in the presence or absence of sulfate. If sulfate is added back to cultures of zygotes grown without sulfate, fucoidin is detected at the rhizoid tip by RCA(I)-FITC several hours later. These results support the conclusion that the enzymatic sulfation of the fucan is a modification of the polysaccharide required for its localization and/or assembly into a specific region of the cell wall.

Cell-Wall-Associated Oxidases from the Lignifying Xylem of Angiosperms and Gymnosperms: Monolignol Oxidation

Holzforschung ◽  
1999 ◽  
Vol 53 (5) ◽  
pp. 503-510 ◽  
Author(s):  
Nigel Deighton ◽  
Andrew Richardson ◽  
Derek Stewart ◽  
Gordon J. McDougall
Keyword(s):  
Cell Wall ◽  
Peroxidase Activity ◽  
Oxidase Activity ◽  
Cell Walls ◽  
Specific Activity ◽  
Coniferyl Alcohol ◽  
Liquid Chromatography Mass Spectrometry ◽  
Sinapyl Alcohol ◽  
Marked Preference ◽  
Dehydrogenation Polymers

Summary Cell-wall-associated oxidases extracted from the lignifying xylem of Sitka spruce and ash oxidise sinapyl alcohol at a greater rate than coniferyl alcohol and p-coumaryl alcohol (SA > CA > pCA). The enzyme from ash shows a marked preference, on a specific activity basis, for the oxidation of SA over CA and pCA (SA ≫ CA ≥ pCA) and has a particularly low affinity for the oxidation of coniferyl alcohol compared to the enzyme from spruce (SA > CA > pCA). This difference in monolignol preference between the spruce and ash enzymes may relate to their required functions during lignification, in that the hardwood enzyme would be supplied mainly SA and the softwood enzyme would be supplied mainly CA. The spruce enzyme also displayed a marked preference for the oxidation of sinapyl alcohol over sinapyl aldehyde even when the two compounds were presented in a mixture. Purified cell walls from the lignifying xylem of spruce could oxidise CA by the action of bound oxidase activity and dissolved oxygen (~ 240 μM) but CA oxidation was increased many fold by the action of the bound peroxidase activity and 240 μM H2O2. However, the initial dimeric and trimeric products of the peroxidase- and oxidase-catalysed reactions detected by liquid chromatography-mass spectrometry were the same and present in similar proportions. This indicates that the oxidation of CA by oxidase or by peroxidase proceeds via the same intermediates and occurs by a similar mechanism. Insoluble dehydrogenation polymers (DHPs) of CA were formed in similar yields by spruce extracts in the absence (oxidase activity) or presence (peroxidase activity) of H2O2. The peroxidase-catalysed DHPs and the oxidase-catalysed DHPs gave Fourier transform infra-red spectra with maxima that were characteristic of DHPs of CA. However, differences in the comparative intensities of some maxima suggest that the oxidase-catalysed DHPs were less condensed than the peroxidase-catalysed polymers. These findings are discussed with respect to the possible contribution of oxidases to lignin structure in developing wood.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

scholarly journals Ibudilast Mitigates Delayed Bone Healing Caused by Lipopolysaccharide by Altering Osteoblast and Osteoclast Activity

2021 ◽  
Vol 22 (3) ◽  
pp. 1169
Author(s):  
Yuhan Chang ◽  
Chih-Chien Hu ◽  
Ying-Yu Wu ◽  
Steve W. N. Ueng ◽  
Chih-Hsiang Chang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bone Formation ◽  
Cancellous Bone ◽  
Bone Healing ◽  
Bone Bridge ◽  
Cell Wall Components ◽  
Osteoclast Activity ◽  
Wall Components

Bacterial infection in orthopedic surgery is challenging because cell wall components released after bactericidal treatment can alter osteoblast and osteoclast activity and impair fracture stability. However, the precise effects and mechanisms whereby cell wall components impair bone healing are unclear. In this study, we characterized the effects of lipopolysaccharide (LPS) on bone healing and osteoclast and osteoblast activity in vitro and in vivo and evaluated the effects of ibudilast, an antagonist of toll-like receptor 4 (TLR4), on LPS-induced changes. In particular, micro-computed tomography was used to reconstruct femoral morphology and analyze callus bone content in a femoral defect mouse model. In the sham-treated group, significant bone bridge and cancellous bone formation were observed after surgery, however, LPS treatment delayed bone bridge and cancellous bone formation. LPS inhibited osteogenic factor-induced MC3T3-E1 cell differentiation, alkaline phosphatase (ALP) levels, calcium deposition, and osteopontin secretion and increased the activity of osteoclast-associated molecules, including cathepsin K and tartrate-resistant acid phosphatase in vitro. Finally, ibudilast blocked the LPS-induced inhibition of osteoblast activation and activation of osteoclast in vitro and attenuated LPS-induced delayed callus bone formation in vivo. Our results provide a basis for the development of a novel strategy for the treatment of bone infection.

scholarly journals Co-operative action by endo- and exo-β-(1→3)-glucanases from parasitic fungi in the degradation of cell-wall glucans of Sclerotinia sclerotiorum (Lib.) de Bary

1974 ◽  
Vol 140 (1) ◽  
pp. 47-55 ◽  
Author(s):  
David Jones ◽  
Alex. H. Gordon ◽  
John S. D. Bacon
Keyword(s):  
Cell Wall ◽  
Sclerotinia Sclerotiorum ◽  
Culture Filtrate ◽  
Cell Walls ◽  
Trichoderma Viride ◽  
Major Constituent ◽  
Coniothyrium Minitans ◽  
Parasitic Fungi ◽  
Complete Breakdown

1. Two fungi, Coniothyrium minitans Campbell and Trichoderma viride Pers. ex Fr., were grown on autoclaved crushed sclerotia of the species Sclerotinia sclerotiorum, which they parasitize. 2. in vitro the crude culture filtrates would lyse walls isolated from hyphal cells or the inner pseudoparenchymatous cells of the sclerotia, in which a branched β-(1→3)-β-(1→6)-glucan, sclerotan, is a major constituent. 3. Chromatographic fractionation of the enzymes in each culture filtrate revealed the presence of several laminarinases, the most active being an exo-β-(1→3)-glucanase, known from previous studies to attack sclerotan. Acting alone this brought about a limited degradation of the glucan, but the addition of fractions containing an endo-β-(1→3)-glucanase led to almost complete breakdown. A similar synergism between the two enzymes was found in their lytic action on cell walls. 4. When acting alone the endo-β-(1→3)-glucanase had a restricted action, the products including a trisaccharide, tentatively identified as 62-β-glucosyl-laminaribiose. 5. These results are discussed in relation to the structure of the cell walls and of their glucan constituents.

Peroxidase: a multifunctional enzyme in grapevines

2003 ◽  
Vol 30 (6) ◽  
pp. 577 ◽  
Author(s):  
Alfonso Ros Barceló ◽  
Federico Pomar ◽  
Matías López-Serrano ◽  
Maria Angeles Pedreño
Keyword(s):  
Cell Wall ◽  
Metabolic Regulation ◽  
Trichoderma Viride ◽  
Visible Spectrum ◽  
H2o2 Production ◽  
Class Iii ◽  
Absorption Bands ◽  
Peroxidase Isoenzyme ◽  
Wide Range ◽  
Uv C

Peroxidases are heme-containing enzymes that catalyse the one-electron oxidation of several substrates at the expense of H2O2. They are probably encoded by a large multigene family in grapevines, and therefore show a high degree of polymorphism. Grapevine peroxidases are glycoproteins of high thermal stability, whose molecular weight usually ranges from 35 to 45 kDa. Their visible spectrum shows absorption bands characteristic of high-spin class III peroxidases. Grapevine peroxidases are capable of accepting a wide range of natural compounds as substrates, such as the cell wall protein extensin, plant growth regulators such as IAA, and phenolics such as benzoic acids, stilbenes, flavonols, cinnamyl alcohols and anthocyanins. They are located in cell walls and vacuoles. These locations are in accordance with their key role in determining the final cell wall architecture, especially regarding lignin deposition and extensin insolubilization, and the turnover of vacuolar phenolic metabolites, a task that also forms part of the molecular program of disease resistance. Although peroxidase is a constitutive enzyme in grapevines, its levels are strongly modulated during plant cell development and in response to both biotic and abiotic environmental factors. To gain an insight into the metabolic regulation of peroxidase, several authors have studied how grapevine peroxidase and H2O2 levels change in response to a changing environment. Nevertheless, the results obtained are not always easy to interpret. Despite such difficulties, the response of the peroxidase–H2O2 system to both UV-C radiation and Trichoderma viride elicitors is worthy of study. Both UV-C and T. viride elicitors induce specific changes in peroxidase isoenzyme / H2O2 levels, which result in specific changes in grapevine physiology and metabolism. In the case of T. viride-elicited grapevine cells, they show a particular mechanism for H2O2 production, in which NADPH oxidase-like activities are apparently not involved. However, they offer a unique system whereby the metabolic regulation of peroxidase by H2O2, with all its cross-talks and downstream signals, may be elegantly dissected.

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