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PubMed1990

Fractionation and characterization of mitogenic and anti-complementary active fractions from kampo (Japanese Herbal) medicine "juzen-taiho-to".

"Juzen-Taiho-To" (TJ-48), which is a kampo (Japanese herbal) medicine prepared by decocting a prescription of ten kinds of herbs, has several immunostimulating activities. In order to characterize the active substances for anti-complementary and mitogenic activities, TJ-48 was fractionated. Anti-complementary activity was observed in the water- and methanol-insoluble fraction (F-2) and the crude polysaccharide fraction (F-5), whereas mitogenic activity was only found in F-5. However, other low molecular mass fractions did not show both activities. Methylation analysis indicated that F-2 mainly contained amylopectin-like polysaccharides. Both Pronase digestion and periodate oxidation decreased the anti-complementary activity of F-2, and the beta-amylase-resistant fraction of F-2 still retained the potent anti-complementary activity. When F-5, which has the most potent of both activities, was further fractionated, only the major acidic polysaccharide fraction, F-5-2, showed potent mitogenic activity. Endo-alpha-(1----4)-polygalacturonase digestion showed that F-5-2 mainly contained pectic polysaccharides, and the endo-polygalacturonase treatment of F-5-2 reduced the mitogenic activity but not the anti-complementary activity. F-2 and F-5 each activated the complement system by a different mode of action.

Animals↗
PubMed2002

Purification and partial characterization of exopolygalacturonase I from Penicillium frequentans.

A polygalacturonase with a molecular mass of 74 kDa, an isoelectric point around pH 4.2 and pH--and temperature optima of 3.9 and 50 degrees C, respectively, was purified from a culture fluid of Penicillium frequentans. The enzyme was characterized as an exo-alpha-1,4-polygalacturonase (exo-PG I). Km and Vmax for sodium polypectate hydrolysis were 0.68 g/l and 596.8 U x mg(-1), respectively. The enzyme, a glycoprotein with a carbohydrate content of 81%, is probably the main pectinase of Penicillium frequentans responsible for cleaving monomer units from the non-reducing end of pectin.

Brazil↗
PubMed2004

Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression.

The isolation and characterization of fruit-specific promoters are critical for the manipulation of the nutritional value and quality of fruits by genetic engineering. The analysis of regulatory sequences of many ripening-related genes has remained elusive for many species due to their low transformation efficiency and/or lengthy regeneration of a small number of transgenic plants. Strawberry is an important crop and represents one of the most widely studied non-climacteric model systems. However, until recently, its difficult regeneration has limited the functional study of promoters by stable transformation. A protocol based on biolistic transient transformation has been developed in order to study the function of promoters in a fast and efficient manner in strawberry fruits. The protocol has been applied to the study of the GalUR promoter, a gene involved in the biosynthesis of vitamin C in this fruit. The activity of the GalUR promoter is restricted to the fruit, being strictly dependent on light. The analysis of deletion series revealed the presence of a minimum activation region 397 bp upstream of the gene with a putative G-box motif, and a negative regulatory region between -397 and -518 bp, where an I-box was identified. The transient assay has been used to study the activity of the tomato polygalacturonase and the pepper fibrillin promoters in strawberry fruits. Whereas slight activity was observed with the fibrillin promoter, no significant activity was found with the polygalacturonase promoter. The GalUR promoter in transiently transformed ripe tomato fruits showed no activity, indicating the presence of regulatory sequences specific for its function in strawberry fruit.

Alcohol Oxidoreductases↗
PubMed2001

Effects of the two-component system comprising GacA and GacS of Erwinia carotovora subsp. carotovora on the production of global regulatory rsmB RNA, extracellular enzymes, and harpinEcc.

Posttranscriptional regulation mediated by the regulator of secondary metabolites (RSM) RsmA-rsmB pair is the most important factor in the expression of genes for extracellular enzymes and HarpinEcc in Erwinia carotovora subsp. carotovora. RsmA is a small RNA-binding protein, which acts by lowering the half-life of a mRNA species. rsmB specifies an untranslated regulatory RNA and neutralizes the RsmA effect. It has been speculated that GacA-GacS, members of a two-component system, may affect gene expression via RsmA. Because expA, a gacA homolog, and expS (or rpfA), a gacS homolog, have been identified in E. carotovora subsp. carotovora, we examined the effects of these gacA and gacS homologs on the expression of rsmA, rsmB, and an assortment of exoprotein genes. The gacA gene of E. carotovora subsp. carotovora strain 71 stimulated transcription of genes for several extracellular enzymes (pel-1, a pectate lyase gene; peh-1, a polygalacturonase gene; and celV, a cellulase gene), hrpNEcc (an E. carotovora subsp. carotovora gene specifying the elicitor of hypersensitive reaction), and rsmB in GacA+ and GacS+ E. carotovora subsp. carotovora strains. Similarly, the E. carotovora subsp. carotovora gacA gene stimulated csrB (rsmB) transcription in Escherichia coli. A GacS- mutant of E. carotovora subsp. carotovora strain AH2 and a GacA- mutant of E. carotovora subsp. carotovora strain Ecc71 compared with their parent strains produced very low levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts but produced similar levels of rsmA RNA and RsmA protein as well as transcripts of hyperproduction of extracellular enzymes (Hex) hexA, kdgR (repressor of genes for uronate and pectate catabolism), rsmC, and rpoS (gene for Sigma-S, an alternate Sigma factor). The levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts as well as production of pectate lyase, polygalacturonase, cellulase, protease, and HarpinEcc proteins were stimulated in GacS- and GacA- mutants by GacS+ or GacA+ plasmids, respectively. The GacA effect on exoenzyme genes and hrpNEcc was abrogated in E. carotovora subsp. carotovora mutants deficient in RsmA and RsmC or RsmA, RsmC, and rsmB RNA. The expression of lacZ transcriptional fusions of rsmB of Erwinia amylovora and Erwinia herbicola pv. gypsophilae was markedly reduced in a GacA- and a GacS- mutant of Pseudomonas syringae pv. syringae. Southern blot hybridization revealed the presence of gacA and gacS homologs in all tested strains of soft-rotting Erwinia spp. and several nonsoft-rotting Erwinia species such as E. amylovora, E. rhapontici, E. herbicola, E. stewartii, and E. herbicola pv. gypsophilae. These findings establish that the GacA-GacS system controls transcription of rsmB of E. carotovora subsp. carotovora, E. amylovora, and E. herbicola pv. gypsophilae and support the hypothesis that the effects of this two-component system on extracellular protein production in E. carotovora subsp. carotovora is mediated, at least in part, via the levels of rsmB transcripts.

Bacterial Outer Membrane Proteins↗
PubMed1996

Reversible Inhibition of Tomato Fruit Gene Expression at High Temperature (Effects on Tomato Fruit Ripening).

The reversible inhibition of three ripening-related processes by high-temperature treatment (38[deg]C) was examined in tomato (Lycopersicon esculentum L. cv Daniella) fruit. Ethylene production, color development, and softening were inhibited during heating and recovered afterward, whether recovery took place at 20[deg]C or fruit were first held at chilling temperature (2[deg]C) after heating and then placed at 20[deg]C. Ethylene production and color development proceeded normally in heated fruit after 14 d of chilling, whereas the unheated fruit had delayed ethylene production and uneven color development. Levels of mRNA for 1-aminocyclopropane-1-carboxylic acid oxidase, phytoene synthase, and polygalacturonase decreased dramatically during the heat treatment but recovered afterward, whereas the mRNA for HSP17 increased during the high-temperature treatment and then decreased when fruit were removed from heat. As monitored by western blots, the HSP17 protein disappeared from fruit tissue after 3 d at 20[deg]C but remained when fruit were held at 2[deg]C. The persistence of heat-shock proteins at low temperature may be relevant to the protection against chilling injury provided by the heat treatment. Protein levels of 1-aminocyclopropane-1-carboxylic acid oxidase and polygalacturonase also did not closely follow the changes in their respective mRNAs. This implied both differences in relative stability and turnover rates of mRNA compared to protein and nontranslation of the message that accumulated in low temperature. The results suggest that high temperature inhibits ripening by inhibiting the accumulation of ripening-related mRNAs. Ripening processes that depend on continuous protein synthesis including ethylene production, lycopene accumulation, and cell-wall dissolution are thereby diminished.

Journal Article↗
PubMed1979

Ultrastructural changes in the cell walls of ripening apple and pear fruit.

Ultrastructural changes in the cell walls of "Calville de San Sauveur" apples (Malus sylvestris Mill) and "Spadona" pear (Pyrus communis L.) fruit were followed during ripening. In apple, structural alterations in cell walls became apparent at advanced stages of softening and showed predominantly dissolution of the middle lamella. In pears softening was also associated with the dissolution of the middle lamella, and in addition a gradual disintegration of fibrillar material throughout the cell wall. In fully ripe fruit almost all of the fibrillar arrangement in the cell wall was lost. Application of enzyme solutions containing polygalacturonase and cellulase to tissue discs from firm pear fruit led to ultrastructural changes observed in naturally ripening pears. In apple polygalacturonase alone was sufficient to dissolve the middle lamella region of the cell walls, as was also found to occur in naturally ripening fruit. In both apple and pear the cell wall areas containing plasmodesmata maintained their structural integrity throughout the ripening process. At advanced stages of ripening vesicles appeared in the vicinity of plasmodesmata.

Journal Article↗
PubMed1980

Cell Wall Metabolism in Ripening Fruit: II. CHANGES IN CARBOHYDRATE-DEGRADING ENZYMES IN RIPENING ;BARTLETT' PEARS.

Mature ;Bartlett' pear (Pyrus communis) fruits were ripened at 20 C. Fruits at different stages of ripeness were homogenized, and extracts of the low speed pellet (crude cell wall) were prepared. These extracts contained polygalacturonase, pectin esterase, and activity against seven p-nitrophenyl glycoside substrates. Polygalacturonase, alpha-galactosidase, and alpha-mannosidase increased in activity as the fruit ripened. Cellulase and activities against pear wall xylan and arabinan were absent from the extracts.

Journal Article↗
PubMed1984

Pectic polysaccharide breakdown of cell walls in cucumber roots grown with calcium starvation.

Pectic polysaccharides from the roots of cucumber (Cucumis sativus L.) grown in liquid culture medium with or without calcium (1 mm CaCl(2)) were studied after extraction successively by hot water and Na hexametaphosphate solution. The Ca(2+) starvation-treatment caused a striking reduction in content of extracted pectic polysaccharide; from an equivalent weight of cell walls, only 33.1% of the control level was extracted from root cell walls of plants cultured under Ca(2+) deficiency. The extracted pectic polysaccharides were fractionated into neutral and acidic polymers by a DEAE-Sephadex column. The acidic polymers, which represented more than 76% of the yield, appeared to be a major fraction of extracted pectic polysaccharides. The changes of molecular size and glycosyl residue composition of this fraction were compared for the control and Ca(2+)-deprived samples. The results indicate that Ca(2+) deficiency caused structural changes which could involve both branching pattern and extent of contiguous galacturonosyl units in the water-solubilized pectic polysaccharides. Ca(2+) starvation also led to a notable decrease in molecular size of the hexametaphosphate-solubilized polysaccharides and, to a lesser extent, of the water-solubilized fraction as well. In addition, polygalacturonase activity in tissue homogenates increased remarkably with the Ca(2+) deficiency, whereas beta-galactosidase activity did not undergo a change. Thus, it appears that one major effect of Ca(2+) deprivation was to stimulate polygalacturonase activity, an effect which could be involved in the control of the breakdown of pectic polysaccharides in the cell walls.

Journal Article↗
PubMed1987

Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells.

The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-alpha-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-alpha-1,4-polygalacturonase-treated walls by treatment with an endo-beta-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-beta-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides.

Journal Article↗
PubMed1988

Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit.

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO(2) and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.

Journal Article↗
PubMed1989

Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides.

Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-alpha-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-alpha-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na(2)CO(3) at 1 and 22 degrees C. These previously uncharacterized polysaccharides accounted for approximately 4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO(3)-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na(2)CO(3) at 1 degrees C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.

Journal Article↗
PubMed2004

Antisense phenotypes reveal a role for SHY, a pollen-specific leucine-rich repeat protein, in pollen tube growth.

SHY, a pollen-specific gene identified in a screen for genes upregulated at pollen germination, encodes a leucine-rich repeat (LRR) protein that is predicted to be secreted. To test if SHY plays an important role during pollen germination, we generated transgenic plants expressing an antisense (AS) copy of the SHY cDNA in pollen. Primary transformants exhibited poor seed set, but homozygous lines could be identified. In these lines, nearly all pollen tubes failed to reach the ovules; tube growth was arrested at the apex of the ovary and the pollen tubes exhibited abnormal callose deposits throughout the tube and in the tips. We show that a SHY::eGFP fusion protein is targeted to the cell wall. The structure of the SHY protein is nearly identical to other extracellular matrix glycoproteins that are composed of LRRs, such as the polygalacturonase inhibitor proteins (PGIP) of plants. PGIPs may function as defense proteins by inhibiting fungal endo-polygalacturonases, but enzyme assays with extracts of AS-SHY pollen do not support such an inhibitor role for SHY. The tomato ortholog of SHY interacts with a tomato receptor kinase (LePRK2) in yeast two-hybrid and pull-down assays; this, and the AS-SHY phenotypes, suggest instead that SHY might function in a signal transduction pathway mediating pollen tube growth.

Amino Acid Sequence↗
PubMed2005

A case study from the interaction of strawberry and Botrytis cinerea highlights the benefits of comonitoring both partners at genomic and mRNA level.

Strawberry Fragaria x ananassa (cv. Korona) was inoculated with Botrytis cinerea by dipping berries in a conidial suspension. Colonization by the pathogen was monitored using real-time PCR, ELISA and ergosterol assays, the first showing the highest sensitivity. The expression of pathogen beta-tubulin and six polygalacturonases (Bcpg1-6) and three host defence genes (polygalacturonase-inhibiting protein (FaPGIP) and two class II chitinases) were monitored using real-time RT-PCR. The maximum transcript levels of the host defence genes occurred at 16 h postinoculation (hpi) at the presumed initial penetration stage. The unique transcript profile of Bcpg2 over the 96-h incubation time and its high transcript levels relative to those of the other Bcpgs at 8-24 hpi suggest that the gene has a specific role in the penetration stage. Bcpg1 was expressed constitutively at a relatively high level in actively growing mycelia throughout the experimental period. Comparison of the transcript profiles indicated that Bcpg1 and Bcpg3-6 were coordinately regulated.

Base Sequence↗
PubMed1969

Some pink yeasts associated with softening of olives.

Pink yeasts identified as Rhodotorula glutinis var. glutinis, R. minuta var. minuta, and R. rubra produce polygalacturonases which cause a slow softening of olive tissue. Both pectin methyl esterase and polygalacturonase are produced when cultures are grown in appropriate media. Crude, cell-free dialyzed enzyme preparations measured viscosimetrically exhibited optimal activity on sodium polygalacturonate at pH 6.0 and 40 C, and were active in the range of pH 4.0 to 9.0 and 10 to 50 C. Cultures grown in sterilized olives and brine at pH 4.0 with sterile glucose added aseptically caused a slow softening of tissue as measured with a Christel texturometer. Similar results were obtained when crude, cell-free enzyme preparations were added to olives in buffer solution at pH 6.0 with Merthiolate. Commercial control of these yeasts is easy if anaerobic conditions can be provided. Otherwise, the industry has to resort to manual removal of the film from the brine surface, either by skimming or by flagellation.

Journal Article↗
PubMed1999

Cloning, sequence analysis and overexpression of a Saccharomyces cerevisiae endopolygalacturonase-encoding gene (PGL1).

Only a few yeast strains produce pectin-degrading enzymes such as pectin esterases and depolymerases (hydrolases and lyases). Strain SCPP is the only known Saccharomyces strain to produce these pectinases. One of these pectolytic enzymes. PGL1-encoded endopolygalacturonase (EC 3.2.1.15), hydrolyses the alpha-1,4-glycosidic bonds within the rhamnogalacturonan chains in pectic substances. This paper presents the cloning and sequencing of the first S. cerevisiae gene involved in pectin degradation. Few differences were found between the two deduced amino acid sequences encoded by PGL1-1 from a pectolytic (PG+) strain (SCPP) and PGL1-2 from a non-pectolytic (PG-) strain (X2180-1B). Similarities were found with other polygalacturonases from plants and other microorganisms. Of the two S. cerevisiae genes, only the one isolated from strain SCPP was able, by overexpression, to confer endopolygalacturonase activity to a laboratory strain of S. cerevisiae. Overexpression of PGL1-1 gene in a non-pectolytic strain resulted in halo formation on polygalacturonic acid-containing agar plates stained with ruthenium red.

Amino Acid Sequence↗
PubMed1999

Cloning, targeted disruption and heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1).

The yeast Kluyveromyces marxianus strain BKM Y-719 produces an efficient pectin-degrading endopolygalacturonase (EPG) that cleaves the internal alpha-1,4-D-glycosidic linkages to yield oligomers of varying sizes. The EPG1 gene encoding this industrially important EPG was cloned by using the polymerase chain reaction (PCR) technique and degenerate primers to generate a 135 bp DNA fragment with which a genomic library was screened. The cloned fragment contained an open reading frame (ORF) of 1083 bp, encoding a 361 amino acid polypeptide. The predicted amino acid (aa) sequence of EPG showed similarity with polygalacturonases (PGs) of fungi. Analysis of the aa sequence indicated that the first 25 aa constitute a signal sequence and a motif (C218XGGHGXSIGSVG230) that is usually associated with a PG active site. Pulsed-field gel electrophoresis resolved chromosomal bands for K. marxianus BKM Y-719 and using chromoblotting it seems that EPG1 is present as only a single copy in the genome.

Amino Acid Sequence↗
PubMed2000

Genetic improvement of Aspergillus carbonarius for pectinase overproduction during solid state growth.

Low pectinase production by Aspergillus carbonarius growing on wheat bran solid substrate was found to be due to reduced colonizational ability of the fungus. Since A. niger showed higher growth rates on wheat bran, strain improvement to obtain higher pectinase production in solid state was carried out by inter-specific fusion of protoplasts of A. carbonarius and A. niger. One of the mutants selected for higher activities of alpha-glucosidase showed improved growth rates on wheat bran solid substrate together with increased pectinase production. Size similarities of amplified polymorphic DNA of the mutant with the two parents and identification of a 66 kDa polygalacturonase specific to A. niger suggested genetic recombination in the mutant.

Aspergillus↗
PubMed2006

Degree of blockiness of amide groups as indicator for difference in physical behavior of amidated pectins.

Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo-polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI-TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used.

Amides↗