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Chitinase
Kinds of Chitinase Terms modified by Chitinase Selected AbstractsA Thermostable Chitinase with Chitin-Binding Activity from Phaseolus limensisJOURNAL OF FOOD SCIENCE, Issue 6 2008S.Y. Wang ABSTRACT:, A 28.6-kDa chitinase with chitin-binding activity was isolated from the large lima bean (Phaseolus limensis) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, and high-performance liquid chromatography (HPLC) on SP-Toyopearl. There was an almost 108-fold increase in specific activity of the purified chitinase compared with that of the crude extract. The enzyme exhibited a pI of 7.8 by isoelectric focusing electrophoresis. The optimum pH and the optimum temperature for activity toward N-acetyld-glucosamine were 5.4 and 40 to 50 °C, respectively. The enzyme was stable up to 55 °C. It exerted a potent inhibitory action toward fungal species, including Fusarium solani, Pythium aphanidermatum, and Sclerotium rolfsii. [source] Soil microbial activity along an arctic-alpine altitudinal gradient from a seasonal perspectiveEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 5 2008U. C. M. Löffler Summary The knowledge on dynamics of soil microbial activity and its correlation to climate and vegetation is still poor but essential for predicting climatic changes scenarios. Seasonal dynamics of soil microbial activity and cell counts were studied along an arctic-alpine altitudinal gradient. The gradient comprised 12 ridges from 1000 to 1600 m altitude. Soil samples were collected during March, May, July and September 2005. The effect of temperature, snow depth and vegetation, all of which changed with altitude, on soil microbial activity and bacterial cell counts was analysed. The potential activities of phosphatase and chitinase were determined using fluorescent 4-methylumbelliferyl labelled analogues. Total and live bacterial cell counts were determined by live-dead-staining. We detected marked differences in soil microbial variables along the altitudinal gradient, forming three major clusters: a low alpine belt, a middle alpine belt, and an intermediate transition zone. Our results demonstrated that more frequent occurrence of shrubs and bryophytes would also increase microbial activity. Furthermore, we detected a clear relation (R2 = 0.6; P < 0.02) between high soil temperatures and greater soil microbial activity during summer. As higher temperatures are predicted to promote shrubs and higher humidity to promote bryophytes we expect microbial activity in dry heath tundra soils will increase with anticipated warmer, and in the case of Scandinavia, more humid climates. We did not find winter microbial activity to be less at snow-free sites than at sites covered by snow up to depths of 30 cm; hence, possible future decreases in snow depth will not result in reduced winter microbial activity. We demonstrate that shrubs support winter microbial activity not only by trapping snow but also directly by increasing the amount of organic carbon. [source] The chitinolytic system of Lactococcus lactis ssp. lactis comprises a nonprocessive chitinase and a chitin-binding protein that promotes the degradation of ,- and ,-chitinFEBS JOURNAL, Issue 8 2009Gustav Vaaje-Kolstad It has recently been shown that the Gram-negative bacterium Serratia marcescens produces an accessory nonhydrolytic chitin-binding protein that acts in synergy with chitinases. This provided the first example of the production of dedicated helper proteins for the turnover of recalcitrant polysaccharides. Chitin-binding proteins belong to family 33 of the carbohydrate-binding modules, and genes putatively encoding these proteins occur in many microorganisms. To obtain an impression of the functional conservation of these proteins, we studied the chitinolytic system of the Gram-positive Lactococcus lactis ssp. lactis IL1403. The genome of this lactic acid bacterium harbours a simple chitinolytic machinery, consisting of one family 18 chitinase (named LlChi18A), one family 33 chitin-binding protein (named LlCBP33A) and one family 20 N -acetylhexosaminidase. We cloned, overexpressed and characterized LlChi18A and LlCBP33A. Sequence alignments and structural modelling indicated that LlChi18A has a shallow substrate-binding groove characteristic of nonprocessive endochitinases. Enzymology showed that LlChi18A was able to hydrolyse both chitin oligomers and artificial substrates, with no sign of processivity. Although the chitin-binding protein from S. marcescens only bound to ,-chitin, LlCBP33A was found to bind to both ,- and ,-chitin. LlCBP33A increased the hydrolytic efficiency of LlChi18A to both ,- and ,-chitin. These results show the general importance of chitin-binding proteins in chitin turnover, and provide the first example of a family 33 chitin-binding protein that increases chitinase efficiency towards ,-chitin. [source] Biochemical characteristics of C-terminal region of recombinant chitinase from Bacillus licheniformis, implication of necessity for enzyme propertiesFEBS JOURNAL, Issue 9 2008Hsu-Han Chuang The functional and structural significance of the C-terminal region of Bacillus licheniformis chitinase was explored using C-terminal truncation mutagenesis. Comparative studies between full-length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, kcat/Km, was slightly increased for the truncated enzymes toward the soluble 4-methylumbelliferyl- N-N,-diacetyl chitobiose or 4-methylumbelliferyl- N - N,- N,-triacetyl chitotriose or insoluble ,-chitin substrate. By contrast, changes to substrate affinity, Km, and turnover rate, kcat, varied considerably for both types of chitin substrates between the full-length and truncated enzymes. Both truncated enzymes exhibited significantly higher thermostabilities than the full-length enzyme. The truncated mutants retained similar substrate-binding specificities and abilities against the insoluble substrate but only had approximately 75% of the hydrolyzing efficiency of the full-length chitinase molecule. Fluorescence spectroscopy indicated that both C-terminal deletion mutants retained an active folding conformation similar to the full-length enzyme. However, a CD melting unfolding study was able to distinguish between the full-length and truncated mutant molecules by the two phases of apparent transition temperatures in the mutants. These results indicate that up to 145 amino acid residues, including the putative C-terminal chitin-binding region and the fibronectin (III) motif of B. licheniformis chitinase, could be removed without causing a seriously aberrant change in structure and a dramatic decrease in insoluble chitin hydrolysis. The results of the present study provide evidence demonstrating that the binding and hydrolyzing of insoluble chitin substrate for B. licheniformis chitinase was not dependent solely on the putative C-terminal chitin-binding region and the fibronectin (III) motif. [source] Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymesFEBS JOURNAL, Issue 21 2006Ingunn A. Hoell We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single-domain family 19 chitinase from the Gram-positive bacterium Streptomyces coelicolor A3(2). Although chitinase G was not capable of releasing 4-methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases. The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl-chitin,remazol,brilliant violet) and a small, presumably amorphous, subfraction of ,-chitin and ,-chitin, but was not capable of degrading crystalline chitin completely. The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472,484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate-binding grooves in family 19 chitinases from plants. In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites (, 2 to +,2), as opposed to six (, 3 to +,3) for plant enzymes. The most prominent structural difference leading to reduced size of the substrate-binding groove is the deletion of a 13-residue loop between the two putatively catalytic glutamates. The importance of these two residues for catalysis was confirmed by a site-directed mutagenesis study. [source] Emergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18FEBS JOURNAL, Issue 7 2005Anne Durand The xylanase inhibitor protein I (XIP-I), recently identified in wheat, inhibits xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). Sequence and structural similarities indicate that XIP-I is related to chitinases of family GH18, despite its lack of enzymatic activity. Here we report the identification and biochemical characterization of a XIP-type inhibitor from rice. Despite its initial classification as a chitinase, the rice inhibitor does not exhibit chitinolytic activity but shows specificities towards fungal GH11 xylanases similar to that of its wheat counterpart. This, together, with an analysis of approximately 150 plant members of glycosidase family GH18 provides compelling evidence that xylanase inhibitors are largely represented in this family, and that this novel function has recently emerged based on a common scaffold. The plurifunctionality of GH18 members has major implications for genomic annotations and predicted gene function. This study provides new information which will lead to a better understanding of the biological significance of a number of GH18 ,inactivated' chitinases. [source] Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensisFEBS JOURNAL, Issue 3 2002Evert Bokma Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the ,1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with ,,2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower kcat and an approximately twofold higher Km than the wild-type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X-ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild-type hevamine, the carbonyl oxygen atom of the N -acetyl group of the ,1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125,and Tyr183 contribute to catalysis by positioning the,carbonyl oxygen of the N -acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate-assisted catalysis. [source] Bacterial quorum sensing and nitrogen cycling in rhizosphere soilFEMS MICROBIOLOGY ECOLOGY, Issue 2 2008Kristen M. DeAngelis Abstract Plant photosynthate fuels carbon-limited microbial growth and activity, resulting in increased rhizosphere nitrogen (N) mineralization. Most soil organic nitrogen is macromolecular (chitin, protein, nucleotides); enzymatic depolymerization is likely rate limiting for plant nitrogen accumulation. Analyzing Avena (wild oat) planted in microcosms containing sieved field soil, we observed increased rhizosphere chitinase and protease-specific activities, bacterial cell densities, and dissolved organic nitrogen (DON) compared with bulk soil. Low-molecular-weight (MW) DON (<3000 Da) was undetectable in bulk soil but comprised 15% of rhizosphere DON. Extracellular enzyme production in many bacteria requires quorum sensing (QS), cell-density-dependent group behavior. Because proteobacteria are considered major rhizosphere colonizers, we assayed the proteobacterial QS signals N -acyl-homoserine lactones (AHLs), which were significantly increased in the rhizosphere. To investigate the linkage between soil signaling and nitrogen cycling, we characterized 533 bacterial isolates from Avena rhizosphere: 24% had chitinase or protease activity and AHL production; disruption of QS in seven of eight isolates disrupted enzyme activity. Many Alphaproteobacteria were newly found with QS-controlled extracellular enzyme activity. Enhanced specific activities of nitrogen-cycling enzymes accompanied by bacterial density-dependent behaviors in rhizosphere soil gives rise to the hypothesis that QS could be a control point in the complex process of rhizosphere nitrogen mineralization. [source] Interactions between engineered tomato plants expressing antifungal enzymes and nontarget fungi in the rhizosphere and phyllosphereFEMS MICROBIOLOGY LETTERS, Issue 1 2008Mariangela Girlanda Abstract The introduction of genetically modified (GM) plants in agroecosystems raises concern about possible effects on nontarget species. The impact of a tomato line transformed for constitutive expression of tobacco ,-1,3-glucanase and chitinase on indigenous nonpathogenic fungi was investigated. In greenhouse experiments, no significant differences were found in the colonization by arbuscular mycorrhizal fungi. Diversity indices computed from over 20 500 colonies of culturable rhizosphere and phyllosphere saprotrophic microfungi, assigned to 165 species (plus>80 sterile morphotypes), showed no significant differences between GM and wild-type plants. Differences were found by discriminant analysis in both the rhizosphere and the phyllosphere, but such effects were minor compared with those linked to different plant growth stages. [source] Targeted gene analysis in Ulmus americana and U. pumila tissuesFOREST PATHOLOGY, Issue 2 2008C. Nasmith Summary Steady-state gene expression was compared between Dutch elm disease (DED)-susceptible Ulmus americana and DED-resistant U. pumila callus, leaf midrib, root and inner bark tissues. Stress-related cDNAs including phenylalanine ammonia-lyase (PAL), chitinase (CHT) and polygalacturonase-inhibiting protein (PGIP) were isolated and compared following RT-PCR of elm tissues. Complete CHT and partial PAL and PGIP cDNA transcripts were identified, each displaying sequence variation between elm species. These transcripts were Dig-labelled and subsequently used for northern analyses of the elm tissues. Midrib and root tissue displayed highest steady-state gene expression compared with inner bark and callus tissues. A modified nucleic acid isolation technique was necessary for downstream RNA analyses. Lithium chloride and polyvinylpyrrolidone were critical for efficient removal of polysaccharides and phenolics associated with some of the elm tissues. Steady-state gene expression is discussed in relation to the tissues investigated. The use of tissues other than in vitro callus culture more closely represents the tissues associated with the elm's vascular response to DED. [source] Genetic polymorphisms of chitotriosidase in Caucasian children with bronchial asthmaINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 3 2006S. Bierbaum Summary In humans, two types of chitinases have been identified: chitotriosidase I (CHIT1) and acid mammalian chitinase (AMCase). They are enzymes that cleave chitin, a polysaccharide contained in many different human parasites. So far, only little is known about their function in human and especially in human diseases. Recently we have described association of polymorphisms of AMCase with bronchial asthma in a pediatric population. In this study we were interested in whether CHIT1 is also involved in the genetics of asthma. The amino acid variants Gly102Ser and Ala442Gly, as well as a 24 bp duplication within CHIT1, were typed by means of restriction fragment length polymorphisms on 322 children with asthma and 270 randomly chosen adult controls. Statistical analyses made use of the Armitage's trend test; haplotypes were calculated by famhap and fastehplus. The amino acid variants showed no association with bronchial asthma. The 24 bp duplication, previously shown to completely demolish CHIT1 activity, was also evenly distributed between asthmatics and controls. Finally, the haplotype showed no association with the disease. We conclude from our results that CHIT1 does not play a major role in the development of bronchial asthma in Caucasian children. The results might also imply that the two human chitinases that have been identified so far have quite distinct functions in human diseases even though they have the same substrate. [source] Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinasesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007G. Ahmadian Abstract Aims: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. Methods and Results: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N -terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4·5 and 5·1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. Conclusions: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. Significance and Impact of the Study: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt. [source] Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activityJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2002M. Liu Aims: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. Methods and Results: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7·0, and so did 17 strains at pH 10·0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml,1 in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2·35-fold. Conclusion: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. Significance and Impact of the Study: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide. [source] Plant growth promotion and induction of resistance in Camellia sinensis by Bacillus megateriumJOURNAL OF BASIC MICROBIOLOGY, Issue 3 2006Usha Chakraborty Dr. Bacillus megaterium de Bary TRS-4 was isolated from tea rhizosphere and tested for its ability to promote growth and cause disease reduction in tea plants. In vivo studies revealed the ability of this bacterium to promote growth of tea plants very significantly. Brown root rot disease, caused by Fomes lamaoensis was markedly reduced by application of the bacterium to the soil. Population of F. lamaoensis in soil before and after application of B. megaterium, as determined by ELISA and dot-blot using PAb raised against the pathogen, was shown to be greatly reduced in presence of the bacterium. Biochemical changes induced in tea plants were also examined. Root colonization by B. megaterium and subsequent inoculation with F. lamaoensis also led to an increase in polyphenolics, as well as in defense related enzymes-peroxidase, chitinase, , -1,3-glucanase and phenyl alanine ammonia lyase. Determination of mechanism of action of this bacterium revealed it to be able to solubilize phosphate, produce IAA, siderophore and antifungal metabolite. The plant growth promotion and reduction of disease intensity have been shown to be due to a combination of several mechanisms. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Classically and alternatively activated macrophages contribute to tissue remodelling after myocardial infarctionJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009C. Troidl Abstract An important goal in cardiology is to minimize myocardial necrosis and to support a discrete but resilient scar formation after myocardial infarction (MI). Macrophages are a type of cells that influence cardiac remodelling during MI. Therefore, the goal of the present study was to investigate their transcriptional profile and to identify the type of activation during scar tissue formation. Ligature of the left anterior descending coronary artery was performed in mice. Macrophages were isolated from infarcted tissue using magnetic cell sorting after 5 days. The total RNA of macrophages was subjected to microarray analysis and compared with RNA from MI and LV-control. mRNA abundance of relevant targets was validated by quantitative real-time PCR 2, 5 and 10 days after MI (qRT-PCR). Immunohistochemistry was performed to localize activation type-specific proteins. The genome scan revealed 68 targets predominantly expressed by macrophages after MI. Among these targets, an increased mRNA abundance of genes, involved in both the classically (tumour necrosis factor ,, interleukin 6, interleukin 1,) and the alternatively (arginase 1 and 2, mannose receptor C type 1, chitinase 3-like 3) activated phenotype of macrophages, was found 5 days after MI. This observation was confirmed by qRT-PCR. Using immunohistochemistry, we confirmed that tumour necrosis factor ,, representing the classical activation, is strongly transcribed early after ligature (2 days). It was decreased after 5 and 10 days. Five days after MI, we found a fundamental change towards alternative activation of macrophages with up-regulation of arginase 1. Our results demonstrate that macrophages are differentially activated during different phases of scar tissue formation after MI. During the early inflammatory phase, macrophages are predominantly classically activated, whereas their phenotype changes during the important transition from inflammation to scar tissue formation into an alternatively activated type. [source] Lifestyle impacts on the aging-associated expression of biomarkers of DNA damage and telomere dysfunction in human bloodAGING CELL, Issue 4 2010Zhangfa Song Summary Cellular aging is characterized by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and activation of DNA damage responses. There is some evidence that DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage, and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging-associated expression of serum markers of DNA damage (CRAMP, EF-1,, stathmin, n -acetyl-glucosaminidase and chitinase) in comparison with other described markers of cellular aging (p16INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age-independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age-independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16INK4a expression and negatively with telomere length in peripheral blood T-lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging. [source] A Thermostable Chitinase with Chitin-Binding Activity from Phaseolus limensisJOURNAL OF FOOD SCIENCE, Issue 6 2008S.Y. Wang ABSTRACT:, A 28.6-kDa chitinase with chitin-binding activity was isolated from the large lima bean (Phaseolus limensis) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, and high-performance liquid chromatography (HPLC) on SP-Toyopearl. There was an almost 108-fold increase in specific activity of the purified chitinase compared with that of the crude extract. The enzyme exhibited a pI of 7.8 by isoelectric focusing electrophoresis. The optimum pH and the optimum temperature for activity toward N-acetyld-glucosamine were 5.4 and 40 to 50 °C, respectively. The enzyme was stable up to 55 °C. It exerted a potent inhibitory action toward fungal species, including Fusarium solani, Pythium aphanidermatum, and Sclerotium rolfsii. [source] Phage display screening for peptidic chitinase inhibitors,JOURNAL OF MOLECULAR RECOGNITION, Issue 6 2008Cordula Petter Abstract A phage display library with disulfide-cyclized peptides was screened for peptides binding to chitinases from Serratia marcescens. One of those peptides was found to efficiently inhibit chitinase A and two others were inhibitors of chitinase B. Complete substitutional analysis of all three peptides using cellulose-bound peptide spot synthesis revealed key interaction positions and allowed optimization of the chitinase B inhibitory peptides towards higher affinity, with inhibitory constants in the lower nanomolar range. Inhibition by all peptides proved to be competitive and highly specific for the chitinase used to select them, as shown with a series of chitinases from different organisms. Copyright © 2008 John Wiley & Sons, Ltd. [source] Gene expression in endoprosthesis loosening: Chitinase activity for early diagnosis?,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2008L. Morawietz Abstract The aim of the study was to identify markers for the early diagnosis of endoprosthesis loosening, for the differentiation between wear particle,induced and septic loosening and to gather new insights into the pathogenesis of endoprosthesis loosening. Gene expression profiles were generated from five periprosthetic membranes of wear particle,induced and five of infectious (septic) type using Affymetrix HG U133A oligonucleotide microarrays. The results of selected differentially expressed genes were validated by RT-PCR (n,=,30). The enzyme activity and the genotype of chitinase-1 were assessed in serum samples from 313 consecutive patients hospitalized for endoprosthesis loosening (n,=,54) or for other reasons, serving as control subjects (n,=,259). Eight hundred twenty-four genes were differentially expressed with a fold change greater than 2 (data sets on http://www.ncbi.nlm.nih.gov/geo/ GSE 7103). Among these were chitinase 1, CD52, calpain 3, apolipoprotein, CD18, lysyl oxidase, cathepsin D, E-cadherin, VE-cadherin, nidogen, angiopoietin 1, and thrombospondin 2. Their differential expression levels were validated by RT-PCR. The chitinase activity was significantly higher in the blood from patients with wear particle,induced prosthesis loosening (p,=,0.001). However, chitinase activity as a marker for early diagnosis has a specificity of 83% and a sensitivity of 52%, due to a high variability both in the disease and in the control group. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:394,403, 2008 [source] Lipid transfer proteins from Brassica campestris and mung bean surpass mung bean chitinase in exploitabilityJOURNAL OF PEPTIDE SCIENCE, Issue 10 2007Peng Lin Abstract Antifungal peptides with a molecular mass of 9 kDa and an N -terminal sequence demonstrating remarkable similarity to those of nonspecific lipid transfer proteins (nsLTPs) were isolated from seeds of the vegetable Brassica campestris and the mung bean. The purified peptides exerted an inhibitory action on mycelial growth in various fungal species. The antifungal activity of Brassica and mung bean nsLTPs were thermostable, pH-stable, and stable after treatment with pepsin and trypsin. In contrast, the antifungal activity of mung bean chitinase was much less stable to changes in pH and temperature. Brassica LTP inhibited proliferation of hepatoma Hep G2 cells and breast cancer MCF 7 cells with an IC50 of 5.8 and 1.6 µM, respectively, and the activity of HIV-1 reverse transcriptase with an IC50 of 4 µM. However, mung bean LTP and chitinase were devoid of antiproliferative and HIV-1 reverse transcriptase inhibitory activities. In contrast to the mung bean LTP, which exhibited antibacterial activity, Brassica LTP was inactive. All three antifungal peptides lacked mitogenic activity toward splenocytes. These results indicate that the two LTPs have more desirable activities than the chitinase and that there is a dissociation between the antifungal and other activities of these antifungal proteins. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Effect of periodontal treatment on the activity of chitinase in whole saliva of periodontitis patientsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2002G. J. Van Steijn Human salivary chitinase could play a role in the defence against chitin-containing oral pathogens. The activity levels of chitinase in the whole saliva of periodontitis patients were significantly higher than those in saliva from controls. Periodontal treatment for a period of 5,6 months resulted in a three- to fourfold decrease in this enzyme activity. The activity of ,- N -acetylhexosaminidase, which is another enzyme that hydrolyses glycosidic linkages, also decreased as a result of treatment, although to a lesser extent. The decrease in chitinase activity upon treatment of the disease did not correlate with the decrease that was seen in clinical attachment loss and bleeding on probing, and only a weak correlation was observed with the changes in probing pocket depth and plaque index. No correlations were found between the above clinical parameters and the decrease in ,- N -acetylhexosaminidase activity. [source] Induced Resistance by , -Aminobutyric Acid in Artichoke against White Mould Caused by Sclerotinia sclerotiorumJOURNAL OF PHYTOPATHOLOGY, Issue 10 2010Emanuela Marcucci Abstract ,-aminobutyric acid (BABA) was assessed for the ability to protect two artichoke cultivars, C3 and Exploter, against white mould caused by Sclerotinia sclerotiorum, which represents a major problem in the cultivation of this crop in many growing areas of Central Italy. Changes in the activity and isoenzymatic profiles of the pathogenesis-related (PR) proteins ,-1,3-glucanase, chitinase and peroxidase in plantlets upon BABA treatment and following inoculation of the pathogen in plantlets and leaves detached from adult plants were also investigated as molecular markers of induced resistance and priming. BABA treatments by soil drenching induced a high level of resistance against S. sclerotiorum in artichoke plantlets of both cultivars C3 and Exploter with a similar level of protection and determined a consistent increase in peroxidase activity paralleled with the differential induction of alkaline isoenzyme with a pI 8.6. A consistent change was found in Exploter in the peroxidase activity following BABA treatments and pathogen inoculation and was paralleled with the expression of an anionic band in plantlets and both anionic and cationic bands in leaves. Our results showed a correlation between BABA-induced resistance (BABA-IR) and a augmented capacity to express basal defence responses, more pronounced in cultivar C3 and associated ,-1,3-glucanase accumulation in both plantlets and leaves inoculated with the pathogen, whereas chitinase resulted affected only at plantlet stage. The present results represent the first one showing the effect of BABA in inducing resistance in artichoke and associated accumulation of selected PRs. If confirmed in field tests, the use of BABA at early plant stages may represent a promising approach to the control soilborne pathogens, such as the early infection of S. sclerotiorum. [source] Induced Resistance in Yali Pear (Pyrus bretschneideri Rehd.) Fruit against Infection by Penicillium expansum by Postharvest Infiltration of Acibenzolar-S-methylJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2005J. Cao Abstract The objective of the present study was to evaluate how disease resistance in Yali pear fruit (Pyrus bretschneideri Rehd.) was affected by the infiltration of acibenzolar-S-methyl (ASM) after harvest. The disease incidence and lesion area in/on the fruit inoculated with Penicillium expansum significantly (P < 0.05) decreased by the infiltration with 0.5 mm ASM, and the duration of protection conferred by ASM lasted over 15 days. ASM did not directly inhibit the mycelial growth of P. expansum in vitro. However, ASM treatment significantly enhanced activities of the main defence enzymes including peroxidase, phenylalanine ammonia-lyase and chitinase, and activities of antioxidant enzymes including superoxide dismutase and catalase in the fruit during the infection. Two kinds of second metabolites, total phenolic compounds and flavonoids, and two productions of lipid peroxidation, H2O2 and malondialdehyde, were also involved in the resistance and significantly accumulated in ASM-treated fruit in the infection. The inhibitory effect of ASM on the disease may be related to its ability to enhance defence responses in the fruit. The application of ASM in inducing resistance in fruit possesses promising in control of postharvest diseases alternative to fungicides. [source] Synthesis of Fluorinated Chitin Derivatives via Enzymatic PolymerizationMACROMOLECULAR BIOSCIENCE, Issue 10 2006Akira Makino Abstract Summary: Synthesis of fluorinated chitin derivatives has been achieved using chitinase from Bacillus sp. as a catalyst. 6,-Fluoro- (1a), 6-fluoro- (1b) and 6,6,-difluoro- (1c) chitobiose oxazoline derivatives were newly prepared as TSAS monomers for chitinase. Ring-opening polyaddition of these monomers proceeded effectively at pH 8.0,9.0 and 30,40,°C, giving rise to alternatingly 6-fluorinated chitin derivatives (2a and 2b) from 1a and 1b, and fully 6-fluorinated chitin derivative (2c) from 1c under total control of regioselectivity and stereochemistry. XRD measurements revealed that polysaccharides 2a and 2b had crystalline structures similar to that of , -chitin. [source] Interactions of Enzymes and a Lectin with a Chitin-Based Graft Copolymer Having Polysarcosine Side ChainsMACROMOLECULAR BIOSCIENCE, Issue 6 2004Rikiya Nakamura Abstract Summary: The molecular-recognition abilities of a water-soluble chitin derivative, chitin- graft -polysarcosine (2) were investigated using chitinase, lysozyme, and wheat germ agglutinin (WGA). The enzymatic degradabilities of 2 were evaluated using chitinase and lysozyme. The molecular weight of those compounds of 2 with a higher affinity toward water decreased rapidly, as compared with partially deacetylated chitin (1). The 1H NMR spectrum of the low-molecular-weight fraction, yielded after lysozymic hydrolysis, indicated that saccharide residues in the chitinous backbone were specifically recognized by the lysozyme, then , -glycosidic linkages in the backbone were selectively hydrolyzed. Furthermore, the molecular-recognition ability of the chitinous backbone of graft copolymer 2 toward the lectin WGA was elucidated by the enzyme-linked lectin-binding assay (ELLA). It was revealed that the graft copolymer with a lower degree of substitution (DS) value efficiently interacted with WGA. Interestingly, a graft copolymer having longer polysarcosine side chains showed higher recognition ability toward WGA than that having short side chains. The structure of the graft copolymer, chitin- graft -polysarcosine 2, used here. [source] Cytological and enzymatic responses to aluminium stress in root tips of Norway spruce seedlingsNEW PHYTOLOGIST, Issue 3 2004Nina Elisabeth Nagy Summary ,,Aluminium (Al) stress reduces plant growth. However, some species such as Norway spruce (Picea abies) seem to tolerate high Al concentrations. The aim of this study was to investigate characteristics possibly involved in Al tolerance in Norway spruce seedlings. ,,Seedlings (10-d-old) were exposed to Al3+ concentrations of 0.5 and 5 mm for up to 168 h. The effect of Al stress on root growth, cell morphology and Al distribution, callose production, and peroxidase and chitinase activity was analysed. ,,Root growth decreased after 1 d and 2 d with 5 and 0.5 mm Al, respectively. Callose concentration increased strongly after 6 h treatment with 5 mm Al. The activity of many peroxidase and chitinase isoforms decreased after 1,24 h exposure of both treatments. Several isoforms increased after 48,168 h exposure to 5 mm Al. ,,We postulate that, with external Al concentrations 0.5 mm or lower, an increased production above constitutive levels of peroxidase or chitinase is not required for Al tolerance in young Norway spruce seedlings. High constitutive levels of peroxidase and chitinase in this species may be part of this Al tolerance. [source] Isolation and characterization of cgchi3, a nodule-specific gene from Casuarina glauca encoding a class III chitinasePHYSIOLOGIA PLANTARUM, Issue 3 2007Ana Fortunato Chitinases (EC 3.2.1.14) catalyse the hydrolysis of chitin, a homopolymer of ,-1,4-linked N -acetyl- d -glucosamine residues. Plant chitinases are involved in a wide variety of processes; in particular, their expression has been found to be enhanced in symbiotic and pathogenic plant,microbe interactions. During this work we have cloned and characterized a gene encoding a class III chitinase from actinorhizal nodules of Casuarina glauca (cgchi3). CGCHI3 was found to be encoded by a single gene that was specifically activated in nodules as compared with uninoculated control roots and leaves. The expression of this gene was further enhanced in nodules after salicylic acid treatment and completely repressed after wounding. In situ hybridisation analysis revealed that cgchi3 is an early nodulin gene, being expressed in the meristem and in the uninfected cortical cells of young nodules. Based on the obtained results we suggest that this gene is involved in nodule development. This is the first report on a class III chitinase coding gene that is specifically activated during actinorhizal symbiosis. [source] White lupin has developed a complex strategy to limit microbial degradation of secreted citrate required for phosphate acquisitionPLANT CELL & ENVIRONMENT, Issue 5 2006LAURE WEISSKOPF ABSTRACT White lupins (Lupinus albus L.) respond to phosphate deficiency by producing special root structures called cluster roots. These cluster roots secrete large amounts of carboxylates into the rhizosphere, mostly citrate and malate, which act as phosphate solubilizers and enable the plant to grow in soils with sparingly available phosphate. The success and efficiency of such a P-acquisition strategy strongly depends on the persistence and stability of the carboxylates in the soil, a parameter that is influenced to a large extent by biodegradation through rhizosphere bacteria and fungi. In this study, we show that white lupin roots use several mechanisms to reduce microbial growth. The abundance of bacteria associated with cluster roots was decreased at the mature state of the cluster roots, where a burst of organic acid excretion and a drastic pH decrease is observed. Excretion of phenolic compounds, mainly isoflavonoids, induced fungal sporulation, indicating that vegetative growth, and thus potential citrate consumption, is reduced. In addition, the activity of two antifungal cell wall-degrading enzymes, chitinase and glucanase, were highest at the stage preceding the citrate excretion. Therefore, our results suggest that white lupin has developed a complex strategy to reduce microbial degradation of the phosphate-solubilizing agents. [source] Effects of foliar- and root-applied silicon on the enhancement of induced resistance to powdery mildew in Cucumis sativusPLANT PATHOLOGY, Issue 5 2005Y. C. Liang Two cucumber (Cucumis sativus) cultivars differing in their resistance to powdery mildew, Ningfeng No. 3 (susceptible) and Jinchun No. 4 (resistant), were used to study the effects of foliar- and root-applied silicon on resistance to infection by Podosphaera xanthii (syn. Sphaerotheca fuliginea) and the production of pathogenesis-related proteins (PRs). The results indicated that inoculation with P. xanthii significantly suppressed subsequent infection by powdery mildew compared with noninoculation, regardless of Si application. Root-applied Si significantly suppressed powdery mildew, the disease index being lower in Si-supplied than in Si-deprived plants, regardless of inoculation treatment. The resistant cultivar had a more constant lower disease index than the susceptible cultivar, irrespective of inoculation or Si treatment. Moreover, with root-applied Si, activities of PRs (for example peroxidase, polyphenoloxidase and chitinase) were significantly enhanced in inoculated lower leaves or noninoculated upper leaves in inoculated plants of both cultivars. Root-applied Si significantly decreased the activity of phenylalanine ammonia-lyase in inoculated leaves, but increased it in noninoculated upper leaves. However, Si treatment failed to change significantly the activity of PRs in plants without fungal attack. Compared to the control (no Si), foliar-applied Si had no effects either on the suppression of subsequent infection by P. xanthii or on the activity of PRs, irrespective of inoculation. Based on the findings in this study and previous reports, it was concluded that foliar-applied Si can effectively control infections by P. xanthii only via the physical barrier of Si deposited on leaf surfaces, and/or osmotic effect of the silicate applied, but cannot enhance systemic acquired resistance induced by inoculation, while continuously root-applied Si can enhance defence resistance in response to infection by P. xanthii in cucumber. [source] Plant defence reactions against fusarium wilt in chickpea induced by incompatible race 0 of Fusarium oxysporum f.sp. ciceris and nonhost isolates of F. oxysporumPLANT PATHOLOGY, Issue 6 2002J. M. Cachinero Germinated seeds of ,kabuli' chickpea cv. ICCV 4 were inoculated with a conidial suspension of the incompatible race 0 of Fusarium oxysporum f.sp. ciceris (Foc) or of nonhost F. oxysporum resistance ,inducers', and 3 days later were challenged by root dip with a conidial suspension of highly virulent Foc race 5. Prior inoculation with inducers delayed the onset of symptoms and/or significantly reduced the final amount of fusarium wilt caused by race 5. However, the extent of disease suppression varied with the nature of the inducing agent; the nonhost isolates of F. oxysporum were more effective at disease suppression than the incompatible Foc race 0. Inoculation with the inducers gave rise to synthesis of maackiain and medicarpin phytoalexins in inoculated seedlings; these did not accumulate in plant tissues but were released into the inoculum suspension. Inoculation with inducers also resulted in accumulation of chitinase, ,-1,3-glucanase and peroxidase activities in plant roots. These defence-related responses were induced more consistently and intensely by nonhost isolates of F. oxysporum than by incompatible Foc race 0. The phytoalexins and, to a lesser extent, the antifungal hydrolases, were also induced after challenge inoculation with Foc race 5. However, in this case the defence responses were induced in both preinduced and noninduced plants infected by the pathogen. It is concluded that the suppression of fusarium wilt in this study possibly involved an inhibitory effect on the pathogen of preinduced plant defences, rather than an increase in the expression of defence mechanisms of preinduced plants following a subsequent challenge inoculation. [source] | ||||||||||||||||||||||||||||