Mammalian chitinases and the related chilectins(Chi Ls) belong to the GH18 family, which hydrolyse the glycosidic bond of chitin by a substrate-assisted mechanism. Chitin the fundamental component in the coating of nu...Mammalian chitinases and the related chilectins(Chi Ls) belong to the GH18 family, which hydrolyse the glycosidic bond of chitin by a substrate-assisted mechanism. Chitin the fundamental component in the coating of numerous living species is the most abundant natural biopolymer. Mounting evidence suggest that the function of the majority of the mammalian chitinases is not exclusive to catalyze the hydrolysis of chitin producing pathogens, but include crucial role specific in the immunologic activities. The chitinases and chitinaselike proteins are expressed in response to different proinflammatory cues in various tissues by activated macrophages, neutrophils and in different monocytederived cell lines. The mechanism and molecular interaction of chitinases in relation to immune regulation embrace bacterial infection, inflammation, dismetabolic and degenerative disease. The aim of this review is to update the reader with regard to the role of chitinases proposed in the recent innate and adaptive immunity literature. The deep scrutiny of this family of enzymes could be a useful base for further studies addressed to the development of potential procedure directing these molecules as diagnostic and prognostic markers for numerous immune and inflammatory diseases.展开更多
Chitinases catalyze the hydrolysis of chitin, a linear homopolymer of β-(1,4)-linked N-acetylglucosamine. The broad range of applications of chitinolytic enzymes makes their identification and study very promising. M...Chitinases catalyze the hydrolysis of chitin, a linear homopolymer of β-(1,4)-linked N-acetylglucosamine. The broad range of applications of chitinolytic enzymes makes their identification and study very promising. Metagenomic approaches offer access to functional genes in uncultured representatives of the microbiota and hold great potential in the discovery of novel enzymes, but tools to extensively explore these data are still scarce. In this study, we develop a chitinase mining pipeline to facilitate the comprehensive search of these enzymes in environmental metagenomic databases and also to explore phylogenetic relationships among the retrieved sequences. In order to perform the analyses, UniprotKB fungal and bacterial chitinases sequences belonging to the glycoside hydrolases (GH) family-18, 19 and 20 were used to generate 15 reference datasets, which were then used to generate high quality seed alignments with the MAFFT program. Profile Hidden Markov Models (pHMMs) were built from each seed alignment using the hmmbuild program of HMMER v3.0 package. The best-hit sequences returned by hmmsearch against two environmental metagenomic databases (Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis—CAMERA and Integrated Microbial Genomes—IMG/M) were retrieved and further analyzed. The NJ trees generated for each chitinase dataset showed some variability in the catalytic domain region of the metagenomic sequences and revealed common sequence patterns among all the trees. The scanning of the retrieved metagenomic sequences for chitinase conserved domains/signatures using both the InterPro and the RPS-BLAST tools confirmed the efficacy and sensitivity of our pHMM-based approach in detecting putative chitinases sequences. These analyses provide insight into the potential reservoir of novel molecules in metagenomic databases while supporting the chitinase mining pipeline developed in this work. By using our chitinase mining pipeline, a larger number of previously unannotated metagenomic chitinase sequences can be classified, enabling further studies on these enzymes.展开更多
Vibrio pacini synthesizes multiple chitinases, of which three have been purified in this study by ammonium sulphate fractionation, chitin affinity chromatography and gel chromatography. Molecular weights of the three ...Vibrio pacini synthesizes multiple chitinases, of which three have been purified in this study by ammonium sulphate fractionation, chitin affinity chromatography and gel chromatography. Molecular weights of the three chitinases, Chi1, Chi2 and Chi3 are 27×103, 39×103 and 46×103 respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzymes have optimal activity at pH 7–8, and retain 50% enzymatic activity pH 4–9. The activities of chitinases are inhibited by Pb2+, Fe3+ and Cu2+, and increased by Ca2+, Mg2+ and Mn2+. Chi3 is found to inhibit the growth of six species of fungi. Such characters of chitinase are different from those of any other chitinase that were reported before. Key words chitinase - Vibrio pacini - purification - inhibit CLC number Q 55 Foundation item: Supported by the Key Technologies Research and Development Programme of the Tenth Five-Year Plan of the Nation Scientific and Technological Development (2001 BA708B04-07)Biography: HAN Bao-qin (1963-), female, Professor.展开更多
Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased toler- ance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinasel (CERK1/LysM R...Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased toler- ance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinasel (CERK1/LysM RLK1) gene, known to play a critical role in signaling defense responses induced by exogenous chitin. Arabidopsis plants overexpress- ing the endochitinase chit36 and hexoaminidase excyl genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity, heavy-metal stresses, and Botrytis cinerea infection. Resistant lines, overexpressing fungal chitinases at different levels, were outcrossed to lysm rlkl mutants. Independent homozygous hybrids lost resistance to biotic and abiotic stresses, despite enhanced chitinase activity. Expression analysis of 270 stress-related genes, including those induced by reactive oxygen species (ROS) and chitin, revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlkl knockout mutant or the hybrids harboring the mutation. These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.展开更多
Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthe...Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.展开更多
The objective to this work was to evaluate the enzymatic activity in the culture of Solanum lycopersicum L.infected with Fusarium oxysporum after the combined application of Beauveria bassiana and plant extracts.Solan...The objective to this work was to evaluate the enzymatic activity in the culture of Solanum lycopersicum L.infected with Fusarium oxysporum after the combined application of Beauveria bassiana and plant extracts.Solanum lycopersicum plantlets were transplanted 15 days after the emergency.Five days after transplanting,Beauveria bassiana spores were applied at a concentration of 1×10^(7)spores mL^(−1)onto soil(along with A.indica(N)and P.auritum(H)leaf extracts)where S.lycopersicum plants were planted.Eight days after transplanting,spores of F.oxysporum strain were applied at a concentration of 1×10^(6)spores mL^(−1)to soil where S.lycopersicum plants were growing.The development of S.lycopersicum plants was monitored for 114 days,whereby a randomized complete block treatment design was used,the roots of the plants were crushed with liquid nitrogen,after which UV/VIS spectrophotometry was used to determine protein concentration,as well as the activity ofβ-1,3-glucanases,peroxidases(POX,EC1.11.1.7),catalase and chitinases.The treatments combining B.bassiana and A.indica and P.auritum extracts,had a significant difference in enzymatic activity levels,thus contributing to better defense mechanisms and a greater protection to S.lycopersicum plants in the presence of F.oxysporum.The application of B.bassiana and plant extracts to the ground mitigated damage caused by F.oxysporum on S.lycopersicum plants.展开更多
The objective of this work was to evaluate the effect of Beauveria bassiana(Bb 1205)on controlling Fusarium oxysporum f.sp.lycopersici(Fol 17108)in tomato plants in greenhouse conditions.Inoculation of Bb 1205 was the...The objective of this work was to evaluate the effect of Beauveria bassiana(Bb 1205)on controlling Fusarium oxysporum f.sp.lycopersici(Fol 17108)in tomato plants in greenhouse conditions.Inoculation of Bb 1205 was the most promising among the agronomic variables and expression of the activity of the enzymesβ-1,3-glucanases and chitinases.Inoculation of Bb 1205 occurred at a concentration of 1×108 conidia·mL−1,which was administered onto the leaves,directly into the soil and via injection.Infection with Fol 17108 occurred with 1×106 spores·mL−1,which were added directly to the soil.Spectrophotometry was used for measuring agronomic parameters,namely activity of chitinases andβ-1,3-glucanases in foliage and roots.When Bb 1205 was added to the soil,the chlorophyll index and aerial part length showed significant differences.In addition,it was determined that root length,fresh weight of foliage,flower,and fruit count increased 82 days after inoculation(dai).Chitinase activity induced by Bb 1205 in leaves and roots of tomato plants infected with Fol 17108 was observed when injected into the stem at 32 dai(41.8 and 11.6-fold,respectively).Inoculation on the foliage showed a 10-fold increase ofβ-1,3-glucanases in the roots after 82 dpi.As for leaves,a 3.8-fold increase was found when the stem was inoculated.In the different in vivo applications,Bb 1205 activated its defenses by expressing the chitinase enzymes andβ-1,3-glucanase,thus reducing the damage caused by Fol 17108,demonstrating increase plant growth thereafter.展开更多
AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-compleme...AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2(scAAV2)vector technologies.METHODS:An scAAV2 vector with C3 transferase(C3)as the reporter gene(scAAV2-C3)was selected.The scAAV2-C3 vectors were driven by Ch3L1(scAAV2-Ch3L1-C3),MGP(scAAV2-MGP-C3),enhanced MGP(scAAV2-eMGP-C3)and cytomegalovirus(scAAV2-CMV-C3),respectively.The cultured primary human TM cells were treated with each vector at different multiplicities of infections.Changes in cell morphology were observed by phase contrast microscopy.Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining,real-time quantitative polymerase chain reaction and Western blot.Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively.In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope.Intraocular pressure(IOP)was monitored using a rebound tonometer.Ocular responses were evaluated by slit-lamp microscopy.Ocular histopathology analysis was examined by hematoxylin and eosin staining.RESULTS:In TM cell culture studies,the vectormediated C3 expression induced morphologic changes,disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rhoassociated protein kinase signaling pathway.At the same dose,these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3,but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3.At lowinjected dose,the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGPC3-injected and scAAV2-eMGP-C3-injected eyes.At highinjected dose,significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes.Similar to scAAV2-CMV-C3,scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium.In anterior segment tissues of scAAV2-eMGP-C3-injected eyes,no obvious morphological changes were found except for the TM.Inflammation was absent.CONCLUSION:In scAAV2-transduced TM cells,the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus,but obviously higher than that of MGP.In the anterior chamber of rat eye,the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter,but not by Ch3L1 promoter.These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.展开更多
Various strains of powdery mildew(PM),a notorious plant fungal disease,are prevalent and pose a significant threat to plant health.To control PM,transgenic technology can be used to cultivate more resistant plant vari...Various strains of powdery mildew(PM),a notorious plant fungal disease,are prevalent and pose a significant threat to plant health.To control PM,transgenic technology can be used to cultivate more resistant plant varieties.In the present study,we utilized the rapid amplification of cDNA ends(RACE)technique to clone the full-length cDNA sequence of the EuCHIT30.7 gene to explore plant genes with disease resistance functions.Bioinformatics analysis revealed that this gene belongs to the GH18 family and is classified as a class III chitinase.The EuCHIT30.7 gene is expressed throughout the Eucommia ulmoides plant,with the most abundant expression in male flowers.Subcellular localization analysis indicated that the protein encoded by this gene was detected within both the cell membrane and cytoplasm.Upon PM inoculation,overexpression of EuCHIT30.7 in tobacco plants led to a significantly reduced relative lesion area and a decreased spore count compared to both wild-type and empty vector control plants.Activities of the protective enzymes,namely,peroxidase(POD),superoxide dismutase(SOD),catalase(CAT),and phenylalaninammo-nialyase(PAL),in tobacco plants overexpressing EuCHIT30.7 were significantly greater than those in wild-type and empty vector tobacco plants.Furthermore,the rate of increase in malondialdehyde(MDA)content was significantly lower in tobacco plants expressing EuCHIT30.7 compared to control tobacco plants.In EuCHIT30.7 transgenic tobacco,the expression of pathogen-related protein genes,namely,PR2,PR5,PR1a,PDF1.2,and MLP423,along with the tobacco PM negative regulatory gene,MLO2,were significantly higher compared to control tobacco plants.These findings suggested that EuCHIT30.7 significantly enhances the resistance of tobacco to PM.展开更多
BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by th...BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.展开更多
[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to expre...[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to express in Trichoderma atroviride cells. The cDNA fragment of chitinase gene was cloned by RT-PCR approach. [Result] The activity of chitinase induced reached 40.17 μg/10 min; and the specific fragment amplified was 834 bp in length and proved to be the fragment of chitinase gene by sequencing and sequence analysis. [Conclusion] The result showed the feasibility of isolating the full length of chitinase gene and its transformation, and further producing chitinase.展开更多
Entomopathogenic fungi (EF) are recognized biological control agents of insects. Basically, the entomopathogenic fungi pathogen activity depends on the ability of its enzymatic equipment, consisting of lipases, protea...Entomopathogenic fungi (EF) are recognized biological control agents of insects. Basically, the entomopathogenic fungi pathogen activity depends on the ability of its enzymatic equipment, consisting of lipases, proteases and chitinases, which are in charge of breaking down the insect’s integument. Lipases are the first enzymes synthesized by the entomopathogenic fungi. Recently, a cytochrome P450 subfamily, referred as CYP52XI and MrCYP52 has been identified in Beauveria bassiana and Metarhizium robertsii, respectively. These break down long-chain alkenes and fatty acids to become initial nutrients. Subsequently, subtilisin type (Pr1) proteases sintetize;these enzymes are considered as virulence indicators and they are regulated by a signal transduction mechanism activated by the protein kinase A (PKA) mediated by AMPc. Through the employment of genetic engineering, it has been possible to increase virulence producing Pr1 recombinants with Androctonus australis neurotoxins or with chitinases, reducing the insect’s time of death. In the course of time, the Pr1 protease gene has presented evolutionary adaptations by gene duplication or horizontal transfer infecting different orders of insects. In the same way, the entomopathogenic fungi chitinases have presented a functional diversification. Currently, these have been phylogenetically classified into three subgroups, in accordance to the catalytic site domain and the chitin binding domain. The chitinolytic activity has increased through a directed evolution processes and genetic recombination with Bombyx mori chitinase. Recently, enzymes have been employed as control agents for insects and phytopathogenic fungi (disease originator) opening new potentialities in order to improve the entomopathogenic fungi use. Solid state fermentation is a bioprocess that would produce at great scale enzymes and some other metabolites in grade of increasing the entomopathogenic fungi virulence, in the control of insects and potentially in some diseases affecting plants.展开更多
AIM: To assess blood chitinase 3-like 1(CHi3L1) levels for 2 mo after minimally invasive colorectal resection(MICR) for colorectal cancer(CRC). METHODS: CRC patients in an Institutional Review Board approved data/plas...AIM: To assess blood chitinase 3-like 1(CHi3L1) levels for 2 mo after minimally invasive colorectal resection(MICR) for colorectal cancer(CRC). METHODS: CRC patients in an Institutional Review Board approved data/plasma bank who underwent elective MICR for whom preoperative(PreO p), early postoperative(PostO p), and 1 or more late PostO p samples [postoperative day(POD) 7-27] available were included. Plasma CHi3L1 levels(ng/m L) were determined in duplicate by enzyme linked immunosorbent assay. RESULTS: PreOp and PostOp plasma sample were available for 80 MICR cancer patients for the study. The median PreOp CHi3L1 level was 56.8 CI: 41.9-78.6 ng/mL(n = 80). Significantly elevated(P < 0.001) median plasma levels(ng/mL) over PreOp levels were detected on POD1(667.7 CI: 495.7, 771.7; n = 79), POD 3(132.6 CI: 95.5, 173.7; n = 76), POD7-13(96.4 CI: 67.7, 136.9; n = 62), POD14-20(101.4 CI: 80.7, 287.4; n = 22), and POD 21-27(98.1 CI: 66.8, 137.4; n = 20, P = 0.001). No significant difference in plasma levels were noted on POD27-41. CONCLUSION: Plasma CHi3L1 levels were significantly elevated for one month after MICR. Persistently elevated plasma CHi3L1 may support the growth of residual tumor and metastasis.展开更多
A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular...A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer (pH 2.5). The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2+, Ba^2+, Na^+, and K^+ enhanced the enzyme activity, whereas Fe^2+, Ag^+, Hg^2+, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.展开更多
文摘Mammalian chitinases and the related chilectins(Chi Ls) belong to the GH18 family, which hydrolyse the glycosidic bond of chitin by a substrate-assisted mechanism. Chitin the fundamental component in the coating of numerous living species is the most abundant natural biopolymer. Mounting evidence suggest that the function of the majority of the mammalian chitinases is not exclusive to catalyze the hydrolysis of chitin producing pathogens, but include crucial role specific in the immunologic activities. The chitinases and chitinaselike proteins are expressed in response to different proinflammatory cues in various tissues by activated macrophages, neutrophils and in different monocytederived cell lines. The mechanism and molecular interaction of chitinases in relation to immune regulation embrace bacterial infection, inflammation, dismetabolic and degenerative disease. The aim of this review is to update the reader with regard to the role of chitinases proposed in the recent innate and adaptive immunity literature. The deep scrutiny of this family of enzymes could be a useful base for further studies addressed to the development of potential procedure directing these molecules as diagnostic and prognostic markers for numerous immune and inflammatory diseases.
文摘Chitinases catalyze the hydrolysis of chitin, a linear homopolymer of β-(1,4)-linked N-acetylglucosamine. The broad range of applications of chitinolytic enzymes makes their identification and study very promising. Metagenomic approaches offer access to functional genes in uncultured representatives of the microbiota and hold great potential in the discovery of novel enzymes, but tools to extensively explore these data are still scarce. In this study, we develop a chitinase mining pipeline to facilitate the comprehensive search of these enzymes in environmental metagenomic databases and also to explore phylogenetic relationships among the retrieved sequences. In order to perform the analyses, UniprotKB fungal and bacterial chitinases sequences belonging to the glycoside hydrolases (GH) family-18, 19 and 20 were used to generate 15 reference datasets, which were then used to generate high quality seed alignments with the MAFFT program. Profile Hidden Markov Models (pHMMs) were built from each seed alignment using the hmmbuild program of HMMER v3.0 package. The best-hit sequences returned by hmmsearch against two environmental metagenomic databases (Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis—CAMERA and Integrated Microbial Genomes—IMG/M) were retrieved and further analyzed. The NJ trees generated for each chitinase dataset showed some variability in the catalytic domain region of the metagenomic sequences and revealed common sequence patterns among all the trees. The scanning of the retrieved metagenomic sequences for chitinase conserved domains/signatures using both the InterPro and the RPS-BLAST tools confirmed the efficacy and sensitivity of our pHMM-based approach in detecting putative chitinases sequences. These analyses provide insight into the potential reservoir of novel molecules in metagenomic databases while supporting the chitinase mining pipeline developed in this work. By using our chitinase mining pipeline, a larger number of previously unannotated metagenomic chitinase sequences can be classified, enabling further studies on these enzymes.
文摘Vibrio pacini synthesizes multiple chitinases, of which three have been purified in this study by ammonium sulphate fractionation, chitin affinity chromatography and gel chromatography. Molecular weights of the three chitinases, Chi1, Chi2 and Chi3 are 27×103, 39×103 and 46×103 respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzymes have optimal activity at pH 7–8, and retain 50% enzymatic activity pH 4–9. The activities of chitinases are inhibited by Pb2+, Fe3+ and Cu2+, and increased by Ca2+, Mg2+ and Mn2+. Chi3 is found to inhibit the growth of six species of fungi. Such characters of chitinase are different from those of any other chitinase that were reported before. Key words chitinase - Vibrio pacini - purification - inhibit CLC number Q 55 Foundation item: Supported by the Key Technologies Research and Development Programme of the Tenth Five-Year Plan of the Nation Scientific and Technological Development (2001 BA708B04-07)Biography: HAN Bao-qin (1963-), female, Professor.
文摘Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased toler- ance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinasel (CERK1/LysM RLK1) gene, known to play a critical role in signaling defense responses induced by exogenous chitin. Arabidopsis plants overexpress- ing the endochitinase chit36 and hexoaminidase excyl genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity, heavy-metal stresses, and Botrytis cinerea infection. Resistant lines, overexpressing fungal chitinases at different levels, were outcrossed to lysm rlkl mutants. Independent homozygous hybrids lost resistance to biotic and abiotic stresses, despite enhanced chitinase activity. Expression analysis of 270 stress-related genes, including those induced by reactive oxygen species (ROS) and chitin, revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlkl knockout mutant or the hybrids harboring the mutation. These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.
文摘Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.
基金funded by the Tecnológico Nacional de México(TECNM):Project No.6602.18-P.
文摘The objective to this work was to evaluate the enzymatic activity in the culture of Solanum lycopersicum L.infected with Fusarium oxysporum after the combined application of Beauveria bassiana and plant extracts.Solanum lycopersicum plantlets were transplanted 15 days after the emergency.Five days after transplanting,Beauveria bassiana spores were applied at a concentration of 1×10^(7)spores mL^(−1)onto soil(along with A.indica(N)and P.auritum(H)leaf extracts)where S.lycopersicum plants were planted.Eight days after transplanting,spores of F.oxysporum strain were applied at a concentration of 1×10^(6)spores mL^(−1)to soil where S.lycopersicum plants were growing.The development of S.lycopersicum plants was monitored for 114 days,whereby a randomized complete block treatment design was used,the roots of the plants were crushed with liquid nitrogen,after which UV/VIS spectrophotometry was used to determine protein concentration,as well as the activity ofβ-1,3-glucanases,peroxidases(POX,EC1.11.1.7),catalase and chitinases.The treatments combining B.bassiana and A.indica and P.auritum extracts,had a significant difference in enzymatic activity levels,thus contributing to better defense mechanisms and a greater protection to S.lycopersicum plants in the presence of F.oxysporum.The application of B.bassiana and plant extracts to the ground mitigated damage caused by F.oxysporum on S.lycopersicum plants.
基金funded by the Tecnológico Nacional de México(TECNM):Project No.6602.18-P.
文摘The objective of this work was to evaluate the effect of Beauveria bassiana(Bb 1205)on controlling Fusarium oxysporum f.sp.lycopersici(Fol 17108)in tomato plants in greenhouse conditions.Inoculation of Bb 1205 was the most promising among the agronomic variables and expression of the activity of the enzymesβ-1,3-glucanases and chitinases.Inoculation of Bb 1205 occurred at a concentration of 1×108 conidia·mL−1,which was administered onto the leaves,directly into the soil and via injection.Infection with Fol 17108 occurred with 1×106 spores·mL−1,which were added directly to the soil.Spectrophotometry was used for measuring agronomic parameters,namely activity of chitinases andβ-1,3-glucanases in foliage and roots.When Bb 1205 was added to the soil,the chlorophyll index and aerial part length showed significant differences.In addition,it was determined that root length,fresh weight of foliage,flower,and fruit count increased 82 days after inoculation(dai).Chitinase activity induced by Bb 1205 in leaves and roots of tomato plants infected with Fol 17108 was observed when injected into the stem at 32 dai(41.8 and 11.6-fold,respectively).Inoculation on the foliage showed a 10-fold increase ofβ-1,3-glucanases in the roots after 82 dpi.As for leaves,a 3.8-fold increase was found when the stem was inoculated.In the different in vivo applications,Bb 1205 activated its defenses by expressing the chitinase enzymes andβ-1,3-glucanase,thus reducing the damage caused by Fol 17108,demonstrating increase plant growth thereafter.
基金Supported by the National Natural Science Foundation of China(No.81900829,No.82070963)the Xiamen Medical and Health Guiding Project Fund Project(No.3502Z20214ZD1214)+1 种基金the Guangdong Basic and Applied Basic Research Foundation(No.2019A1515011234)the Science and Technology Innovation Committee of Shenzhen(No.JCYJ20210324125614039)。
文摘AIM:To evaluate the potential of two trabecular meshwork(TM)-specific promoters,Chitinase 3-like 1(Ch3L1)and matrix gla protein(MGP),for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2(scAAV2)vector technologies.METHODS:An scAAV2 vector with C3 transferase(C3)as the reporter gene(scAAV2-C3)was selected.The scAAV2-C3 vectors were driven by Ch3L1(scAAV2-Ch3L1-C3),MGP(scAAV2-MGP-C3),enhanced MGP(scAAV2-eMGP-C3)and cytomegalovirus(scAAV2-CMV-C3),respectively.The cultured primary human TM cells were treated with each vector at different multiplicities of infections.Changes in cell morphology were observed by phase contrast microscopy.Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining,real-time quantitative polymerase chain reaction and Western blot.Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively.In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope.Intraocular pressure(IOP)was monitored using a rebound tonometer.Ocular responses were evaluated by slit-lamp microscopy.Ocular histopathology analysis was examined by hematoxylin and eosin staining.RESULTS:In TM cell culture studies,the vectormediated C3 expression induced morphologic changes,disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rhoassociated protein kinase signaling pathway.At the same dose,these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3,but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3.At lowinjected dose,the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGPC3-injected and scAAV2-eMGP-C3-injected eyes.At highinjected dose,significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes.Similar to scAAV2-CMV-C3,scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium.In anterior segment tissues of scAAV2-eMGP-C3-injected eyes,no obvious morphological changes were found except for the TM.Inflammation was absent.CONCLUSION:In scAAV2-transduced TM cells,the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus,but obviously higher than that of MGP.In the anterior chamber of rat eye,the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter,but not by Ch3L1 promoter.These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.
基金the NSCF(Nos.31360272&31870285)Talent Special Project of Guizhou Academy of Agricultural Sciences(No.2022-02)Talent Base for Germplasm Resources Utilization and Innovation of Characteristic Plant in Guizhou(Grant No.RCJD2018-14).
文摘Various strains of powdery mildew(PM),a notorious plant fungal disease,are prevalent and pose a significant threat to plant health.To control PM,transgenic technology can be used to cultivate more resistant plant varieties.In the present study,we utilized the rapid amplification of cDNA ends(RACE)technique to clone the full-length cDNA sequence of the EuCHIT30.7 gene to explore plant genes with disease resistance functions.Bioinformatics analysis revealed that this gene belongs to the GH18 family and is classified as a class III chitinase.The EuCHIT30.7 gene is expressed throughout the Eucommia ulmoides plant,with the most abundant expression in male flowers.Subcellular localization analysis indicated that the protein encoded by this gene was detected within both the cell membrane and cytoplasm.Upon PM inoculation,overexpression of EuCHIT30.7 in tobacco plants led to a significantly reduced relative lesion area and a decreased spore count compared to both wild-type and empty vector control plants.Activities of the protective enzymes,namely,peroxidase(POD),superoxide dismutase(SOD),catalase(CAT),and phenylalaninammo-nialyase(PAL),in tobacco plants overexpressing EuCHIT30.7 were significantly greater than those in wild-type and empty vector tobacco plants.Furthermore,the rate of increase in malondialdehyde(MDA)content was significantly lower in tobacco plants expressing EuCHIT30.7 compared to control tobacco plants.In EuCHIT30.7 transgenic tobacco,the expression of pathogen-related protein genes,namely,PR2,PR5,PR1a,PDF1.2,and MLP423,along with the tobacco PM negative regulatory gene,MLO2,were significantly higher compared to control tobacco plants.These findings suggested that EuCHIT30.7 significantly enhances the resistance of tobacco to PM.
基金Supported by the German Federal Ministry of Education and Research(BMBF-Wachstumskern-PRAEMED.BIO),03WKDB2Csupported by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences,BO/00232/17/5+1 种基金Research Grants of National Research Development and Innovation Office,K115818/2015/1New National Excellence Program of the Ministry of Human Capacities,ÚNKP-18-4 Bolyai Plus.
文摘BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.
基金Supported by Science Foundation from Southwest Forestry College(200524M)Natural Science Foundation of Yunan Province(2002C0047M)Key Scientific and Technological Project of Yunan Province(2003NG12)~~
文摘[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to express in Trichoderma atroviride cells. The cDNA fragment of chitinase gene was cloned by RT-PCR approach. [Result] The activity of chitinase induced reached 40.17 μg/10 min; and the specific fragment amplified was 834 bp in length and proved to be the fragment of chitinase gene by sequencing and sequence analysis. [Conclusion] The result showed the feasibility of isolating the full length of chitinase gene and its transformation, and further producing chitinase.
文摘Entomopathogenic fungi (EF) are recognized biological control agents of insects. Basically, the entomopathogenic fungi pathogen activity depends on the ability of its enzymatic equipment, consisting of lipases, proteases and chitinases, which are in charge of breaking down the insect’s integument. Lipases are the first enzymes synthesized by the entomopathogenic fungi. Recently, a cytochrome P450 subfamily, referred as CYP52XI and MrCYP52 has been identified in Beauveria bassiana and Metarhizium robertsii, respectively. These break down long-chain alkenes and fatty acids to become initial nutrients. Subsequently, subtilisin type (Pr1) proteases sintetize;these enzymes are considered as virulence indicators and they are regulated by a signal transduction mechanism activated by the protein kinase A (PKA) mediated by AMPc. Through the employment of genetic engineering, it has been possible to increase virulence producing Pr1 recombinants with Androctonus australis neurotoxins or with chitinases, reducing the insect’s time of death. In the course of time, the Pr1 protease gene has presented evolutionary adaptations by gene duplication or horizontal transfer infecting different orders of insects. In the same way, the entomopathogenic fungi chitinases have presented a functional diversification. Currently, these have been phylogenetically classified into three subgroups, in accordance to the catalytic site domain and the chitin binding domain. The chitinolytic activity has increased through a directed evolution processes and genetic recombination with Bombyx mori chitinase. Recently, enzymes have been employed as control agents for insects and phytopathogenic fungi (disease originator) opening new potentialities in order to improve the entomopathogenic fungi use. Solid state fermentation is a bioprocess that would produce at great scale enzymes and some other metabolites in grade of increasing the entomopathogenic fungi virulence, in the control of insects and potentially in some diseases affecting plants.
基金Supported by Mr.Wade Thompson and family donation funds to the Divisions of Colon and Rectal surgery,Department of Surgery,Mount Sinai West Hospital,New York,NY 10019
文摘AIM: To assess blood chitinase 3-like 1(CHi3L1) levels for 2 mo after minimally invasive colorectal resection(MICR) for colorectal cancer(CRC). METHODS: CRC patients in an Institutional Review Board approved data/plasma bank who underwent elective MICR for whom preoperative(PreO p), early postoperative(PostO p), and 1 or more late PostO p samples [postoperative day(POD) 7-27] available were included. Plasma CHi3L1 levels(ng/m L) were determined in duplicate by enzyme linked immunosorbent assay. RESULTS: PreOp and PostOp plasma sample were available for 80 MICR cancer patients for the study. The median PreOp CHi3L1 level was 56.8 CI: 41.9-78.6 ng/mL(n = 80). Significantly elevated(P < 0.001) median plasma levels(ng/mL) over PreOp levels were detected on POD1(667.7 CI: 495.7, 771.7; n = 79), POD 3(132.6 CI: 95.5, 173.7; n = 76), POD7-13(96.4 CI: 67.7, 136.9; n = 62), POD14-20(101.4 CI: 80.7, 287.4; n = 22), and POD 21-27(98.1 CI: 66.8, 137.4; n = 20, P = 0.001). No significant difference in plasma levels were noted on POD27-41. CONCLUSION: Plasma CHi3L1 levels were significantly elevated for one month after MICR. Persistently elevated plasma CHi3L1 may support the growth of residual tumor and metastasis.
基金the Science Technology Plan Foundation of Hebei Province, China (07225533)the Doctor Foundation from Agricultural University of Hebei (050031)
文摘A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer (pH 2.5). The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2+, Ba^2+, Na^+, and K^+ enhanced the enzyme activity, whereas Fe^2+, Ag^+, Hg^2+, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.