[Objective] This study aimed to explore optimum conditions for papain-as- sisted extraction of polysaccharides from Agrocybe aegirit. [Method] Because enzyme is capable of destroying organizational structure of cells,...[Objective] This study aimed to explore optimum conditions for papain-as- sisted extraction of polysaccharides from Agrocybe aegirit. [Method] Because enzyme is capable of destroying organizational structure of cells, papain was selected to cat- alyze cells to let polysaccharides out. An orthogonal experiment was carried out to investigate the effects of hydrolysis temperature, hydrolysis time, enzyme concentra- tion and solid to liquid ratio on extraction of polysaccharides from A. aegirit. [Result] Through the orthogonal experiment, the optimum papain-assisted extraction conditions were obtained： hydrolysis temperature, 45 ℃; hydrolysis time, 2 h; enzyme concen- tration 2%; solid to liquid ratio, 1：80. [Conclusion] This study will provide a reference for extraction of polysaccharides from A. aegirit.展开更多
Aim Cysteine proteases are closely associated with many human and non-human pathological processes and are potential targets for metal ions especially Hg^2＋ and the related species. In the present work, on the basis ...Aim Cysteine proteases are closely associated with many human and non-human pathological processes and are potential targets for metal ions especially Hg^2＋ and the related species. In the present work, on the basis of to the general study on the effects of some metal ions on the activity of papain, a well-known representative of cysteine protease family, the inhibitory effects of Hg^2＋ and polysulfide complexes were studied. Results All the metal ions tested （Hg^2＋, Cu^2＋, Ag^＋, Au^3＋, Zn^2＋, Cd^2＋, Fe^3＋, Mn^2＋, Pb^2＋, Yb^3＋） inhibit the activity of papain anda good correlation between the inhibitory potency and softness-and-hardness was observed. Among the metals, Hg^2＋ was shown to be a potent inhibitor of papain with a Kiof 2 × 10^-7 mol·L^-1 among. Excessive amounts of glutathione and cysteine could reactivate the enzyme activity of papain deactivated by Hg^2＋. These evidences supported that Hg^2＋ might bind to the catalytic site of papain. Interestingly, Hg （Ⅱ） polysulfide complexes were for the first time found to inhibit papain with a Ki of 7 × 10^-6 mol·L^-1, whose potency is close to a well known mercury compound, thimerosal （Ki=2.7 × 10^-6）. In addition, Hg （Ⅱ） polysulfide complexes exhibit good permeability （ 1.9 × 10-5 cm· s^-1） to caco-2 monolayer. Conclusion These results suggested that mercury polysulfide complexes might be potential bioactive species in the interaction with cysteine proteases and other- SH-content proteins, providing a new clue to understand the mechanism of the toxicological and pharmacological actions of cinnabar and other insoluble mercury compounds.展开更多
A novel method was developed for papain immobilization through a biomimetic silicification process induced by papain. By incubating papain in a silica precursor solution, the papain-silica composite formed rapidly and...A novel method was developed for papain immobilization through a biomimetic silicification process induced by papain. By incubating papain in a silica precursor solution, the papain-silica composite formed rapidly and oanain was encansulated. The encansulation efficiency and the recovery activity were 82.60% and 83.09%, re-spectively. Compared with enzymes and biomolecules immobilized in biosilica matrix in the presence of additaonal silica-precipitating species, this papaln encapsulation process, a biomimetic approach, realized high encapsulation efficiency by its autosilification activity under mild conditions （near-neutral pH and ambient temperature）. Fur-thermore, the encapsulated papain exhibits enhanced thermal, pH, recycling and storage stabilities. Kinetic analysis showed that the biomimetic silica matrix did not significantly hinder the mass transport of substrate or the release of product.展开更多
As one of the most effective enzymatic modification methods of protein, papain hydrolysis is applied widely in food production, accompanying starch pasting frequently in order to improve industrial quality. Effects of...As one of the most effective enzymatic modification methods of protein, papain hydrolysis is applied widely in food production, accompanying starch pasting frequently in order to improve industrial quality. Effects of the papain hydrolysis on flour pasting properties were investigated in five papain/flour concentrations and five time-treatments. The structure of starch and protein networks in slurry was investigated under microscope before and after pasting. Results showed that papain hydrolysis influenced the pasting properties of wheat flour significantly through affecting structural characteristics, amylase activity and exotbermic transition, especially during the early stage of hydrolysis. Peak viscosity, trough, final, integral area, and setback significantly decreased along with the increasing concentration of papain. Both hydrolysis time and concentration of papain had obviously effect on the breakdown. Pasting temperature and pasting time increased significantly with the enhancement of papain concentration. Hydrolysis time exerted minor effect on the pasting temperature and pasting time. The average peak time was slightly prolonged by lower concentration of papain, otherwise slightly shortened by higher concentration.展开更多
Complexation and interaction between silver and amino group were applied to induce an efficient immobilization of papain on silica spheres.Tbe silver nanoparticles were deposited on the silica spheres before p apainwa...Complexation and interaction between silver and amino group were applied to induce an efficient immobilization of papain on silica spheres.Tbe silver nanoparticles were deposited on the silica spheres before p apainwas coupled to the silica spheres. The silica spheres with silver nanoparticles were characterized by high resolution transmission electron microscopy （HR-TEM）, Fournier transform infrared spectroscopy （FT-IR）, and UV-Vis scanning spectrometer. FT-IR spectrum was also used to characterize the immobilized and free papain. Effect of some factors on the activities of the immobilized papain was investigated. It was observed that the coupled yield and relative activity of the papain on Ag/SiO2 were 1.17 and 1.86 times of those on the bare SiO2, respectively. At an optimum concentration of silver, theobserved activity of the immobilized papain was 2.1 timesof that on the bare.silica.In addition, the maximum specific activity of papain immobilized on Ag/SiO2 was 819.9 U·mg·^-1, which is slightly lower than that of the free papain, 906.2 U·mg^-1 . Stability of the immobilized papain was also examined. The resuits indicate that the silver nanoparticles successfully induce a fine immobilization of papain.展开更多
Papain (Papainase, EC 3.4.22.2) was immobilized on porous silica beads by cross linking with glutaraldehyde. The thermal activation of this immobilized papain in aqueous system was found at, a temperature range from 5...Papain (Papainase, EC 3.4.22.2) was immobilized on porous silica beads by cross linking with glutaraldehyde. The thermal activation of this immobilized papain in aqueous system was found at, a temperature range from 50 to 90 degrees C. The higher the temperature, the more active the immobilized papain will possess. At the same time, the durability of the immobilized papain on heating was greatly improved. The effect of additives and salts on the activity of the immobilized papain were also studied. The results showed that the additives and some of the salts studied could markedly enhance the activity of the immobilized papain at elevated temperature.展开更多
A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde ...A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde as the couplingagent. The enzymatic activity of the immobilized papain was compared with free papain using casein as a substrate, and theeffects of glutaraldehyde concentration, pH, temperature, time and papain amount added on the activity recovery were alsoinvestigated. The results show that the MR-AA carrier contains reactive primary amine groups, hydrophilic amido links andhydroxyl groups, as well as macroporous structures based on its matrix (MR-AV matrix), furthermore, the activity recoveryof papain in the immobilization could reach 48%/～58%. In comparison with free papain, the resulting immobilized papainexhibits a remarkable thermostability and better reusability.展开更多
A new cross-linked heptapeptide matrix(CHM)was prepared and the adsorption performance for papain was investigated.Firstly,a phage-displayed heptapeptide(Ile-Gln-SerPro-His-Phe-Phe)with high affinity to papain was fou...A new cross-linked heptapeptide matrix(CHM)was prepared and the adsorption performance for papain was investigated.Firstly,a phage-displayed heptapeptide(Ile-Gln-SerPro-His-Phe-Phe)with high affinity to papain was found.Secondly,the heptapeptide was cross-linked to a matrix for papain adsorption.Finally,the adsorption conditions were optimized by response surface methodology.The results indicated that the optimum adsorptionconditionsweredeterminedasinitialpH 6.9,temperature 40℃,and the adsorption capacity of CHM for papain was found to be 55.52 mg/g.Moreover,the papain was purified 78-fold in a single step determined by affinity precipitation.More than87%of the adsorbed papain was desorbed using the eluent solventtriethylamine at pH 7.4.The results show that the CHM is a promising adsorbent for papain purification from crude papaya powder.展开更多
High concentration of polymer solution in cosmetics has the essential role in keeping water on peel of skin. This study includes the papain activity in the high concentrated dextran solution for keratin hydrolysis. Pa...High concentration of polymer solution in cosmetics has the essential role in keeping water on peel of skin. This study includes the papain activity in the high concentrated dextran solution for keratin hydrolysis. Papain is the industrial and a representative hydrolase. The concentration of dextran was ranged to 100 g/L. In the increasing concentration of dextran, the activity of papain decreased due to the low diffusivity of papain and substrate. Even in concentrated dextran solution, keratin powder was possible to be hydrolyzed at the high efficiency.展开更多
Using amino-poly(ethylene glycol) and amino-propyl silica gel as initiator cores, two dendrimers were prepared by a two-step procedure. The progress of dendrimers during each step was monitored by infrared spectroscop...Using amino-poly(ethylene glycol) and amino-propyl silica gel as initiator cores, two dendrimers were prepared by a two-step procedure. The progress of dendrimers during each step was monitored by infrared spectroscopy and elemental analysis. Papain was crosslinked to the solid dendrimer with silica gel core by glutaraldehyde, and the enzymatic activity of the immobilized papain kept almost unchanged after repeated usage for fifteen times.展开更多
In this study,we prepared cross-linked enzyme crystals(CLECs)of papain to further broaden the application of the enzyme with high activity in extreme environments.Initially,papain crystals were successfully obtained b...In this study,we prepared cross-linked enzyme crystals(CLECs)of papain to further broaden the application of the enzyme with high activity in extreme environments.Initially,papain crystals were successfully obtained based on the micro-batch,batch,and expanded batch crystallization experiments.Specifically,ammonium sulfate and polyethylene glycol 6000(PEG6000)were synergistically used as the precipitants,while L-cysteine was applied to enhance the activity of papain.Furthermore,the interaction between L-cysteine and papain was modeled by molecular docking technique.It was found that L-cysteine could form a hydrogen bond with aspartic acid residue(Asp)at site 158,and the electrostatic attraction with lysine residue(Lys)at site 156 was also quite obvious.Then the enzyme crystals were cross-linked by glutaraldehyde at optimized conditions.The papain CLECs were identified by various methods,and it was found that the thermal stability and enzymatic activity both increased compared to the raw enzyme.More importantly,it could be applied at more rigorous conditions,for example,pH of 4.展开更多
OBJECTIVE:To evaluate the molecular mechanism underlying the beneficial effect of Bushen Qiangjin capsule(补肾强筋胶囊,BSQJ),a Traditional Chinese Medicine,on knee osteoarthritis(KOA).METHODS:In the present study,32 f...OBJECTIVE:To evaluate the molecular mechanism underlying the beneficial effect of Bushen Qiangjin capsule(补肾强筋胶囊,BSQJ),a Traditional Chinese Medicine,on knee osteoarthritis(KOA).METHODS:In the present study,32 female Sprague-Dawley rats were randomly divided into four groups:control,KOA,high-dose BSQJ(H-BSQJ),and low-dose BSQJ(L-BSQJ).After successfully establishing the KOA model by intra-articular injection of papain,H-BSQJ and L-BSQJ groups were intragastrically administered 0.243 and 0.122 g/kg BSQJ,respectively,daily for 6 weeks.At the end of the experiment,knee articular cartilage tissues of rats were collected for evaluation by hematoxylin and eosin staining,Safranin O-Fast Green staining,and terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay.Serum interleukin-1βand tumor necrosis factor-αlevels of rats were detected with an enzyme-linked immunosorbent assay method.Gene expression of Wnt-4,β-catenin,Frizzled-2,glycogen synthase kinase-3β(GSK-3β),cysteinyl aspartate-specific proteinases 3 and 9(caspases 3 and 9),collagen typeⅡalpha 1(Col2 a1),and matrix metalloproteinases 1 and 13(MMP-1 and MMP-3)of rat knee articular cartilage was quantified by reverse transcription-quantitative polymerase chain reaction analysis.Wnt-4,β-catenin,Frizzled-2,GSK-3β,cleaved caspase-3,and cleaved caspase-9 protein expression in rat knee articular cartilage was determined by western blot analysis.RESULTS:BSQJ obviously reduced pathological damage and matrix degradation of articular cartilage in KOA rats.Compared with the KOA group,H-BSQJ rats exhibited downregulated m RNA and protein expression of Wnt-4,β-catenin,Frizzled-2,and caspase-3,as well as upregulated m RNA and protein expression of GSK-3β.In addition,H-BSQJ significantly increased m RNA expression of Col2 a1 and decreased m RNA expression of MMP-1 and MMP-13.CONCLUSION:BSQJ exerted a beneficial effect on KOA by a mechanism involving downregulation of the Wnt/β-catenin pathway,which inhibited both cartilage extracellular matrix degradation and chondrocyte apoptosis to ameliorate KOA in rats.展开更多
Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophag...Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 （PLP2-TM） of coronaviruses acts as a novel autophagy- inducing protein. Intriguingly, PLP2-TM induces incom- plete autophagy process by increasing the accumula- tion of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2- TM interacts with the key autophagy regulators, LC3 and Beclinl, and promotes Beclinl interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclinl partially reverses PLP2-TM＇s inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results sug- gested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclinl, which in turn modulates coronavirus replication and antiviral innate immunity.展开更多
基金Supported by Youth Fund of National Natural Science Foundation of China(31201242/C130405)Science Research Program of Education Department of Hubei Province(Q20143004)~~
文摘[Objective] This study aimed to explore optimum conditions for papain-as- sisted extraction of polysaccharides from Agrocybe aegirit. [Method] Because enzyme is capable of destroying organizational structure of cells, papain was selected to cat- alyze cells to let polysaccharides out. An orthogonal experiment was carried out to investigate the effects of hydrolysis temperature, hydrolysis time, enzyme concentra- tion and solid to liquid ratio on extraction of polysaccharides from A. aegirit. [Result] Through the orthogonal experiment, the optimum papain-assisted extraction conditions were obtained： hydrolysis temperature, 45 ℃; hydrolysis time, 2 h; enzyme concen- tration 2%; solid to liquid ratio, 1：80. [Conclusion] This study will provide a reference for extraction of polysaccharides from A. aegirit.
文摘Aim Cysteine proteases are closely associated with many human and non-human pathological processes and are potential targets for metal ions especially Hg^2＋ and the related species. In the present work, on the basis of to the general study on the effects of some metal ions on the activity of papain, a well-known representative of cysteine protease family, the inhibitory effects of Hg^2＋ and polysulfide complexes were studied. Results All the metal ions tested （Hg^2＋, Cu^2＋, Ag^＋, Au^3＋, Zn^2＋, Cd^2＋, Fe^3＋, Mn^2＋, Pb^2＋, Yb^3＋） inhibit the activity of papain anda good correlation between the inhibitory potency and softness-and-hardness was observed. Among the metals, Hg^2＋ was shown to be a potent inhibitor of papain with a Kiof 2 × 10^-7 mol·L^-1 among. Excessive amounts of glutathione and cysteine could reactivate the enzyme activity of papain deactivated by Hg^2＋. These evidences supported that Hg^2＋ might bind to the catalytic site of papain. Interestingly, Hg （Ⅱ） polysulfide complexes were for the first time found to inhibit papain with a Ki of 7 × 10^-6 mol·L^-1, whose potency is close to a well known mercury compound, thimerosal （Ki=2.7 × 10^-6）. In addition, Hg （Ⅱ） polysulfide complexes exhibit good permeability （ 1.9 × 10-5 cm· s^-1） to caco-2 monolayer. Conclusion These results suggested that mercury polysulfide complexes might be potential bioactive species in the interaction with cysteine proteases and other- SH-content proteins, providing a new clue to understand the mechanism of the toxicological and pharmacological actions of cinnabar and other insoluble mercury compounds.
基金Supported by the National Natural Science Foundation of China （21006020, 21276060, 21276062）, the Natural Science Foundation of Hebei Province （B2010000035, B2011202095）, the Science and Technology Research Key Project of Higher School in Hebei Province （ZD2010118）, the Application Basic Research Plan Key Basic Research Project of Hebei Province （11965150D） and Open Funding Project of ~e National Key Laboratory ofBiochemi＇cal Engineering （China）.
文摘A novel method was developed for papain immobilization through a biomimetic silicification process induced by papain. By incubating papain in a silica precursor solution, the papain-silica composite formed rapidly and oanain was encansulated. The encansulation efficiency and the recovery activity were 82.60% and 83.09%, re-spectively. Compared with enzymes and biomolecules immobilized in biosilica matrix in the presence of additaonal silica-precipitating species, this papaln encapsulation process, a biomimetic approach, realized high encapsulation efficiency by its autosilification activity under mild conditions （near-neutral pH and ambient temperature）. Fur-thermore, the encapsulated papain exhibits enhanced thermal, pH, recycling and storage stabilities. Kinetic analysis showed that the biomimetic silica matrix did not significantly hinder the mass transport of substrate or the release of product.
基金supported by the National Natural Science Foundation of China(31171554)the National Basic Research Program of China(2009CB118300)the Improved Variety Project of Shandong Province, China(LN2008-167)
文摘As one of the most effective enzymatic modification methods of protein, papain hydrolysis is applied widely in food production, accompanying starch pasting frequently in order to improve industrial quality. Effects of the papain hydrolysis on flour pasting properties were investigated in five papain/flour concentrations and five time-treatments. The structure of starch and protein networks in slurry was investigated under microscope before and after pasting. Results showed that papain hydrolysis influenced the pasting properties of wheat flour significantly through affecting structural characteristics, amylase activity and exotbermic transition, especially during the early stage of hydrolysis. Peak viscosity, trough, final, integral area, and setback significantly decreased along with the increasing concentration of papain. Both hydrolysis time and concentration of papain had obviously effect on the breakdown. Pasting temperature and pasting time increased significantly with the enhancement of papain concentration. Hydrolysis time exerted minor effect on the pasting temperature and pasting time. The average peak time was slightly prolonged by lower concentration of papain, otherwise slightly shortened by higher concentration.
基金Supported by the National High Technology Research and Development Program of China （2006AA02Z211）, the National Natural Science Foundation of China （20376034）, the Natural Science Foundation of Jiangsu Province （BK2006181）, and the Foundation of Jiangsu Province for College Postgraduate Students in Innovation Engineering （2007）.
文摘Complexation and interaction between silver and amino group were applied to induce an efficient immobilization of papain on silica spheres.Tbe silver nanoparticles were deposited on the silica spheres before p apainwas coupled to the silica spheres. The silica spheres with silver nanoparticles were characterized by high resolution transmission electron microscopy （HR-TEM）, Fournier transform infrared spectroscopy （FT-IR）, and UV-Vis scanning spectrometer. FT-IR spectrum was also used to characterize the immobilized and free papain. Effect of some factors on the activities of the immobilized papain was investigated. It was observed that the coupled yield and relative activity of the papain on Ag/SiO2 were 1.17 and 1.86 times of those on the bare SiO2, respectively. At an optimum concentration of silver, theobserved activity of the immobilized papain was 2.1 timesof that on the bare.silica.In addition, the maximum specific activity of papain immobilized on Ag/SiO2 was 819.9 U·mg·^-1, which is slightly lower than that of the free papain, 906.2 U·mg^-1 . Stability of the immobilized papain was also examined. The resuits indicate that the silver nanoparticles successfully induce a fine immobilization of papain.
基金This research was supported by the Foundation of State Education Committee of China.
文摘Papain (Papainase, EC 3.4.22.2) was immobilized on porous silica beads by cross linking with glutaraldehyde. The thermal activation of this immobilized papain in aqueous system was found at, a temperature range from 50 to 90 degrees C. The higher the temperature, the more active the immobilized papain will possess. At the same time, the durability of the immobilized papain on heating was greatly improved. The effect of additives and salts on the activity of the immobilized papain were also studied. The results showed that the additives and some of the salts studied could markedly enhance the activity of the immobilized papain at elevated temperature.
基金This work was supported by the Lanzhou University Foundation for Middle-aged & Yonger Specialists (LD810718).
文摘A kind of macroporous bead carrier of copolymer containing monomer units of N-aminoethyl acrylamide and vinylalcohol was synthesized, i.e. the MR-AA carrier. Papain was immobilized on the carrier using glutaraldehyde as the couplingagent. The enzymatic activity of the immobilized papain was compared with free papain using casein as a substrate, and theeffects of glutaraldehyde concentration, pH, temperature, time and papain amount added on the activity recovery were alsoinvestigated. The results show that the MR-AA carrier contains reactive primary amine groups, hydrophilic amido links andhydroxyl groups, as well as macroporous structures based on its matrix (MR-AV matrix), furthermore, the activity recoveryof papain in the immobilization could reach 48%/～58%. In comparison with free papain, the resulting immobilized papainexhibits a remarkable thermostability and better reusability.
基金Fundamental Research Funds for the Central Universities,China(No.2232015D3-15)Shanghai Natural Science Foundation,China(No.14ZR1401300)+1 种基金Shanghai Pujiang Program,China(No.16PJD007)“111 Project”Biomedical Textile Materials Science and Technology,China(No.B07024)
文摘A new cross-linked heptapeptide matrix(CHM)was prepared and the adsorption performance for papain was investigated.Firstly,a phage-displayed heptapeptide(Ile-Gln-SerPro-His-Phe-Phe)with high affinity to papain was found.Secondly,the heptapeptide was cross-linked to a matrix for papain adsorption.Finally,the adsorption conditions were optimized by response surface methodology.The results indicated that the optimum adsorptionconditionsweredeterminedasinitialpH 6.9,temperature 40℃,and the adsorption capacity of CHM for papain was found to be 55.52 mg/g.Moreover,the papain was purified 78-fold in a single step determined by affinity precipitation.More than87%of the adsorbed papain was desorbed using the eluent solventtriethylamine at pH 7.4.The results show that the CHM is a promising adsorbent for papain purification from crude papaya powder.
文摘High concentration of polymer solution in cosmetics has the essential role in keeping water on peel of skin. This study includes the papain activity in the high concentrated dextran solution for keratin hydrolysis. Papain is the industrial and a representative hydrolase. The concentration of dextran was ranged to 100 g/L. In the increasing concentration of dextran, the activity of papain decreased due to the low diffusivity of papain and substrate. Even in concentrated dextran solution, keratin powder was possible to be hydrolyzed at the high efficiency.
文摘Using amino-poly(ethylene glycol) and amino-propyl silica gel as initiator cores, two dendrimers were prepared by a two-step procedure. The progress of dendrimers during each step was monitored by infrared spectroscopy and elemental analysis. Papain was crosslinked to the solid dendrimer with silica gel core by glutaraldehyde, and the enzymatic activity of the immobilized papain kept almost unchanged after repeated usage for fifteen times.
基金supported by the Natural Science Foundation of Hebei Province(grant Nos.B2020202015,B2020202066)National Natural Science Foundation of China(grant Nos.21878066,22008050).
文摘In this study,we prepared cross-linked enzyme crystals(CLECs)of papain to further broaden the application of the enzyme with high activity in extreme environments.Initially,papain crystals were successfully obtained based on the micro-batch,batch,and expanded batch crystallization experiments.Specifically,ammonium sulfate and polyethylene glycol 6000(PEG6000)were synergistically used as the precipitants,while L-cysteine was applied to enhance the activity of papain.Furthermore,the interaction between L-cysteine and papain was modeled by molecular docking technique.It was found that L-cysteine could form a hydrogen bond with aspartic acid residue(Asp)at site 158,and the electrostatic attraction with lysine residue(Lys)at site 156 was also quite obvious.Then the enzyme crystals were cross-linked by glutaraldehyde at optimized conditions.The papain CLECs were identified by various methods,and it was found that the thermal stability and enzymatic activity both increased compared to the raw enzyme.More importantly,it could be applied at more rigorous conditions,for example,pH of 4.
基金Supported by Guangdong Basic and Applied Basic Research Foundation,China(No.2018A030313584)Science and Technology Planning Project of Guangzhou,China(No.201804010217)Project of Administration of Traditional Chinese Medicine of Guangdong Province,China(No.20194002,20183001,and 20164002)。
文摘OBJECTIVE:To evaluate the molecular mechanism underlying the beneficial effect of Bushen Qiangjin capsule(补肾强筋胶囊,BSQJ),a Traditional Chinese Medicine,on knee osteoarthritis(KOA).METHODS:In the present study,32 female Sprague-Dawley rats were randomly divided into four groups:control,KOA,high-dose BSQJ(H-BSQJ),and low-dose BSQJ(L-BSQJ).After successfully establishing the KOA model by intra-articular injection of papain,H-BSQJ and L-BSQJ groups were intragastrically administered 0.243 and 0.122 g/kg BSQJ,respectively,daily for 6 weeks.At the end of the experiment,knee articular cartilage tissues of rats were collected for evaluation by hematoxylin and eosin staining,Safranin O-Fast Green staining,and terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay.Serum interleukin-1βand tumor necrosis factor-αlevels of rats were detected with an enzyme-linked immunosorbent assay method.Gene expression of Wnt-4,β-catenin,Frizzled-2,glycogen synthase kinase-3β(GSK-3β),cysteinyl aspartate-specific proteinases 3 and 9(caspases 3 and 9),collagen typeⅡalpha 1(Col2 a1),and matrix metalloproteinases 1 and 13(MMP-1 and MMP-3)of rat knee articular cartilage was quantified by reverse transcription-quantitative polymerase chain reaction analysis.Wnt-4,β-catenin,Frizzled-2,GSK-3β,cleaved caspase-3,and cleaved caspase-9 protein expression in rat knee articular cartilage was determined by western blot analysis.RESULTS:BSQJ obviously reduced pathological damage and matrix degradation of articular cartilage in KOA rats.Compared with the KOA group,H-BSQJ rats exhibited downregulated m RNA and protein expression of Wnt-4,β-catenin,Frizzled-2,and caspase-3,as well as upregulated m RNA and protein expression of GSK-3β.In addition,H-BSQJ significantly increased m RNA expression of Col2 a1 and decreased m RNA expression of MMP-1 and MMP-13.CONCLUSION:BSQJ exerted a beneficial effect on KOA by a mechanism involving downregulation of the Wnt/β-catenin pathway,which inhibited both cartilage extracellular matrix degradation and chondrocyte apoptosis to ameliorate KOA in rats.
基金This research was supported by grants from the National Natural Science Foundation of China （Grant Nos. 81273231,81172799 to Z. C. and 81102478, 81471947 to Y. X.）.
文摘Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 （PLP2-TM） of coronaviruses acts as a novel autophagy- inducing protein. Intriguingly, PLP2-TM induces incom- plete autophagy process by increasing the accumula- tion of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2- TM interacts with the key autophagy regulators, LC3 and Beclinl, and promotes Beclinl interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclinl partially reverses PLP2-TM＇s inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results sug- gested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclinl, which in turn modulates coronavirus replication and antiviral innate immunity.
