Large-scale production of cell culture-based classical swine fever virus(CSFV)vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium.Hence,we have developed an efficient ...Large-scale production of cell culture-based classical swine fever virus(CSFV)vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium.Hence,we have developed an efficient method for purifying CSFV from cell-culture medium.Pure viral particles were obtained with two steps of tangential-flow filtration(TFF)and size-exclusion chromatography(SEC),and were compared with particles from ultracentrifugation by transmission electron microscopy(TEM),infectivity and recovery test,and real time fluorescent quantitative PCR(FQ-PCR).TFF concentrated the virus particles effectively with a retention rate of 98.5%,and 86.2%of viral particles were obtained from the ultrafiltration retentate through a Sepharose 4 F F column on a biological liquid chromatography system.CSFV purified by TFF-SEC or ultracentrifugation were both biologically active from 1.0×10–4.25 TCID50·mL^(–1) to 3.0×10^(–6.25) TCID50·mL^(–1),but the combination of TFF and SEC produced more pure virus particles than by ultracentrifugation alone.In addition,pure CSFV particles with the expected diameter of 40–60 nm were roughly spherical without any visible contamination.Mice immunized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultracentrifugation-purified CSFV(P<0.05).The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.展开更多
The effects of variety and growth location on grain composition and starch structures were investigated using three rice (Oryza sativa L.) cultivars (Phka Romduol, Sen Pidao and IR66) with different amylose conten...The effects of variety and growth location on grain composition and starch structures were investigated using three rice (Oryza sativa L.) cultivars (Phka Romduol, Sen Pidao and IR66) with different amylose contents. All the three cultivars were planted in three different agro-climatic zones (Phnom Penh, Coastal and Plateau) of Cambodia. The protein content of polished grains increased when rice was planted at a location with higher average temperature, but their lipid content decreased. The amylose content and degree of branching were not greatly affected by the minor temperature differences among the growing locations. Starch fine structures characterized by the chain-length distribution were significantly different among the cultivars, but not significantly among different locations. The results suggested that protein and lipid biosyntheses were more sensitive to the environmental temperature than that of starch in rice grains.展开更多
Humic acids (HA) are one of the main environmental factors controlling the fate and behavior of the compounds released into the environment. In particular, they are universally considered of great importance in determ...Humic acids (HA) are one of the main environmental factors controlling the fate and behavior of the compounds released into the environment. In particular, they are universally considered of great importance in determining soil extracellular enzyme activity and stability via association with essential soil enzymes. The objective of this study was to investigate the interaction of coal HA with an extracellular multicopper oxidase laccase (EC 1.10.3.2) that catalyze the oxidation of a wide range of reducing substances in the environment. Using size-exclusion chromatography analysis and monitoring laccase activity, the formation of a stable and an enzymatically active complex between HA and laccase was shown. Basing the data obtained by isoelectric focusing of HA-laccase complex, non-covalent character of laccase association with HA was considered and binding of laccase to HA by weak dispersive forces such as van der Waals, hydrophobic, π-π, CH-π and others was hypothesized.展开更多
Protein complexes are important for almost all biological processes.Hence,to fully understand how cells work,it is also necessary to characterize protein complexes and their dynamics in response to various cellular cu...Protein complexes are important for almost all biological processes.Hence,to fully understand how cells work,it is also necessary to characterize protein complexes and their dynamics in response to various cellular cues.Moreover,the dynamics of protein interaction play crucial roles in regulating the(dis)association of protein complexes and,in turn,regulating biological processes such as metabolism.Here,mitochondrial protein complexes were investigated by blue native PAGE and size-exclusion chromatography under conditions of oxidative stress in order to monitor their dynamic(dis)associations.Rearrangements of enzyme interactions and changes in protein complex abundance were observed in response to oxidative stress induced by menadione treatment.These included changes in enzymatic protein complexes involving g-amino butyric acid transaminase(GABA-T),D-ornithine aminotransferase(D-OAT),or proline dehydrogenase 1(POX1)that are expected to affect proline metabolism.Menadione treatment also affected interactions between several enzymes of the tricarboxylic acid(TCA)cycle and the abundance of complexes of the oxidative phosphorylation pathway.In addition,we compared the mitochondrial complexes of roots and shoots.Considerable differences between the two tissues were observed in the mitochondrial import/export apparatus,the formation of super-complexes in the oxidative phosphorylation pathway,and specific interactions between enzymes of the TCA cycle that we postulate may be related to the metabolic/energetic requirements of roots and shoots.展开更多
Dextran-modified iron oxide nanoparticles were prepared by precipitation of Fe(Ⅱ) and Fe(Ⅲ) salts with ammonium hydroxide by two methods. Iron oxide was precipitated either in the presence of dextran solution, o...Dextran-modified iron oxide nanoparticles were prepared by precipitation of Fe(Ⅱ) and Fe(Ⅲ) salts with ammonium hydroxide by two methods. Iron oxide was precipitated either in the presence of dextran solution, or the dextran solution was added after precipitation. In the second method, the iron oxide particle size and size distribution could be controlled depending on the concentration of dextran in the solution. The nanoparticles were characterized by size-exclusion chromatography, transmission electron microscopy and dynamic light scattering. Optimal conditions for preparation of stable iron oxide colloid particles were determined, The dextran/iron oxide ratio 0-0,16 used in precipitation of iron salts can be recommended for synthesis of nanoparticles suitable for biomedical applications, as the colloid does not contain excess dextran and does not coagulate.展开更多
Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in productio...Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in production of recombinant products Convenlional dilution refolding technique suffers from disadvantages of low recovery and low concentration Various chromatographic refolding techniques have been developed over the last few years These include size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and different affinity chromatography. A successful strategy is the use of gradient elution in column control which provides a gentle and gradual change of the solution environment for the macromolecule to rsfold at nano-scale, The gradient refolding at column scale could minimize misfolding and aggregation which are induced by sudden change of the solution in conventional refolding operation.展开更多
Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an (-helix and a (-strand, was suggested to be essential for subunit interactions. To examine the critical ami...Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an (-helix and a (-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and Ile8Glu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6-0.8 mol/L GdmCl. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCl (0.2 mol/L). In aqueous solution, the Ile8Glu variant exists predominantly in the dimeric form with increased KM and decreased kcat as compared with those of WT and L3P. However, the activity of Ile8Glu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less stable than WT in oligomer structure. Results also support the fact that some residues in the N-terminal arm, especially the Leu8 in the (-structure, contribute the important binding energies to the dimerization of dimers, which might affect the assembly of the enzyme as well as the catalytic function.展开更多
基金the Special Fund for Agro-scientific Research in the Public Interest(201203039)China Agriculture Research System(CARS-36).
文摘Large-scale production of cell culture-based classical swine fever virus(CSFV)vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium.Hence,we have developed an efficient method for purifying CSFV from cell-culture medium.Pure viral particles were obtained with two steps of tangential-flow filtration(TFF)and size-exclusion chromatography(SEC),and were compared with particles from ultracentrifugation by transmission electron microscopy(TEM),infectivity and recovery test,and real time fluorescent quantitative PCR(FQ-PCR).TFF concentrated the virus particles effectively with a retention rate of 98.5%,and 86.2%of viral particles were obtained from the ultrafiltration retentate through a Sepharose 4 F F column on a biological liquid chromatography system.CSFV purified by TFF-SEC or ultracentrifugation were both biologically active from 1.0×10–4.25 TCID50·mL^(–1) to 3.0×10^(–6.25) TCID50·mL^(–1),but the combination of TFF and SEC produced more pure virus particles than by ultracentrifugation alone.In addition,pure CSFV particles with the expected diameter of 40–60 nm were roughly spherical without any visible contamination.Mice immunized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultracentrifugation-purified CSFV(P<0.05).The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.
基金partially supported by Australian Research Council Linkage (Grant No. LP0990769)
文摘The effects of variety and growth location on grain composition and starch structures were investigated using three rice (Oryza sativa L.) cultivars (Phka Romduol, Sen Pidao and IR66) with different amylose contents. All the three cultivars were planted in three different agro-climatic zones (Phnom Penh, Coastal and Plateau) of Cambodia. The protein content of polished grains increased when rice was planted at a location with higher average temperature, but their lipid content decreased. The amylose content and degree of branching were not greatly affected by the minor temperature differences among the growing locations. Starch fine structures characterized by the chain-length distribution were significantly different among the cultivars, but not significantly among different locations. The results suggested that protein and lipid biosyntheses were more sensitive to the environmental temperature than that of starch in rice grains.
文摘Humic acids (HA) are one of the main environmental factors controlling the fate and behavior of the compounds released into the environment. In particular, they are universally considered of great importance in determining soil extracellular enzyme activity and stability via association with essential soil enzymes. The objective of this study was to investigate the interaction of coal HA with an extracellular multicopper oxidase laccase (EC 1.10.3.2) that catalyze the oxidation of a wide range of reducing substances in the environment. Using size-exclusion chromatography analysis and monitoring laccase activity, the formation of a stable and an enzymatically active complex between HA and laccase was shown. Basing the data obtained by isoelectric focusing of HA-laccase complex, non-covalent character of laccase association with HA was considered and binding of laccase to HA by weak dispersive forces such as van der Waals, hydrophobic, π-π, CH-π and others was hypothesized.
基金supported by funding from the Max Planck Society(S.M.J.,A.G.,A.R.F.,and Y.Z.)the European Union’s Horizon 2020 research and innovation program,project PlantaSYST(SGA-CSA no.739582 under FPA no.664620)for supporting their researchfinancial support from the IMPRS-PMPG program.
文摘Protein complexes are important for almost all biological processes.Hence,to fully understand how cells work,it is also necessary to characterize protein complexes and their dynamics in response to various cellular cues.Moreover,the dynamics of protein interaction play crucial roles in regulating the(dis)association of protein complexes and,in turn,regulating biological processes such as metabolism.Here,mitochondrial protein complexes were investigated by blue native PAGE and size-exclusion chromatography under conditions of oxidative stress in order to monitor their dynamic(dis)associations.Rearrangements of enzyme interactions and changes in protein complex abundance were observed in response to oxidative stress induced by menadione treatment.These included changes in enzymatic protein complexes involving g-amino butyric acid transaminase(GABA-T),D-ornithine aminotransferase(D-OAT),or proline dehydrogenase 1(POX1)that are expected to affect proline metabolism.Menadione treatment also affected interactions between several enzymes of the tricarboxylic acid(TCA)cycle and the abundance of complexes of the oxidative phosphorylation pathway.In addition,we compared the mitochondrial complexes of roots and shoots.Considerable differences between the two tissues were observed in the mitochondrial import/export apparatus,the formation of super-complexes in the oxidative phosphorylation pathway,and specific interactions between enzymes of the TCA cycle that we postulate may be related to the metabolic/energetic requirements of roots and shoots.
文摘Dextran-modified iron oxide nanoparticles were prepared by precipitation of Fe(Ⅱ) and Fe(Ⅲ) salts with ammonium hydroxide by two methods. Iron oxide was precipitated either in the presence of dextran solution, or the dextran solution was added after precipitation. In the second method, the iron oxide particle size and size distribution could be controlled depending on the concentration of dextran in the solution. The nanoparticles were characterized by size-exclusion chromatography, transmission electron microscopy and dynamic light scattering. Optimal conditions for preparation of stable iron oxide colloid particles were determined, The dextran/iron oxide ratio 0-0,16 used in precipitation of iron salts can be recommended for synthesis of nanoparticles suitable for biomedical applications, as the colloid does not contain excess dextran and does not coagulate.
基金The Natural Science Foundation of China(NSFC No.20136020,20125616)Chinese Academy of Sciences are gratefully acknowledged for financial supports to this research.
文摘Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in production of recombinant products Convenlional dilution refolding technique suffers from disadvantages of low recovery and low concentration Various chromatographic refolding techniques have been developed over the last few years These include size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and different affinity chromatography. A successful strategy is the use of gradient elution in column control which provides a gentle and gradual change of the solution environment for the macromolecule to rsfold at nano-scale, The gradient refolding at column scale could minimize misfolding and aggregation which are induced by sudden change of the solution in conventional refolding operation.
基金This work was supported in part by grants from the National Pandeng Program and the NationalNatural Science Foundation of China (Grant No. 39930060). We thank Dr. S. B. Xiong for some technical help in cloning the LDHA gene and Prof. K. C. Ruan for the
文摘Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an (-helix and a (-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and Ile8Glu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6-0.8 mol/L GdmCl. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCl (0.2 mol/L). In aqueous solution, the Ile8Glu variant exists predominantly in the dimeric form with increased KM and decreased kcat as compared with those of WT and L3P. However, the activity of Ile8Glu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less stable than WT in oligomer structure. Results also support the fact that some residues in the N-terminal arm, especially the Leu8 in the (-structure, contribute the important binding energies to the dimerization of dimers, which might affect the assembly of the enzyme as well as the catalytic function.