Polyacrylamide gel electrophoresis (PAGE) and biochemical staining method were used in this study for the analysis on malate dehydrogenase (MDH,E.C. 1.1.1.37) isozymes zymogram in 11 different types of tissues of male...Polyacrylamide gel electrophoresis (PAGE) and biochemical staining method were used in this study for the analysis on malate dehydrogenase (MDH,E.C. 1.1.1.37) isozymes zymogram in 11 different types of tissues of male and female Varicorhinus macrolepis. It had been found for the first time that the phenotype of malate dehydrogenase (MDH),acid phosphatase (ACP) and superoxide dismutase (SOD) showed difference between male and female V. macrolepis,and there was no difference among different individuals in the same sex. Therefore,the electrophoresis band of malate dehydrogenase,acid phosphatase and superoxide dismutase could be used as an indicator for the identification of gender and tissues of V. macrolepis,which would provide basic data for the developmental genetics,variety improvement and directed breeding of V. macrolepis groups,thus facilitating the development and protection of this valuable fish species.展开更多
A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold pur...A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50oC,respectively.The protease activity is largely enhanced by Ca2 +,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml -1and 21.32μg·min -1·ml -1, respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.展开更多
AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control...AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction(PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10 3-10 4 white cells/μL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28Bassociated polymorphisms(SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159 bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray.First,30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray.Full agreement between plasmids and the BBM assay was observed,with 30/30 correct matches(100%).The kappa value for the BBM assay with plasmids was 1.00(P < 0.05).For the 40 clinical blood samples,the BBM assay hybridization and direct sequencing results were compared for each amplicon.For patient blood samples,agreement was 28/28 for rs8099917T/T,9/11 for rs8099917T/G,1/1 for rs8099917G/G,24/24 for rs12979860C/C,11/14 for rs12979860C/T,and 2/2 for rs12979860T/T.Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes:2/11 rs8099917T/G and 3/14 rs12979860C/T.The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%,respectively;and the corresponding kappa values were 0.88 and 0.85(A kappa value > 0.75 was defined as substantial agreement).The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles.The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10 2 white blood cells/μL.CONCLUSION:This biosensor microarray assay was highly specific,sensitive,rapid and easy to perform.It is compatible with clinical practice for detection of rs8099917 and rs12979860.展开更多
Using poly（acrylic acid） （PAA） aqueous solution, NaOH aqueous solution, aniline （An） and ammonium persulfate （APS）, PAn-PAA hydrogels with a semi-interpenetrating structure connected by physical interlocks, ch...Using poly（acrylic acid） （PAA） aqueous solution, NaOH aqueous solution, aniline （An） and ammonium persulfate （APS）, PAn-PAA hydrogels with a semi-interpenetrating structure connected by physical interlocks, chemical ion bonds and hydrogen bonds were prepared. The swelling properties of the hydrogels in solutions of different pH values （adjusted by adding NaOH or HCI） were studied. All the hydrogels prepared have similar swelling curves （the curves of equilibrium swelling ratio vs. pH value） and reach their maximum swelling at pH of 8 - 10. The maximum swelling ratio of the hydrogels is dependent on composition, including molecular weight of PAA, polymer content of the hydrogel, and molar ratios of AA to An, APS to An, and NaOH to AA, And the compositional dependence of the swelling capacity of PAn-PAA hydrogels was also studied.展开更多
Mostly fed with grass in fresh or conserved form, cattle and other livestock have to cope with silicate defence bodies from plants （phytoliths） and environmental silicates （grit）, which abrade tooth enamel and cou...Mostly fed with grass in fresh or conserved form, cattle and other livestock have to cope with silicate defence bodies from plants （phytoliths） and environmental silicates （grit）, which abrade tooth enamel and could additionally interact with various salivary proteins. To detect potential candidates for silicate-binding proteins, bovine whole saliva was incubated with grass-derived phytoliths and silicates. Interactions of salivary proteins with pulverized bovine dental enamel and dentine were additionally analysed. After intense washing, the powder fractions were loaded onto 1D-polyacrylamide gels, most prominent adhesive protein bands were cut out and proteins were identified by mass spectrometry within three independent replicates. All materials were mainly botmd by bovine odorant-binding protein, bovine salivary protein 30× 10^3 and carbonic anhydrase VI. The phytolith/silicate fraction showed additional stronger interaction with haemoglobin β and lactoperoxidase. Conceivably, the binding of these proteins to the surfaces may contribute to biological processes occurring on them.展开更多
In this paper,the poly(acrylamide)hydrogel used to immobilize saccharomyces cerevisiae for asymmetric synthesis of R(-)-mandelic acid was prepared with free radical ploymerization in deionized water at room temperatur...In this paper,the poly(acrylamide)hydrogel used to immobilize saccharomyces cerevisiae for asymmetric synthesis of R(-)-mandelic acid was prepared with free radical ploymerization in deionized water at room temperature under nitrogen atmosphere.The influence of the composition of hydrogel,loading amount of cells and culture conditions on the asymmetric synthesis was investigated.Results show that PAAm hydrogel is a feasible carrier for immobilization of cells which is a potential alternative method to prepare enantiomerically pure R(-)-mandelic acid.展开更多
The difference in accumulation of high molecular weight glutenin subunits （HMW-GS） in superior and inferior grains results in the non-uniformity of grain quality in winter wheat （Triticum aestivum L.）. The HMW-GS ...The difference in accumulation of high molecular weight glutenin subunits （HMW-GS） in superior and inferior grains results in the non-uniformity of grain quality in winter wheat （Triticum aestivum L.）. The HMW-GS accumulation and glutenin macropolymer （GMP） content were studied in superior and inferior grains during the grain filling period, using the sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）. Compared to inferior grains, HMW-GS was formed earlier and total accumulation amount was higher in superior grains. The total HMW-GS content was higher in superior grain than that in inferior grain except at maturity. For individual HMW-GS types, the accumulation and content of subunit 7 were the highest, followed by subunit 12, while that of subunit 8 were the lowest, followed by subunit 2 in superior grain. In contrary, the accumulation and content of subunit 7 at maturity were significantly higher than subunit 8, but they were similar between subunit 2 and subunit 12 in inferior grain. Moreover, the accumulation of subunit 7 and 12 in superior grain were significantly higher than that in inferior grain. However, compared to the inferior grain, the GMP accumulation was higher, while content was lower in superior grain at maturity.展开更多
To characterize the antigenicity of nucleocapsid proteins(NP) derived from canine coronavirus(CCo V) and canine respiratory coronavirus(CRCo V) in China, the N genes of CCo V(CCo V-BJ70) and CRCo V(CRCo V-BJ202) were ...To characterize the antigenicity of nucleocapsid proteins(NP) derived from canine coronavirus(CCo V) and canine respiratory coronavirus(CRCo V) in China, the N genes of CCo V(CCo V-BJ70) and CRCo V(CRCo V-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs(r NPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCo V-BJ70 and CRCo V-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) results showed that r NPs of CCo V and CRCo V were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the r NP from CRCo V could cross-react with mice antisera against human coronavirus(HCo V-229 E, NL63, OC43, HKU1), while r NP of CCo V had cross-reactivity with only anti-sera against viruses belonging to the same group(HCo V-229 E and NL63). In summary, CCo V and CRCo V r NPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.展开更多
基金Supported by National Natural Science Foundation of China(30700071 )Natural Science Foundation of Shandong Province(Y2008D03 )Science and Technology Program of Qingdao City(08-1-27-jch)~~
文摘Polyacrylamide gel electrophoresis (PAGE) and biochemical staining method were used in this study for the analysis on malate dehydrogenase (MDH,E.C. 1.1.1.37) isozymes zymogram in 11 different types of tissues of male and female Varicorhinus macrolepis. It had been found for the first time that the phenotype of malate dehydrogenase (MDH),acid phosphatase (ACP) and superoxide dismutase (SOD) showed difference between male and female V. macrolepis,and there was no difference among different individuals in the same sex. Therefore,the electrophoresis band of malate dehydrogenase,acid phosphatase and superoxide dismutase could be used as an indicator for the identification of gender and tissues of V. macrolepis,which would provide basic data for the developmental genetics,variety improvement and directed breeding of V. macrolepis groups,thus facilitating the development and protection of this valuable fish species.
基金Supported by the National High Technology Research and Development Program of China（2007AA021506） the Natural Science Foundation of Zhejiang Province（R207609）
文摘A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50oC,respectively.The protease activity is largely enhanced by Ca2 +,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml -1and 21.32μg·min -1·ml -1, respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.
文摘AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction(PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10 3-10 4 white cells/μL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28Bassociated polymorphisms(SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159 bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray.First,30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray.Full agreement between plasmids and the BBM assay was observed,with 30/30 correct matches(100%).The kappa value for the BBM assay with plasmids was 1.00(P < 0.05).For the 40 clinical blood samples,the BBM assay hybridization and direct sequencing results were compared for each amplicon.For patient blood samples,agreement was 28/28 for rs8099917T/T,9/11 for rs8099917T/G,1/1 for rs8099917G/G,24/24 for rs12979860C/C,11/14 for rs12979860C/T,and 2/2 for rs12979860T/T.Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes:2/11 rs8099917T/G and 3/14 rs12979860C/T.The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%,respectively;and the corresponding kappa values were 0.88 and 0.85(A kappa value > 0.75 was defined as substantial agreement).The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles.The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10 2 white blood cells/μL.CONCLUSION:This biosensor microarray assay was highly specific,sensitive,rapid and easy to perform.It is compatible with clinical practice for detection of rs8099917 and rs12979860.
文摘Using poly（acrylic acid） （PAA） aqueous solution, NaOH aqueous solution, aniline （An） and ammonium persulfate （APS）, PAn-PAA hydrogels with a semi-interpenetrating structure connected by physical interlocks, chemical ion bonds and hydrogen bonds were prepared. The swelling properties of the hydrogels in solutions of different pH values （adjusted by adding NaOH or HCI） were studied. All the hydrogels prepared have similar swelling curves （the curves of equilibrium swelling ratio vs. pH value） and reach their maximum swelling at pH of 8 - 10. The maximum swelling ratio of the hydrogels is dependent on composition, including molecular weight of PAA, polymer content of the hydrogel, and molar ratios of AA to An, APS to An, and NaOH to AA, And the compositional dependence of the swelling capacity of PAn-PAA hydrogels was also studied.
基金supported by the German Research Foundation (DFG, SU 124/15-1)
文摘Mostly fed with grass in fresh or conserved form, cattle and other livestock have to cope with silicate defence bodies from plants （phytoliths） and environmental silicates （grit）, which abrade tooth enamel and could additionally interact with various salivary proteins. To detect potential candidates for silicate-binding proteins, bovine whole saliva was incubated with grass-derived phytoliths and silicates. Interactions of salivary proteins with pulverized bovine dental enamel and dentine were additionally analysed. After intense washing, the powder fractions were loaded onto 1D-polyacrylamide gels, most prominent adhesive protein bands were cut out and proteins were identified by mass spectrometry within three independent replicates. All materials were mainly botmd by bovine odorant-binding protein, bovine salivary protein 30× 10^3 and carbonic anhydrase VI. The phytolith/silicate fraction showed additional stronger interaction with haemoglobin β and lactoperoxidase. Conceivably, the binding of these proteins to the surfaces may contribute to biological processes occurring on them.
文摘In this paper,the poly(acrylamide)hydrogel used to immobilize saccharomyces cerevisiae for asymmetric synthesis of R(-)-mandelic acid was prepared with free radical ploymerization in deionized water at room temperature under nitrogen atmosphere.The influence of the composition of hydrogel,loading amount of cells and culture conditions on the asymmetric synthesis was investigated.Results show that PAAm hydrogel is a feasible carrier for immobilization of cells which is a potential alternative method to prepare enantiomerically pure R(-)-mandelic acid.
基金This study is supported by the projects of Priority Academic Program Developmem （PAPD）, the National Natural Science Foundation of China （31171484, 31471445）, the National Natural Science Foundation for Distinguished Young Scientists （31325020）, Jiangsu Agriculture Science and Technology Innovation Fund （CX （14） 2002）, the Specialized Research Fund for the Doctoral Program of Higher Education （20090097110009）, the National Non-profit Program by Ministry of Agriculture （200903003）, and the China Agriculture Research System （CARS-03）.
文摘The difference in accumulation of high molecular weight glutenin subunits （HMW-GS） in superior and inferior grains results in the non-uniformity of grain quality in winter wheat （Triticum aestivum L.）. The HMW-GS accumulation and glutenin macropolymer （GMP） content were studied in superior and inferior grains during the grain filling period, using the sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）. Compared to inferior grains, HMW-GS was formed earlier and total accumulation amount was higher in superior grains. The total HMW-GS content was higher in superior grain than that in inferior grain except at maturity. For individual HMW-GS types, the accumulation and content of subunit 7 were the highest, followed by subunit 12, while that of subunit 8 were the lowest, followed by subunit 2 in superior grain. In contrary, the accumulation and content of subunit 7 at maturity were significantly higher than subunit 8, but they were similar between subunit 2 and subunit 12 in inferior grain. Moreover, the accumulation of subunit 7 and 12 in superior grain were significantly higher than that in inferior grain. However, compared to the inferior grain, the GMP accumulation was higher, while content was lower in superior grain at maturity.
基金supported by the Mega project for Infectious Disease Research of China (2014ZX10004001-002, 2013ZX10004601)National Basic Research Program of China (2011CB504704)
文摘To characterize the antigenicity of nucleocapsid proteins(NP) derived from canine coronavirus(CCo V) and canine respiratory coronavirus(CRCo V) in China, the N genes of CCo V(CCo V-BJ70) and CRCo V(CRCo V-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs(r NPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCo V-BJ70 and CRCo V-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) results showed that r NPs of CCo V and CRCo V were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the r NP from CRCo V could cross-react with mice antisera against human coronavirus(HCo V-229 E, NL63, OC43, HKU1), while r NP of CCo V had cross-reactivity with only anti-sera against viruses belonging to the same group(HCo V-229 E and NL63). In summary, CCo V and CRCo V r NPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.
