许多蛋白质二聚化或多聚化在调节其功能方面发挥重要作用,研究蛋白质的聚合有助于阐明相关的生物学过程.本文使用酶联免疫吸附法,对分子量11 k D的马传染性贫血病毒核壳蛋白(nucleocapsid protein 11 k D,NCp11)的聚合进行检测.首先利...许多蛋白质二聚化或多聚化在调节其功能方面发挥重要作用,研究蛋白质的聚合有助于阐明相关的生物学过程.本文使用酶联免疫吸附法,对分子量11 k D的马传染性贫血病毒核壳蛋白(nucleocapsid protein 11 k D,NCp11)的聚合进行检测.首先利用亲和层析分别纯化了NCp11以及N-端或C-端融合有FLAG标签的NCp11.然后将NCp11包被于聚苯乙烯96孔板底,加入带FLAG标签的NCp11与之聚合,再依次加入抗FLAG抗体、辣根过氧化物酶标记的二抗及底物,反应终止后于450 nm波长下读取吸收值.结果表明,酶联免疫吸附法适用于NCp11聚合的检测,可对聚合的特异性、剂量依赖效应和影响因素等进行定量评价.利用该方法不仅能检测蛋白质的聚合,而且具有灵敏度高、特异性好、通量高、成本低和快速简便等优点,有望获得广泛应用.展开更多
Objective To investigate the variability of human cytomegalovirus （HCMV） UL138 open reading frame （ORF） in clinical strains. Methods HCMV UL138 ORF was amplified by polymerase chain reaction （PCR） and PCR amplif...Objective To investigate the variability of human cytomegalovirus （HCMV） UL138 open reading frame （ORF） in clinical strains. Methods HCMV UL138 ORF was amplified by polymerase chain reaction （PCR） and PCR amplification products were sequenced directly, and the data were analyzed in 19 clinical strains. Results LIL138 ORF in all 30 clinical strains was amplified successfully. Compared with that of Toledo strain, the nucleotide and amino acid sequence identifies of LIL138 ORF in all strains were 97.41% to 99.41% and 98.24% to 99.42%, respectively. All of the nucleofide mutations were substitutions. The spatial structure and post-translational modification sites of HL138 encoded proteins were conserved. The result of phylogenetic tree showed that HCMV HL138 sequence variations were not definitely related with different clinical symptoms. Conclusion HCMV UL138 ORF in clinical strains is high conservation, which might be helpful for UL138 encoded protein to play a role in latent infection of HCMV.展开更多
Some proteins secreted by microorganisms have large molecular weights. We report here an approach to prepare coating by multilayer polymers for antifouling of proteins, especially the proteins with a large molecular w...Some proteins secreted by microorganisms have large molecular weights. We report here an approach to prepare coating by multilayer polymers for antifouling of proteins, especially the proteins with a large molecular weight.Stainless steel was used as the model substrate. The substrate was first coated with a hybrid polymer film, which was formed by simultaneous hydrolytic polycondensation of 3-aminopropyltriethoxysilane and polymerization of dopamine(HPAPD). After grafting the macroinitiator 2-bromoisobutyryl bromide, the block polymer brushes PMMA-b-PHEMA were grafted. Three proteins were used to test protein adsorption and antifouling behavior of the coating, including recombinant green fluorescent(54 k Da), recombinant R-transaminase(2 × 90 k Da), and recombinant catalase(4 × 98 k Da). It is demonstrated that the block polymer brushes not only can prevent the adsorption of small molecular weight proteins, but also can significantly reduce the adsorption of the large molecular weight proteins.展开更多
Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed i...Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEG ylated hirudin were investigated, wh.en the PEGylation react, on.wasconducted under 4℃ after 10h, in the borate buffer at pH 8.5 .with the molar ratio 230 ： 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro.Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.展开更多
AIM:To investigate the expression profile of miRNA in esophageal squamous cell carcinoma(ESCC).METHODS:The expression profile of miRNA in ESCC tissues was analyzed by miRNA microarray.The expression levels of miR-143 ...AIM:To investigate the expression profile of miRNA in esophageal squamous cell carcinoma(ESCC).METHODS:The expression profile of miRNA in ESCC tissues was analyzed by miRNA microarray.The expression levels of miR-143 and miR-145 in 86 ESCC patients were determined by real-time polymerase chain reaction(PCR) using TaqMan assay.The mobility effect was estimated by wound-healing using esophageal carcinoma cells transfected with miRNA expression plasmids.RESULTS:A set of miRNAs was found to be deregulated in the ESCC tissues,and the expression levels of miR-143 and-145 were significantly decreased in most of the ESCC tissues examined.Both miR-143 and miR-145 expression correlated with tumor invasion depth.The transfection of human esophageal carcinoma cells with miR-143 and miR-145 expression plasmids resulted in a greater inhibition of cell mobility,however,the protein level of the previously reported target of miR-145,FSCN1,did not show any significant downregulation.CONCLUSION:These findings suggest that the deregulation of miRNAs plays an important role in the progression of ESCC.Both miR-143 and miR-145 might act as anti-oncomirs common to ESCC.展开更多
Objective To assess the expression level of D-Tyr-tRNATyr deacylase(DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease(AD).Methods Altogether 12 SAMP8 mice and 12...Objective To assess the expression level of D-Tyr-tRNATyr deacylase(DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease(AD).Methods Altogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study.Semi-quantita-tive reverse transcription-polymerase chain reaction(RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice.Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice.The behavior of the mice was tested by using a Step-through Test System.Results Both mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice(P<0.05).In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice.Conclusions DTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.展开更多
AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） tra...AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） transfected cell line using the MKN-45 gastric cancer cell line.After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer.RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9（MMP9）,Akt,p-Akt.To confirm the relationship between PI3KAkt and MMP9,the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS：Notch2 expression was dramatically decreased after Notch2 siRNA transfection（100.00% ± 9.74% vs 11.61% ± 3.85%,P 〈 0.01 by qRT-PCR）.There was also a marked reduction of Notch target gene Hes1（100.00% ± 4.74% vs 61.61% ± 3.58%,P 〈 0.05） at the mRNA,indicating an inhibition of Notch signaling.Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels（100.00% ± 9.74% vs 65.61% ± 7.58%,P 〈 0.05）.Down-regulation of Notch2 by siRNA enhanced tumor cell invasion（100.00% ± 21.64% vs 162.22% ± 16.84%,P 〈 0.05） and expression of MMP9（1.56 fold,P 〈 0.05）,and activated the pro-MMP9 protein to its active form（1.48 fold,P 〈 0.05）.There was no significant difference in the protein levels of Akt between the two groups（100.00% ± 10.87% vs 96.61% ± 7.33%,P 〉 0.05）,while down-regulation of Notch2 elevated p-Akt expression（100.00% ± 9.87% vs 154.61% ± 13.10%,P 〈 0.05）.Furthermore,p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002（p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%,P 〈 0.05;MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%,P 〈 0.05）.CONCLUSION：Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway展开更多
The process to obtain a protein-containing ingredient from the remaining whey of Argentinean ＂Cuatriolo＂ cheese production was studied. In order to optimize the protein recovery, physical and chemical treatments wer...The process to obtain a protein-containing ingredient from the remaining whey of Argentinean ＂Cuatriolo＂ cheese production was studied. In order to optimize the protein recovery, physical and chemical treatments were investigated. Two protocols with different sequences of application of heating step and acid addition were assayed in the presence and absence of fat and CaCl2. The results were evaluated by the yield, water retention and particle size. The results showed that the highest yield of the process （76.6%） and an increase in water retention （39.8 w/w） were achieved when the acid was added after boiled and incubated for 30 min at 90 ℃. In these working conditions, the presence of calcium shows a lower yield of recovery （72.8 %） and this behaviour correlates with a smaller particle size. Additionally, the presence of fat reduces the particle size and decreases the performance of the process （69.4%）. Thus, the yield of protein recovery is related to the particle size of the aggregates, i.e., the recovery of proteins increases when increase the particle size. The simultaneous presence of fat and CaCI2 increases the amount of water retained in the aggregated protein （47.62 w/w）. In conclusion, the process of aggregation in whey protein should take into account both the design of suitable protocol and the presence of fat and additives.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
文摘许多蛋白质二聚化或多聚化在调节其功能方面发挥重要作用,研究蛋白质的聚合有助于阐明相关的生物学过程.本文使用酶联免疫吸附法,对分子量11 k D的马传染性贫血病毒核壳蛋白(nucleocapsid protein 11 k D,NCp11)的聚合进行检测.首先利用亲和层析分别纯化了NCp11以及N-端或C-端融合有FLAG标签的NCp11.然后将NCp11包被于聚苯乙烯96孔板底,加入带FLAG标签的NCp11与之聚合,再依次加入抗FLAG抗体、辣根过氧化物酶标记的二抗及底物,反应终止后于450 nm波长下读取吸收值.结果表明,酶联免疫吸附法适用于NCp11聚合的检测,可对聚合的特异性、剂量依赖效应和影响因素等进行定量评价.利用该方法不仅能检测蛋白质的聚合,而且具有灵敏度高、特异性好、通量高、成本低和快速简便等优点,有望获得广泛应用.
基金Supported by the National Natural Science Foundation of China (30801254)
文摘Objective To investigate the variability of human cytomegalovirus （HCMV） UL138 open reading frame （ORF） in clinical strains. Methods HCMV UL138 ORF was amplified by polymerase chain reaction （PCR） and PCR amplification products were sequenced directly, and the data were analyzed in 19 clinical strains. Results LIL138 ORF in all 30 clinical strains was amplified successfully. Compared with that of Toledo strain, the nucleotide and amino acid sequence identifies of LIL138 ORF in all strains were 97.41% to 99.41% and 98.24% to 99.42%, respectively. All of the nucleofide mutations were substitutions. The spatial structure and post-translational modification sites of HL138 encoded proteins were conserved. The result of phylogenetic tree showed that HCMV HL138 sequence variations were not definitely related with different clinical symptoms. Conclusion HCMV UL138 ORF in clinical strains is high conservation, which might be helpful for UL138 encoded protein to play a role in latent infection of HCMV.
基金Supported by the National Natural Science Foundation of China(21476023)
文摘Some proteins secreted by microorganisms have large molecular weights. We report here an approach to prepare coating by multilayer polymers for antifouling of proteins, especially the proteins with a large molecular weight.Stainless steel was used as the model substrate. The substrate was first coated with a hybrid polymer film, which was formed by simultaneous hydrolytic polycondensation of 3-aminopropyltriethoxysilane and polymerization of dopamine(HPAPD). After grafting the macroinitiator 2-bromoisobutyryl bromide, the block polymer brushes PMMA-b-PHEMA were grafted. Three proteins were used to test protein adsorption and antifouling behavior of the coating, including recombinant green fluorescent(54 k Da), recombinant R-transaminase(2 × 90 k Da), and recombinant catalase(4 × 98 k Da). It is demonstrated that the block polymer brushes not only can prevent the adsorption of small molecular weight proteins, but also can significantly reduce the adsorption of the large molecular weight proteins.
文摘Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEG ylated hirudin were investigated, wh.en the PEGylation react, on.wasconducted under 4℃ after 10h, in the borate buffer at pH 8.5 .with the molar ratio 230 ： 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro.Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.
基金Supported by Grants from the NSFC-Guangdong Joint Fund,No.U0932001the National Natural Science Foundation of China,No.30900560
文摘AIM:To investigate the expression profile of miRNA in esophageal squamous cell carcinoma(ESCC).METHODS:The expression profile of miRNA in ESCC tissues was analyzed by miRNA microarray.The expression levels of miR-143 and miR-145 in 86 ESCC patients were determined by real-time polymerase chain reaction(PCR) using TaqMan assay.The mobility effect was estimated by wound-healing using esophageal carcinoma cells transfected with miRNA expression plasmids.RESULTS:A set of miRNAs was found to be deregulated in the ESCC tissues,and the expression levels of miR-143 and-145 were significantly decreased in most of the ESCC tissues examined.Both miR-143 and miR-145 expression correlated with tumor invasion depth.The transfection of human esophageal carcinoma cells with miR-143 and miR-145 expression plasmids resulted in a greater inhibition of cell mobility,however,the protein level of the previously reported target of miR-145,FSCN1,did not show any significant downregulation.CONCLUSION:These findings suggest that the deregulation of miRNAs plays an important role in the progression of ESCC.Both miR-143 and miR-145 might act as anti-oncomirs common to ESCC.
基金Supported by National Natural Science Foundation of China (30721063)
文摘Objective To assess the expression level of D-Tyr-tRNATyr deacylase(DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease(AD).Methods Altogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study.Semi-quantita-tive reverse transcription-polymerase chain reaction(RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice.Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice.The behavior of the mice was tested by using a Step-through Test System.Results Both mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice(P<0.05).In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice.Conclusions DTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.
基金Supported by The National Natural Science Foundation of China,No. 30870364Fund for Key Laboratory of Digestive System Tumors,Gansu Province,No. lzujbky-2011-t03
文摘AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） transfected cell line using the MKN-45 gastric cancer cell line.After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer.RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9（MMP9）,Akt,p-Akt.To confirm the relationship between PI3KAkt and MMP9,the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS：Notch2 expression was dramatically decreased after Notch2 siRNA transfection（100.00% ± 9.74% vs 11.61% ± 3.85%,P 〈 0.01 by qRT-PCR）.There was also a marked reduction of Notch target gene Hes1（100.00% ± 4.74% vs 61.61% ± 3.58%,P 〈 0.05） at the mRNA,indicating an inhibition of Notch signaling.Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels（100.00% ± 9.74% vs 65.61% ± 7.58%,P 〈 0.05）.Down-regulation of Notch2 by siRNA enhanced tumor cell invasion（100.00% ± 21.64% vs 162.22% ± 16.84%,P 〈 0.05） and expression of MMP9（1.56 fold,P 〈 0.05）,and activated the pro-MMP9 protein to its active form（1.48 fold,P 〈 0.05）.There was no significant difference in the protein levels of Akt between the two groups（100.00% ± 10.87% vs 96.61% ± 7.33%,P 〉 0.05）,while down-regulation of Notch2 elevated p-Akt expression（100.00% ± 9.87% vs 154.61% ± 13.10%,P 〈 0.05）.Furthermore,p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002（p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%,P 〈 0.05;MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%,P 〈 0.05）.CONCLUSION：Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway
文摘The process to obtain a protein-containing ingredient from the remaining whey of Argentinean ＂Cuatriolo＂ cheese production was studied. In order to optimize the protein recovery, physical and chemical treatments were investigated. Two protocols with different sequences of application of heating step and acid addition were assayed in the presence and absence of fat and CaCl2. The results were evaluated by the yield, water retention and particle size. The results showed that the highest yield of the process （76.6%） and an increase in water retention （39.8 w/w） were achieved when the acid was added after boiled and incubated for 30 min at 90 ℃. In these working conditions, the presence of calcium shows a lower yield of recovery （72.8 %） and this behaviour correlates with a smaller particle size. Additionally, the presence of fat reduces the particle size and decreases the performance of the process （69.4%）. Thus, the yield of protein recovery is related to the particle size of the aggregates, i.e., the recovery of proteins increases when increase the particle size. The simultaneous presence of fat and CaCI2 increases the amount of water retained in the aggregated protein （47.62 w/w）. In conclusion, the process of aggregation in whey protein should take into account both the design of suitable protocol and the presence of fat and additives.
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.
