Understanding the thermal stability of the proteins in human serum is essential since human serum is the important source of pharmaceutical proteins. Near-infrared（NIR） spectroscopy was applied to the investigation ...Understanding the thermal stability of the proteins in human serum is essential since human serum is the important source of pharmaceutical proteins. Near-infrared（NIR） spectroscopy was applied to the investigation of thermal changes in secondary structure and hydration of human serum proteins.However, as a multicomponent system, the overlap of the broad NIR bands makes the structural analysis very difficult directly using the spectra of serum samples. Therefore, continuous wavelet transform（CWT） was used to improve the resolution of NIR spectra, and Monte Carlo-uninformative variable elimination（MC-UVE） method was applied to the selection of the variables associated with the proteins for the structural analysis. The variables（5956, 5867, 5815, 5747, 4525, 4401, 4359 and 4328 cm^-1） related to protein secondary structures and those（7074, 6951, 6827 and 6700 cm 1） connected with water species were selected. Then, the thermal stability was analyzed through the intensity variations of the selected variables with temperature from 30℃ to 80 ℃. It was found that the variation of the spectral variables related to both a-helix and b-sheet changes apparently around 60 ℃, indicating the beginning of the thermal denaturation and the transition from a-helix to b-sheet. Moreover, an obvious change was found around 60℃for the content of the water specie S3, i.e., the water cluster containing three hydrogen bonds. The result demonstrates that MC-UVE can identify the protein-related NIR spectral variables, and the water species may be a marker for investigation of the structural change of proteins in biochemical systems.展开更多
Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is ...Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.展开更多
The role of serum and glucocorticoid-induced kinase 1 （SGK1） pathway in the connective tissue growth factor （CTGF） expression was investigated in cultured human mesangial cells （HMCs） under high glucose. By usin...The role of serum and glucocorticoid-induced kinase 1 （SGK1） pathway in the connective tissue growth factor （CTGF） expression was investigated in cultured human mesangial cells （HMCs） under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 （SD） could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP （FP） under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 （KN） could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.展开更多
Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). ...Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). These protein samples were 99% pure HSA (termed HSA<sub>99</sub>) and 96% pure HSA (termed HSA<sub>96</sub>). According to the supplier, the 3% difference in purity between HSA<sub>96</sub> and HSA<sub>99</sub> is primarily attributed to the presence of globulins and fatty acids. Our primary aim was to investigate the utility of DSC in discerning changes in HSA that occur when the protein is specifically adducted, and determine how adduct formation manifests itself in HSA denaturation curves, or thermograms, measured by DSC. Effects of site specific covalent attachment of biotin (the adduct) on the thermodynamic stability of HSA were investigated. Each of the HSA preparations was modified by biotinylation targeting a single site, or multiple sites on the protein structure. Thermograms of both modified and unmodified HSA samples successfully demonstrated the ability of DSC to clearly discern the two HSA preparations and the presence or absence of covalent modifications. DSC thermogram analysis also provided thermodynamic characterization of the different HSA samples of the study, which provided insight into how the two forms of HSA respond to covalent modification with biotin. Consistent with published studies [1] HSA<sub>96</sub>, the preparation with contaminants that contain globulins and fatty acids seems to be comprised of two forms, HSA<sub>96-L</sub> and HSA<sub>96-H</sub>, with HSA<sub>96-L</sub> more stable than HSA<sub>99</sub>. The effect of multisite biotinylation is to stabilize HSA<sub>96-L</sub> and destabilize HSA<sub>96-H</sub>. Thermodynamic analysis suggests that the binding of ligands comprising the fatty acid and globulin-like contaminant contributes approximately 6.7 kcal/mol to the stability HSA<sub>96-L</sub>.展开更多
Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pa...Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.展开更多
Objectives To investigate the effects and mechanism of glycated serum albumin(GSA) on expression of Monocyte chemoattratant protein-1(MCP-1) in Endothelial Cells. Methods Human Umbilical Vein Endothelial Cells (HUVEC)...Objectives To investigate the effects and mechanism of glycated serum albumin(GSA) on expression of Monocyte chemoattratant protein-1(MCP-1) in Endothelial Cells. Methods Human Umbilical Vein Endothelial Cells (HUVEC)are cultured with GSA of different concentrations and interfered by glycosylation products inhibitor Aminoguanidine (AG) and anti-oxidant N-acetylcy-steine (NAC), The expression of MCP-1 are evaluated by Immunocytochemistry and Sandwich ELISA. MDA content and SOD activity are determined by the technique of TBA and XOD respectively. Results GSA can stimulate MCP-1 production and secretion. Immunocytochemistry showed that after HUVECs were cultured with 50 mg/L GSA, expression of MCP-1 in group 4hrs, 8hrs and 12hrs was 1.3, 1.9 and 2.8 fold as much as that in control group (P < 0.01), and there was significant difference among the experiment groups(P < 0.01). Sandwich ELISA showed that expression of MCP-1 in three different groups was 1.6, 2.4 and 3.0 fold as much as that in control group(P < 0.01), and there was significant difference among the experiment groups(P < 0.01); GSA can cause the decrease of SOD activity(P < 0.05) and increase of MDA content(P < 0.01); AG and NAC can restrain obviously the expression of MCP-1 of HUVECs stimulated by GSA(P < 0.01); NAC can restrain the effect of GSA on SOD activity and MDA content in HUVECs (P < 0.05). Conclusions GSA can stimulate the expression of MCP-1 of endothelial cells by inducing endothelial cells oxidative stress.展开更多
目的:分析血清人附睾分泌蛋白4(Human epididymis protein 4,HE4)水平和子宫内膜癌患者临床病理特征的相关性.方法:回顾性分析2019年1月至2022年6月医院收治的120例子宫内膜癌患者临床资料.所有患者均实施子宫全切术治疗,收集患者的临...目的:分析血清人附睾分泌蛋白4(Human epididymis protein 4,HE4)水平和子宫内膜癌患者临床病理特征的相关性.方法:回顾性分析2019年1月至2022年6月医院收治的120例子宫内膜癌患者临床资料.所有患者均实施子宫全切术治疗,收集患者的临床病理特征资料,检测所有患者HE4水平,分析血清HE4水平与子宫内膜癌患者临床病理特征的相关性.结果:120例子宫内膜癌患者中,病理类型:子宫内膜样腺癌46例、子宫内膜样透明细胞癌36例、子宫内膜样鳞癌38例;淋巴结转移:发生35例、未发生85例;肌层浸润深度:>1/2肌层40例、≤1/2肌层80例;TNM分期:Ⅰ期22例、Ⅱ期43例、Ⅲ期55例.病灶所处部位:子宫底55例、子宫角37例、宫颈管28例.不同病理类型、TNM分期、病灶所处部位患者的血清HE4水平比较,差异无统计学意义;肌层浸润深度>1/2肌层患者的血清HE4水平高于肌层浸润深度≤1/2肌层的患者,发生淋巴结转移患者的血清HE4水平较未发生淋巴结转移的患者明显增加(P<0.05).经Logistic回归显示,血清HE4水平升高是子宫内膜癌患者肌层浸润深度>1/2肌层的风险因子(OR>1,P<0.05);血清HE4水平升高是子宫内膜癌患者发生淋巴结转移的风险因子(OR>1,P<0.05).结论:血清HE4水平升高增加可加剧肌层浸润深度,促使子宫内膜癌患者发生淋巴结转移.展开更多
Currently there is no successful platform technology for the sustained release of protein drugs.It seems inevitable to specifically develop new materials for such purpose, and hence the understanding of protein–mater...Currently there is no successful platform technology for the sustained release of protein drugs.It seems inevitable to specifically develop new materials for such purpose, and hence the understanding of protein–material interactions is highly desirable. In this study, we synthesized cholesterol-grafted polyglutamate(PGA-g-Chol) as a hydrophobically-modified polypeptide, and thoroughly characterized its interaction with a model protein(human serum albumin) in the aqueous solution by using circular dichroism, fluorescence methods, and light scattering. With the protein concentration fixed at 5 μmol/L,adding PGA-g-Chol polymers into the solution resulted in continuous blue shift of the protein fluorescence(from 339 to 332 nm), until the polymer molar concentration reached the same value as the protein. In contrast, the un-modified polyglutamate polymers apparently neither affected the protein microenvironment nor formed aggregates. Based on the experimental data, we proposed a physical picture for such protein–polymer systems, where the polymer first bind with the protein in a 1:1 molar ratio via a fraction of their hydrophobic pendant cholesterol resides along the polymer chain. In this protein/polymer complex, there are excess unbound cholesterol residues. As the polymer concentration increases, the polymers form multi-polymer aggregates around 200 nm in diameter via the same hydrophobic cholesterol residues. The protein/polymer complex also participate in the aggregation via their excess cholesterol residues, and consequently the proteins are encapsulated into the nanoparticles. The encapsulation was also found to increase the thermal stability of the model protein.展开更多
基金supported by National Natural Science Foundation of China(No.21475068)
文摘Understanding the thermal stability of the proteins in human serum is essential since human serum is the important source of pharmaceutical proteins. Near-infrared（NIR） spectroscopy was applied to the investigation of thermal changes in secondary structure and hydration of human serum proteins.However, as a multicomponent system, the overlap of the broad NIR bands makes the structural analysis very difficult directly using the spectra of serum samples. Therefore, continuous wavelet transform（CWT） was used to improve the resolution of NIR spectra, and Monte Carlo-uninformative variable elimination（MC-UVE） method was applied to the selection of the variables associated with the proteins for the structural analysis. The variables（5956, 5867, 5815, 5747, 4525, 4401, 4359 and 4328 cm^-1） related to protein secondary structures and those（7074, 6951, 6827 and 6700 cm 1） connected with water species were selected. Then, the thermal stability was analyzed through the intensity variations of the selected variables with temperature from 30℃ to 80 ℃. It was found that the variation of the spectral variables related to both a-helix and b-sheet changes apparently around 60 ℃, indicating the beginning of the thermal denaturation and the transition from a-helix to b-sheet. Moreover, an obvious change was found around 60℃for the content of the water specie S3, i.e., the water cluster containing three hydrogen bonds. The result demonstrates that MC-UVE can identify the protein-related NIR spectral variables, and the water species may be a marker for investigation of the structural change of proteins in biochemical systems.
文摘Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.
基金a grant from the National Natural Sciences Foundation of China (No. 30600810)
文摘The role of serum and glucocorticoid-induced kinase 1 （SGK1） pathway in the connective tissue growth factor （CTGF） expression was investigated in cultured human mesangial cells （HMCs） under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 （SD） could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP （FP） under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 （KN） could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.
文摘Thermal denaturation and stability of two commercially available preparations of Human Serum Albumin (HSA), differing in their advertised level of purity, were investigated by differential scanning calorimetry (DSC). These protein samples were 99% pure HSA (termed HSA<sub>99</sub>) and 96% pure HSA (termed HSA<sub>96</sub>). According to the supplier, the 3% difference in purity between HSA<sub>96</sub> and HSA<sub>99</sub> is primarily attributed to the presence of globulins and fatty acids. Our primary aim was to investigate the utility of DSC in discerning changes in HSA that occur when the protein is specifically adducted, and determine how adduct formation manifests itself in HSA denaturation curves, or thermograms, measured by DSC. Effects of site specific covalent attachment of biotin (the adduct) on the thermodynamic stability of HSA were investigated. Each of the HSA preparations was modified by biotinylation targeting a single site, or multiple sites on the protein structure. Thermograms of both modified and unmodified HSA samples successfully demonstrated the ability of DSC to clearly discern the two HSA preparations and the presence or absence of covalent modifications. DSC thermogram analysis also provided thermodynamic characterization of the different HSA samples of the study, which provided insight into how the two forms of HSA respond to covalent modification with biotin. Consistent with published studies [1] HSA<sub>96</sub>, the preparation with contaminants that contain globulins and fatty acids seems to be comprised of two forms, HSA<sub>96-L</sub> and HSA<sub>96-H</sub>, with HSA<sub>96-L</sub> more stable than HSA<sub>99</sub>. The effect of multisite biotinylation is to stabilize HSA<sub>96-L</sub> and destabilize HSA<sub>96-H</sub>. Thermodynamic analysis suggests that the binding of ligands comprising the fatty acid and globulin-like contaminant contributes approximately 6.7 kcal/mol to the stability HSA<sub>96-L</sub>.
基金supported by the National Natural Science Foundation of China,No.30360100,30760234,30860260,81160373,81360458
文摘Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.
文摘Objectives To investigate the effects and mechanism of glycated serum albumin(GSA) on expression of Monocyte chemoattratant protein-1(MCP-1) in Endothelial Cells. Methods Human Umbilical Vein Endothelial Cells (HUVEC)are cultured with GSA of different concentrations and interfered by glycosylation products inhibitor Aminoguanidine (AG) and anti-oxidant N-acetylcy-steine (NAC), The expression of MCP-1 are evaluated by Immunocytochemistry and Sandwich ELISA. MDA content and SOD activity are determined by the technique of TBA and XOD respectively. Results GSA can stimulate MCP-1 production and secretion. Immunocytochemistry showed that after HUVECs were cultured with 50 mg/L GSA, expression of MCP-1 in group 4hrs, 8hrs and 12hrs was 1.3, 1.9 and 2.8 fold as much as that in control group (P < 0.01), and there was significant difference among the experiment groups(P < 0.01). Sandwich ELISA showed that expression of MCP-1 in three different groups was 1.6, 2.4 and 3.0 fold as much as that in control group(P < 0.01), and there was significant difference among the experiment groups(P < 0.01); GSA can cause the decrease of SOD activity(P < 0.05) and increase of MDA content(P < 0.01); AG and NAC can restrain obviously the expression of MCP-1 of HUVECs stimulated by GSA(P < 0.01); NAC can restrain the effect of GSA on SOD activity and MDA content in HUVECs (P < 0.05). Conclusions GSA can stimulate the expression of MCP-1 of endothelial cells by inducing endothelial cells oxidative stress.
文摘目的:分析血清人附睾分泌蛋白4(Human epididymis protein 4,HE4)水平和子宫内膜癌患者临床病理特征的相关性.方法:回顾性分析2019年1月至2022年6月医院收治的120例子宫内膜癌患者临床资料.所有患者均实施子宫全切术治疗,收集患者的临床病理特征资料,检测所有患者HE4水平,分析血清HE4水平与子宫内膜癌患者临床病理特征的相关性.结果:120例子宫内膜癌患者中,病理类型:子宫内膜样腺癌46例、子宫内膜样透明细胞癌36例、子宫内膜样鳞癌38例;淋巴结转移:发生35例、未发生85例;肌层浸润深度:>1/2肌层40例、≤1/2肌层80例;TNM分期:Ⅰ期22例、Ⅱ期43例、Ⅲ期55例.病灶所处部位:子宫底55例、子宫角37例、宫颈管28例.不同病理类型、TNM分期、病灶所处部位患者的血清HE4水平比较,差异无统计学意义;肌层浸润深度>1/2肌层患者的血清HE4水平高于肌层浸润深度≤1/2肌层的患者,发生淋巴结转移患者的血清HE4水平较未发生淋巴结转移的患者明显增加(P<0.05).经Logistic回归显示,血清HE4水平升高是子宫内膜癌患者肌层浸润深度>1/2肌层的风险因子(OR>1,P<0.05);血清HE4水平升高是子宫内膜癌患者发生淋巴结转移的风险因子(OR>1,P<0.05).结论:血清HE4水平升高增加可加剧肌层浸润深度,促使子宫内膜癌患者发生淋巴结转移.
基金supported by the National Natural Science Foundation of China(Grant No.21434008)
文摘Currently there is no successful platform technology for the sustained release of protein drugs.It seems inevitable to specifically develop new materials for such purpose, and hence the understanding of protein–material interactions is highly desirable. In this study, we synthesized cholesterol-grafted polyglutamate(PGA-g-Chol) as a hydrophobically-modified polypeptide, and thoroughly characterized its interaction with a model protein(human serum albumin) in the aqueous solution by using circular dichroism, fluorescence methods, and light scattering. With the protein concentration fixed at 5 μmol/L,adding PGA-g-Chol polymers into the solution resulted in continuous blue shift of the protein fluorescence(from 339 to 332 nm), until the polymer molar concentration reached the same value as the protein. In contrast, the un-modified polyglutamate polymers apparently neither affected the protein microenvironment nor formed aggregates. Based on the experimental data, we proposed a physical picture for such protein–polymer systems, where the polymer first bind with the protein in a 1:1 molar ratio via a fraction of their hydrophobic pendant cholesterol resides along the polymer chain. In this protein/polymer complex, there are excess unbound cholesterol residues. As the polymer concentration increases, the polymers form multi-polymer aggregates around 200 nm in diameter via the same hydrophobic cholesterol residues. The protein/polymer complex also participate in the aggregation via their excess cholesterol residues, and consequently the proteins are encapsulated into the nanoparticles. The encapsulation was also found to increase the thermal stability of the model protein.
