红细胞羰基毒化及谷胱甘肽的保护作用

探索丙二醛（MDA）对红细胞的羰基毒化过程,以及谷胱甘肽（GSH）的拮抗作用．采用血液粘度测量、扫描电镜观察、羰基化蛋白含量测定以及荧光偏振度检测等方法,从红细胞表观粘度、红细胞形态、红细胞膜上羰基化蛋白含量以及红细胞膜脂流动性4个指标上进行研究．MDA导致的羰基应激造成红细胞损伤和血液粘度增加,而GSH可阻止羰基应激或还原羰．氨反应产物,且具有浓度依赖性．揭示了羰基应激可能是血瘀证的关键生化原因之一．为抗应激、抗衰老提供了理论和实验的重要依据．     

水溶性壳聚糖与牛血清白蛋白相互作用的研究

采用紫外和荧光光谱研究了水溶性壳聚糖(CS)与牛血清白蛋白(BSA)之间的相互作用。结果表明:随CS浓度的增加,BSA的紫外吸收光谱表现出明显的增色效应和较小的紫移;CS可以猝灭BSA的内源荧光,其猝灭机理是CS与BSA形成复合物的静态猝灭。并且测定了在不同温度下,该反应的结合常数KA分别为6.92×106(298 K),5.01×106(308 K),3.31×106(318 K),CS与BSA以摩尔比1∶1结合。同时采用同步荧光光谱法探讨了CS对BSA构象的影响。     

华蟾素对MDA - MB -231细胞线粒体膜电位及活性氧的影响

目的:研究华蟾素诱导乳腺癌MDA - MB - 231细胞凋亡过程中,细胞内活性氧(ROS)及线粒体膜电位(△Ψm)的变化,探讨华蟾素对乳腺癌细胞的作用机制.方法:用不同浓度的华蟾素作用于MDA - MB - 231细胞24h后,分别用荧光探针罗丹明123和荧光探针DCFH-DA进行荧光染色,用流式细胞仪检测细胞内线粒体膜电位和活性氧的变化.结果:不同浓度的华蟾素作用于MDA - MB - 231细胞后,随着药物浓度的增加(0、12.5、25、37.5、50μg/ml),细胞内的ROS水平显著升高,荧光强度从3 609±24上升为6 263±35;同时,线粒体膜电位(△Ψm)显著下降,荧光强度从242±6降低到173±4.结论:华蟾素作用细胞后,使得细胞内活性氧水平显著升高,同时,线粒体膜电位显著下降,推测华蟾素对MDA - MB - 231细胞通过线粒体途径诱发细胞凋亡.     

胶毒素与BSA的相互作用

应用荧光、圆二色和紫外—可见吸收等波谱法研究胶毒素与牛血清白蛋白(BSA)的相互作用。荧光光谱实验结果表明胶毒素主要靠疏水作用与BSA结合, 而对其内源荧光产生猝灭作用,其淬灭方式为静态猝灭, 胶毒素与BSA的结合常数为7.2×103 L/mol。圆二色光谱检测发现, 随着胶毒素浓度的增加, BSA的a-螺旋数量也增加, 当胶毒素浓度为BSA浓度的100倍时, BSA的a-螺旋增加40.1%, 表明胶毒素与BSA的结合改变了BSA的空间构象。     

金霉素单克隆抗体的制备及检测方法的建立

采用羰基二咪唑法,将半抗原金霉素(AM)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制备金霉素免疫抗原AM-BSA和检测抗原AM-OVA,通过紫外光谱扫描检测偶联产物。采用细胞杂交瘤技术,制备抗金霉素单克隆抗体杂交瘤细胞株,建立了金霉素竞争ELISA检测方法,其灵敏度达到50ng/ml,且呈现良好的线性关系(r=0.9812),并且与其他抗生素无交叉反应。     

叶绿素荧光动力学检测塑化剂对尖细栅藻的毒性效应

以汉江水华藻类尖细栅藻Scenedesmus acuminatus为材料, 研究了塑化剂(邻苯二甲酸二异辛酯, DEHP)对其光合系统的毒性效应。以不同浓度的DEHP处理S.acuminatus, 分析其光合系统电子传递特征及相关参数, 以及快速叶绿素荧光诱导动力曲线, 发现低浓度的DEHP (10 mg/L)对S.acuminatus活性反应中心的电子传递链中QA-的还原有促进作用, 而高浓度(50100 mg/L)则显著抑制; 光合活性则与DEHP浓度呈效应-剂量关系, 最大光化学效率降低; 同时DEHP显著降低SOD活性, 增加MDA含量, 而且随DEHP浓度升高, 处理时间的延长, 效果越明显。研究结果表明, DEHP处理对S.acuminatus的光合系统传递链和抗氧化系统有一定的影响,说明快速荧光动力方法能够应用于DEHP毒性效应的快速检测。     

CLIC1通过干扰线粒体动力学平衡促进内皮细胞损伤的研究

该文通过研究H2O2诱导人脐静脉内皮细胞(HUVEC)中氯离子通道蛋白1(chloride intracellular channel 1, CLIC1)对线粒体动力学平衡的影响,探讨CLIC1在内皮细胞损伤中的作用及机制。体外培养HUVEC细胞,分别用CLIC1抑制剂IAA94(40μmol/L)、H2O2(0.9 mmol/L)、IAA94(40μmol/L)和H2O2(0.9 mmol/L)联合处理,荧光法检测细胞活性氧(reactive oxygen species,ROS)和丙二醛(malondialdehyde, MDA)的含量; JC-1染色法检测细胞线粒体膜电位的变化;定量PCR技术检测CLIC1、线粒体动力相关蛋白1(dynamin-related protein 1, Drp1)以及线粒体融合蛋白1(mitofusin 1, Mfn1)的mRNA表达;免疫印迹技术检测CLIC1、Drp1蛋白的水平。结果显示:与正常组相比, H2O2处理的内皮细胞中ROS、MDA含量增加(P0.05), CLIC1表达量上调(P0.05),三磷酸腺苷(ATP)含量减少(P0.05),线粒体膜电位降低(P0.001),线粒体融合蛋白Mfn1表达显著降低(P0.05),线粒体分裂蛋白Drp1表达显著升高(P0.05);而IAA94预处理2 h后,内皮细胞中ROS、MDA含量减少(P0.05),线粒体融合蛋白Mfn1表达显著增加(P0.05),线粒体分裂蛋白Drp1表达显著降低(P0.05),线粒体膜电位升高(P0.001)。以上结果表明, CLIC1在H2O2诱导的内皮细胞线粒体损伤中发挥重要作用,其机制可能与CLIC1干扰线粒体动力学平衡有关。     

食物氧化蛋白对小鼠肠道菌群及氧化还原状态的影响

目的研究摄食不同方式氧化酪蛋白对小鼠肠道菌群和氧化还原状态的影响。方法分别以H2O2/Cu2+、HClO处理酪蛋白,丙二醛(MDA)处理酪蛋白、大豆蛋白。雄性KM小鼠分为6组:酪蛋白组,H2O2-Cu2+氧化酪蛋白组,HClO氧化酪蛋白组,MDA氧化酪蛋白组,大豆蛋白组和MDA氧化大豆蛋白组,饲喂10周。结果酪蛋白和大豆蛋白经氧化处理后羰基含量显著上升(P0.05),体外消化率下降。饲喂氧化蛋白饲粮的小鼠结肠内容物乳杆菌数量均显著低于对照组(P0.05);HClO和MDA氧化酪蛋白组大肠埃希菌、肠球菌数量显著高于对照组(P0.05),MDA氧化大豆蛋白组大肠埃希菌数量显著高于对照组(P0.05)。氧化蛋白处理引起小鼠结肠组织MDA上升,其中MDA氧化蛋白处理达显著水平(P0.05);结肠过氧化氢酶(CAT)活力上升,其中H2O2/Cu2+和MDA氧化蛋白组达显著水平(P0.05);H2O2/Cu2+氧化酪蛋白处理引起结肠GSH-Px显著升高(P0.05);氧化蛋白引起结肠总抗氧化能力(T-AOC)下降,其中H2O2/Cu2+、HClO氧化酪蛋白和MDA氧化大豆蛋白处理达显著水平(P0.05)。蛋白质体外消化率与结肠肠球菌呈负相关(R=-0.81,P=0.051);蛋白羰基含量与结肠乳杆菌呈显著负相关(R=-0.94,P0.01);大肠埃希菌(R=0.93,P0.01)和肠球菌(R=0.85,P0.05)分别与蛋白羰基含量呈正相关。结论氧化后蛋白消化率降低、羰基含量增高,导致肠道乳杆菌减少,大肠埃希菌和肠球菌上升;结肠黏膜脂质过氧化,氧化损伤程度与蛋白氧化处理方式有关。     

莲心碱通过抑制NF-κB激活Nrf2/HO-1通路发挥抗炎抗氧化作用

目的 探讨莲心碱(liensinine, lie)在脂多糖(lipid polysaccharides, lPS)诱导的RAW264.7细胞炎症模型中的抗炎抗氧化作用机制。方法 将细胞分为对照组(Control group),LPS组(LPS group),莲心碱(0.5、1、2μmol/L)组。采用CCK-8法筛选莲心碱的无细胞毒性浓度,Griess法检测一氧化氮(NO)的浓度,酶联免疫法(ELISA)检测前列腺素2(PGE2)的浓度以及促炎因子(TNF-α、IL-6和IL-1β)的含量、实时荧光定量PCR(qRT-PCR)检测COX-2和iNOS mRNA的表达,DCFH-DA探针检测细胞中活性氧(ROS)含量,试剂盒检测细胞中超氧化物歧化酶(SOD)的活性以及谷胱甘肽(GSH)和丙二醛(MDA)的含量,蛋白质免疫印迹法(Western Blot)检测NF-κB、MAPK和Nrf2/HO-1通路中关键蛋白的表达情况。结果 0～4μmol/L莲心碱对细胞活力无影响,与LPS组相比,莲心碱组(0.5、1、2μmol/L)细胞中的促炎因子含量下降(P<0.05),NO和PGE2浓度...     

葎草幼苗的生理生化特征对盐胁迫的响应

采用盆栽法研究葎草(Humulus scandens)幼苗的生物量、气体交换参数、叶水势、叶绿素含量、叶绿素荧光参数、抗氧化酶活性及丙二醛(MDA)含量对不同浓度盐(NaCl)(分别为0、100、200、300、400mmol/L)胁迫的响应,以探讨葎草幼苗对盐胁迫的适应机制及耐性机理。结果表明:(1)随着NaCl胁迫浓度的增加,葎草幼苗生长受到严重抑制,显著降低了根、茎、叶的干重、单叶面积、比叶面积(SLA)和根冠比(R/S)。(2)NaCl胁迫后,葎草幼苗的净光合速率(Pn)、气孔导度(Gs)、胞间CO_2浓度(Ci)、蒸腾速率(Tr)和叶水势(Ψw)均随盐浓度增加呈显著下降趋势,气孔限制值(Ls)和瞬时水分利用效率(WUEi)却显著增加。(3)NaCl胁迫下,叶绿素a(Chl a)和叶绿素b(Chl b)等光合色素含量以及过氧化氢酶(CAT)活性均显著降低,但超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性和丙二醛(MDA)含量显著升高,说明植株的抗氧化酶系统对NaCl胁迫具有积极的响应策略。(4)NaCl胁迫显著提高了幼苗最小荧光(F_o)、可变荧光(F_v)、最大荧光(F_m)、表观光合电子传递速率(ETR)和光化学淬灭系数(qP),有效降低了PSⅡ有效光化学量子产量(F_m′/F_o′)、非光化学淬灭系数(q N),并在不同盐浓度梯度下存在着显著的效果差异。研究表明,低浓度(100mmol/L)NaCl胁迫对葎草幼苗生长没有显著影响,但随着浓度的增加,葎草幼苗的气体交换、叶绿素含量、PSⅡ反应能力、抗氧化酶系统均受到抑制,植株的生长发育受到严重影响。     

Reaction of pyridoxamine with malondialdehyde: Mechanism of inhibition of formation of advanced lipoxidation end-products

Summary. Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) are implicated in many age-related chronic diseases and in protein aging. Recent studies suggest that pyridoxamine (PM) is an efficient AGEs/ALEs inhibitor in various biological systems. Because malondialdehyde (MDA) is an important intermediate in the formation of ALEs during lipid peroxidation, the purpose of this study is to determine whether PM can trap MDA directly and thereby prevent ALEs formation. PM reacted readily with MDA under physiological conditions. Within 6 h, a 1-pyridoxamino-propenal adduct derived from reaction of equimolar PM + MDA was detected. A 1-amino-3-iminopropene complex and a dihydropyridine-pyridinium complex were also identified after 7 d incubation. PM also greatly inhibited the lipofuscin-like fluorescence formation induced by MDA reaction with bovine serum albumin (BSA). Our results showed clearly that PM inhibited the formation of ALEs by trapping MDA directly under physiological condition, and provide insight into the mechanism of action of PM in protecting proteins against carbonyl stress.     

Spectral diagnostics of the interaction between pyridoxine hydrochloride and bovine serum albumin in vitro

The interaction between pyridoxine hydrochloride (VB6) and bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence spectroscopy and UV-visible absorption spectra. The quenching mechanism of fluorescence of BSA by VB6 was discussed. The number of binding sites n and observed binding constant K(b) was measured by fluorescence quenching method. The thermodynamic parameters DeltaH(theta), DeltaG(theta), DeltaS(theta) at different temperatures were calculated and the results indicate the binding reaction is mainly entropy-driven and hydrophobic interaction played major role in the reaction. The distance r between donor (BSA) and acceptor (VB6) was obtained according to FOrster theory of non-radiation energy transfer. Synchronous fluorescence and three-dimensional fluorescence spectra were used to investigate the structural change of BSA molecules with addition of VB6, the result indicates that the secondary structure of BSA molecules is changed in the presence of VB6.     

Experimental,computational and chemometrics studies of BSA-vitamin B6 interaction by UV–Vis,FT-IR,fluorescence spectroscopy,molecular dynamics simulation and hard-soft modeling methods

The interaction of pyridoxine (Vitamin B6) with bovine serum albumin (BSA) is investigated under pseudo-physiological conditions by UV–Vis, fluorescence and FTIR spectroscopy. The intrinsic fluorescence of BSA was quenched by VB6, which was rationalized in terms of the static quenching mechanism. According to fluorescence quenching calculations, the bimolecular quenching constant (k_q), dynamic quenching (K_SV) and static quenching (K_LB) at 310 K were obtained. The efficiency of energy transfer and the distance between the donor (BSA) and the acceptor (VB6) were calculated by Foster’s non-radiative energy transfer theory and were equal to 41.1% and 2.11 nm.The collected UV–Vis and fluorescence spectra were combined into a row-and column-wise augmented matrix and resolved by multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS helped to estimate the stoichiometry of interactions, concentration profiles and pure spectra for three species (BSA, VB6 and VB6-BSA complex) existed in the interaction procedure. Based on the MCR-ALS results, using mass balance equations, a model was developed and binding constant of complex was calculated using non-linear least squares curve fitting. FT-IR spectra showed that the conformation of proteins was altered in presence of VB6. Finally, the combined docking and molecular dynamics (MD) simulations were used to estimate the binding affinity of VB6 to BSA. Five-nanosecond MD simulations were performed on bovine serum albumin (BSA) to study the conformational features of its ligand binding site. From MD results, eleven BSA snapshots were extracted, at every 0.5 ns, to explore the binding affinity (GOLD score) of VB6 using a docking procedure. MD simulations indicated that there is a considerable flexibility in the structure of protein that affected ligand recognition. Structural analyses and docking simulations indicated that VB6 binds to site I and GOLD score values depend on the conformations of both BSA and ligand. Molecular modeling results showed that VB6–BSA complex formed not only on the basis of electrostatic forces, but also on the basis of π–π staking and hydrogen bond. There was an excellent agreement between the experimental and computational results. The results presented in this paper, will offer a reference for detailed and systematic studies on the biological effects and action mechanism of small molecules with proteins.     

吡哆胺-一种天然的AGEs/ALEs抑制剂

衰老及老年相关疾病,如：糖尿病、动脉粥状硬化、各种神经退行性疾病等,与组织蛋白氧化修饰密切相关．在造成蛋白质氧化修饰的反应中,非酶糖基化和脂质过氧化是最重要的两类,它们最终形成非酶糖基化终产物(AGEs)和脂过氧化终产物(ALEs)．基于羰基毒害衰老理论,具有强烈反应活性的羰基类化合物是非酶糖基化和脂质过氧化的共同中间产物,它们是造成蛋白修饰的直接原因之一．吡哆胺是维生素B6的一种天然成分;由于它能直接清除羰基类化合物,从而抑制AGEs／ALEs的生成;又因为吡哆胺对人体副作用很小．因此吡哆胺有望成为一种新型的防治多种老年相关疾病的药物．     

Escherichia coli Heat-Labile Enterotoxin Binds to Glycosylated Proteins with Lactose by Amino Carbonyl Reaction

The binding of Escherichia coli heat-labile enterotoxin (LT) type I to glycosylated proteins with lactose (Galβ1-4Glc) by amino carbonyl reaction was studied by the Western blot assay and by the microtiter well binding assay. LT bound to a lactose-α-lactalbumin amino carbonyl product (Lac-LA), whereas cholera toxin did not. The binding ability of Lac-LA was abolished by β-galactosidase treatment, indicating that the terminal galactose is essential for the binding of LT. The binding of LT to Lac-LA was inhibited by galactose and lactose, and most effectively inhibited by lactulose (Galβ1-4Fru), which is a structural analog of the Amadori rearrangement product of the amino carbonyl reaction between lactose and an ε-amino group of a lysine residue (lactuloselysine). The results suggest that LT recognizes the portion of lactuloselysine in Lac-LA. LT also bound to a melibiose (Galα1-6Glc)-α-lactalbumin amino carbonyl product (Mel-LA), but the binding ability of Mel-LA was weaker than that of Lac-LA, suggesting that the β1-4 linked terminal galactose is dispensable but preferable for the binding. Furthermore, LT bound to the amino carbonyl products of lactose with β-lactoglobulin, caseins, bovine serum albumin, and ovalbumin. These results indicate that LT binds to the amino carbonyl products between proteins and sugars containing the terminal galactose, such as lactose.     

Sensitive determination of protein based on the fluorescence enhancement effect of terbium (III)–epinephrine–protein–sodium dodecylsulfate system

It was found that the fluorescence of Tb³⁺–epinephrine (E) complex can be enhanced by both bovine serum albumin (BSA) and sodium dodecylsulfate (SDS), and stabilized by ascorbic acid (AA). It is considered that the fluorescence enhancement of the Tb³⁺–E–BSA–AA–SDS system originates not only from the hydrophobic microenvironment provided by BSA–SDS, but also from the energy transfer from BSA to Tb³⁺ in this system. Therefore, a new fluorescence method for the determination of protein concentrations as low as 1.3 × 10^?9 g mL^?1 BSA is established using Tb³⁺–epinephrine complex as probe. The method has been applied for the determination of BSA and human serum albumin in actual samples, and the results obtained are satisfactory. Compared with other fluorescence methods, this method is simpler and more sensitive for the determination of protein. The mechanism of the fluorescence enhancement of the system is studied in detail. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct reaction of taurine with malondialdehyde: evidence for taurine as a scavenger of reactive carbonyl species

Though taurine has been reported very useful in preventing oxidative stress and various age-related diseases, the detailed biochemical mechanism of its biological functions is not well understood. Direct reaction of malondialdehyde (MDA) with taurine was studied using spectrofluorometry, spectrophotometry and liquid chromatography online with mass spectrometry (LC/MS). The results indicated that taurine reacted readily with MDA at supraphysiological conditions to yield mainly two products: a fluorescent 1,4-dihydropyridine and non-fluorescent enaminal derivatives. Taurine also significantly inhibited the formation of lipofuscin-like fluorescence induced by MDA-modified bovine serum albumin. These findings suggested that taurine effectively reduces carbonyl stress due to the amino group in its molecular structure, and we propose that it should be the mechanism related with the pathophysiological functions of taurine in the biological system.     

Detection of an advanced glycosylation product bound to protein in situ

Protein amino groups can react with glucose without the aid of enzymes to form stable Amadori products containing 1-amino-1-deoxyketose residues. These adducts can undergo subsequent rearrangements and dehydrations to form various brown and fluorescent pigments. Recently, a chromophore, 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI), was isolated from acid hydrolysates of bovine serum albumin (BSA) and poly-L-lysine which had been incubated with glucose. To confirm the presence of FFI in situ, a radioimmunoassay was developed. A derivative of FFI, 4-furanyl-2-furoyl-1H-imidazole-1-hexanoic acid, was coupled to BSA and used to immunize rabbits. A radioactive FFI derivative was synthesized by reaction of 2-furyl-glyoxal with gamma-amino-[2,3-3H]butyric acid to form FFI-[3H]butyric acid. The resultant antiserum showed binding affinity to FFI and cross-reactivity for related compounds. FFI bound to proteins was liberated by acid hydrolysis or digestion by proteinase K prior to measurement. A linear relationship was seen between the amount of FFI equivalent detected and the amount of acid hydrolysate or enzymatic digest assayed. Poly-L-lysine and BSA incubated with glucose showed a time-dependent increase in the amounts of fluorescence and FFI equivalence. The detection of a time-related increase in the amount of FFI or a closely related structure in enzymatically digested proteins implicates it as an in situ product on proteins which have undergone the Maillard reaction with glucose. Of physiological significance is that FFI could also be detected in human globin and serum albumin from normal individuals. Thus, proteins exposed to glucose in vitro and in vivo form FFI as an in situ glycosylation product.     

Conversion of lysine to N(epsilon)-(carboxymethyl)lysine increases susceptibility of proteins to metal-catalyzed oxidation.

Metal-catalyzed oxidation (MCO) of proteins leads to the conversion of some amino acid residues to carbonyl derivatives, and may result in loss of protein function. It is well documented that reactions with oxidation products of sugars, lipids, and amino acids can lead to the conversion of some lysine residues of proteins to N(epsilon)-(carboxymethyl)lysine (CML) derivatives, and that this increases their metal binding capacity. Because post-translational modifications that enhance their metal binding capacity should also increase their susceptibility to MCO, we have investigated the effect of lysine carboxymethylation on the oxidation of bovine serum albumin (BSA) by the Fe(3+)/ascorbate system. Introduction of approximately 10 or more mol CML/mol BSA led to increased formation of carbonyls and of the specific oxidation products glutamic and adipic semialdehydes. These results support the view that the generation of CML derivatives on proteins may contribute to the oxidative damage that is associated with aging and a number of age-related diseases.     

Direct reaction of taurine with malondialdehyde: evidence for taurine as a scavenger of reactive carbonyl species

Abstract

Though taurine has been reported very useful in preventing oxidative stress and various age-related diseases, the detailed biochemical mechanism of its biological functions is not well understood. Direct reaction of malondialdehyde (MDA) with taurine was studied using spectrofluorometry, spectrophotometry and liquid chromatography online with mass spectrometry (LC/MS). The results indicated that taurine reacted readily with MDA at supraphysiological conditions to yield mainly two products: a fluorescent 1,4-dihydropyridine and non-fluorescent enaminal derivatives. Taurine also significantly inhibited the formation of lipofuscin-like fluorescence induced by MDA-modified bovine serum albumin. These findings suggested that taurine effectively reduces carbonyl stress due to the amino group in its molecular structure, and we propose that it should be the mechanism related with the pathophysiological functions of taurine in the biological system.