亚甲蓝与多聚阴离子化合物结合反应的研究

对光谱探针亚甲蓝(MB)分别与硫酸化茯苓多糖(SP)、硫酸软骨素(CS)、透明质酸(HA)等多聚阴离子化合物相互作用的吸收光谱进行了比较研究,探讨了CS等多聚阴离子化合物与MB相互作用机理及其与MB结合的功能基团,计算了MB与SP、CS最大结合数分别为54和73。研究结果表明MB与多聚阴离子化合物的磺酸基发生明显的结合反应,与羧基不能发生明显的结合反应,多聚阴离子化合物与MB结合的功能基团是磺酸基。     

硫酸化茯苓多糖急性毒性实验研究

按10、8、6.4、5.12、4.1g/kg体重剂量的硫酸化茯苓多糖(SP)分别灌胃给药,通过观察小鼠的活动和急性毒性反应,记录小鼠的死亡数,用综合计算法计算半数致死量(LD50)等相关数据.结果表明硫酸化茯苓多糖灌胃给药的LD50为7.358/kg,LD50的95%平均可信限为(7.358±0.894)g/kg,属低毒性物质.该研究为开发应用硫酸化茯苓多糖新药提供科学的药理学依据.     

多糖硫酸酯化修饰及其抗凝血作用研究进展

硫酸多糖是一种单糖分子上的一个或多个羟基被硫酸根取代的多糖,近年来由于其良好的抗凝血活性及其较低的副作用被人们所关注.硫酸多糖包括天然硫酸多糖和化学法硫酸化多糖两种,研究证实两者都具有较好的抗凝血活性.对于硫酸多糖的抗凝血活性的构效关系的研究是改进其功能性质的核心,也是国内外研究的热点.本文综述了硫酸多糖的硫酸化方法,硫酸多糖的抗凝血活性以及机理,并对硫酸多糖的研究方向进行了展望.     

过硫酸化岩藻多糖硫酸酯

岩藻多糖硫酸酯(fucoidan或fucan sulfate,FS)是一种硫酸多糖,具有多种生物活性。硫酸基团对多糖活性起重要作用,并且多糖活性与硫酸基团含量呈正相关。近些年,国内外研究者对SF进行硫酸化修饰以获得高硫酸基团含量和高活性的过硫酸化岩藻多糖硫酸酯。为促进对FS硫酸化修饰的深入了解,本文对FS活性与硫酸基团含量及位置的关系、过硫酸化方法及过硫酸化岩藻多糖硫酸酯活性的研究进展进行了综述。     

硫酸化茯苓多糖对人乳腺癌细胞株MCF-7凋亡的影响

为了探讨硫酸化茯苓多糖(SP)对人乳腺癌细胞株MCF-7凋亡的影响,采用MTT法检测不同浓度、作用时间SP对乳腺癌MCF-7细胞的抑制作用,倒置显微镜观察MCF-7细胞的形态学变化,RT-PCR检测SP处理MCF-7细胞凋亡相关基因(Bcl-2,Bax)的表达;Western blotting技术检测SP对乳腺癌细胞凋亡蛋白Bcl-2、Bax表达变化。结果表明,SP对MCF-7细胞增殖有抑制作用,且在一定范围内呈剂量效应;细胞贴壁能力减弱,细胞间隙增大,胞膜褶皱;Bcl-2基因表达水平和蛋白表达水平明显降低(p0.05),Bax基因表达水平和蛋白表达水平明显升高(p0.05)。基于以上研究,SP通过促凋亡基因Bax的表达,抑制抗凋亡基因Bcl-2的表达来下调Bcl-2/Bax比值,激活凋亡途径,诱导MCF-7细胞的凋亡。     

灰树花子实体多糖D-组分的提取、硫酸化及免疫测定

从灰树花(Maitake)子实体中提取分离得到子实体多糖D组分,并分别考察了提取中的几个关键因素对D组分得率的影响,包括加水倍数、浸提时间、醇沉浓度、浓缩倍数。然后,对D组分进行了硫酸化修饰,得到了两种不同取代度的硫酸化多糖。最后,研究了灰树花多糖D组分及硫酸化灰树花多糖D组分对小鼠的免疫器官重量、腹腔巨噬细胞吞噬功能、血清溶血素形成及淋巴细胞转化率的影响。结果表明,在10mgkg剂量下,灰树花多糖D组分及硫酸化灰树花多糖D组分具有免疫增强作用,并且在合适的取代度下硫酸化灰树花多糖D组分比灰树花多糖D组分表现了更强的免疫调节作用。     

薤白多糖的硫酸化修饰及体外抗氧化活性

为探讨薤白多糖硫酸化修饰的最佳条件,以及硫酸化修饰提高薤白多糖活性的可能性,采用氯磺酸-吡啶法对醇沉法得到的薤白多糖和柱层析纯化的3种分级薤白多糖进行硫酸化修饰,以氯磺酸-吡啶配比、反应温度和反应时间为自变量,修饰产物的硫酸基取代度(DS)为响应值,应用响应面设计法确定硫酸化修饰的最佳条件,用H2O2/Fe2+体系法和邻苯三酚自氧化法测定修饰产物的抗氧化活性。结果表明:薤白多糖氯磺酸-吡啶法修饰的最佳条件为氯磺酸∶吡啶=1∶3,反应温度65℃,反应时间2 h,此条件下硫酸根取代度为0.470,硫酸化修饰能提高薤白多糖的体外抗氧化活性。     

多糖硫酸化修饰和多糖硫酸酯的研究进展

硫酸多糖是一类糖羟基上带有硫酸根的多糖,包括从植物中提取的各种硫酸多糖、肝素、天然多糖的硫酸衍生物及人工合成的各种硫酸多糖。硫酸多糖具有抗病毒、抗肿瘤、抗凝血和增强免疫等生物活性,为提高中药多糖的生物活性,可通过硫酸化修饰的方法进行结构改造,获取多糖硫酸酯。本文就多糖的硫酸化修饰方法、多糖硫酸酯的生物学活性及其影响因素、作用机制和临床应用进行了综述。     

糖枣多糖经硫酸化修饰前后对酪氨酸酶抑制作用分析

为促进湖南地区枣果资源的开发与利用,探索糖枣多糖对酪氨酸酶活性的影响。以三年生糖枣为试材,将其水提多糖依次采用DEAE-52纤维素和Sephadex G-100葡聚糖柱层析,分离出多糖组分;分别测定各组分多糖经硫酸化修饰前后对酪氨酸酶的抑制作用,并对抑制活性最强的部分进行动力学研究。结果表明:DT_(B3)的酶活性抑制率最高,达到77.94%,DT_C的酶活性抑制率次之为69.93%,其余组分对酶活性抑制率均小于50%;经硫酸化修饰后,多糖组分DT_(B3)和DT_C对酪氨酸酶抑制率变化较小,DT_A、DT_(B1)、DT_(B2)对酪氨酸酶抑制率在原有的基础上都有较大的提高,分别提高了60.19%、41.66%、29.55%,其抑制率分别达到了56.54%、58.53%、61.60%,但都远低于70%。同时DT_(B3)能在短时间内与酶快速的结合,并显示出较强的活性抑制能力,且呈现非竞争性抑制类型。由上可知,各组分多糖对酪氨酸酶活性抑制率的大小存在一定的差异,多糖经硫酸化修饰后对酪氨酸酶的抑制率具有一定的影响,糖枣多糖中DT_(B3)对酪氨酸酶具有较高的抑制作用,存在一定的开发利用价值。     

响应面法优化猴头菌硫酸化多糖

[目的]为了获得猴头菌多糖的最佳硫酸化修饰条件,提高猴头菌硫酸化多糖硫酸基团的取代度。[方法]采用单因素比较氯磺酸-吡啶摩尔比、反应温度和反应时间对猴头菌硫酸化多糖取代度的影响,利用响应面法对各因素的最佳水平以及各因素之间的交互作用进一步研究。[结果]猴头菌多糖的最佳硫酸化修饰条件为:氯磺酸-吡啶摩尔比为1:4、反应温度为59℃、反应时间为2. 6 h,取代度为0. 457,与模型的预测值基本相符。[结论]响应面法优化得到猴头菌多糖的硫酸化修饰条件参数准确,该模型可以用于猴头菌多糖硫酸化修饰条件的优化。     

Secondary structure of substance P bound to liposomes in organic solvents and in solution from Raman and CD spectroscopy.

The membrane lipid phase may be an important mediator of the peptide-receptor interaction. In order to understand the mechanism of this interaction, it is important to know the peptide structure, not only in the hydrophobic lipid bilayer environment, but also at the bilayer surface and in solution. To investigate this problem we have measured the secondary structure of the 11-residue neuropeptide substance P (SP) and its fragments in aqueous solutions, in membrane mimetic solvents, and associated with lipid bilayers using Raman and CD spectroscopy. Raman and CD spectra of SP bound to liposomes indicate a less than 20% helix content. We interpret these results to indicate that SP contains virtually no helix when bound to negatively charged liposomes. These spectra are similar to spectra of peptides in type I and III beta-turns. SP forms between 10 and 30% (1-3 residues) helical structure in sodium dodecyl sulfate micelles and less than 10% helix in methanol and trifluoroethanol. The binding of SP to negatively charged liposomes significantly changes the structure of the lipid acyl chains, decreasing order in some cases and increasing it in others. Raman spectra of SP in water indicates that SP near 30 mM forms an ensemble of structures in water that is distinct from completely unfolded peptide and from the aggregated beta-sheet form observed in saline solutions. We conclude from our CD results that methods used to quantitate secondary structure from CD spectra of short peptides cannot be used to distinguish between very short helical segments and beta-turns.     

The interaction between cholesterol and human serum albumin

The interaction between cholesterol and Human Serum Albumin (HSA) was studied by fluorescence technique. Addition of cholesterol causes decreasing of the fluorescence intensity of HSA and the mechanism can be attributed to static quenching. Both negative enthalpy and entropy change indicate this binding was an "enthalpy-driven" reaction. The number of binding site and distance between residues and ligands were also calculated: n = 0.98, r = 3.84 nm. UV-vis spectra showed HSA molecules unfolded to some extent and the hydrophobicity was decreased in the presence of cholesterol.     

Investigation of the Interaction Between Human Serum Albumin and Antitumor Palladium(II) Complex Containing 1,10-Phenanthroline and Dithiocarbamate Ligands

The interaction between [Pd(But-dtc)(phen)]NO₃ (where But-dtc = butyldithiocarbamate and phen = 1,10-phenanthroline) with HSA (Human Serum Albumin) was investigated by applying fluorescence, UV–Vis and circular dichroism techniques under physiological conditions. The results of fluorescence spectra indicated that the Pd(II) complex could effectively quench the fluorescence intensity of HSA molecules via static mechanism. The number of binding sites and binding constant of HSA–Pd(II) complex were calculated. Analysis of absorption titration data on the interaction between Pd(II) complex and HSA revealed the formation of HSA–Pd(II) complex with high-binding affinity. Thermodynamic parameters indicated that hydrophobic forces play a major role in this interaction. Furthermore, CD measurements were taken to explore changes in HSA secondary structure induced by the Pd(II) complex.     

Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14(+) peripheral blood monocytes.

Activation of T cells by Ag or stimulation of monocytes with inflammatory cytokines induces CD44 to bind to hyaluronan (HA), an adhesion event implicated in leukocyte-leukocyte, leukocyte-endothelial cell, and leukocyte-stromal cell interactions. We have previously shown that TNF-alpha induces CD44 sulfation in a leukemic cell line, which correlated with the induction of HA binding and CD44-mediated adhesion. In this study, we establish that TNF-alpha and IFN-gamma induce HA binding and the sulfation of CD44 in CD14(+) PBMC, whereas no induced HA binding or CD44 sulfation was observed in CD14(-) PBMC stimulated with TNF-alpha. Treatment of cells with NaClO(3), an inhibitor of sulfation, prevented HA binding in a significant percentage of CD14(+) PBMC induced by TNF-alpha, LPS, IL-1beta, or IFN-gamma. Furthermore, stimulation with TNF-alpha or IFN-gamma in the presence of NaClO(3) reduced the ability of isolated CD44H to bind HA, demonstrating a direct effect of CD44H sulfation on HA binding. In contrast, the transient induction of HA binding in T cells by PHA was not affected by NaClO(3), suggesting that activated T cells do not use sulfation as a mechanism to regulate HA binding. Overall, these results demonstrate that inducible sulfation of CD44H is one mechanism used by CD14(+) peripheral blood monocytes to induce HA binding in response to inflammatory agents such as TNF-alpha and IFN-gamma.     

Study on the binding of cerium to bovine serum albumin

The interaction of Ce(3+) to bovine serum albumin (BSA) has been investigated mainly by fluorescence spectra, UV-vis absorption spectra, and circular dichroism (CD) under simulative physiological conditions. Fluorescence data revealed that the quenching mechanism of BSA by Ce(3+) was a static quenching process, the binding constant is 6.70 × 10(5) , and the number of binding site is 1. The thermodynamic parameters (ΔH = -29.94 kJ mol(-1) , ΔG = -32.38 kJ mol(-1) , and ΔS = 8.05 J mol(-1) K(-1) ) indicate that electrostatic effect between the protein and the Ce(3+) is the main binding force. In addition, UV-vis, CD, and synchronous fluorescence results showed that the addition of Ce(3+) changed the conformation of BSA.     

Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor

The acidic region of the Factor VIII light chain was studied with regard to structural requirements for the formation of a functional von Willebrand factor (vWF)-binding site. Factor VIII mutants lacking the B domain, with additional deletions and an amino acid replacement within the sequence 1649-1689 were constructed using site-directed mutagenesis and expressed in Cos-1 cells. These mutants, which were recovered as single-chain molecules with similar specific activities, were compared in their binding to immobilized vWF. Deletion of amino acids 741-1648 or 741-1668 did not affect the binding of Factor VIII to vWF. However, a mutant with a deletion of residues 741-1689 was no longer capable of interacting with vWF. This indicates a role for residues within the sequence 1669-1689 in the formation of a vWF-binding site. When recombinant Factor VIII was expressed in the presence of chlorate, an inhibitor of protein sulfation, the resulting Factor VIII displayed strongly reduced binding to vWF. vWF binding was completely abolished when within the sequence 1669-1689 the tyrosine residue Tyr1680, which is part of a consensus tyrosine sulfation sequence, was replaced by phenylalanine. The Factor VIII sequence 1673-1689 was identified as a high affinity substrate for tyrosylprotein sulfotransferase (Km = 57 microM) in cell-free sulfation studies. It is concluded that sulfation of Tyr1680 is required for the interaction of Factor VIII with vWF. Two synthetic peptides that represent the sequence 1673-1689, but differ with respect to sulfation of Tyr1680 are shown to have vWF binding affinity that is considerably lower than the Factor VIII protein. Several models to accommodate our findings are discussed.     

Surface-enhanced Raman and steady fluorescence study of interaction between antitumoral drug 9-aminoacridine and trypsin-like protease related to metastasis processes, guanidinobenzoatase

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) were applied to study the interaction of the antitumoral drug 9-aminoacridine (9AA) with a trypsin-like protease guanidinobenzoatase (GB) extracted from a mouse Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission was detected in the emission fluorescence spectra at certain drug and enzyme concentrations. A SERS study was accomplished on silver colloids at several excitation wavelengths in order to obtain more information about the interaction mechanism. The results derived from Raman spectroscopy indicated that 9AA in the amino monomeric form may interact with the enzyme by means of two different bonds: an ionic bond with a negatively charged amino acid and a ring stacking interaction with an aromatic residue placed in the catalytic site of GB. This interaction mechanism was responsible for a strong exciplex emission detected at a longer wavelength than the expected value of the normal fluorescence emission. Moreover, the GB concentration dependence of the interaction suggested that the drug was sensitive to the quaternary structure of the enzyme.     

Study on the interaction between the inclusion complex of hematoxylin with β-cyclodextrin and DNA

Ultraviolet-visible (UV-vis) spectra, fluorescence spectra, electrochemistry, and the thermodynamic method were used to discuss the interaction mode between the inclusion complex of hematoxylin with β-cyclodextrin and herring sperm DNA. On the condition of physiological pH, the result showed that hematoxylin and β-cyclodextrin formed an inclusion complex with binding ratio n(hematoxylin):n(β-cyclodextrin) = 1:1. The interaction mode between β-cyclodextrin-hematoxylin and DNA was a mixed binding, which contained intercalation and electrostatic mode. The binding ratio between β-cyclodextrin-hematoxylin and DNA was n(β-cyclodextrin -hematoxylin):n(DNA) = 2:1, binding constant was K(?)(298.15K) = 5.29 × 10? L·mol?1, and entropy worked as driven force in this action.