树舌多糖结构修饰前后CD谱比较研究

树舌多糖ＣＦ２ａ经１５ｍｍｏｌ／Ｌ１０＾－４ｈ选择化,继以氯酸氧化、得修饰树舌多糖ＣＦ２ａｍ,其结构分析表明侧链氧化率为６５％,以Ｊ－５００Ｃ圆二色性仪比较测定ＣＦ２ａ,ＣＦ２ａ一刚果红复合物及ＣＦ２ａｍ在不同ｐＨ下的ＣＤ谱,结果表明紫外区中性多糖子整体修饰、即其与刚果红形成复事物时,除提供很强的外源Ｃｏｔｔｏｎ效应外也提供了一些有序结构信息。     

箬叶多糖及其化学修饰物、亚硒酸钠和GSH对Cu~(2+)诱导的LDL氧化修饰的保护作用

研究了箬叶多糖ＦⅢ－ａ及其化学修饰物、亚硒酸钠和ＧＳＨ对Ｃｕ２＋诱导的低密度脂蛋白氧化修饰的保护作用．其结果表明箬叶多糖、硫酸酯多糖、硒酸酯多糖可显著抑制脂质过氧化产物（ＴＢＡＲＳ）及荧光物质的生成,彼此之间无明显差异．但对ＶＥ的消耗有着不同的保护作用,其顺序是ＦⅢ－ａ＞Ｓ－ＦⅢ－ａ＞Ｓｅ－ＦⅢ－ａ,并且具有明显的量效关系．硒或ＧＳＨ对Ｃｕ２＋诱导的ＬＤＬ氧化修饰无明显的抑制,但联合使用在０．１２５ｍｍｏｌ／ＬＮａ２ＳｅＯ３和０．２ｍｍｏｌ／ＬＧＳＨ及１２．５μｍｏｌ／ＬＮａ２ＳｅＯ３和０．０２ｍｍｏｌ／ＬＧＳＨ的浓度下能强烈地抑制ＴＢＡＲＳ的生成,甚至比正常的ＬＤＬ还要低．但是对ＶＥ的消耗只有较弱的保护作用,硒酸酯多糖与此相似．Ｎａ２ＳｅＯ３在０．１２５ｍｍｏｌ／Ｌ时可以明显抑制荧光物质的生成．     

树舌多糖CF2a溶液中构象行为的研究

根据刚果红实验,不同ＮａＣｌ浓度中分子量测定,不同ＮａＯＨ浓度,不同ｐＨ下［ａ］ｄ,ＣＤ谱及ηＳＤ／Ｃ的变化,推测ＣＦ２ａ在纯水中为三股螺旋,在稀盐溶液２中为单股螺旋,在ＤＭＳＯ－Ｈ２Ｏ体系中,体积分数φＨ２Ｏ降至０．２后三股螺旋解离为单股螺旋。在ＤＭＳＯ中单股螺旋随脲浓度增加转为无序结构,脲,酸,碱等影响氢键因素对ＣＦ２ａ水溶液之［ａ］Ｄ,ηＳＤ／Ｃ均有显著影响,表明维持ＣＦ２ａ在溶液中有序构象     

芦丁和槲皮素对低密度脂蛋白氧化修饰的抑制作用

以低密度脂蛋白（ＬＤＬ）氧化修饰为模型和以硫代巴比妥酸反应物质（ＴＢＡＳ）生成量以及ＬＤＬ的α－Ｔｏｃｏｐｈｅｒｏｌ和荧光物质含量为指标,以时间效应和浓度效应说明槲皮素和芦丁能明显地抑制Ｃａ＾２＋诱导的ＬＤＬ氧化修饰但其抗氧化修饰的程度无明显差异。它们对已受到Ｃｕ＾２＋氧化修饰的ＬＤＬ的过氧化无明显地终止作用。     

罗丹明B标记树舌多糖的条件

通过对树舌多糖进行荧光标记,解决多糖本身缺少易于检测发光基团的难题,为进一步研究多糖的生物学活性提供一种新型可靠的辅助手段。利用树舌多糖还原性末端与罗丹明B活泼的氨基发生反应,形成树舌多糖-罗丹明B复合物,再经流水透析和分子筛进一步纯化得到罗丹明B标记的树舌多糖。对树舌多糖与罗丹明B的反应时间、反应温度和p H进行了单因素考察。试验结果证明:罗丹明B可以有效的标记树舌多糖,在反应时间24 h、p H 5、温度25℃的条件下标记效果最好。在单因素试验的基础上对罗丹明B标记树舌多糖的条件进行响应面优化,最终得到罗丹明B标记树舌多糖的最优条件为:反应时间23.10 h、温度25.83℃、p H 5.23,预测标记后样品在552 nm波长处的最大OD值为0.135。     

钙调素对细胞周期的调节

ＲＣ３细胞是一种用真核表达载体Ｐ＾ｃａｍ转染ＮＩＨ３Ｔ３细胞建成的可调钙调素（Ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）高表达细胞模型。通过分子杂交及蛋白免疫印迹方法证实在地塞米松（Ｄｅｘａｍｅｔｈａｓｏｍｅ,ＤＸＭ）作用下,ＲＣ３细胞可高表达ＣａＭ．ＣａＭ拮抗剂三氟拉嗪（ｔｒｉｆｌｕｏｐｒａｚｉｎｅ,ＴＦＰ）则使Ｇ１期细胞增加,Ｓ期细胞减少。高表达ＣａＭ使细胞分裂指数提高,Ｇ２期细胞减少,有丝分裂前期细胞     

尖吻蝮蛇毒抗凝血因子Ⅱ与活化凝血因子X的结合性质

皖南尖吻蝮蛇毒抗凝血因子Ⅱ（ＡＣＦⅡ）与活化凝血因子Ⅹ（ＦＸａ）在钙离子作用下形成了１：１的复合物,从而延长凝血时间。这个复合反应是依赖于钙离子的,ＡＣＦⅡ在没有钙离子存在条件下不能与ＦＸａ形成复合物。ＡＣＦⅡ是凝血因子Ⅸ或凝血因子Ⅹ结合蛋白家族中一个新发现的成员,其相对分子质量为２９４６８．１。ＡＣＦⅡ分子中含有２５１个氨基酸残基,其氨基酸组成与这一家族中其他成员十分相似。ＡＣＦⅡ在抗凝血反应中     

天花粉蛋白与FMP复合物的晶体结构

用浸泡法得到了天花粉蛋白（ＴＣＳ）与ＦＭＰ复合物的晶体,在ＳＩＭＥＮＮＳＸ－２００Ｂ面探测器系统上收集了一套２．０分辨率的Ｘ射线衍射数据。用同晶差值傅立叶法解析了复合物的结构,经Ｘ—ＰＬＯＲ程序修正得到了ＴＣＳ—ＦＭＰ复合物的分子结构并找出了１９７个水分子,最后的Ｒ因子为０．１７２,键长和键角的ＲＭＳ偏差分别为０．０１５和２．９２２度。ＴＣＳ—ＦＭＰ复合物中,ＦＭＰ与天花粉蛋白分子有较好的结合,其结合位置正处于根据三维结构和突变体信息推测的Ｎ一糖苷酶活性口袋之中。它的类嘌呤环夹在Ｙ７０和Ｙ１１１两个侧链环之间,与Ｙ７０环近乎平行,其Ｎ７和Ｎ６分别与ＴＣＳ分子的Ｇ１０９４羰基氧和Ｉ７１的Ｎ成氢键,Ｎ３靠近Ｒ１６３的侧链,其磷酸根则伸向活性口袋的底部,与Ｅ１８９、Ｅ１６０和Ｒ１６３等残基作用。     

槲皮素和芦丁抑制Fe~（2＋）和Cu~（2＋）诱导LDL氧化修饰的比较

槲皮素和芦丁抑制Ｆｅ￣（２＋）和Ｃｕ￣（２＋）诱导ＬＤＬ氧化修饰的比较阎道广,周玫,陈瑗（第一军医大学自由基医学研究室广州５１０５１５）关键词低密度脂蛋白的氧化修饰,桷皮素,芦丁,Ｆｅ￣（２＋）,Ｃｕ￣（２＋）黄酮类物质是自由基清除剂,能与超氧阴离子...     

He—Ne激光照射对成纤维细胞内[Ca^2＋]i浓度影响的流式细胞计测定

目的：Ｈｅ－Ｎｅ激光照射治疗的机理不明,激光照射引起细胞内Ｃａ＾２＋水平变化,为治疗机理提供理论依据。方法：Ｈｅ－Ｎｅ激光照射引起鼠成纤维细胞Ｌ９２９内［Ｃａ＾２＋］ｉ的变化,用ＨＯ３４２对细胞ＤＮＡ活性染色,Ｆｌｕｏ－３ＡＭ对细胞内Ｃａ＾２＋染色,利用ＦＣＭ同时定量分析细胞ＤＮＡ和细胞内Ｃａ＾２＋的变化。结果：激光照射１５ｍｉｎ（光剂量１１．８１Ｊ／ｃｍ＾２后,ＦＣＭ分析可见ＤＮＡ分布直方图右移     

Induced circular dichroism as a probe of Cibacron Blue and Congo Red bound to dehydrogenases

We have investigated the circular dichroism induced in Cibacron Blue and Congo Red upon binding to several dehydrogenases to probe the conformation of the bound dyes. The circular dichroism spectra of Congo Red are quite similar when the dye is bound to lactic dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase but has bands of opposite sign when bound to cytoplasmic malic dehydrogenase. The circular dichroism spectra of Cibacron Blue bound to these same dehydrogenases are quite different from one another. Since circular dichroism is sensitive to the conformation of bound dye, these differences argue for at least local changes in dye conformation or environment when bound to different dehydrogenases. Congo Red appears to be less sensitive to these effects than Cibacron Blue.     

Is Congo red an amyloid-specific dye?

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.     

Mechanism of A beta(1-40) fibril-induced fluorescence of (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene (K114)

K114, (trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene, is a fluorescent Congo Red analogue that binds tightly to amyloid fibrils, but not the monomeric proteins, with a concomitant enhancement in fluorescence. The mechanism for the low aqueous fluorescence and the subsequent enhancement by A beta(1-40) fibrils was investigated by fluorescence spectroscopy and binding analysis. K114's unusually low buffer fluorescence is due to self-quenching in sedimentable aggregates or micelles which upon interacting with amyloid fibrils undergo an enhancement in fluorescence intensity and shifts in the excitation and emission spectra. These spectral changes are suggestive of a stabilization of the phenolate anion, perhaps by hydrogen bonding, rather than an increase in the microenvironment dielectric constant or dye immobilization. 1,4-Bis(4-aminophenylethenyl)-2-methoxybenzene, which lacks the phenol moiety, and X-34, which contains a stabilized phenol (pK approximately 13.4), do not display the phenolate anion fluorescence in the presence of fibrils. The apparent affinity of K114 for fibril binding is 20-30 nM with a stoichiometry of 2.2 mol of K114/mol of A beta(1-40) monomer. Competition studies indicate that K114 and Congo Red share a site, but K114 does not bind to sites on A beta(1-40) fibrils for neutral benzothiazole (BTA-1), cationic thioflavin T, or the hydrophobic (S)-naproxen and (R)-ibuprofen molecules. Comparison of benzothiazole binding stoichiometry which has been suggested to reflect disease-relevant amyloid structures to that of Congo Red analogues which reflect total fibril content may be useful in defining biologically pertinent conformational forms of amyloid.     

Aggregation characteristics of ovalbumin in beta-sheet conformation determined by spectroscopy

Protein misfolding and aggregation are involved in a number of the so-called "conformational" diseases (e.g., transmissible spongiform encephalopathies and Alzheimer disease). The development of rational strategies to interfere with aggregation is a potential therapeutic approach that requires complete knowledge of the aggregation process. We studied the aggregation of ovalbumin in beta-sheet conformation using mainly the spectral changes in the spectra of two dyes (Congo Red and pinacyanol) caused by the aggregates. We assumed a linear model of polymerization that fit to the experimental data. The critical aggregation constant, concentration of half-aggregation, nucleation parameter, growth parameter, and number of aggregation and free energy changes (total and per residue) were determined as aggregation-related parameters. Beta-Ovalbumin aggregates in a cooperative way. Moreover, the differences between such parameters obtained with Congo Red and pinacyanol suggest that each dye interacts with the protein in its own way.     

Enlargement of anode for enhanced simultaneous azo dye decolorization and power output in air-cathode microbial fuel cell

Air-cathode, microbial fuel cells (MFC) with different anode surface areas were evaluated for simultaneous decolorization of Congo Red and bioelectricity production. Doubling the anode area from 18 to 36?cm² increased net power by 150?% (0.16–0.4?mW), normalized power (per anode surface area) by 22?% (88–107?mW?m^?2) and Congo Red decolorization by 163?% (1.6–4.2?mg?l^?1?h^?1). Quadrupling the original anode area induced an additional 5?% increase (up to 4.2?mW) in net power and 174?% increase (up to 11.5?mg?l^?1?h^?1) in Congo Red decolorization; however, normalized power decreased by 85?% (down to 58?mW?m^?2). Increased bacterial attachment could account for both the enhanced power and Congo Red decolorization in larger anode MFCs. The limited effect on power output likely arises from cathode limitation or inefficient utilization of anodes.     

N-acetoxy-2-acetylaminofluorene modification of a deoxyoligonucleotide duplex.

The carcinogen N-acetoxy-2-acetylaminofluorene was reacted with d (CCACGCACC) to form a covalent adduct with attachment at the single guanine. The sample was purified, mixed 1:1 with d (GGTGCGTGG) and studied by thermal denaturation experiments. The Tm for the mixture was 35 +/- 3 degrees C, consistent with duplex formation. The method of continuous variation shows that the modified oligomer, d (CCACGAAFCACC), forms a 1:1 duplex with d (GGTGCGTGG). Circular dichroism spectra also indicate the formation of a duplex and suggest that the modified duplex has a left-handed conformation. Addition of the intercalating drug ethidium alters the CD spectrum of the modified duplex, resulting in a CD spectrum similar to that of ethidium bound to right-handed DNA.     

Self-assembly of Congo Red—A theoretical and experimental approach to identify its supramolecular organization in water and salt solutions

The supramolecular organization of Congo Red molecules was studied to approach an understanding of the unusual complexation characteristics associated with the liquid crystalline nature of this dye. Differential scanning calorimetry (DSC) and nmr data indicate that Congo Red assembly arrangements differ in water and salt solutions. Compact, highly ordered material with a distinct melting transition is created, but not below 0.3% sodium chloride concentration. The twist in the assembly arrangement of Congo Red molecules, caused in water by repulsion, decreases when the charges are shielded, allowing for more overlapping of the naphthalene rings and their engagement in stacking interaction. The crystallization transition observed in DSC analysis of Congo Red fast-assembled by cooling in salt solutions indicates that the formation of compact crystalline mesophase material is a time-consuming process in which coplanarity and a highly ordered organization must be achieved. Two different superposition variants, called “direct” and “reversed” here, were considered fundamental to compact Congo Red organization. They correspond to optimal face-to-face ring stackings, and are formed by simple direct translation or alternative imposition of reversed (180° rotated) molecules, respectively. In NaCl solution (2.8%) there is a significant downfield chemical shift alteration of the nmr signal related to proton 8, which is in the naphthalene ring on the side opposite to the charged sulfonic group. It was associated selectively with the transition of Congo Red to compact form. This effect confirms the close approach of the sulfonic groups and proton 8, and indicates that formation of the reversed arrangement is favored in the Congo Red supramolecular organization. Molecular dynamics simulation based on AMBER 4.1 force field and analysis of electrostatic field densities around the molecule were used for comparative modeling. Molecular dynamics (150 ps) were simulated for two eight-molecule micelle models constructed to reflect direct and reversed arrangements of Congo Red molecules. Although both versions generally preserved their initial assembly structure in the simulations, the reversed version proved more stable. The proximity of the sulfonic group and proton 8, confirmed by computer analysis, explains the correlation between the formation of Congo Red micellar organization and the distinct shift alteration related to this proton, as found by nmr. © 1998 John Wiley & Sons, Inc. Biopoly 46: 267–281, 1998     

Differential Congo Red staining: The effects of pH, non-aqueous solvents and the substrate

Summary Congo Red is an acid-base indicator dye. In free solution the colour and absorption characteristics of Congo Red depend not only on the pH but are also governed by the nature of the solvent environment. In tissue sections stained by Congo Red, alteration of the pH and the use of non-aqueous solvents can effect differential colouring of the tissue components. Stained sections of unmodified and chemically substituted celluloses show that differential red or blue coloration reflects the acidic or basic character of the substrate. In stained tissue sections, secondary protein structure and porosity of the substrate may also influence their colour. The effect of non-aqueous solvents is probably to modify the ionization state of the dye- substrate complex, thus altering the colour of the Congo Red. Such solvents may also change the aggregation or solvation states of the dye, with consequent modification in the colour of tissue components.     

Approaches to discovery and characterization of inhibitors of amyloid beta-peptide polymerization

Polymerization of the amyloid beta-peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease (AD). Inhibition of the formation of these toxic polymers of Abeta has thus emerged as an approach to developing therapeutics for AD. Techniques for studying Abeta polymerization include the use of fibril nucleation and extension assays in a variety of formats. Detection of polymeric forms of Abeta has been achieved using turbidity, dye binding, light scattering and toxicity among other methods. Direct and indirect methods have been described for the measurement of binding affinities for Abeta fibrils. Imaging techniques include electron microscopy, X-ray diffraction and atomic force microscopy. These techniques have been used to characterize different classes of compounds that inhibit the formation of Abeta polymers. These compounds include dyes such as Congo Red, the antibiotic rifampicin, the anthracycline 4'-iodo-4'-deoxydoxorubicin, and a large variety of Abeta-derived peptides and modified peptides, among other reported inhibitors.     

Effect of Congo red on the motility of the bacterium Azospirillum brasilense

In semiliquid laboratory media, the bacterium Azospirillum brasilense migrates with the formation of swarming rings. It is demonstrated that adsorption of the sulfonated azodye Congo Red confers on A. brasilense the ability to consistently spread in a semiliquid agar and form microcolonies. Spontaneous variants of A. brasilense with rapid swarming are described, as well as variants that swarm in the presence of Congo Red. It is assumed that at least two types of compounds are formed, which are (a) necessary for swarming and/or spreading with the formation of microcolonies and (b) capable of interacting with Congo Red.