激光在研究生物大分子结构与功能中的应用：酵母甘露聚糖波谱性 …

为了进一步了解酵母甘露聚糖的精细结构信息，以便研究其功能，我们采用本实验室“拟规模化制备酵母甘露聚糖新工艺”，从废啤酒酵母和市售鲜酵母细胞壁中制取了纯化的酵母甘露聚糖。     

生物大分子功能的研究:--酵母甘露聚糖对8.2Gy60Coγ-射线辐照小鼠的辐射防护作用

目的：为了解酵母甘露聚糖对小鼠辐射损伤的保护作用。方法：６０ Ｃｏ γ射线以８２ Ｇｙ 照射 ６０ 只小鼠,雌雄各半。其中 ３０ 只在辐照前 １０ 分钟腹腔注射 ４００ｍ ｇ Ｍ ａｎ／ｋｇ,另 ３０ 只注射生理盐水。结果：从比较研究项目：鼠的存活率,成活天数;４０ 天体重的动态变化;外同血 Ｗ Ｂ Ｃ、 Ｒ Ｂ Ｃ及 Ｐ Ｌ Ｔ 及脾指数等的数值差异上,均显示酸母甘露聚糖对６０ Ｃｏ γ射线辐射损伤有保护作用。同时也发现该保护作用存在性别敏感性差异。结论：酵母甘露聚糖是一种比较好的辐射防护剂;酵母甘露聚糖对雄性鼠辐射损伤的保护作用显著,有统计学意义（ Ｐ＜ ００１）。     

生物信息大分子功能的研究——酵母甘露聚糖对鼠Sarcoma-180瘤生长的抑制效应

酵母甘露聚糖（Mannan,简称Man）是能参与生物信息流影响生物体、特别是在糖基化方面起着重要调控作用的生物信息大分子.它是否能在抑制生物机体中肿瘤生长方面具有重要作用？研究结果表明：酵母甘露聚糖既能使患S-180瘤鼠的体质增强的同时又有抑制其体内所患S-180瘤生长的功效.Man抑制患鼠机体内的S-180瘤生长的功效（抑瘤率）随用量的增加而提高,当Man用量达360 mg（40 mg/Kg/d·9d）时,其抑瘤率达98.4％的最高水平,此时鼠体重增加1.66倍.Man的抑瘤功效有最佳适用量并存在性别敏感性,通常是雄性鼠的抑瘤率高于雌性鼠.Man抑制鼠S-180肿瘤生长的作用优于市售5-氟尿嘧啶的作用.     

半夏凝集素的糖结合活性研究

半夏凝集素(PTL)可与甘露聚糖结合。本文以PTL与~(125)I标记的甘露聚糖的结合活性为指标,观察了一些金属离子对PTL的糖结合活性的影响,井对PTL的糖结合专一性作了较系统的研究。结果表明常见的金属离子或EDTA对其糖结合活性无显著影响,但K~+可明显增加PTL的糖结合活性。大多数单糖,二糖不抑制PTL与甘露聚糖的结合,但一些疏水配基形成的糖苷可产生显著的抑制效应。PTL专一与高甘露糖型糖链结合。     

生物信息大分子结构修饰对其功能的影响——酵母甘露聚糖硫酸酯化衍生物的制备及其对HIV-1细胞生长抑制效应的初步研究

研究由安徽大学ADF实验室以拟规模化环保型循环工艺生产流程分离、提取、纯化制得的生物多糖-酵母甘露聚糖(Yeast Mannan,简称Man),在对其结构、性质和生物活性研究的基础上,进一步对其结构进行硫酸酯化修饰,制得新产品硫酸酯化酵母甘露聚糖(Man-Sulfated),并探索Man、Man-Sulfated对HIV-1病毒细胞生长的抑制作用。研究结果表明:1)Man对宿主细胞C8166无毒性;2)实验用HIV-1病毒细胞对宿主细胞C8166有毒性,TCID50为1.1×10~6Cells/m L;3)用氯磺酸∶吡啶(2∶1)甲酰胺法制得的Man-Sulfated对宿主细胞C8166也无毒性,并具有抑制HIV-1病毒细胞生长的功能,而Man则无此新功能;4)Man-Sulfated具有抑制HIV-1生长的效能,其与硫酸酯化修饰Man结构时使用的硫酸酯化试剂类别、方法及反应条件的不同,而引起对Man结构、构象的修饰变化程度不同密切相关。浓硫酸酯化法对Man结构修饰太强烈,构象变化过大,影响其生物功能。     

大鼠大脑皮质与纹状体显微拉曼光谱的研究

用Spex-1428显微激光拉曼光谱仪测定正常大鼠大脑皮质和纹状体的激光拉曼光谱的变化,发现正常大鼠大脑皮质和纹状体的激光拉曼光谱在模式上大同小异,包含有十分丰富的生物大分子结构信息。结果如下：（1）在大脑皮质和纹状体同时出现且性状亦相似的有以下一些特征峰：808cm^-1的特征峰,对应于A型DNA的特征峰;832cm^-1和836cm^-1对就于酪氨酸（Tyr)环的振动峰;1020cm^-1和1046cm^-1相当于蛋白质中氨基酸内的C－N伸缩振动峰;1330cm^-1相当于腺嘌呤环的C＝C和C－N伸缩振动峰;1544cm^-1相当于酰胺Ⅱ的N－H平面内弯曲振动和C－N伸缩援峰;1684cm^-1对应蛋白质二级结构中的转角（turn)结构。（2）大脑皮质较纹状体明显的特征峰：1092cm^-1的DNA骨架的对3称振动峰;1350cm^-1的色氨酸峰;1690cm^-1为尿嘧啶的C＝O振动峰。（3）纹状体较大脑皮质明显的特征峰;1450－1463cm^-1为蛋白质CH2弯曲振动的特征峰带,纹状体在此区段有1454cm^-1峰,而大脑皮质在此区域比较低平;1490cm^-1,为鸟嘌呤的特征峰。结果显示：显微拉曼光谱揭示的大脑皮质和纹状体的生物大分子的结构成分信息十分丰富,既有共性也有差异,显微拉曼光谱是一种十分灵敏的研究手段。     

大鼠大脑皮质梗塞后显微激光拉曼光谱的变化

用Ｓｐｅｘ－１４２８显微激乐拉曼光谱仪测定局灶性脑梗塞后大鼠大脑以质的激光拉曼光谱的变化,以正常侧大脑皮质作对照,研究大鼠大脑皮质垢生物大分子的结构及化学成分的变化,得到如下结果：（１）特征峰１０７６ｃｍ＾－１增大,表明梗塞区的ＤＮＡ的结构出现变化,其有序性降低（２）１５７２ｃｍ＾－１为与蛋白南侧链有关的特性峰,脑梗塞后此峰明显降低亦出的蛋白质结构亦出现变化;（３）８３２ｃｍ＾－１（Ｔｙｒ）峰降低     

试论生物信息流与激光生物效应的相关性--激光生物效应分子机理研究

为了探索激光生物学效应的分子机理,以便更好的掌控和利用激光生物效应,用XeCl（308 nm）准分子紫外激光以相同变化的激光参量直接辐照有生物活性的生物大分子BTG DNA、BSA（v）蛋白质和糖Mannan.实验结果分析告知：电镜法观察到受辐照的三类生物大分子的表观结构、构象（含结构信息）和光谱法（IR、Vis-UV、FR、CD）分析指出生物大分子的内在结构部件的相关的特征峰的峰位、峰值都受影响,其变迁都与激光参量的变化相呼应;与三类生物大分子中分子内、分子间沟通与信息传递相关的氢键、糖苷键等的形成与否的类似或相同的结构部件（如-C-H、-N-H、-CH-OH、-C-O、-C =O等）其特征峰的变迁都更敏感于激光参量的改变.激光辐照生物体时,激光似生物信号分子通过它的能量以粒子性、电磁波相干性影响生物大分子的分子结构、构象（含结构信息）的稳定性、增加分子内、分子间相互沟通、信息传递,亦增加了结构部件的被修饰的可能性.进而影响着生物信息流的流量与流向和细胞信号转导的协同与整体表达,产生相应的生物效应.掌握获得功能生物大分子结构构象信息与使用适宜的激光参量的相关的关系值（阈值）是重要的关键.     

XeCl（308nm）准分子紫外激光对生物大分子酵母甘露聚糖分子结构的辐照效应

激光在农业和医疗等方面已有广泛应用,并且取得增产和一定疗效,但也有费介现象(暂称后继效应或潜在效应).激光辐照生物体虽然是局部的有限时间,但对由多种生物大分子组成的牛物体的影响可能是综合的.为了探索激光生物学效应的分子机理,以便进一步认识和掌握激光的生物学效应,对激光直接辐照有生物活性的生物大分子一酵母甘露聚糖产生的效应进行电镜扫描观察和紫外及红外光谱法分析研究.实验结果表明:308 nm 激光(单脉冲25 mJ,33.3 ml-34.4 ml,脉冲频率2次/s)无论是固定辐照时间(45 s)变化样晶所置距离,或反之固定样品所置距离(290 mm)改变辐照时广开J为15 s、30 s、45 s或60 s,都能引起受照样品的结构(构象)变化,特别足糖分子结构中富含的-OH、-CH-OH、-C-O-c-、-N-C=0、-C=0等结构部件处的IR谱线对激光能最变化影响敏感.它们既是糖分子内或分子间形成氧键和糖苷键的结构基础部件,又是甘露聚糖(Man-nan)糖基化其他生物大分子进而影响它们在生物信息流中作用的重要结构部件.结果显示糖在激光牛物效应中起了不可忽视的作用,这为综合探索激光生物效应分子机理和糖在生物信息传递中的作用提供了重要线索,为选择甘露聚糖作为生物导弹载体提供了参考依据.     

甘露聚糖结合凝集素相关丝氨酸蛋白酶

甘露聚糖结合凝集素（MBL）是一种重要的天然免疫防御分子,通过激活MBL相关丝氨酸蛋白酶（MAST）而启动补体激活凝集素途径来清除病原体。本文就MASP的基因、分子结构及功能等方面研究概况作一介绍。     

Studies on the Discrimination of SCP-related Yeast by Proton Magnetic Resonance Spectroscopy: Structural Changes in Cell Wall Mannan of Candida subtropicalis Grown in Different Media

Applicability of PMR spectroscopy to the discrimination of the SCP-related yeast was investigated because the yeast was inactivated by heat treatment. When Candida subtropicalis was cultured in a medium containing glucose as a sole source of carbon, its cell wall mannan (mannan A) showed a PMR spectral pattern characterized by three intense peaks at δ: 4.97, 5.10, 5.29 and two small peaks at δ: 4.90, 5.19. Mannan (mannan B) from the yeast cultured in a medium containing n-pentadecane and triton X–100 showed a different spectral pattern in which the signals were observed at δ: 4.97, 5,10, 5.29, the intensity ratios of the signals were also different from those of mannan A. Acetolysis mannan was analyzed to compare the difference between two specified structures by using a gel elution method, methylation analysis and PMR spectra. Mannan B contained a less amount of the a (1→3) linkage than mannan A did, and differed from mannan A in its distribution pattern of side chain units. Our previous results together with the present ones proved the PMR method to be effective for the discrimination of the yeast.     

Antioxidant and antimutagenic activity of mannan neoglycoconjugates: mannan-human serum albumin and mannan-penicillin G acylase

The antioxidant and antimutagenic activity of the yeast cell-wall mannan and mannan conjugates--in particular the mannan of Saccharomyces cerevisiae (M-S.c.) and conjugates of mannan S. cerevisiae with human serum albumin (M-HSA1, M-HSA2) and the microbial enzyme penicillin G acylase (M-PGA)--were evaluated in vitro in the unicellular flagellate Euglena gracilis exposed to the genotoxic agents ofloxacin and acridine orange (AO). M-S.c., M-HSA1, M-HSA2 and M-PGA show a statistically significant, concentration-dependent protective antigenotoxic activity against both compounds. M-PGA was the most efficient inhibitor of ofloxacin- and AO-induced chloroplast DNA damage, whereas M-HSA2 and M-HSA1 were less effective and M-S.c. had the lowest antigenotoxic activity. It is suggested that different mechanisms may be involved in their protective effect--antioxidant activity in the case of ofloxacin-induced DNA damage and direct adsorption of AO on mannan conjugates as possible mechanisms of protection, based on spectrophotometric measurements. The important characteristics of yeast cell-wall mannans and mannan conjugates, such as their high water solubility, their broad spectrum of biological activity, low toxicity, stability and their antimutagenic effects via different modes of action, appear to be promising features for their practical application as antioxidants and antimutagenic agents.     

Candidacidal activity of myeloperoxidase: characterization of myeloperoxidase-yeast complex formation

We have previously demonstrated the ability of human neutrophil myeloperoxidase to bind to cell wall mannan polysaccharide isolated from Candida albicans. This binding capacity provides for association of the enzyme with target yeast which is essential for efficient candidacidal activity. In this report, we further consider the role of the mannan-binding property of myeloperoxidase in the candidacidal activity of the enzyme. Solubilized mannan antagonizes binding of the enzyme to yeast, suggesting that mannan may be a primary component of the fungal cell wall which serves as a target for binding of myeloperoxidase. Myeloperoxidase is shown to form complexes with both solubilized mannan and Candida yeast, with Kds of 0.97 x 10(-5) M and 1.2 x 10(-5) M, respectively. The interaction between myeloperoxidase and mannan does not allow the enzyme to readily dissociate from the surface of target yeast. As a result, the enzyme may be unable to dissociate from dead yeast to become available for binding to additional fungal targets.     

锌酵母中酵母甘露多糖组分的特征和结构

本文研究从锌酵母中分离出的酵母甘露多糖ＸＰ的特征和结构。ＸＰ经全水解和＾１３ＣＮＭＲ谱显示除甘露糖基外,还有少量Ｌ－鼠李糖基和甲氧基。甲基化分析、过碘酸盐氧化、Ｓｍｉｔｈ降解、乙酰解和部分酸水解显示ＸＰ的主链是１→６连接的甘露糖,侧链是１→２连接的甘露糖。＾１Ｈ及＾１３Ｃ ＮＭＲ谱表明所有糖苷键均为α型,结合元素分析ＸＰ基本是酵母甘露多糖和蛋白质以及锌的络合物。     

Identification and properties of a sterol-binding polysaccharide isolated from Saccharomyces Cerevesiae

A sterol-solubilizing polysaccharide isolated from yeast has been described (Adams, B. G. and Parks L. W. (1967) Biochem. Biophys. Res. Commun. 28,490–494) which greatly simplifies the addition of sterolsto aqueous media. The polysaccharide has now been identified by gas-liquid chromatography and carbazole reactions as yeast cell wall mannan. The cell wall mannan has been purified and binding constants for several sterols have been determined. Binding of sterols is a biphasic function of mannan concentration and is independent of pH over a range of 5 to 8.     

Secretion of cell-wall glycoproteins by yeast protoplasts. Effect of 2-deoxy-d-glucose and cycloheximide

The effect of 2-deoxy-d-glucose and cycloheximide on the synthesis and secretion of the cell-wall constituents protein and mannan in yeast protoplasts was examined in detail. Although the 2-deoxy-d-glucose hardly influenced protein synthesis, a significant parallel inhibition of carbohydrate and protein secretion into the medium was observed. The mechanism of this inhibition is considered as an interference of metabolites of 2-deoxy-d-glucose with the synthesis of yeast mannan. Cycloheximide, which is an effective inhibitor of protein synthesis in yeast (Kerridge, 1958), inhibited the secretion of non-diffusible carbohydrate in yeast protoplasts, but on the other hand had no effect on the activity of particulate yeast mannan synthetase. Our results clearly show that blocking the synthesis of either part of the mannan-protein complex prevents the extracellular appearance of the other component. The nature of this phenomenon is discussed.     

Identification of two Saccharomyces cerevisiae cell wall mannan chemotypes

We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 --> 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 --> 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. alpha-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties.     

Yeast mannan increases Bacteroides thetaiotaomicron abundance and suppresses putrefactive compound production in in vitro fecal microbiota fermentation

ABSTRACT

Yeast mannan is a part of yeast cell wall and can potentially affect gut microflora as a soluble dietary fiber. We demonstrated that yeast mannan suppressed putrefactive production and increased the relative abundance of Bacteroides thetaiotaomicron in in vitro fecal fermentation. These results suggest that yeast mannan can be used as a novel prebiotic food ingredient.     

Study of real-time lectin-carbohydrate interactions on the surface of a quartz crystal microbalance

A quartz crystal microbalance (QCM) biosensor system for lectin-carbohydrate interactions has been developed. Yeast mannan was immobilised on polystyrene-coated quartz crystals, and interactions tested with the lectin concanavalin A (Con A). The biosensor could be easily operated, where mannan immobilisation and all binding analyses were performed in real-time using a flow-through system. The apparent binding constant for yeast mannan to Con A was estimated to be 0.4 microM, well in accordance to reported literature values. In addition, the effective concentration values (EC50-values) for a series of mannose/mannoside ligands, acting as competitors to the mannan/Con A interaction, were determined to range from 0.18 to 5.3 mM, in good correlation with a related enzyme-labelled lectin assay (ELLA) protocol.     

C. albicans increases cell wall mannoprotein, but not mannan, in response to blood, serum and cultivation at physiological temperature

The cell wall of Candida albicans is central to the yeasts ability to withstand osmotic challenge, to adhere to host cells, to interact with the innate immune system and ultimately to the virulence of the organism. Little is known about the effect of culture conditions on the cell wall structure and composition of C. albicans. We examined the effect of different media and culture temperatures on the molecular weight (Mw), polymer distribution and composition of cell wall mannan and mannoprotein complex. Strain SC5314 was inoculated from frozen stock onto yeast peptone dextrose (YPD), blood or 5% serum agar media at 30 or 37°C prior to mannan/mannoprotein extraction. Cultivation of the yeast in blood or serum at physiologic temperature resulted in an additive effect on Mw, however, cultivation media had the greatest impact on Mw. Mannan from a yeast grown on blood or serum at 30°C showed a 38.9 and 28.6% increase in Mw, when compared with mannan from YPD-grown yeast at 30°C. Mannan from the yeast pregrown on blood or serum at 37°C showed increased Mw (8.8 and 26.3%) when compared with YPD mannan at 37°C. The changes in Mw over the entire polymer distribution were due to an increase in the amount of mannoprotein (23.8-100%) and a decrease in cell wall mannan (5.7-17.3%). We conclude that C. albicans alters the composition of its cell wall, and thus its phenotype, in response to cultivation in blood, serum and/or physiologic temperature by increasing the amount of the mannoprotein and decreasing the amount of the mannan in the cell wall.