糖蛋白糖链的分析

糖蛋白是蛋白与糖类的共价复合物,结合于蛋白肽链上的糖链具有重要的生物学功能。对糖蛋白上的糖链进行分析,要经过糖链释放、分离不同类型糖链、相对分子质量测定及糖链测序等步骤。本简要介绍糖蛋白糖链分析各步骤的常用方法、各种方法的特点、适用范围等。     

光架桥法可获得糖链阵列

日本东洋纺公司和Summit glycro research公司通过将糖链固定在金表面的糖链阵列，成功地利用表面等离子共振（SPR）法检测出糖链结合蛋白质和糖链的相互作用。[第一段]     

视黄酸对皮肤上皮基底培养细胞表面膜糖蛋白N-连接型糖链结构的影响

维生素A类化合物对糖蛋白N-连接型糖链(简称N-糖链)结构的影响,近年来在研究其作用机制中颇受重视。本文研究视黄酸(RA)对大鼠皮肤上皮基底培养细胞表面膜糖蛋白糖链结构作用,发现RA促进N-糖链合成,使~3H-甘露糖掺入糖链量增加43.5％,RA可改变N-糖链的类型,促进复杂型N-糖链合成,表现为增加三、四天线复杂型N-糖链合成而不是二天线;RA还使含分叉性GIeNAc和核心Fuc的百分比上升。本文还用细胞电泳方法研究膜表面唾液酸相对量,发现RA可引起唾液酸含量下降。结果提示RA对N-糖链结构的影响,是其多种生物学作用的可能途径之一。     

糖链的化学合成

作为生物大分子之一,糖链的研究还没有像蛋白质和核酸那样深入。现阶段糖链的获得仍然存在很大的挑战,阻碍了糖生物学的发展。鉴于通过分离手段得到所需的糖链很困难,酶法合成糖链亦存在着诸多问题,因此目前化学方法合成糖链是最佳的选择。对近年来糖链的化学合成所取得的最新进展进行简要的介绍,主要包括一釜合成、固相合成和标签辅助的合成三个方面。     

认识糖链的最后机会

基础技术综合研究所糖链丁程研究中心从2001年起开始进行糖链分析毖础技术的开发，以该研究中心为主将启动旨在糖链的医疗应用的项目。我们就有父将糖链研究推出的前景，采访了20年前任肚界上首次克隆糖转移酶以来，一直从事糖链研究的成松久副主任。[编者按]     

科研快讯

《临床研究期刊》:胃部一种糖链缺失可致胃癌日本一个研究小组最新报告说,实验鼠胃黏膜中的一种糖链缺失会引发胃癌,早期胃癌患者胃部这一糖链也出现减少甚至消失,因此可据此寻找预防胃癌的方法。糖链是葡萄糖、半乳糖等糖类分子按特定序列形成的链状物。日本信州大学医学部教授中山淳率领的研究小组在美国《临床研究期刊》网络版上刊文说,胃黏膜产生的液体中含有一种糖链,这种糖链含有α型N乙酰氨基葡萄糖。研究人员通过基因技术,培育出胃黏膜不产生这种糖     

双丁酰环烯酸腺苷对人肝癌细胞株SMMC—7721表面N—糖…

本文采用系列凝集素柱层析法,并配合外切糖苷酶研究了在双丁酰环磷酸腺苷（ｄＢ－ｃＡＭＰ）作用１－５过程中人肝癌细胞株ＳＭＭＣ－７７２１细胞表面Ｎ－糖链类型及复杂型糖链天线数的变化。结果表明,ｄＢ－ｃＡＭＰ促进＾３Ｈ－Ｍａｎ参人细胞表面Ｎ－糖链,使高甘露糖型Ｎ－糖链的百分比下降,并促进二天线Ｎ－糖链的生物合成,使多天线特别是四天线和Ｃ２Ｃ２Ｃ６三天线Ｎ－糖链的百分比减少。结果提示,Ｎ－糖链结构的这些变     

基于超滤膜辅助的糖蛋白全N-连接糖链的富集和质谱解析

糖基化作为一种常见的蛋白质翻译后修饰,对蛋白质的空间结构、生物功能等具有重要的影响.解析糖蛋白糖链结构有助于更清楚地认识糖蛋白及其功能.本研究建立了一种基于超滤膜富集血清中糖蛋白全N-连接糖链,并利用质谱技术对糖链结构进行分析的方法.根据糖蛋白及其糖链结构之间的分子质量差异,利用Millipore公司的10 ku超滤膜富集血清糖蛋白上酶解(PNGase F)释放的全N-连接糖链,并使用MALDI-TOF/TOF-MS解析糖链结构.通过该技术可以从血清中富集并鉴定到23种独特的N-连接的糖链结构,并且利用二级质谱进行了结构确认.该方法可以被用于从大量生物样本中富集糖蛋白全N-连接糖链,可以达到快速、高通量地解析糖蛋白N-连接糖链的目的.     

糖组学:破解生命信息的第3种途径

糖组学是随着糖生物学而兴起的研究糖链的表达、调控和生理功能的科学。糖链由于结构的多样性和复杂性而成为细胞的信息分子,是生物体基因组信息的延续,因此糖组学研究是后基因组时代阐明基因功能的必由之路。糖组学的内容主要包括对糖链的结构研究和在细胞信号传递、细胞识别方面的功能分析,以及通过糖蛋白组建糖组学数据库,从而建立起一套从基因组蛋白组到糖组的研究体系,对糖链的生物学功能的认识将有助于基因组学和蛋白质组学的研究。     

多天线糖链对运铁蛋白与受体结合及内吞的研究

应用系列凝集素柱层析法（伴刀豆球蛋白,小扁豆凝集素,欧曼陀罗凝集素）分别从正常人血清及孕妇血清中提纯含有二天线无核心岩藻糖复杂型糖链的运铁蛋白及含有多天线无核心岩藻糖复杂型糖链的运铁蛋白,与正常的含有二天线糖链的运铁蛋白相比,含有多天线糖链的运铁蛋白,与正常的含有二天线糖链的运铁蛋白相比,含有多天线糖链的运铁蛋白与ＳＭＭＣ－７７２１细胞膜表面的运铁蛋白受体的亲和力下降,但最大结合量不变,此外,其在     

Efficacy of a prostaglandin analogue in reproduction in the anestrous mare

A prostaglandin F analogue was studied in anestrous mares: a dose-response study; a study in mares presumed pregnant; and a field evaluation of effective doses in breeding establishments. A dose of 2.0mg given by single subcutaneous injection to mares with initial plasma progesterone levels greater than 1.0ng/ml, caused luteolysis on the basis of decline in plasma progesterone concentrations. Follicle maturation leading to ovulation, accompanied by estrus, was observed, and fertility at mating either by natural service or artificial insemination was satisfactory. A dose of 1.0mg was generally effective for luteolysis, but pregnancy rates were lower than after 2.0mg. A proportion of mares which had less than 1.0mg of plasma progesterone at the time of injection ovulated and became pregnant.     

水杉愈伤组织诱导及植株再生

通过愈伤组织诱导器官发生途径,建立了水杉（Metasequoia glyptostroboides）的植株再生体系,探讨了不同外植体（种胚、幼叶切块、茎段、根段）和植物生长调节剂对不定芽直接再生和愈伤组织诱导器官发生的影响。结果表明：以种胚、无菌苗叶片、茎段和根作为外植体,在MS补加2,4-D、NAA和6-BA不同组合的培养基上都能诱导得到愈伤组织,其中种胚诱导愈伤组织效果最好,诱导率可达100%,茎诱导效果次之,诱导率为97.1%。诱导愈伤组织效果较好的培养基有：MS＋1.0mg·L-12,4-D＋0.5mg·L-16-BA、MS＋0.1mg·L-16-BA＋1.0mg·L-1NAA、MS＋0.5mg·L-16-BA＋1.0mg·L-1NAA、MS＋1.0mg·L-16-BA＋1.0mg·L-1NAA、MS＋0.5mg·L-16-BA＋2.0mg·L-1NAA、MS＋1.0mg·L-16-BA＋2.0mg·L-1NAA和MS＋0.5mg·L-12,4-D＋0.5mg·L-1NAA。以愈伤组织在MS培养基上植株再生效果最好,再生率为62.5%。     

Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32

X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-prolyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40°C. The enzyme was incubated at 40°C for 15 min with various metal ions. It was activated by Mg²⁺ (2.5 mM), Ca²⁺ (0.1 to 2.5 mM), Na⁺ (10 to 50 mM), and K⁺ (10 to 50 mM) and was inhibited by Hg²⁺ (0.1 to 2.5 mM), Cu²⁺ (0.1 to 2.5 mM), and Zn²⁺ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40°C), 1,10-phenanthroline (1.0 mM, 15 min at 40°C), phenylmethylsulfonyl fluoride (1.0 mM), N-ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40°C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40°C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).     

Requirement for protein synthesis for survival of unmodified bacteriophage T1 in a restricting host.

At high multiplication of infection, a substantial fraction of restricting cells (P1 lysogens) could be productively infected by unmodified coliphage T1 (T1.0) provided that protein synthesis was uninhibited during the first 5 min of infection. Successful infection under restricting conditions was accompanied by more genetic recombination than was seen under nonrestricting host, the recombination frequency declined for markers on T1.0 genomes; no effect was seen on recombination between markers on modified (T1.P) genomes. This suggested that recombination between unmodified genomes may be essential for their survival under conditions of host restriction. In a restricting host, genetic markers on T1.0 could recombine with T1.P even when the rescuing phage was added 6 min after T1.0 infection. However, even marker rescue recombination was diminished when protein synthesis was inhibited during early infection. Since DNA restriction is an early event, protein synthesis may be required soon after infection of a restricting host by T1.0 in order to preserve restriction-damaged DNA in a form that can participate in recombination. Experiments are also described that rule out some possibilities for the role of such a protein(s).     

Structure of a -D-glucan from the mycelial wall of Basidiomycete QM 806

A water-soluble β-D-glucan has been isolated from the mycelial wall of Basidiomycete QM 806. The structure of this glucan was investigated by methylation, periodate, and enzymic studies. Hydrolysis of the methylated glucan gave 2,3,4,6-tetra-, 2,4,6-, 2,3,4- and 2,3,6-tri-, and 2,4-di-O-methyl-D-glucose in the following molar proportions: 1.0:1.0:O.8:1.2:1.0. Periodate oxidation of the glucan followed by reduction and mild acid hydrolysis gave glycerol, erythritol, and D-glucose in the molar proportions, 2.1, 1.0, and 2.0, respectively. The glucan was degraded to the extent of 38% by an exo-β-(1→3)-glucanase isolated from the same organism, though the branch points (joined through O-1, O-3, and O-6) appeared to be resistant to the enzyme whereas the (1→4) linkages were not. On the basis of these findings, the structure of the glucan and the possible role of the glucanase are discussed.     

Specific and abundant secretion of a novel hydroxyproline-rich glycoprotein from salt-adapted winged bean cells

Winged bean callus was adapted to increasing concentrations of NaCl by sequential transfer to medium with 0, 0.5, 1.0, 1.5, and 2.0% (w/v) NaCl. When the culture media, after cell suspension cultures of callus adapted to 0.5 (SA-0.5), 1.0 (SA-1.0), 1.5 (SA-1.5), or 2.0% (w/v) NaCl (SA-2.0), were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, six specific or enhanced polypeptide bands (SAP1, -2, -3, -4, -5, and -6) were observed. SAP1, with a molecular weight of 84,000, was abundantly secreted in suspension cultures of SA-1.0 and SA-1.5, and was observed as the most striking polypeptide band. The SAP1 yield was about 4 mg/g cells fresh weight. SAP1 was abundantly secreted after the suspension culture of SA-1.0 in the presence of AlCl₃, but little was secreted in the presence of KCl, LiCl, CaCl₂, MgCl₂, mannitol, sucrose, or abscisic acid. SAP1 was purified from the culture medium after suspension culture of SA-1.0 in the presence of 1.0% (w/v) NaCl. Two steps, ammonium sulfate fractionation and CM-cellulose chromatography, were sufficient for purification to homogeneity. Finally, about 5 mg of SAP1 could be isolated from 7 g of fresh callus cells. Of the amino-terminal 32 amino acid residues of SAP1, 10 and 5 were found to be hydroxyproline and proline, respectively. SAP1 on an acrylamide gel was stained by the periodic acid-Schiff method. It is interesting that SAP1 has pentahydroxyproline blocks (Hyp₅) instead of tetrahydroxyproline blocks (Hyp₄) common to many hydroxyproline-rich glycoproteins in dicotyledons. Thus, this novel hydroxyproline-rich glycoprotein was shown to be abundantly secreted from NaCl-adapted winged bean cells.     

Compositional consistency of a heteropolysaccharide-7 produced by Beijerinckia indica

The component sugars of heteropolysaccharide-7 (PS-7) produced by Beijerinckia indica were rhamnose and glucose (1.0:4.8, mol:mol) by gas chromatographic analysis. Galacturonic acid, previously reported as a repeat unit of PS-7, was not found in purified PS-7. The yield of PS-7 varied with physiological conditions, such as concentration of carbon source and initial pH of medium, but the molar ratio of rhamnose to glucose stayed within 1.0 to 4.6-5.1. B. indica utilized glucose and some glucose analogs as carbon sources and produced exopolymers, although there was no direct incorporation of these sugars into PS-7. The molar ratio of rhamnose to glucose in each polymer synthesized from glucose-related sugars showed no significant variation (1.0 to 4.5-4.7)     

The viral protein Egf1.0 is a dual activity inhibitor of prophenoloxidase-activating proteinases 1 and 3 from Manduca sexta

Some pathogens are capable of suppressing the melanization response of host insects, but the virulence factors responsible are largely unknown. The insect pathogen Microplitis demolitor bracovirus encodes the Egf family of small serine proteinase inhibitors. One family member, Egf1.0, was recently shown to suppress melanization of hemolymph in Manduca sexta in part by inhibiting the enzymatic activity of prophenoloxidase activating proteinase 3 (PAP3). However, other experiments suggested this viral protein suppresses melanization by more than one mechanism. Here we report that Egf1.0 inhibited the amidolytic activity of PAP1 and dose-dependently blocked processing of pro-PAP1 and pro-PAP3. Consistent with its PAP inhibitory activity, Egf1.0 also prevented processing of pro-phenoloxidase, serine proteinase homolog (SPH) 1, and SPH2. Isolation of Egf1.0-protein complexes from plasma indicated that Egf1.0 binds PAPs through its C-terminal repeat domain. Egf1.0 also potentially interacts with SPH2 and two other proteins, ferritin and gloverin, not previously associated with the phenoloxidase cascade. Overall, our results indicate that Egf1.0 is a dual activity PAP inhibitor that strongly suppresses the insect melanization response.     

Development of Salt-Resistant Active Transport in a Moderately Halophilic Bacterium

The moderately halophilic bacterium Vibrio costicola accumulates α-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na⁺ ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCl as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCl) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCl. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.     

On the response of a cell population to low-dose irradiation

The cell composition of a population of human blood lymphocytes was studied after irradiation at doses of 5 cGy, 1.0 Gy and 5 cGy + 1.0 Gy and the use of a cytokinesis block. The frequencies of uni-, bi- and multinucleate lymphocytes with and without micronuclei (MN) were taken into account. By the standard criterion the frequency of binucleate lymphocytes with MN among binucleate lymphocytes--the donors were characterized as follows: in with reduction of radiosensitivity after irradiation with 5 cGy + 1.0 Gy as compared to the values of radiosensitivity after irradiation with 1.0 Gy only (an adaptive response, AR); in with no change of radiosensitivity after exposure to these doses (no AR); and with an increased ofradiosensitivity after exposure to these doses (syndrome of increased radiosensitivity, IRS). It was found that upon exposure to 1.0 Gy and 5 cGy + 1.0 Gy in some donors with AR, without AR and with IRS the total numbers of damaged cells in the population and the number of binucleate cells with MN were equal. This result calls in question the involvement of the repair mechanism in the alteration of radiosensitivity of lymphocytes in these donors. It was also observed that in the same donors a simultaneous increase (or a decrease in the case of IRS) of the portion of undamaged binucleate cells in the population took place. Our results demonstrate the existence of a new, populational, mechanism involved in the alteration of radiosensitivity after exposure to the adaptive and challenge doses.