海洋红细菌Lentibacter algarum胞外多糖的分离纯化及结构解析

从青岛潮间带的海水中分离得到的红细菌科细菌Lentibacter algarum的发酵液中提取胞外多糖,对其进行离子交换色谱分离纯化,得到水洗和0.1、0.5、1.0 mol/L NaCl溶液4个洗脱组分。对含量最高的0.1 mol/L NaCl洗脱组分La0.1进行进一步的凝胶排阻柱层析纯化,得到组分La0.1-1。通过化学测定和高效液相色谱(HPLC)、高效凝胶渗透色谱法(HPGPC)等分析方法对其理化性质、分子量、单糖组成、连接方式及初步结构进行研究。结果表明,La0.1-1总糖含量为66%,平均分子量为12.0 kDa。其单糖组成主要为半乳糖、甘露糖和氨基葡萄糖,比例为Gal∶Man∶GlcN=1.35∶1.1∶1.0。对La0.1-1进行气相色谱质谱联用(GC-MS)和一维核磁(1-D NMR)分析结果显示,La0.1-1的连接方式是以β构型为主,主要存在→2)-Manp(1→,→3)-Galp(1→连接,还存在少量的→4)-Galp(1→和→4)-Manp(1→等连接方式,表明该多糖以直链为主,还存在一定的分支,分支发生在→2)-Manp(1→的O-6位和→3)-Galp(1→的O-4或O-6位。氨基葡萄糖主要为→4)GlcN(1→和末端连接方式。核磁分析还显示La0.1-1存在一定的乙酰基取代,初步判断主要取代在氨基葡萄糖的N-2位上,也可能存在于甘露糖和半乳糖上。本研究是首次对Lentibacter属细菌的胞外多糖进行测定,获得了结构较为新颖的胞外多糖资源,为开发海洋多糖资源提供物质基础。     

紫球藻多糖化学结构解析

为明确紫球藻多糖的化学结构,本文采用化学分析和光谱分析方法对紫球藻多糖的一级糖链结构进行了分析。GC分析表明该多糖由木糖、葡萄糖和半乳糖组成,为一种杂多糖,其摩尔比为:2.96∶1.25∶3.06;红外光谱分析结果显示紫球藻多糖为硫酸化多糖,糖苷键类型为β构型;化学分析结果推断紫球藻多糖糖链连接方式以1→3为主,存在少量1→2,1→4,1→6键型,且半乳糖在支链或主链末端有较大量的存在,木糖和葡萄糖在主链或靠近主链区域有特定分布;NMR分析显示紫球藻多糖的硫酸酯基连在C-6上,且多糖的糖苷键为β型;GC-MS联机分析进一步确定紫球藻多糖为一种主要含有1→3糖苷键,并含有1→4,1→6糖苷键的杂多糖。综合上述分析,推断出紫球藻多糖的糖链主链的重复单元结构。     

中药巴戟天中水溶性多糖的分离及结构鉴定(英文)

中药巴戟天(Morinda officinalis)的根经过水提、醇沉、脱色和离子交换得到水溶性多糖(MOHP-1),经过FIIR、HPLC、NMR和GC-MS分析,最后确定MOHP-1是由果糖以(2→1)糖苷键连接的菊淀粉性多糖,其结构为α-Glcp-(1→2)-[β-Fruf-(2→1)-β-Frucf]n。     

丹皮多糖对α-葡萄糖苷酶作用的影响

研究丹皮多糖(PSM2b)对α-葡萄糖苷酶作用的影响,探索其降血糖作用的途径。通过建立体外酶-抑制剂模型,测定丹皮多糖PSM2b及其分级分离组分对酶作用的抑制率。结果显示PSM2b的分级分离组分PSM2b-1,PSM2b-2,PSM2b-3在体外对α-葡萄糖苷酶有一定的抑制作用,PSM2b对酶的抑制作用不明显。     

茶多糖TGC的结构表征

采用气相色谱-质谱联用技术(GC-MS)分析均一茶多糖TGC的单糖组成, 并与NMR, 圆二色谱、紫外扫描等其他分析方法结合, 对茶多糖TGC的一级结构及其在溶液中的构象加以探讨. 结果表明: 茶多糖TGC是由鼠李糖、阿拉伯糖、木糖、葡萄糖、甘露糖和半乳糖等6种单糖组成, 它在水溶液中应以有序的螺旋构象存在, 其一级结构为: 主链的骨架结构由鼠李糖、葡萄糖和半乳糖构成, 这3种单糖都有可能连接支链, 不接支链时其连接方式为β1→3, 支链主要由阿拉伯糖构成, 其连接方式可为β1→2, β1→3, β2→3三种, 木糖以β1→存在于主链和支链的末端.     

蜗牛黏液多糖的提取、表征及抗氧化性和免疫活性

利用高效凝胶色谱串联紫外、示差和多角度激光散射检测器(HPSEC-UV-RI-MALLS)对蜗牛黏液中的多糖进行分子量分布表征。利用气相色谱串联质谱(GC-MS)和红外光谱(FT-IR)对其化学结构进行表征;并对所获得的多糖进行抗氧化及免疫活性的评价。结果显示:蜗牛黏液中主要含有5个组分的多糖,是其重要的活性成分。对这5个多糖进行鉴定,其平均分子量为4.549×10~6、1.392×10~5、6.291×10~4、5.262×10~(4 )和4.153×10~(4 )。单糖由岩藻糖、甘露糖、葡萄糖和半乳糖组成,摩尔比为1.69∶2.46∶0.12∶1。多糖主链为→2) Manp-(1→,→3) Fucp-(1→和→3) Manp-(1→。此外,蜗牛黏液多糖具有明显的抗氧化作用,能很好地清除ABTS·~+和·OH,IC_(50)值分别为2.35和4.70 mg/mL;且能明显增强巨噬细胞(RAW264.7)的吞噬能力,促进NO和白细胞介素(IL-6)、肿瘤坏死因子(TNF-α)等免疫细胞因子的释放。     

食用菌多糖的提取纯化及生物活性研究进展

食用菌多糖因具有抗氧化、免疫调节、抗肿瘤、降血糖、降血脂等生物活性而备受关注.食用菌多糖的结构影响其生物活性,具有(1→3)、(1→4)、(1→6)及混合糖苷键的β-D-葡聚糖是高活性食用菌多糖的结构特征之一,展现出提高抗氧化酶活性、促进分泌抗癌因子、刺激脾脏和胸腺细胞增殖等不同功能.酸碱、超声波及微波、Sevag法、...     

蛹虫草胞外多糖的研究 Ⅰ.半乳甘露聚糖(CM-I)的纯化和结构研究

从蛹虫草菌体培养液中提取水溶性粗多糖经分离纯化,得一种含有少量蛋白的半乳甘露聚糖CM-Ⅰ,分子量2.7×10~4,[α]_D~(19)=+54.7°。糖组成摩尔比,半乳糖:甘露糖=6∶5。经高碘酸氧化,Smith降解,部分酸水解,半乳糖苷酶解,~1H-NMR分析,完全甲基化与GC及GC-MS分析,证明:多糖CM-Ⅰ具有高度分枝结构,其以β-(1→2)连接的甘露糖为主干,枝链由较大量的β-(1→6)半乳糖和较大量的β-(1→2)呋喃半乳糖构成,分别连接在主干的0—4和0—6上。     

菊花中一个新的多糖的研究

通过热水提取、离子交换层析和凝胶层析,从菊花(Chrysanthemum morifolium Ramat.)中分离得到一个新的多糖.根据糖组成分析、甲基化分析、高碘酸氧化、部分酸水解和NMR谱图分析,确定其结构如下:[→4)-Galp-(1→4)-Galp-(1→4)-Galp-(1→4)-Galp-(1→6)-Galp-(1]→F3L2Araf-(1→5)-Araf-(1→4)-Glcp-(1→4)-Glcp1→6 F3[→4)-Galp-(1→4)-Galp-(1→4)-Galp-(1→4)-Ga6p-(1→6)-Galp-(1]→n     

玉米花粉多糖PMA Ⅰ的结构解析

从玉米花粉中分离得到一种多糖PMAⅠ,经鉴定为单一纯多糖,通过IR、NMR、高碘酸氧化、Smith降解等手段对其结构进行了研究,确定PMAⅠ主要以Rha,Xyl和GlcOMe组成,分子摩尔比RhaXylGlcOMe=12.72.11,以α(1→3)键为主链,以α(1→6)键构成分支结构.     

Submaxillary mucins. Intermolecular interactions and gel-forming potential of concentrated solutions.

The intermolecular interactions in concentrated solutions of pig submaxillary mucin (PSM) and sheep submaxillary mucin (SSM) were studied by mechanical spectroscopy. PSM and SSM were purified from detectable protein and nucleic acid by equilibrium centrifugation in a CsCl density gradient. PSM and SSM isolated in the presence of proteinase inhibitors showed distinct differences from preparations isolated in the presence of 0.2 M-NaCl alone, the latter having a carbohydrate and amino acid analysis similar to other preparations isolated by precipitation or ion-exchange techniques. Gel-filtration studies showed that preparations isolated in the presence of 0.2 M-NaCl alone were dissociated into smaller-sized glycoprotein units by 3.5 M-CsCl or 2.0 M-NaCl (SSM), pH 2.0 (PSM) or heating at 100 degrees C for 10 min (PSM and SSM). Preparations isolated in the presence of proteinase inhibitors were not dissociated by these treatments. Proteolysis fragmented all submaxillary mucin preparations into small glycopeptides of Mr 13,700 for PSM and of Mr 14,000 and 15,000 for SSM. PSM preparations when concentrated formed viscoelastic gels, as determined by mechanical spectroscopy. In contrast, SSM showed characteristics of a weak viscoelastic liquid under comparable conditions (coil overlap). PSM glycoprotein isolated in proteinase inhibitors formed weak viscoelastic gels at concentrations between 5 and 15 mg/ml. Preparations of PSM glycoprotein isolated in the presence of 0.2 M-NaCl (concentration 10-97 mg/ml) had the same overall mechanical gel structure as those preparations extracted in the presence of proteinase inhibitors. This gel structure was seen to collapse following proteolysis of both preparations or after acid treatment of the glycoprotein isolated in the presence of 0.2 M-NaCl, consistent with the breakdown in size of the polymeric glycoprotein. Treatment of PSM gel with 0.2 M-2-mercaptoethanol caused a surprising increase in gel strength, which was further markedly increased on removal of the reducing agent by dialysis. An association of reduced subunits of PSM was observed by gel filtration after removal of 0.2 M-2-mercaptoethanol. These results point to intermolecular disulphide exchange occurring on reduction of these PSM glycoprotein preparations. These results demonstrate that gel formation in PSM glycoprotein is similar to that for other gastrointestinal mucus glycoproteins from stomach to colon. Gel formation in PSM, as in other mucins, depends on polymerization of subunits.(ABSTRACT TRUNCATED AT 400 WORDS)     

安徽某铁矿酸性矿山废水中真核生物的群落结构特征

【目的】研究酸性矿山废水中真核生物的群落结构特征以及群落结构与环境因子之间的关系。【方法】利用分子生物学方法,通过构建18S rRNA基因克隆文库进行系统发育分析;利用典范对应分析(CCA)方法解析环境因子对真核生物群落结构的影响。【结果】系统发育分析表明:子囊菌门(Ascomycota)普遍存在于4个样品中,并在样品1和样品3中占统治地位,而绿藻门(Chlorophyta)和担子菌门(Basidiomycota)分别为样品2和样品4的优势类群。该酸性矿山废水中的克隆与许多已知的耐酸耐重金属真核生物亲缘关系较近,如Sarcinomyces petricola、Penicillium janthinellum、Coniochaeta velutina、Trichoderma viride、Chlorellaprotothecoides var.acidicola、Ochromonas sp.等。此外,样品中还存在大量的已知人类病原菌,如Lecythophorahoffmannii、Cryptococcus neoformans。CCA分析表明:TN、SO24-、Fe2+、Eh是影响真核生物群落空间分布的主要因素。【结论】所研究的酸性矿山废水中真核生物的群落结构在时间和空间上均有较大差异,这可能与水体的理化性质有关;高含量人类致病菌的存在是之前研究所未发现的;酸性环境中真核生物的生态学研究有助于开发高效处理酸性矿山废水的方法。     

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.     

The SpoMBe pathway drives membrane bending necessary for cytokinesis and spore formation in yeast meiosis

Precise control over organelle shapes is essential for cellular organization and morphogenesis. During yeast meiosis, prospore membranes (PSMs) constitute bell-shaped organelles that enwrap the postmeiotic nuclei leading to the cellularization of the mother cell's cytoplasm and to spore formation. Here, we analysed how the PSMs acquire their curved bell-shaped structure. We discovered that two antagonizing forces ensure PSM shaping and proper closure during cytokinesis. The Ssp1p-containing coat at the leading edge of the PSM generates a pushing force, which is counteracted by a novel pathway, the spore membrane-bending pathway (SpoMBe). Using genetics, we found that Sma2p and Spo1p, a phospholipase, as well as several GPI-anchored proteins belong to the SpoMBe pathway. They exert a force all along the membrane, responsible for membrane bending during PSM biogenesis and for PSM closure during cytokinesis. We showed that the SpoMBe pathway involves asymmetric distribution of Sma2p and does not involve a GPI-protein-containing matrix. Rather, repulsive forces generated by asymmetrically distributed and dynamically moving GPI-proteins are suggested as the membrane-bending principle.     

Structure-activity relationship of sphingomyelin analogs with sphingomyelinase from Bacillus cereus

The aim of this study was to examine how structural properties of different sphingomyelin (SM) analogs affected their substrate properties with sphingomyelinase (SMase) from Bacillus cereus. Using molecular docking and dynamics simulations (for SMase-SM complex), we then attempted to explain the relationship between SM structure and enzyme activity. With both micellar and monolayer substrates, 3O-methylated SM was found not to be degraded by the SMase. 2N-methylated SM was a substrate, but was degraded at about half the rate of its 2NH-SM control. PhytoPSM was readily hydrolyzed by the enzyme. PSM lacking one methyl in the phosphocholine head group was a good substrate, but PSM lacking two or three methyls failed to act as substrates for SMase. Based on literature data, and our docking and MD simulations, we conclude that the 3O-methylated PSM fails to interact with Mg(2+) and Glu53 in the active site, thus preventing hydrolysis. Methylation of 2NH was not crucial for binding to the active site, but appeared to interfere with an induced fit activation of the SMase via interaction with Asp156. An OH on carbon 4 in the long-chain base of phytoPSM appeared not to interfere with the 3OH interacting with Mg(2+) and Glu53 in the active site, and thus did not interfere with catalysis. Removing two or three methyls from the PSM head group apparently increased the positive charge on the terminal N significantly, which most likely led to ionic interactions with Glu250 and Glu155 adjacent to the active site. This likely interaction could have misaligned the SM substrate and hindered proper catalysis.     

Phosphorus Recovery from Phosphate Rocks Using Phosphate-Solubilizing Bacteria

In this paper, we are presenting a biological process to recover phosphorus by solubilizing low-grade phosphate rocks. To this end, the efficiency of different phosphate-solubilizing microorganism (PSM) species for solubilizing P from phosphate rocks using both pure cultures and associations. Nutritional conditions, phosphate rock concentrations, and reactor designs were tested. The genus Bacillus, especially Bacillus megaterium (ATCC 14581), was found to be the most promising PSM for solubilizing P. Production of organic acids and acidic pH values were shown to be directly related to P solubilizing. However, associations between tested microorganisms did not significantly enhance process efficiency. We conclude that nutritional factors of the medium are important to solubilization, and lower phosphate rock concentrations lead to better solubilization. The Air Lift reactor was promising for B. megaterium (ATCC 14581), but adaptations are needed for further tests.     

PSM/SH2B1 splice variants: critical role in src catalytic activation and the resulting STAT3s-mediated mitogenic response

A role of PSM/SH2B1 had been shown in mitogenesis and extending to phenotypic cell transformation, however, the underlying molecular mechanism remained to be established. Here, four alternative PSM splice variants and individual functional protein domains were compared for their role in the regulation of Src activity. We found that elevated cellular levels of PSM variants resulted in phenotypic cell transformation and potentiated cell proliferation and survival in response to serum withdrawal. PSM variant activity presented a consistent signature pattern for any tested response of highest activity observed for gamma, followed by delta, alpha, and beta with decreasing activity. PSM-potentiated cell proliferation was sensitive to Src inhibitor herbimycin and PSM and Src were found in the same immune complex. PSM variants were substrates of the Src Tyr kinase and potentiated Src catalytic activity by increasing the V(max) and decreasing the K(m) for ATP with the signature pattern of variant activity. Dominant-negative PSM peptide mimetics including the SH2 or PH domains inhibited Src catalytic activity as well as Src-mediated phenotypic cell transformation. Activation of major Src substrate STAT3 was similarly potentiated by the PSM variants in a Src-dependent fashion or inhibited by PSM domain-specific peptide mimetics. Expression of a dominant-negative STAT3 mutant blocked PSM variant-mediated phenotypic cell transformation. Our results implicate an essential role of the PSM variants in the activation of the Src kinase and the resulting mitogenic response--extending to phenotypic cell transformation and involving the established Src substrate STAT3.     

丹皮多糖PSM_(2b)体外对小鼠免疫细胞功能的影响

中药丹皮提取的丹皮多糖有效部位PSM2b在体外能直接促进小鼠脾细胞增殖 ,并能协同ConA诱导的脾细胞增殖作用。对小鼠腹腔巨噬细胞亦有激活作用 ,可增强小鼠腹腔巨噬细胞吞噬中性红 ;诱导巨噬细胞合成一氧化氮。结论 :PSM2b可增强T淋巴细胞功能 ,并对巨噬细胞具有激活作用     

Importance of the phosphocholine linkage on sphingomyelin molecular properties and interactions with cholesterol; a study with phosphate oxygen modified sphingomyelin-analogues

We have characterized the molecular properties and membrane behavior of synthetically modified sphingomyelin analogues, modified on the oxygen connecting the phosphocholine group to the ceramide backbone. The oxygen was replaced with an S-atom (S-PSM), an NH-group (NH-PSM) or a CH(2)-group (CH(2)-PSM). Diphenylhexatriene and Laurdan anisotropy experiments showed that an S-linkage increased and NH- and CH(2)-linkages decreased the stability of PSM-analogue bilayer membranes as compared to PSM. When the polarity of the interface was probed using Laurdan, S-PSM appeared to have a lower polarity as compared to PSM whereas NH-PSM and CH(2)-PSM had higher polarities of their respective interfaces. Fluorescence quenching-studies with cholestatrienol showed that all compounds formed SM/cholesterol-rich domains. The S-PSM/cholesterol and PSM/cholesterol domains displayed a similar thermostability, whereas NH-PSM/cholesterol and CH(2)-PSM/cholesterol domains were less thermostable. DSC on vesicles containing the PSM-analogues showed a more complex melting behavior as compared to PSM, whereas equimolar mixtures of the PSM-analogues and PSM showed almost ideal mixing with PSM for NH- and S-PSM. Our data show that the properties of the bond linking the phosphocholine head group to the 1-hydroxyl on the ceramide molecule is important for the stability of SM/SM and SM/cholesterol interactions.