化学修饰对金顶侧耳多糖抗病毒（CB5）活性的影响

从金侧耳子实体中分离纯化一半乳甘露聚糖ＰＣ－３,其分子量约为６７ＫＤ,一级结构ａ（１→６）糖苷键相连的Ｇａｌ构成分子的主链,部份残基Ｃ２上带有分支,分支结构为ａ（１－２）Ｍａｎ－ａ（１－２）Ｍａｎ。按高碘酸氧化、部份酸水解、甲基化、硫酸程序对ＰＣ－３的结构进行化学修饰;并分别将ＰＣ－３及其衍生物与柯萨厅病毒Ｂ５进行体外实验,结果表明,ＰＣ－３经硫酸化后,显著地提高了抗病毒活性;而高碘酸氧化,甲基化     

碱提水溶小皮伞多糖B_3的研究

用ＧＣ,ＩＲ,ＮＭＲ,ＧＣ－ＭＳ及高碘酸氧化,Ｓｍｉｔｈ降解,甲基化分析,部分酸水解等方法确定了一个新的碱提水溶小皮伞多糖的一级结构：分子主链α－Ｄ－（１→２）Ｍａｎ,支链α－（１→４）,α－（ａ→６）Ｇｌｃ,且均连在主链的０─６上。     

金顶侧耳多糖PC—4的结构确定与抗肿瘤活性的研究

３％氯乙酸浸提过的金顶侧耳子实体中分离纯化另一水溶性多糖ＰＣ－４。该多糖分子量经为１８９ｋＤ。纸层析与气相层析分析表明其为单一聚糖。经高磺酸氧化,Ｓｍｉｔｈ降解,甲基化,层析,气质联机分析,核磁其振（Ｈ－ＮＭＲ,１３Ｃ－ＮＭＲ）谱及红外光谱测定等,可确定Ｃ－４的主链结构由β－（１→３）糖苷键相连的葡萄糖构成,部份残基Ｃ６,上带有分支。约每５个糖残基有两个侧链,侧链仅为１个葡萄糖残基。     

碱提水溶小皮伞多糖B3的研究

用ＧＣ,ＩＲ,ＮＭＲ,ＧＣ－ＭＳ及高碘酸氧化,Ｓｍｉｔｈ降解,甲基化分析,部分酸水解等方法确定了一个新的碱提水溶小皮伞多糖的一级结构,分子主链α－Ｄ－（１→４）。α－（ａ→６）Ｇｌｅ,且均连在主链的０－６上。     

玉米根细胞中对钒酸盐敏感的H~＋－泵的研究

用奎吖咽（ｑｕｉｎａｃｒｉｎｅ）作荧光指标剂,测定玉米（ＺｅａｍａｙｓＬ．）根尖微粒体（ＭＩＣ）膜囊泡的Ｈ~＋－泵活性,结果表明１ｍｍｏｌ／ＬＮａＮ_３仅抑制该泵活性约８％,而０．８ｍｍｏｌ／Ｌ钒酸盐（Ｖａｎ）则可抑制其活性达８０％,说明ＭＩＣ制剂中Ｈ~＋－泵活性主要由质膜（ＰＭ）Ｈ~＋－ＡＴＰａｓｅ产生。此泵活性严格需要Ｍｇ~（２＋）,二价阳离子作用大小的顺序为Ｍｇ~（２＋）＞Ｍｎ~（２＋）＞Ｚｎ~（２＋）＞Ｃａ~（２＋）＝０;阴离子作用大小的１顺序为Ｂｒ~－＞Ｃｌ~－＞ＮＯ_３~－＞ＳＯ_４~（２－）,并初步证实当质膜同侧发生电子传递时,没有跨膜Ｈ~＋梯度（△μＨ~＋）生成。     

细胞松驰素B促微丝解聚对DNA合成的作用

利用微丝（ｍｉｃｒｏｆｉｌａｍｅｎｔ,ＭＦ）解聚药物细胞松驰素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ,ＣＢ）处理Ｇ_０期小鼠Ｃ_３Ｈ_（１０）Ｔ_（１／２）成纤维细胞,对Ｇ_０至Ｓ期ＤＮＡ合成,胸腺嘧啶核苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,ＴＫ）活性、ＴＫ基因表达、钙调素（ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）水平和一些细胞周期早期基因的表达进行了观察,Ｇ_０期细胞经３ｍｇ／ＬＣＢ处理２ｈ,促ＭＦ解聚增强了血清对Ｓ期细胞ＴＫ活性、ＴＫ基因表达和ＤＮＡ合成的刺激作用,并促进细胞提前进入Ｓ期．血清刺激Ｇ_０期细胞进入晚Ｇ_１期和Ｓ期时,ＣａＭ水平明显升高,而ＣＢ预处理则使ＣａＭ含量进一步增加,特别是ＣＢ处理促使Ｓ期ＣａＭ增加向核内转移．ＣＢ处理明显增强血清对ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ基因表达的刺激作用,而ＰＫＣ抑制剂Ｈ_７则抑制ＣＢ处理对这些基因转录的刺激作用,说明ＣＢ使Ｇ_０期细胞ＭＦ解聚刺激ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ的转录活性与ＰＫＣ的作用有关．结果表明Ｇ_０至Ｓ期早期ＭＦ的重组可促进细胞进入Ｓ期,增强ＤＮＡ合成．     

香椿的组织培养和玻璃苗的防止

１植物名称香椿（Ｔｏｏｎａｓｉｎｅｎｓｉｓ）。２材料类别（１）种子发芽３～５ｄ后的下胚轴及带少许下胚轴的子叶;（２）当年生半木质化的腋芽茎段。３培养条件芽诱导及增殖培养,以ＭＳ为基本培养基,蔗糖３０ｇ·Ｌ~（－１）,琼脂０．５％,附加激素（单位ｍｇ·Ｌ~（－１））：（１）６－ＢＡ０．２;（２）６－ＢＡ０．２、ＧＡ_３２．０;（３）ＩＡＡ０．１、６ＢＡ０．２;（４）ＺＴ０．２、ＧＡ_３２．０。诱导生根培养基为１／ＺＭＳ或仅含ＭＳ有机质（铁盐减半）,附加１．０ｍｇ·Ｌ~（－１）ＩＢＡ、１５ｇ·Ｌ~（－１）…     

宫粉郁金的组织培养和快速繁殖

１植物名称宫粉郁金（Ｃｕｒｃｕｍａ　ｋｗａｎｇｓｉｅｎｓｉｓ）。２材料类别　块茎萌动芽。３培养条件　萌动芽生长培养基：（１）ＭＳ＋６－ＢＡ１ｍｇ·Ｌ－１（单位下同）＋ＮＡＡ０．２。不定芽增殖与愈伤组织诱导培养基：（２）ＭＳ＋６－ＢＡ１０＋ＫＴ５;（３）ＭＳ＋６．ＢＡ１０;（４）ＭＳ＋６－ＢＡ５＋ＫＴ２．５;（５）ＭＳ＋６－ＢＡ５;（６）ＭＳ＋６－ＢＡ２＋ＫＴ１。生根培养基：（７）ＭＳ＋ＮＡＡ０．５;（８）ＭＳ＋６－ＢＡ０．５＋ＮＡＡ０．５;（９）ＭＳ。以上培养基均加０．７％琼脂,３％蔗糖,ｐＨ５…     

灯盏细辛中酚类化合物的化学研究

从灯盏细辛（Ｅｒｉｇｅｒｏｎ ｂｒｅｖｉｓｃａｐｕｓ（Ｖａｎ．）Ｈａｎｄ－Ｍａｚｚ）的乙酸乙酯部分首次分离得到５个二咖啡酰基的酚类化合物（１￣５）和９个其他类型化合物。其中ｅｒｉｇｏｓｔｅｒ Ａ（１）为一全新骨架的化合物。利用波谱方法（＾１Ｈ－ＮＭＲ,＾１３Ｃ－ＮＭＲ,２Ｄ－ＮＭＲ和ＦＡＢ－ＭＳ等）对这些化合物的结构进行了鉴定。     

丙型肝炎病毒C33_c单克隆抗体的研制和鉴定

用丙型肝炎病毒重组蛋白Ｃ３３_ｃ抗原免疫ＢＡＬＢ／ｃ小鼠,运用杂交瘤技术成功地建立了７株能稳定分泌抗Ｃ３３_ｃ单克隆抗体的杂交瘤细胞１Ｈ６Ｄ２、２Ｇ１Ａ６、３Ａ４Ａ８、３Ｅ３Ｅ７、４Ｇ１２Ｃ１０、４Ａ１０Ｃ２、５Ｆ４Ｂ６．试验结果表明,７株ＭｃＡｂｓ具有良好的ＨＣＶ特异性,间接ＥＬＩＳＡ法测得小鼠腹水ＭｃＡｂ效价为１：１０￣４－１：４×１０￣４;竞争抑制实验和相加指数测定证实７株ＭｃＡｂｓ识别相关的抗原表位;７株ＭｃＡｂｓ中１株为ＩｇＭ（５Ｆ４Ｂ６）,其它６株为ＩｇＧ（２ａ）。     

金顶侧耳酸提水溶性多糖的研究——PC-3的分离、纯化与结构确定

金顶侧耳(Pleurotus citrinopileatus)子实体的3%三氯乙酸提取液经甲醇分级,十六烷基三甲基溴化铵等进一步提纯得到水溶性多糖PC-3。经Sepharose CL-4B柱层析,醋酸纤维素薄膜电泳、聚丙烯酰胺凝胶电泳鉴定,PC-3为均一级份。G.C.与P.C.分析表明,Gal、Man为该多糖的组份,其单糖摩尔比为2.5∶1.0。PC-3的分子量约为67000,比旋光度[α]_D~(18°)=+28°。经核磁共振谱等分析,PC-3不含β型糖苷键。部份酸水解、高碘酸氧化、Smith降解、完全甲基化、G.C.及G.C.-M.S.的分析表明,PC-3是由α(1→6)糖苷键相连的半乳糖构成分子的主链,部分残基C_2上带有分支,每5个Gal残基带有1个侧链,侧链为α(1-2)Man-α(1-2)Man。     

硫酸化对金顶侧耳多糖构象及生物活性的影响

从金顶侧耳子实体中分离纯化一半乳甘露聚糖ＰＣ－３。该多糖在水中为无规线团构象,与Ｃｏｎ Ａ可相互结合并产生沉淀,对柯萨奇病毒ＣＢ５有一定的抑制作用,ＰＣ－３经硫酸化修饰后,由于同性电荷的排斥作用,使糖链的无规线团扩展呈伸展状态,局部可能形成螺旋。硫酸化的ＰＣ－３与Ｃｏｎ Ａ不能结合成多糖－蛋白复合物,但显著地提高了抗病毒ＣＢ５的活性。     

1,2-α-D-Mannosidase from a wood-rotting basidiomycete, Pycnoporus sanguineus

An acidic α-D-mannosidase has been isolated from the culture filtrate of a wood-rotting Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme determined. The extracellular mannosidase was homogeneous on PAGE at pH 9.4. The M_r as determined by SDS-polyacrylamide disc gel electrophoresis was 64000, and the pI was pH 4.7 using electrofocusing. The purified enzyme had a pH optimum of 4.5 with Baker's yeast mannan and had no activity towards p-nitrophenyl-α-mannoside. The K_m and k_cat values for Manα1-2Man at pH 4.5 and 30° were 0.9 mM and 1.9 sec. the enzyme had no activity towards Manα1-3Manα1-2Man, and it cleaved specifically the 1,2-α-linked side chain of yeast α-mannan, producing free α-D-mannose.     

Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference

Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1-6Man(α) and Manα1-3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.     

Beneficial effect of intracellular free high-mannose oligosaccharides on cryopreservation of mammalian cells and proteins

The cryoprotective effect of intracellular free high-mannose oligosaccharides (HMOS) on mammalian cells and proteins was examined by monitoring PC-12 cell viability and assaying protein kinase C (PKC)-epsilon activity. 1-Deoxymannojirimycin, an inhibitor of alpha-mannosidase, to cause an increase in intracellular free HMOS, significantly rescued PC-12 cells with 2-h freezing insult at -15 degrees C in a concentration (1-50mM)- and pretreatment time (48-72h)-dependent manner, as compared with unpretreated cells; full rescue from freezing injury was obtained with 1-deoxymannojirimycin at more than 25mM for 48-h pretreatment and more than 3mM for 72- and 96-h pretreatment. For PC-12 cells pretreated with 1-deoxymannojirimycin at 1mM for 72h, thawed cell viability after more than 8-w cryopreservation at -80 degrees C in 10% (v/v) dimethyl sulfoxide was much higher than that for cells without pretreatment. PKC-epsilon activity was well preserved after 16-h cryopreservation at -20 degrees C in the presence of mannose 9-N-acetylglucosamine 2 (Man9-GlcNAc2) (1 mM), an HMOS, while the activity was reduced to 15% without Man9-GlcNAc2. Collectively, the results of the present study suggest that intracellular free HMOS is a key molecule to protect mammalian cells and proteins from freezing injury; in other words, HMOS could be a new target for cryopreservation of mammalian cells and proteins.     

Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.

The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.     

Calystegine B3 as a specific inhibitor for cytoplasmic alpha-mannosidase, Man2C1

Cytoplasmic α-mannosidase (Man2C1) has been implicated in non-lysosomal catabolism of free oligosaccharides derived from N-linked glycans accumulated in the cytosol. Suppression of Man2C1 expression reportedly induces apoptosis in various cell lines, but its molecular mechanism remains unclear. Development of a specific inhibitor for Man2C1 is critical to understanding its biological significance. In this study, we identified a plant-derived alkaloid, calystegine B(3), as a potent specific inhibitor for Man2C1 activity. Biochemical enzyme assay revealed that calystegine B(3) was a highly specific inhibitor for Man2C1 among various α-mannosidases prepared from rat liver. Consistent with this in vitro result, an in vivo experiment also showed that treatment of mammalian-derived cultured cells with this compound resulted in drastic change in both structure and quantity of free oligosaccharides in the cytosol, whereas no apparent change was seen in cell-surface oligosaccharides. Calystegine B(3) could thus serve as a potent tool for the development of a highly specific in vivo inhibitor for Man2C1.     

Structure of pentasaccharide of glycopeptide from TIME-EA4, N-glycoprotein in silkworm diapause eggs

The TIME-EA4, from silkworm diapause eggs of pure strain C108, Bombyx mori, has glycosylated chain as tetrasaccharide (Man(2)GlcNAc(2)) attaching to the Asn(22) of T3 peptide from tryptic digests. On the other hand, from Showa silkworm strain we additionally observed a pentasaccharide (Man(3)GlcNAc(2)) on T3 at the same linkage site. The linkage pattern of the 5-sugar chain was studied through Smith degradation combined with LC-MS and MS/MS analyses. These advanced methods led us to conclude that the pentasaccharide was branching as Man 1-->3(Man 1-->6)Man 1-->4GlcNAc 1-->4GlcNAc.