Carbohydrate moieties of procine 32 kDa enamelin

The structures of asparagine-linked oligosaccharides of porcine 32 kDa enamelin are reported. The oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The pyridylamino oligosaccharides were separated into eight kinds of oligosaccharides. The structures of these oligosaccharides were determined by a combination of a sequential exoglycosidase digestion and a two-dimensional suger mapping technique. The oligosaccharides consisted of fucose, galactose, mannose, N-acetylglucosamine, and N-acetylneuraminic acid, and were classified into two groups according to their core-sugar chain structures; one was a biantennary-type and the other was a triantennary-type oligosaccharide. The variation of the oligosaccharides in each of these groups was caused by the differences in the number, the site, and the mode of linkage of N-acetylneuraminic acid to the core-sugar chains.     

PAI－1糖基化位点突变体的构建及其在哺乳动物细胞中表达

Ⅰ型纤溶酶原激活物抑制剂（ＰＡＩ－１）是一种Ｍｒ为５０×１０３的单链糖蛋白，有３７９个氨基酸残基，３个Ｎ型糖基化位点。构建ＰＡＩ－１糖基化突变体，以便研究糖链的功能。用寡核苷酸定位突变技术将３个糖基化位点２０９，２６５，３２９位进行突变，把３个糖基化位点都发生了突变的ＰＡＩ－１ｃＤＮＡ组装到真核表达载体ｐＳＶ２中，得到真核表达质粒ｐＺＨ－ｐ１－Ｍ３Ｅ；在二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞（ＣＨＯｄｈｆｒ－）中进行短暂表达，用发色底物法和夹心ＥＬＩＳＡ方法检测培养液中ＰＡＩ－１的活性和含量。结果：糖基化位点突变的ＰＡＩ－１能在ＣＨＯ细胞中表达，但表达水平及活性较低。非糖基化ＰＡＩ－１的活性和抗原分别为４．３４ＩＵ／ｍｌ和３．１５μｇ／Ｌ结论：用寡核苷酸定位突变方法获得了ＰＡＩ－１糖基化突变体，并且在ＣＨＯ细胞中得到表达。     

Identification of a frequent variant in ALG6, the cause of Congenital Disorder of Glycosylation-Ic

Congenital Disorder of Glycosylation (CDG) type Ic is caused by mutations in ALG6. This gene encodes an alpha1,3 glucosyltransferase used for synthesis of the lipid linked oligosaccharide (LLO) precursor of the protein N-glycosylation pathway. CDG-Ic patients have moderate to severe psychomotor retardation, seizures, hypotonia, strabismus, and feeding difficulties. We previously identified a typical patient with a heterozygous point mutation, c.391T>C (p.Tyr131His) in ALG6. Using complementation analysis of ALG6-deficient yeast, we show that this alteration is as severe as the most common disease-causing mutation, c998C>T (p. Ala333Val), which occurs in over half of all known CDG-Ic patients. The frequency of c.391T>C (p.Tyr131His) in the US population, is 0.0214, suggesting that homozygotes would occur at a rate of& tilde;1:2,200. We identified one patient with typical CDG-Ic symptoms and a homozygous p.Tyr131His alteration in ALG6. However, in contrast to most CDG patients, her LLO and plasma transferrin glycosylation appeared normal. Thus, it is unclear whether c.391T>C causes CDG-Ic or contributes to the symptoms. Genotyping additional patients with CDG-like symptoms will be required to resolve this issue.     

A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides

A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [³H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Galβ1,3[3OMeGalβ1,4GlcNAcβ1,6]αBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.     

远志化学成分的分离与鉴定

目的对远志的化学成分进行研究,为更好地开发利用远志药材奠定基础.方法采用色谱技术进行分离,以1H-NMR、13C-NMR等波谱技术鉴定化合物的结构.结果分离鉴定了5个化合物,分别是tenuifoliside A(1)、α-D-(6-O-白芥子酰基)-吡喃葡萄糖基(1→2)-β-D-(3-O-白芥子酰基)-呋喃果糖(2)、α-D-(6-O-4-甲基-3,5-二甲氧基肉桂酰基)-吡喃葡萄糖基(1→2)-β-D-(3-O-白芥子酰基)-呋喃果糖(3)、3,4,5-三甲氧基肉桂酸(4)、苯甲酸丙酯(5).结论化合物3、5为首次从该植物中获得,其中化合物3为新化合物.     

Synthesis and bioassay of β-（1,4）-D-mannans as potential agents against Alzheimer＇s disease

Aim： Oligomannurarate 971 derived from a marine plant has shown neuroprotective effects. In this study we synthesized a series of truncated derivatives of the oligosaccharide, and investigated the effect of these derivatives against Aβ peptide toxicity in vitro. Methods： The sulfoxide method was applied to synthesize the derivatives. SH-SY5Y human neuroblastoma cells were treated with Aβ1-40 （2 pmol/L）, and the cell viability was detected using a CCK8 assay. Results： A series of β-（1,4）-D-mannosyl oligosaccharide, ranging from the disaccharide to the hexasaccharide, were synthesized. Addition of 10 pmol/L β-（1,4）-D-mannobiose 6, β-（1,4）-D-mannotriose 9 or β-（1,4）-D-mannotetraose 12 in SH-SY5Y cells significantly attenuated Aβ1-40-induced toxicity. The efficacies were similar to those caused by 10 pmol/L oligomannurarate 971 or alzhemed. Other oligosaccharides including oligomaltoses and oligocelluloses were less active. Conclusion： Synthetic homogeneous short chain β-（1,4）-D-mannans shows neuroprotective effect against Aβ peptide toxicity similar to that of heterogeneous oligomannurarate 971 and alzhemed.     

Conjugation of functional oligosaccharides reduced in vitro allergenicity of β-lactoglobulin

Bovine β-lactoglobulin (βLG) was conjugated with fructooligosaccharides (FOS), galactooligosaccharides (GOS) and isomaltooligosacharides (IMO) by Maillard reaction in an effort to reduce the allergenicity of βLG. Fourier transform infrared spectra indicated that βLG was bound to FOS, GOS and IMO covalently. Structural analyses by dichroism spectra and fluorescence studies suggested that the surface of βLG in each conjugate was covered with oligosaccharides without apparent disruption of native conformation. The βLG-oligosaccharides conjugates almost maintained the retinol-binding activity of βLG. Enzyme-linked immunosorbent assay indicated that, conjugation of βLG with these functional oligosaccharides was effective in reducing the allergenicity of βLG.     

ROS对糖尿病大鼠血脂代谢及肾脏组织中VEGF的影响

目的探讨地黄寡糖(ROS)对糖尿病大鼠血脂代谢及肾脏组织中血管内皮生长因子(VEGF)的影响。方法将40只SPF级大鼠按照随机数字表法分为健康组、DM组、ROS组及药物对照组,每组10只。DM组、ROS组及药物对照组大鼠均建立糖尿病模型。ROS组及药物对照组分别采用200 mg/(kg·d)的ROS及二甲双胍灌胃,剩余两组灌胃等体积生理盐水,治疗22 d。治疗结束后,检测血脂代谢指标、肾脏重量、肾脏病理组织切片、肾脏组织中VEGF阳性率及蛋白。结果与健康组比较,DM组、ROS组、药物对照组总胆固醇(TC)、三酰甘油(TG)升高,高密度脂蛋白胆固醇(HDL-C)降低,经药物干预后,ROS组、药物对照组TC、TG明显低于DM组,HDL-C明显高于DM组,且ROS组TG明显低于药物对照组,差异有统计学意义(P<0.05)。与健康组比较,ROS组、药物对照组大鼠肾脏重量及肾脏指数均明显增加,药物干预后,与DM组比较,ROS组及药物对照组肾脏重量及肾脏指数明显降低,差异有统计学意义(P<0.05)。健康组大鼠肾脏结构完整,肾小球基质分布清晰,未见增生及肥大;DM组大鼠肾小球基质模糊,毛细血管增厚,炎性反应浸润严重,肾小球出现肥大;经药物干预的ROS组及药物对照组上述病理情况均好转,症状减轻。健康组、DM组、ROS组及药物对照组大鼠肾脏组织中VEGF蛋白相对表达量分别为1.00±0.15、3.03±0.33、1.45±0.27、1.50±0.30,组间比较,差异有统计学意义(F=109.100,P<0.001)。DM组VEGF蛋白相对表达量明显高于健康组,差异有统计学意义(t=17.880,P<0.001)。ROS组及药物对照组VEGF蛋白相对表达量明显低于DM组,差异有统计学意义(t=11.870、10.990,P<0.001)。结论灌胃ROS能够改善糖尿病大鼠血脂代谢指标,降低肾脏重量,这与抑制VEGF表达具有相关性。