牛磺酸抑制心肌β受体激动后的蛋白磷酸化

本工作在离休大鼠心脏灌流模型上,观察牛磺酸对异丙肾上腺素（Ｉｓｏ）刺激引起的心肌收缩蛋白（肌钙蛋白Ｉ,Ｔ,Ｃ－蛋白以及Ｐ轻链）和肌浆网的受磷醚蛋白（ＰＬＢ）磷酸化的影响。结果表明：Ｉｓｏ刺激后￣（３２）Ｐｉ参入肌钙蛋白Ｉ和Ｃ蛋白的量校对照组分别增加了５０２．３％和１８６．７％,给予牛磺酸后即明显降低;Ｉｓｏ刺激后￣（３２）ｐｉ参入肌浆网ＰＬＢ的量较对照组增加了５５０．１％,给予牛磺酸后,亦显著降低。表明牛磺酸明显抑制Ｉｓｏ刺激引起的心肌蛋白质磷酸化。提示抑制心肌β受体激动后的蛋白磷酸化,可能是牛磺酸保护心肌的分子机制之一。     

心肌肌钙蛋白I和糖原磷酸化酶同工酶BB金标记免疫层析联合检测法的建立

目的：建立人心肌肌钙蛋白I（cTnI）及糖原磷酸化酶同工酶BB（GPBB）的胶体金免疫层析联合检测法。方法：以纯化的人心肌cTnI和GPBB为免疫原免疫小鼠,制备抗cTnI和抗GPBB单克隆抗体,并用胶体金标记cTnI和GPBB抗体,采用免疫层析技术建立快速准确检测cTnI和GPBB的胶体金免疫层析法。结果：建立的检测方法灵敏度高,可检出血液样品中1ng／mL的cTnI和7ng／mL的GPBB;特异性强,与心肌肌钙蛋白T、心肌肌钙蛋白C、肌酸激酶同工酶均无交叉反应。结论：该方法特异性强,灵敏度高,快速、简便,弥补了传统心肌梗死诊断方法的不足,对急性心肌梗死的早期筛查有重要意义,具有较高的临床应用价值和广泛的应用前景。     

鼠肝细胞核中两型酪氨酸蛋白激酶的分离和部分纯化

鼠肝细胞核中两型酪氨酸蛋白激酶的分离和部分纯化＊张洪陈惠黎＊＊（上海医科大学卫生部糖复合物重点实验室,生化教研室,上海２０００３２）关键词细胞核;酪氨酸蛋白激酶;同工酶;分离纯化收稿日期：１９９６－１２－１８;接受日期：１９９７－０３－０６。＊中华医...     

心肌肌钙蛋白Ⅰ临床应用进展

心肌肌钙蛋白I(Cardiac Troponin I,cTnI)作为诊断心肌损伤的血清标志物之一,同其它检测指标相比,具有出现时间早、诊断窗口期宽、特异性强、诊断阈值明确及检测快速等优点。由于心肌肌钙蛋白I为心肌细胞所特有,因此在心肌缺血性损伤、心肌非缺血性损害的诊断及骨骼肌损伤的鉴别诊断中得到了广泛应用。目前正逐渐取代包括CK-MB在内的其它血清酶检测指标而成为判断心肌损伤,特别是诊断急性心肌梗死(Acute Myocardial Infarction.AMI)的"金标准"。同时心肌肌钙蛋白I对心肌疾病的病情监测、疗效观察及预后评估都具有较高的临床应用价值。本文对cTnI的国内外临床应用研究进展进行综述。     

咖啡因和茶碱对大鼠皂素蜕膜心肌肌钙蛋白Ca^2＋敏感性和肌浆网Ca^2＋…

在低浓度皂素制备的浆膜蜕变（通透性增高）而肌浆网（ＳＲ）膜无损的蜕膜心肌标本上,以其收缩的幅度作为ＳＲ Ｃａ＾２＋释放的半定量指标;高浓度皂素（５００μｇ／ｍｌ）制备的浆膜和ＳＲ膜均蜕变的蜕膜心肌标本,以其张力－ｐＣａ关系曲线以及产生５０％最大张力所需的Ｃａ＾２＋浓度作为肌钙蛋白Ｃａ＾２＋敏感性的定性和定量指标。结果观察到：（１）在低浓度皂素蜕膜心肌标本上,５和１０ｍｍｏｌ／Ｌ咖啡因分别引起约８９     

人心肌肌钙蛋白T的纯化和单克隆抗体的制备

从人左室心肌中成功纯化心肌肌钙蛋白T(cTnT). 经匀浆, 70℃加热处理, 咪唑盐酸透析, DEAE-纤维素层析, 100g心肌获取cTnT 5mg, 纯度为97.6%. 同时采用脾内免疫法, 免疫Balb/C小鼠, 经细胞融合, 筛选, 克隆化得5株稳定分泌抗人cTnT单克隆抗体(McAb)的杂交瘤细胞(G₃, G₈, G₁₀, A₅, A₇), 4株为IgM, 1株为IgG, 染色体数目92～110条. 腹水效价为3.2×10^-6～1. 6×10^-7.     

心肌肌钙蛋白T分子生物学基础研究进展

心肌肌钙蛋白T(cTnT)是调节心脏肌肉收缩的肌钙蛋白复合体组成之一,在人的心脏有4种cTnT异构体,不同的发育阶段表达不同的cTnT异构体。目前发现在心力衰竭终末期cTnT4再度表达。因此,为进一步探讨心衰的发病机制,心肌肌钙蛋白T的分子生物学基因相关研究已成为热点。     

心肌肌钙蛋白T基因突变在心肌病发病机制中的研究进展

肥厚型和扩张型心肌病中,基因缺陷分别占发病的50％和35％,其病理生理机制,主要包括肌小节蛋白基因突变引起的收缩力产生缺陷,细胞骨架蛋白基因突变引起的收缩力传递缺陷等。心肌肌钙蛋白T将肌钙蛋白C和肌钙蛋白I连接到肌动蛋白和原肌球蛋白上,在心肌细胞收缩和舒张过程中发挥重要作用。在肥厚型和扩张型心肌病中发现了多种心肌肌钙蛋白T的基因突变,围绕心肌肌钙蛋白T的研究有助于阐明心肌病的发病机制。本文总结了心肌肌钙蛋白T基因突变在心肌病发病机制中的研究情况。     

心肌肌钙蛋白T在病毒性心肌炎心肌组织中的表达及意义

目的:了解病毒性心肌炎心肌组织中肌钙蛋白T的表达情况,探讨病毒性心肌炎时心肌结构蛋白损伤的机制及意义。方法:运用免疫组化和计算机图像分析技术,观察13例明确性病毒性心肌炎和17例界限性病毒性心肌炎尸检心脏标本中心肌肌钙蛋白T的表达与分布。结果:在正常对照的心肌组织中,蛋白成强阳性表达,分布均匀,未见缺染。在明确性心肌炎及14例界限性心肌炎心肌组织中都存在着不同程度的蛋白表达缺染或脱失。缺染的范围及分布与病毒性心肌炎病变特点基本一致,但其范围往往小于炎症细胞浸润范围。计算机图像分析和数据统计结果显示缺染区域的心肌肌钙蛋白T表达量要明显小于其周边区域和正常心肌细胞(P＜0．01)。结论:病毒性心肌炎患者的心肌损害要早于炎症细胞的浸润,病毒的作用可能是心肌肌钙蛋白T脱失的主要因素。心肌肌钙蛋白T的免疫组化检查可以作为一种有效的手段,来辅助病毒性心肌炎的病理学诊断。     

心肌肌钙蛋白Ⅰ基因突变与心肌病的研究进展

心肌肌钙蛋白I(cTnI)是心肌肌钙蛋白复合物的亚单位之一,与心肌肌钙蛋白T和C相互作用,与肌动蛋白-原肌球蛋白结合从而抑制肌动蛋白-肌球蛋白的收缩作用。在肥厚型、扩张型和限制型心肌病中发现30多种cTnI基因的突变,cTnI基因突变转基因小鼠也反映了心肌病的特征。本文总结了cTnI基因突变在心肌病发病机制中的研究情况。     

红花菜豆凝集素的糖结合专一性

经肼解、Ｂｉｏ－Ｇｅｌ Ｐ－２柱层析、ＮａＢ＾３Ｈ４和ＮａＢＨ４还原,制备各种来源的、氚标记在还原末端的、还原末端为Ｎ－乙酰氨基葡萄糖醇的混合寡糖,经Ｂｉｏ－Ｇｅｌ Ｐ－４凝胶柱分离,以及用糖苷酶酶解,制备了各种不同类型的氚标记的寡糖。这些寡糖在固定化的ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上亲和层析,根据各种类型寡糖在ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上的层析行为,确定红花菜豆（矮生红花变种）凝集素（ＰＣＬ）的     

Comparative study of chicken ovalbumin subfractions having different carbohydrate chain from each other by high performance anion exchange chromatography

1. Ovalbumin was fractionated to six fractions according to their phosphate content by high performance anion exchange chromatography. 2. This method was applied to analyze the phosphate content of ovalbumin subfractions having different carbohydrate chain from each other which were prepared by concanavalin A/Sepharose chromatography from oviduct slices incubated with [2-3H]mannose. 3. Most biosynthetic intermediates bearing a carbohydrate chain of Man8 or 9 GlcNAc2 was not phosphorylated while other fractions bearing differently processed carbohydrate chains such as Man5 or 6 GlcNAc2 or hybrid type carbohydrate chain was phosphorylated at their peptide portion.     

The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae)

The sexuality-inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS-22 and separated by reverse-phase HPLC into several isoforms which differ in the degree of O-glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N-glycans which are clustered in the middle of the polypeptide chain. The N-glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gaschromatographic sugar analysis and two-dimensional HPLC separation to possess the following definite structures: (A) Man beta 1-4GlcNAc beta 1-4GlcNAc; (B) (Man alpha)3 Man beta 1-4GlcNAc beta 1-4GlcNAc Xyl beta; (C) (Man alpha)2 Man beta 1-4GlcNAc beta 1-4GlcNAc; (D) (Man)2Xyl(GlcNAc)2. Xyl beta Two of the three N-glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N-glycosylation site. The O-glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein

The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man=0.2:4.0:1.4:13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man9-GlcNAc2, which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi alpha-mannosidase digestion, and 1H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc1Man9GlcNAc2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc1Man9GlcNAc2 glycan. There were no processed glucosylated oligosaccharides such as Glc1Man5-8GlcNAc2. Furthermore, Glc1Man9GlcNAc2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc1Man9GlcNAc2 may play a role in the folding of arylphorin or in the assembly of hexamers.     

Transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae has transglycosylation activity. Treatment of (Man)6(GlcNAc)2Asn with the enzyme in the presence of free N-acetylglucosamine gave a mixture of (Man)6GlcNAc and (Man)6GlcNAc beta 1----4GlcNAc mixture. N-Acetylglucosamine at the reducing end of the latter sugar chain was found by HPLC of the carbohydrate composition and of an exoglycosidase digest of the pyridylamino derivative of the reducing-end residue, and by 400 MHz 1H-NMR spectroscopy.     

Carbohydrate binding properties of complex-type oligosaccharides on immobilized Datura stramonium lectin

The carbohydrate binding specificity of Datura stramonium agglutinin was studied by analyzing the behavior of a variety of complex-type oligosaccharides on a D. Stramonium agglutinin-Sepharose column. Oligosaccharides which contain Gal beta 1----4GlcNAc-beta 1----4(Gal beta 1----GlcNAc beta 1----2)Man units are retarded in the column so long as the pentasaccharide unit is not substituted by other sugars. Oligosaccharides which contain unsubstituted Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----4GlcNAc beta 1----2)Man groups and those in which there is at least one Gal beta 1----4GlcNAc repeating unit present on an outer chain bind to the column and are eluted with buffer containing N-acetylglucosamine oligomers. Binding was not affected by the inner core portion of complex oligosaccharides nor by the presence of a bisecting N-acetylglucosamine residue. With these principles in mind, the column can be used as an effective tool for the analysis of complex-type, asparagine-linked sugar chains.     

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

Synthesis of 1-N-glycyl beta-oligosaccharide derivatives. Reactivity of Lens culinaris lectin with a fluorescent labeled streptavidin pseudoglycoprotein and immobilized neoglycolipid.

The lectin from Lens culinaris (lentil) has a binding specificity for glycopeptides bearing 6-O-linked fucose on the reducing terminus on complex-type N-linked oligosaccharides. Lentil lectin therefore provides an excellent example of a carbohydrate binding protein in which high-affinity interactions are dependent on the integrity of the oligosaccharide core structure. We report here the synthesis of the 1-N-glycyl beta-derivative of Gal beta 4GlcNAc beta 2Man alpha 6(Gal beta 4GlcNAc beta 2Man alpha 3)Man beta 4GlcNAc beta 4(Fuc alpha 6)-GlcNAc (Gal-2F) and its subsequent biotinylation and palmitoylation. The biotin derivative when bound to a streptavidin-fluorescein isothiocyanate (FITC) conjugate was able to bind to both concanavalin A (ConA) and lentil lectin affinity columns. In contrast, synthesis of the biotin derivative of the glycamine derivative of Gal-2F and subsequent binding to streptavidin-FITC afforded reactivity to a ConA affinity column but not to a lentil lectin affinity column. Lentil lectin also bound to plastic microtiter plates containing the adsorbed palmitoyl-1-N-glycyl beta-derivative. No binding occurred when the homologous glycamine neoglycolipid was used. These results suggest the 1-N-glycyl beta-derivative of oligosaccharides may have general utility as an intermediate in the synthesis of novel glycoconjugate probes.