糖基化终末产物对大鼠肾系膜细胞纤溶酶原激活物抑制物-1表达韵影响

目的：观察糖基化终末产物（AGEs）对大鼠肾系膜细胞纤溶酶原激活物抑制物-1（PAI-1）表达的影响及其与细胞外基质（ECM）成分含量的关系。方法：体外培养正常大鼠肾系膜细胞，分别用糖化牛血清白蛋白（AGEs）及未经糖化的牛血清白蛋白（BSA）处理，以常规培养的肾系膜细胞作为对照，检测不同时间、不同浓度AGEs对纤维连接蛋白（FN）、Ⅳ型胶原、PAI-1表达的影响。MTT法检测AGEs对系膜细胞增殖的作用，ELISA测定条件培养基中FN、Ⅳ型胶原及PAI-1蛋白含量，逆转录聚合酶链式反应（RT—PCR）检测系膜细胞PAI-1 mRNA的表达。结果：与相应浓度的BSA比较，AGEs（0—200mg／L）对系膜细胞增殖无明显影响，但可不同程度地刺激系膜细胞FN、Ⅳ型胶原、PAI-1蛋白的产生。RT—PCR检测显示，给予AGEs（100mg／L）的系膜细胞PAI-1 mRNA的表达明显增加（P〈0．01）。结论：AGEs促进系膜细胞PAI—1的表达，提示AGEs通过上调PAI-1的表达而减少细胞外基质降解，可能是糖尿病肾病细胞外基质积聚的原因之一。     

Caprine arthritis-encephalitis virus-infected goats can generate human immunodeficiency virus-gp120 cross-reactive antibodies(1)

Lentiviruses display surprisingly disparate clinical manifestations in their specific hosts, share complex genetic structures, and exhibit extensive diversity, particularly in their envelope genes. The envelope protein, gp135, of caprine arthritis-encephalitis virus (CAEV) has minimal primary sequence homology to gp120, the envelope protein of human immunodeficiency virus (HIV). Nevertheless, they bear certain similarities in that they both possess five variable regions, both are heavily glycosylated, and both share short sequence motifs. We establish a further relationship and demonstrate that some goats, infected with CAEV, possess gp135-specific antibodies which cross-react with gp120 from several HIV strains, provided the protein is expressed in insect cells. We show that, although the cross-reactivity of these immunoglobulins depends on the level of glycosylation, nevertheless, some antibodies recognize the protein epitopes on gp120, at least some of which are linear in character. Further characterization of this unexpected cross-reaction will define its potential therapeutic utility.     

Isolation of a Trichoderma reesei cDNA encoding GTP: a-d-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation

A cDNA coding for GTP: α-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase. The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids. Sequence comparisons demonstrate 70% identity with the S. cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue. No similarity was found with the MPD synthase encoded by the S. cerevisiae DPM1 gene. The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S. cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores. Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S. cerevisiae dpm1-6 mutant. Received: 21 July 1997 / 24 April 1998     

Increased central adiposity is associated with pro-inflammatory immunoglobulin G N-glycans

Background

Increased body fat may be associated with an increased risk of developing an underlying pro-inflammatory state, thus leading to greater risk of developing certain chronic conditions. Immunoglobulin G has the ability to exert both anti- and pro-inflammatory effects, and the N-glycosylation of the fragment crystallisable portion is involved in mediating this process. Body mass index, a rudimentary yet gold standard indication for body fat, has been shown to be associated with agalactosylated immunoglobulin G N-glycans.

终末糖基化产物对培养的人脐静脉内皮细胞粘附分子表达的影响

目的：探讨终末糖基化产物在糖尿病动脉粥样硬化（AS）形成中的作用机理。 方法： 分离正常人脐静脉内皮细胞（HUVECs），将终末糖基化终产物（AGE）修饰的人血清白蛋白（AGE-HSA）、人血清白蛋白（HSA）与HUVECs在体外共同培养，并用荧光单克隆抗体染色，流式细胞仪定量检测细胞间粘附分子-1（ICAM-1）、血管细胞粘附分子-1（VCAM-1）的表达。 结果： 正常人HUVEC表达ICAM-1和VCAM-1。AGE-HSA能以时间和剂量依赖的方式上调ICAM-1、VCAM-1的表达（P<0.05），而HSA对HUVECs上述粘附分子的表达均无影响。 结论： AGE能上调HUVECs粘附分子的表达，从而促进AS时单核/巨噬细胞的浸润。     

Identification of glycosylation sites in the SU component of the Avian Sarcoma/Leukosis virus Envelope Glycoprotein (Subgroup A) by mass spectrometry

We used enzymatic digestion and mass spectrometry to identify the sites of glycosylation on the SU component of the Avian Sarcoma/Leukosis virus (ASLV) Envelope Glycoprotein (Subgroup A). The analysis was done with an SU(A)-rIgG fusion protein that binds the cognate receptor (Tva) specifically. PNGase F removed all the carbohydrate from the SU(A)-rIgG fusion. PNGase F is specific for N-linked carbohydrates; this shows that all the carbohydrate on SU(A) is N-linked. There are 10 modified aspargines in SU(A) (N17, N59, N80, N97, N117, N196, N230, N246, N254, and N330). All conform to the consensus site for N-linked glycosylation NXS/T. There is one potential glycosylation site (N236) that is not modified. Removing most of the carbohydrate from the mature SU(A)-rIgG by PNGase F treatment greatly reduces the ability of the protein to bind Tva, suggesting that carbohydrate may play a direct role in receptor binding.     

阿魏酸钠对糖尿病大鼠肾脏非酶糖基化和氧化的影响

目的:探讨阿魏酸钠(SF)对糖尿病(DM)大鼠肾脏非酶糖基化和氧化的影响。方法:对链脲佐菌素(STZ)诱导的DM大鼠灌胃给予SF 110 mg·kg^-1·d^-1,治疗8周,测定各组大鼠肾重／体重、肌酐清除率(Ccr)、24 h尿蛋白定量、血清和肾皮质果糖胺(FMN)、血清和肾皮质丙二醛(MDA)含量及抗氧化酶活性,并分别测定肾皮质糖基化终产物(AGEs)含量,观察肾脏病理改变。结果:糖尿病对照组(DM组)大鼠肾重／体重、Ccr、24 h尿蛋白定量、血清FMN、肾皮质FMN和AGEs显著高于正常对照组(N组);SF治疗组(SF组)肾重／体重、Ccr、24 h尿蛋白定量、血清FMN和肾皮质AGEs显著低于DM组;DM组大鼠肾皮质和血清超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性显著低于N组,MDA含量显著高于N组,SF治疗组大鼠肾皮质和血清SOD、CAT活性显著高于DM组,MDA含量显著低于DM组:DM组大鼠肾脏病理改变异常显著,SF组的肾脏病理学改变轻于DM组大鼠。结论: SF通过保护肾脏抗氧化酶,减轻氧化应激,抑制AGEs在肾脏的沉积对DM大鼠肾脏产生保护作用。     

Lipophosphoglycan expression and virulence in Ricin-resistant variants of Leishmania major

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats joined by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the M_r 63 000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies.All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.     

先天性糖基化病的研究进展

先天性糖基化病 (ｃｏｎｇｅｎｉｔａｌｄｉｓｏｒｄｅｒｓｏｆｇｌｙｃｏｓｙｌａ ｔｉｏｎ,ＣＤＧ) ,旧称为糖类缺陷性糖蛋白综合征 (ｃａｒｂｏ ｈｙｄｒａｔｅ-ｄｅｆｉｃｉｅｎｔｇｌｙｃｏｐｒｏｔｅｉｎｓｙｎｄｒｏｍｅｓ,ＣＤＧＳ) ,是一类由于聚糖的合成和结合到其他复合体 (蛋白质和脂质 )过程的缺陷引起的常染色体隐性遗传病[1,2 ] 。绝大多数ＣＤＧ患者有多种系统病变且均有不同程度的精神发育迟缓。迄今发现的ＣＤＧ主要是由于人体内蛋白Ｎ -糖基化途径缺陷引起的 ,不同酶的缺陷产生不同的ＣＤＧ类型。最新的命名系统将脂联寡…     

Biosynthesis of two stage-specific membrane proteins during transformation of Plasmodium gallinaceum zygotes into ookinetes

We have studied the synthesis and expression of surface proteins in zygotes of Plasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [35S]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of Mr 26 000 as observed under reducing conditions on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31 000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26 000 protein declined and little or none was synthesized in the mature ookinetes (greater than 20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-1 and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [3H]glucosamine or [3H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.