葛根素降低糖尿病大鼠血清糖基化终产物水平和单核细胞趋化蛋白1含量

目的　研究葛根素对糖尿病大鼠糖基化终产物 (AGEs)水平和单核细胞趋化蛋白 1(MCP 1)表达的影响。方法　将大鼠随机分为正常对照组 (CON)、糖尿病组 (DM )、糖尿病氨基胍治疗组 (AG )和糖尿病葛根素治疗组 (PU )。腹腔注射链脲佐菌素诱导糖尿病模型 ,检测血清中AGEs(荧光法 )和MCP 1(ELISA)水平 ;病理切片PAS染色及电镜观察肾组织的病理改变。免疫组化检测肾皮质MCP 1的蛋白表达水平。结果　DM组血清AGEs和MCP 1水平较PU组和AG组明显增高 (均P <0 .0 5 ) ;电镜发现两个治疗组的肾小球基底膜和心肌细胞病理改变程度比糖尿病组轻 ;肾小球内皮细胞和系膜区MCP 1免疫组化染色阳性细胞数明显比糖尿病组少。结论　葛根素能降低糖尿病大鼠血清AGEs和MCP 1水平 ,减少肾皮质中MCP 1的表达 ,减轻肾脏和心肌的病变程度     

糖基化终产物对大鼠主动脉平滑肌细胞巨噬细胞炎性蛋白-1α表达的影响

目的　探讨糖基化终产物 (AGEs)对大鼠主动脉平滑肌细胞巨噬细胞炎性蛋白 1α(MIP 1α)mRNA及蛋白表达的影响。　方法　将培养的大鼠主动脉平滑肌细胞用不同浓度 (10 0、2 0 0、4 0 0mg/L)的AGEs孵育 2 4h及同一浓度 (4 0 0mg/L)AGEs孵育 0、12、2 4、36h ,采用RT PCR方法及流式细胞仪检测MIP 1αmRNA及蛋白的表达水平。　结果　对照组平滑肌细胞内MIP 1α呈弱表达 ,10 0、2 0 0、4 0 0mg/LAGEs培养平滑肌细胞 2 4h显著增高MIP 1αmRNA的表达 ,其电泳条带相对积分吸光度值分别为对照组的 1 36、1 75、2 4 5倍 (P <0 0 5 ) ;10 0、2 0 0、4 0 0mg/LAGEs孵育2 4h后 ,各组平滑肌细胞MIP 1α蛋白的平均荧光强度分别为 197± 3、2 6 0± 6及 375± 6 ,分别为对照组(15 8± 4 )的 1 2 4、1 6 3、2 37倍 (P <0 0 5 )。 4 0 0mg/LAGEs培养 12、2 4、36h后 ,各组平滑肌细胞MIP 1αmRNA的表达也明显增高 ,其电泳条带相对积分吸光度值分别为 0h组的 1 5 2、2 38、2 5 3倍(P <0 0 5 ) ;4 0 0mg/LAGEs孵育 12、2 4、36h后 ,各组平滑肌细胞MIP 1α蛋白的平均荧光强度分别为 2 4 4± 5、375± 6及 4 2 5± 3,分别为 0h组 (170± 5 )的 1 4 3、2 2 1、2 4 9倍 (P <0 0 5 )。　结论　糖基化终产物以时间及剂量     

Additional N-glycosylation at Asn(13) rescues the human LHbeta-subunit from disulfide-linked aggregation

CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common -subunit, but differ in their hormone-specific β-subunits. Despite the considerable homology between LHβ and CGβ, we previously demonstrated that, when expressed in GH₃ cells, the secreted form of LHβ showed mispaired disulfide-linked aggregation in addition to monomer, whereas no aggregation was observed in CGβ. To determine the domains which are associated with the LHβ-aggregation and which prevent CGβ-aggregation, mutant β-subunits in glycosylation and carboxy-terminus were expressed in GH₃ cells, and the occurrence of aggregation was assessed by continuous labeling with [³⁵S]methionine/cysteine, immunoprecipitation with anti-hCGβ serum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. No aggregation was seen when N-linked oligosaccharides were attached to the Asn¹³ of LHβ. Removal of the carbohydrate unit at the Asn¹³ of CGβ caused aggregation, although the amount was less than 10% of monomer. The carboxy-terminal regions of neither LHβ nor CGβ were associated with their aggregation. Both CGβ wild-type (WT) and CGβ lacking N-glycosylation at Asn¹³ (CGβ-N13) showed aggregates in lysate. However, in contrast to CGβ-N13, CGβWT revealed no aggregation in medium. These results indicate that the backbone structure consisting of 114 amino acids and N-linked glycosylation at Asn³⁰ is involved in the aggregation of LHβ. Moreover, N-glycosylation at Asn¹³ does not prevent such aggregation, but instead plays an important role in correct folding for both LHβ- and CGβ-subunits to be secreted as monomer.     

The N-Glycosylation Modification of LHBs (Large Surface Proteins of HBV) Effects on Endoplasmic Reticulum Stress,Cell Proliferation and its Secretion

Background

The mutations of LHBs in pre-S, especially in pre-S2, are definitive in hepatocellular carcinoma (HCC) associated with HBV. However, the mechanisms of the N-glycosylation modification in LHBs are unclear. The N-glycosylation modification of LHBs affects Endoplasmic Reticulum stress, cell proliferation and its secretion which was further studied.

Objectives

The objectives of our studies was to indentified that modification of LHBs by N glycosylation modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation.

Materials and Methods

The LHBs was mutated; then expression of proteins related to endoplasmic reticulum stress and EAED path of L02 cells affected by LHBs and its mutations was evaluated. LHBs proteins bound to multiubiquitin chains and its glycosylation motif were studied. The subcellular localization and secretion of LHBs and its mutations were identified. The effect on cell cycle and proliferation by LHBs and its mutations were detected.

Results

These data demonstrated that the N-glycosylation motifs of LHBs were associated with ER stress. The N15S, N123S, and N177S mutated LHBs proteins could induce overexpression of EDEM in L02 cells. LHBs and its mutated proteins contained p62-derived UBA domain, which could affect expression of cyclins. The subcellular localization of LHBs in endoplasmic reticulum was similar to its mutations. The secretion of LHBs was blocked by N320K mutation, which could induce an increase in G1 phase and inhibition of S phase, and inhibited mitotic entry.

Conclusions

In conclusion, our studies powerfully demonstrated that modification of LHBs by N glycosylation could modulate their secretion, affect ER stress or expression of cycling, cell cycle and proliferation. The N320K may be the key sites N-linked glycosylation modification of LHBs. It may be a mechanism of HBV-induced HCC.     

杆状病毒系统表达信号肽序列依赖糖基化的EBVgp350片段

重组的糖基化多肽片断作为一个模型系统用于研究糖基化和识别依赖糖基化功能区或抗原决定簇。作者在杆状病毒系统中表达了完整的gp350及其内部片断。结果表明带有信号肽序列的片断能被糖基化,而不带有信号肽序列的片断不能被糖基化。使用PCR方法将信号肽序列导入EBVgp350的内部序列中,在杆状病毒系统中表达,导致了重组糖蛋白的产生。     

Glycosylation of maternal hair and cord skin during pregnancy in diabetic and non-diabetic women

A study of 15 insulin-dependent diabetics (IDD), 4 gestational diabetics (GDM), and 21 non-diabetic women was undertaken to assess non-enzymatic glycosylation in maternal hair, cord skin, and cord blood. Glycosylation of maternal hair was stable from tip to root in controls. In the IDD group, levels were highest at the tip, but were significantly lower in the middle and scalp segment (p less than 0.01), both tip and middle segments were higher than control values (p less than 0.01). In contrast, only the scalp segment of hair from the GDM group had a higher than normal level of glycosylation (p less than 0.01). Cord skin and cord blood glycosylation were significantly increased in the IDD group compared with normal (0.100 +/- 0.002 vs 0.074 +/- 0.008 mumol fructosamine/100 mg, p less than 0.01, and 0.160 +/- 0.002 vs 0.14 +/- 0.008, p less than 0.05, mean +/- S.E.M.), respectively. Only cord skin glycosylation was increased in the GDM group (0.116 +/- 0.003, p less than 0.01). Thus near normal glycaemic control of maternal insulin-dependent diabetes during pregnancy was associated with small but significant increases in cord skin glycosylation and a fall towards normal in maternal hair glycosylation which may provide a useful retrospective index of diabetic control.     

Non-enzymatically glycosylated serum protein in diabetes mellitus: An index of short-term glycaemia

Summary We measured non-enzymatically-glycosylated serum protein by a colorimetric assay in 107 diabetic and 82 control subjects. The mean level in diabetics was more than twice that in controls. Cross sectional and longitudinal studies in diabetic patients showed that glycosylated serum protein levels correlated with both fasting serum glucose and glycosylated haemoglobin levels. The correlation between glycosylated serum protein and fasting serum glucose was closer in Type 2 than in Type 1 diabetes. Treatment aimed at improving control in eight poorly controlled diabetic patients resulted in a 37% mean fall in glycosylated serum protein within one week, whereas glycosylated haemoglobin decreased only 8%. These studies confirm that non-enzymatic glycosylation of serum proteins is enhanced in diabetes. Measurement of glycosylated serum protein appears to provide an index of glycaemia over the preceding several days. It has the advantage of detecting improvements in glycaemic control much sooner than does glycosylated haemoglobin measurement.     

外敷氨基胍霜剂对糖尿病大鼠皮肤组织的影响

目的 观察外敷氨基胍霜剂对糖尿病大鼠皮肤组织晚期糖基化终末产物(AGE)形成、KC细胞增殖及氧化应激的影响.方法 将硬脂酸、液状石蜡、凡士林、羊毛脂、肉豆蔻酸异丙酯、甘油、50 g/L尼泊金醇、盐酸氨基胍等试剂按一定比例混合制成氨基胍霜剂,以不含有氨基胍的霜剂为基质.取健康大鼠背部皮肤,分别用5、10 g/L氨基胍霜剂和5 g/L氨基胍+10g/L氮酮霜剂处理,于用药后2、4、7、10、12、24 h测定药物透皮效果.将30只SD大鼠按随机数字表法分为正常对照组6只、糖尿病组8只、氨基胍治疗组8只、基质治疗组8只.后3组大鼠腹腔注射链脲佐菌素65 mg/kg,诱导糖尿病模型;对照组大鼠注射0.05 mmol/L柠檬酸缓冲液.注射1周后,正常对照组与糖尿病组大鼠不行任何治疗,氨基胍治疗组与基质治疗组大鼠背部分别连续外用10 g/L氨基胍霜剂与基质治疗4周.取各组皮肤组织,胶原提取液荧光强度检测法测定AGE含量,流式细胞仪分析表皮KC周期,检测氧化应激相关指标超氧化物歧化酶(SOD)、丙二醛、总抗氧化能力、髓过氧化物酶(MPO)含量.对实验数据行t检验.结果 10 g/L氨基胍霜剂透皮效果优于5g/L氨基胍和5 g/L氨基胍+10 g/L氮酮的霜剂.1只基质治疗组大鼠未诱导成功.建模后4周,糖尿病组与氨基胍治疗组大鼠分别死亡4只和1只.糖尿病组大鼠皮肤组织AGE含量为每毫克羟脯氨酸(OHP)中(36.8±2.6)U,明显高于正常对照组的每毫克OHP中(24.6±2.7)U(t=7.2,P＜0.01);氨基胍治疗组AGE含量为每毫克OHP中(28.6±3.7)U,明显低于糖尿病组(t=-3.9,P＜0.05);基质治疗组AGE含量[每毫克OHP中(32.2±5.2)U]与糖尿病组相近(t=1.6,P＞0.05).糖尿病组大鼠S期KC比例为(5.3±0.6)%,低于正常对照组的(7.6±0.9)%(t=4.50,P＜0.01);氨基胍治疗组大鼠S期和G2/M期KC比例均明显高于糖尿病组(t值分别为6.80、3.17,P值均小于0.01);基质治疗组大鼠S期KC比例[(9.2±1.5)%]显著高于糖尿病组(t=4.90,P＜0.01).糖尿病组大鼠皮肤组织氧化应激指标含量均高于正常对照组,其中SOD和MPO差异有统计学意义(t值分别为4.4、3.7,P值均小于0.05);氨基胍治疗组各氧化应激指标含量均较糖尿病组降低,其中SOD含量显著低于糖尿病组(t=-1.4,P＜0.05);基质治疗组MDA、MPO含量显著低于糖尿病组(t值分别为2.6、2.9,P值均小于0.05).结论 外用氨基胍霜剂可以在一定程度上阻碍糖尿病大鼠皮肤组织中AGE的形成,改善表皮KC细胞增殖能力,适当降低皮肤组织氧化应激状态;单用霜剂基质也可适当降低皮肤组织氧化应激状态.

Abstract：

Objective To investigate the effects of aminoguanidine cream on the proliferation of keratinocytes (KC), content of advanced glycosylation end products (AGE) and oxidative stress in skin tissue of rats with diabetes. Methods Stearic acid, liquid paraffin, vaseline, lanolin, isopropyl myristate fat,glycerol, 50 g/L alcohol paraben, aminoguanidine hydrochloride etc. were mixed in certain proportion to make aminoguanidine cream, and cream without aminoguanidine was used as matrix. The dorsal skin of normal rats were harvested and treated by aminoguanidine cream with dose of 5, 10 g/L, or 5 g/L together with 10 g/L azone. The transdermal effect was respectively measured at post treatment hour 2, 4, 7, 10, 12,24. Thirty SD rats were divided into normal control(NC, n = 6) , diabetes(D, n = 8) , aminoguanidine cream-interfered(AI, n = 8), matrix cream-interfered groups(MI, n = 8) according to the random number table. Diabetes was reproduced by intraperitoneal injection of STZ (65 mg/kg) in rats of D, AI, and MI groups, and rats in NC group were injected with 0. 05 mmol/L citrate buffer as control. One week later, dorsal skin of rats in AI and MI groups were respectively treated with 10 g/L aminoguanidine cream and matrix cream by external use for 4 weeks. AGE content was determined with fluorescence detection from skin collagen extract. KC cell cycle was detected by flow cytometry. Skin tissue specimens were obtained for determination of levels of superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and total antioxidant capacity. Data were processed with t test. Results Transdermal effect of aminoguanidine cream with dose of 10 g/L was better than that with 5 g/L or 5 g/L + 10 g/L azone cream. One rat was not induced successfully in MI group. Four weeks after model reproduction, 4 rats died in D group and 1 rat died in AI group. The AGE content in D group was obviously higher than that in NC group [(36.8 ± 2.6),(24. 6 ±2.7) U per milligram hydroxyproline, respectively, t = 7.2, P ＜0. 01], and that in AI group [(28.6 ±3.7) U per milligram hydroxyproline] was also lower as compared with that in D group(t = -3.9,P ＜ 0.05). There was no significant difference in AGE content between MI [( 32.2 ± 5.2) U per milligram hydroxyproline] and D groups(t = 1.6, P ＞ 0. 05). The percentage of KC in S phase was obviously lower in D group than in NC group [(5.3 ±0.6)%, (7.6±0.9)%, respectively, t =4.50, P ＜0. 01], while that in MI group [(9. 2 ± 1.5) %] was higher as compared with that in D group(t = 4.90, P ＜ 0. 01). It was more higher in AI group than in D group on KC percentage in S and G2/M phase (with t value respectively 6.80, 3.17, P values all below 0. 01). The oxidative stress indexes of skin tissue in D group were all higher than those in NC group, in which levels of MPO and SOD showed statistical difference(with t value respectively 4.4, 3.7, P values all below 0. 05). The oxidative stress indexes were all lower in AI group than in D group, especially in SOD level(t = -1.4, P ＜0. 05). Levels of MAD, MPO in MI group were significantly lower than those in D group(with t value respectively 2.6, 2.9, P values all below 0. 05).Conclusions Aminoguanidine cream can promote KC proliferation and appropriately reduce oxidative stress through inhibiting AGE formation to a certain extent in skin tissue of rats with diabetes. Signal use of matrix cream can also reduce oxidative stress in skin tissue of rats with diabetes.     

黏附分子CD44的表达及其糖基化与肝癌转移的相关性

目的 探讨CD44S及其变异体CD44v分子的表达和其糖基化与肝癌细胞侵袭转移的关系. 方法 用免疫组织化学法、量子点、RT-PCR、Western blot、细胞免疫荧光染色、甲基化特异性聚合酶链反应等技术检测转移与非转移性肝癌组织、不同转移潜能人肝癌细胞株中CD44S及其变异体CD44v的定位与表达;并应用多重凝集素印迹法检测这些细胞株中CD44v6的糖基化差异.组间均数比较应用方差分析及t检验,多组间等级资料的比较采用Wilcoxon秩和检验,各组间率的比较采用x2检验.结果 免疫组织化学结果显示,CD44S蛋白定位以细胞质为主; CD44v3、CD44v4/5蛋白定位于细胞膜与细胞质;而CD44v6主要定位于细胞膜.组织芯片结果显示,相对于CD44S及其他CD44v,CD44v6在转移组的表达水平高于非转移组(阳性率为75％与46％),差异具有统计学意义(x2=8.828,P＜0.05);量子点检测(t=2.392,P＜0.05)与血清学检测(t=2.56,P＜0.05)也证实这一结果.Western blot结果显示,CD44v6的表达与肝癌细胞转移潜能呈正相关.此外,在MHCC97L、MHCC97H肝癌细胞中CD44v6基因启动子发生不完全甲基化,而在Hep3B细胞中则发生完全甲基化.而且,相对于Hep3B细胞,MHCC97L及MHCC97H细胞中CD44v6蛋白对朝鲜槐凝集素、黑接骨木凝集素及麦胚凝激素的亲和力较高. 结论 在CD44S及其变异体CD44v中,CD44v6蛋白的高表达与肝癌转移潜能的增强呈正相关;其高表达可能与基因启动子低甲基化有关.此外,CD44v6蛋白唾液酸寡糖链的增加可能与肝癌细胞转移潜能增高有关.     

肝癌转移相关的骨桥蛋白表达及其糖基化的改变

目的 检测骨桥蛋白(OPN)在不同转移潜能肝癌细胞株及肝癌组织中的蛋白表达水平及其糖基化水平,探讨OPN糖基化改变与肝癌转移的相关性及其意义. 方法 用免疫组织化学和Western blot法检测人肝癌组织(非转移组6例、转移组7例)及不同转移潜能人肝癌细胞株(L02、Hep3B、MHCC97L、MHCC97H、HCCLM3、HCCLM6)中OPN蛋白水平;采用免疫沉淀技术纯化肝癌组织中的OPN蛋白,并采用多重凝集素印迹技术检测转移与非转移肝癌组织中OPN糖基化水平差异.数据统计采用t检验和方差分析.结果 OPN在不同转移潜能肝癌细胞株的表达差异具有统计学意义(F=5.04,P＜0.01).在肝癌组织中,转移组OPN蛋白表达水平明显高于非转移组(t=2.447,P＜0.05),相对吸光度值分别为0.69±0.21和0.45±0.14.免疫沉淀技术成功纯化肝癌组织中的OPN蛋白,后续的凝集素印迹结果显示:与非转移组相比,转移组OPN蛋白对凝集素朝鲜槐、红腰果E型、蔓陀罗、刀豆素A的亲和力较低(P值均＜0.05).结论 OPN蛋白表达水平与肝癌转移潜能增强呈正相关;OPN蛋白的α2,3-唾液酸、平分型GlcNAc、多天线、偏二天线的糖链、高甘露糖型N-糖等糖基化水平改变可能与肝癌转移潜能增高有关.