唾液和泪液中主要黏蛋白MUC5B和MUC7协同互补功能

黏蛋白家族中MUC5B和MUC7是人体唾液和泪液中的两种主要黏蛋白成分,早期被认为在非特异性免疫应答、黏液构成以及组织功能的维护等方面发挥作用。近年来,它们的一些新功能如抗HIV、协助非黏蛋白功能的发挥等作用逐渐被发现,故其生理和病理意义也因传统观念的修正而得到扩充。由于MUC5B和MUC7在唾液和泪液中的存在方式并不完全相同,对它们在不同环境条件下功能的相似性、差异性和互补性的研究,具有重要的现实意义。     

Visfatin induces MUC8 and MUC5B expression via p38 MAPK/ROS/NF-κB in human airway epithelial cells

Background

Among a variety of inflammatory mediators, visfatin is a proinflammatory adipocytokine associated with inflammatory reactions in obesity, metabolic syndrome, chronic inflammatory disease, and autoimmune disease. However, the biological role of visfatin in secretion of major mucins in human airway epithelial cells has not been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of visfatin on MUC8 and MUC5B expression in human airway epithelial cells.

Results

Visfatin significantly induced MUC8 and MUC5B expression. Visfatin significantly activated phosphorylation of p38 MAPK. Treatment with SB203580 (p38 MAPK inhibitor) and knockdown of p38 MAPK by siRNA significantly blocked visfatin-induced MUC8 and MUC5B expression.Visfatin significantly increased ROS formation. Treatment with SB203580 significantly attenuated visfatin-induced ROS formation. Treatment with NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression. However, treatment with NAC and DPI did not attenuate visfatin-activated phosphorylation of p38 MAPK. Visfatin significantly activated the phosphorylation of NF-κB. Treatment with PDTC (NF-κB inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression.

Conclusions

These results suggest that visfatin induces MUC8 and MUC5B expression through p38 MAPK/ROS/NF-κB signaling pathway in human airway epithelial cells.     

MUC5B promoter polymorphisms and risk of coal workers’ pneumoconiosis in a Chinese population

Coal workers’ pneumoconiosis (CWP) is characterized by fibrosing nodular lesions that eventually develop into progressive pulmonary fibrosis. Genetic variations have been recognized to be involved in the multi-factorial susceptibility to CWP, and MUC5B is a candidate lung fibrosis susceptibility gene. In the present study, we investigated possible genetic associations between three single nucleotide polymorphisms in MUC5B promoter region and CWP in a case–control study including 686 CWP patients and 680 controls. Genotyping was carried out by TaqMan method. Only rs2672794 allele and genotype frequencies distributions were significantly different between CWP patients and controls (P = 0.017 and 0.046 for allele and genotype, respectively). The MUC5B rs2672794 CC genotype was associated with a significantly increased risk of CWP, compared with the TT genotype. Moreover, individuals with TC/CC genotype had an obviously increased risk of CWP than those with TT genotype, particularly among subgroups of dust exposure <27 years and smokers. This is the first report showing an association between the MUC5B rs2672794 polymorphism and CWP, and our results suggest that MUC5B rs2672794 CC genotype could increase the risk of CWP. Further studies are warranted to confirm our findings.     

γ干扰素抑制幼儿支气管上皮细胞MUC5AC表达

本研究主要探讨了IFN-γ在黏蛋白产生中的作用,特别是在儿童支气管上皮细胞中的MUC5AC转录。通过采用人肺黏液表皮样癌细胞系(NCI-H292)和正常人支气管上皮细胞(NHBE),本研究评估了IFN-γ对MUC5AC转录的影响,发现转化生长因子(TGF)-α和双链RNA (polyI:C)诱导的MUC5AC mRNA和蛋白表达被IFN-γ以浓度依赖性方式抑制。IFN-γ对TGF-α和polyI:C诱导的表皮生长因子受体(EGFR)和细胞外信号调节激酶(ERK)的激活作用有限。染色质免疫沉淀实验表明Sp1与位于MUC5AC启动子上的同源序列结合。Sp1抑制剂光神霉素A抑制MUC5AC mRNA,表明Sp 1在MUC5AC诱导中起关键作用,同时IFN-γ阻碍Sp1与MUC5AC启动子的结合。本研究初步表明,IFN-γ可以抑制MUC5AC的表达,干扰Sp1与其靶序列的结合。     

Galectin-3 binds to MUC1-N-terminal domain and triggers recruitment of β-catenin in MUC1-expressing mouse 3T3 cells

Background

Galectin-3 is expressed in a variety of tumors and its expression level is related with tumor progression. Aberrant expression of MUC1 in various tumors is also associated with a poor prognosis. It has been reported that MUC1 is a natural ligand of galectin-3.

Methods

A stable MUC1 transfectant was produced by introducing MUC1 cDNA into mouse 3T3 fibroblasts (MUC1/3T3 cells). MUC1 was prepared from MUC1/3T3 cells; MUC1-N-terminal domain (MUC1-ND) and -C-terminal domain (MUC1-CD) were separated by CsCl ultracentrifugation, and then the galectin-3-binding domain was determined by co-immuniprecipitation assay. After ligation of galectin-3 to 3T3/MUC1 cells, MUC1-CD was immunoprecipitated from the cell lysate. The immunoprecipitate was subjected to SDS-PAGE and Western blotting, followed by detection of co-immunoprecipitated β-catenin.

Results

Galectin-3 binds to the N-terminal domain of MUC1 but not to the C-terminal one. Galectin-3 present on the cell surface increased with the expression of MUC1 and is colocalized with MUC1. It should be noted that β-catenin was detected in the immunoprecipitate with anti-MUC1-CD Ab from a lysate of galectin-3-treated 3T3/MUC1 cells.

Conclusions

Galectin-3 binds to MUC1-ND and triggers MUC1-mediated signaling in 3T3/MUC1 cells, leading to recruitment of β-catenin to MUC1-CD.

General significance

This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated pathway.     

黏蛋白MUC1的研究进展

黏蛋白MUC1是一种具有高度糖基化胞外区的Ⅰ型跨膜蛋白,存在于正常细胞和多种癌细胞的表面,为公认的血清和细胞肿瘤抗原。本文简述了MUC1的分子生物学结构、生物学功能及其在肿瘤疫苗方面的研究应用进展。     

Synthesis and antibacterial activity of 5′-tetrachlorophthalimido and 5′-azido 5′-deoxyribonucleosides

Reported is an efficient synthesis of adenyl and uridyl 5′-tetrachlorophthalimido-5′-deoxyribonucleosides, and guanylyl 5′-azido-5′-deoxyribonucleosides, which are useful in solid-phase synthesis of phosphoramidate and ribonucleic guanidine oligonucleotides. Replacement of 5′-hydroxyl with tetrachlorophthalimido group was performed via Mitsunobu reaction for adenosine and uridine. An alternative method was applied for guanosine which replaced the 5′-hydroxyl with an azido group. The resulting compounds were converted to 5′-amino-5′-deoxyribonucleosides for oligonucleotide synthesis. Synthetic intermediates were tested as antimicrobials against six bacterial strains. All analogs containing the 2′,3′-O-isopropylidine protecting group demonstrated antibacterial activity against Neisseria meningitidis, and among those analogs with 5′-tetrachlorophthalimido and 5′-azido demonstrated increased antibacterial effect.     

COX-2/PGE2对TNF-α促进MUC5AC分泌的影响

目的：研究环氧化酶-2（COX-2）/前列腺素（PGE2）在肿瘤坏死因子-α（TNF-α）刺激黏液生成过程中的作用。方法：体外培养的BEAS-2B气道上皮细胞系施以TNF-α刺激,以选择性及非选择性COX-2抑制剂为干预因素,比较各干预组与对照组中COX-2、PGE2水平及黏蛋白（MUC）5AC的含量的差异。结果：COX-2选择性抑制剂NS-398能抑制TNF-α引起的MUC5AC mRNA、MUC5AC蛋白含量增高（P〈0.05）,PGE2、COX-2及cAMP的含量也较刺激组减少,非选择性COX-2抑制剂吲哚美辛对MUC5AC mRNA及蛋白含量的影响不大。结论：在BEAS-2B上皮细胞系中,TNF-α能诱导COX-2/PGE2生成而引起黏蛋白分泌增加。     

Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12

Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.     

Synthesis of 5′-Fluoro-5′-deoxy-and 5′-Amino-5′-Deoxytoyocamycin and Sangivamycin and Some Related Derivatives

Abstract

A series of 5′-substituted analogs of toyocamycin were prepared by condensation of silylated 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine with protected 5-azido-5-deoxy- or 5-fluoro-5-deoxyribofuranose followed by debromination and deblocking. Alternatively, 5′-azido-5′-deoxytoyocamycin was prepared by azidation of toyocamycin. Conversion of the 5-nitrile function of the toyocamycin derivatives into a carboxamide or a thiocarboxamide gave the corresponding analogs of sangivamycin or thiosangivamycin while reduction of the 5′-azido-5′-deoxy nucleosides provided 5′-amino-5′-deoxy derivatives.     

Development and characterization of an antibody directed to an α-N-acetyl-D-galactosamine glycosylated MUC2 peptide

In an attempt to raise anti-Tn antibodies, an -N-acetyl-D-galactosamine glycosylated peptide based on the tandem repeat of the intestinal mucin MUC2 was used as an immunogen. The MUC2 peptide (PTTTPISTTTMVTPTPTPTC) was glycosylated in vitro using concentrated -N-acetylgalactosaminyltransferases activity from porcine submaxillary glands which resulted in the incorporation of 8–9 mol of Ga/NAc. Rabbits and mice developed specific anti-MUC2-GalNAc glycopeptide antibodies and no detectable anti-Tn antibodies. Anti-glycopeptide antibodies did not show reactivity with the unglycosylated MUC2 peptide or with other GalNAc glycosylated peptides. A mouse monoclonal antibody (PMH1) representative of the observed immune response was generated and its immunohistological reactivity analysed in normal tissues. PMH1 reacted similarly to other anti-MUC2 peptide antibodies. However, in some cells the staining was not restricted to the supranuclear area but extended to the entire cytoplasm. In addition, PMH1 reacted with purified colonic mucin by Western blot analysis suggesting that PMH1 reacted with some glycoforms of MUC2. The present work presents a useful approach for development of anti-mucin antibodies directed to different glycoforms of individual mucins.     

γ-干扰素对哮喘血清炎症因子及MUC5ac表达的影响分析

为了解析γ-干扰素对哮喘的治疗效果,及对血清炎症因子和MUC5ac表达情况的影响,本研究选取2016年2月至2017年2月在我院接受治疗的哮喘患者为研究对象,根据治疗方式分为对照组和观察组,对照组给予孟鲁司特等常规药物治疗,观察组在此基础上给予γ-干扰素治疗。观察两组患者的治疗效果,比较两组患者治疗前后炎症因子、肺功能和MUC5ac表达水平的差异。研究显示,观察组患者治疗的有效率(97.50%)显著高于对照组(85.00%);两组患者治疗前的炎症因子水平无差异,治疗后,观察组IL-8、IL-17、hs-CRP和TNF-α水平低于对照组;两组患者治疗前的肺功能指标无差异,治疗后,观察组6MWT、FVC、MMEF和PEF水平高于对照组;两组患者治疗前的MUC5ac水平无差异,治疗后,观察组MUC5ac水平低于对照组。本研究表明,哮喘患者采用γ-干扰素进行治疗的效果显著,可有效降低炎症因子及MUC5ac水平,应用价值良好。     

吸烟大鼠肺脏AQP4和MUC5AC的表达及其与一氧化氮的关系

目的：研究烟草烟雾吸入对大鼠肺组织水通道蛋白4（AQP4）和粘蛋白5AC（MUC5AC）表达的影响及其与支气管肺泡灌洗液内一氧化氮代谢物水平的关系,探讨氧化应激对肺部水转运和粘液分泌的影响。方法：免疫组化法观察AQP4在肺组织内的表达,平均光密度法比较模型组和空白组大鼠AQP4的表达差异;半定量RT—PCR法检测肺组织内AQP4及MUC5ACmRNA的表达水平;硝酸还原酶法测定各组大鼠支气管肺泡灌洗液内一氧化氮代谢产物的浓度,分析模型组AQP4、MUC5ACmRNA的表达水平与支气管肺泡灌洗液内一氧化氮代谢物浓度之间的相关关系：结果：AQP4在空白对照组呈强阳性染色,在模型组呈弱阳性染色,两者平均光密度值有显著差异（P〈0．05）。模型组动物肺组织AQP4mRNA的表达降低,MUC5ACmRNA的表达升高,与空白组比较均有显著差异（P〈0．05）,模型组动物支气管肺泡灌洗液内一氧化氮代谢产物的浓度与肺组织AQP4mRNA表达水平呈负相关,相关系数r=-0．798,（P〈0．05）,与MUC5ACmRNA的表达水平呈正相关,相关系数r=0．857,（P〈0．05）。结论：吸烟可导致肺组织AQP4表达下降进而影响气道内水的转运。一氧化氮可能参与了烟雾吸入动物模型中AQP4与MUC5AC基因袁达的调控．     

吸烟大鼠肺脏AQP4 和MUC5AC 的表达及其与 一氧化氮的关系

目的:研究烟草烟雾吸入对大鼠肺组织水通道蛋白4(AQP4)和粘蛋白5AC(MUC5AC)表达的影响及其与支气管肺泡灌洗液内一氧化氮代谢物水平的关系,探讨氧化应激对肺部水转运和粘液分泌的影响。方法:免疫组化法观察AQP4在肺组织内的表达,平均光密度法比较模型组和空白组大鼠AQP4的表达差异;半定量RT-PCR法检测肺组织内AQP4及MUC5AC mRNA的表达水平;硝酸还原酶法测定各组大鼠支气管肺泡灌洗液内一氧化氮代谢产物的浓度,分析模型组AQP4、MUC5AC mRNA的表达水平与支气管肺泡灌洗液内一氧化氮代谢物浓度之间的相关关系。结果:AQP4在空白对照组呈强阳性染色,在模型组呈弱阳性染色,两者平均光密度值有显著差异(P<0.05)。模型组动物肺组织AQP4 mRNA的表达降低,MUC5AC mRNA的表达升高,与空白组比较均有显著差异(P<0.05),模型组动物支气管肺泡灌洗液内一氧化氮代谢产物的浓度与肺组织AQP4 mRNA表达水平呈负相关,相关系数r=-0.798(,P<0.05),与MUC5AC mRNA的表达水平呈正相关,相关系数r=0.857(,P<0.05)。结论:吸烟可导致肺组织AQP4表达下降进而影响气道内水的转运。一氧化氮可能参与了烟雾吸入动物模型中AQP4与MUC5AC基因表达的调控。     

金黄色葡萄球菌肠毒素B诱导人鼻黏膜上皮细胞表达黏蛋白MUC5AC的分子机制

为了观察金黄色葡萄球菌肠毒素B(SEB)对诱导人鼻钻膜上皮细胞分泌粘蛋白MUC5AC的影响,并初步探讨其作用机制,本研究首先在体外培养人鼻黏膜上皮细胞株HNEpC,用不同浓度的SEB孵育细胞0~24 h,ELISA检测培养上清中MUC5AC和转化生长因子-α(TGF-α)的含量;实时定量PCR检测MUC5AC mRNA表达水平;Western blot分析表皮生长因子受体(EGFR)的磷酸化。同时采用肿瘤坏死因子转化酶(TALE)siRNA干扰其表达双察其对TGF-α分泌的影响最后采用TGF-α中和抗体、EGFR抑制剂AG-1478或TALE抑制剂TAPI处理细胞,观察其在介导MUC5AC分泌中的作用。结果显示,1 ng/mL、10 ng/mL和100 ng/mL SEB作用鼻黏膜上皮细胞24 h后,可明显诱导其分泌MUC5AC并表达其mRNA,且其分泌水平随着SEB浓度的增高或时间的延长而逐渐增多。SEB也能诱导鼻黏膜上皮细胞分泌TGF-α,并磷酸化EGFR。给予10μmol/L TALE抑制剂TAPI处理细胞后,TGF-α和MUC5AC分泌水平显著减少,采用TALE siRNA干扰其表达后,TGF-α和MUC5AC也得到了类似结果。采用TGF-α中和抗体预孵育细胞30 min后,可明显抑制EGFR磷酸化。此外,TGF-α和EGFR中和抗体以及AG-1478预处理也可降低MUC5AC分泌。以上结果表明SEB经TALE/TGF-α/EGFR诱导人鼻黏膜上皮细胞表达MUC5AC。     

γ-干扰素对哮喘儿童血清炎症因子及MUC5ac指标的影响

本研究以治疗儿童哮喘类病症为研究对象,通过临床实验研究了γ-干扰素在该类疾病的临床疗效及对炎症因子的影响,具体方法是将106例哮喘患儿随机分入测试组与普通治疗组,测试组:将IFN-γ雾化后吸入进行治疗;普通治疗组:采用目前常规的治疗方法,即口服孟鲁司特,比较两组患儿治疗疗效、血清炎症因子的改变及MUC5ac蛋白水平。结果表明,两组治疗方法都能降低哮喘患儿血清炎症因子及MUC5ac水平,治疗2个月后血清炎症因子都趋于正常水平,但MUC5ac蛋白水平却呈现出差异,其中测试组能恢复到正常水平,而普通治疗组MUC5ac蛋白仍比正常值高20%。测试组诊治后治疗率为94.34%,普通治疗组疗效率为81.13%。本研究的初步结果表明,将IFN-γ雾化吸入治疗法具有更好的治疗效果,治疗周期更短。     

乳腺上皮细胞的特殊粘蛋白MUC1

乳腺上皮细胞的特殊粘蛋白ＭＵＣ１,是乳腺上皮细胞膜结合蛋白,富含唾液酸,含糖量高达５０％并能和许多植物凝集素发生特异性结合。在ＳＤＳ—ＰＡＧＥ电泳上表现为多态性,个体一般为１－２带,源自双亲两个等位基因的遗传和组织中的共同表达。ＭＵＣ１在机体怀孕中期和进入泌乳期在乳腺中水平显著提高,并受体内激素的调节。ＭＵＣ１在机体内有重要的生物学功能     

Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins

Background

The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis.

Methods

The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence.

Results

The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucinShigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells.

Conclusions

Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells.