MG53在运动改善胰岛素抵抗中的作用

胰岛素抵抗(insulin resistance, IR)是多种代谢性疾病的共同病理基础,运动作为改善这一病理过程的重要辅助治疗手段,其干预方式、强度和持续时间等尚未明确。MG53(mitsugumin 53)是一种近年来备受关注的骨骼肌细胞膜修复蛋白,有研究表明其不仅是"效应分子",同时也是重要的信号分子——介导多条信号转导通路发挥广泛的生物学效应。本文通过综述MG53调控胰岛素抵抗发生发展的过程,以及MG53介导运动改善胰岛素抵抗的可能信号转导机制,为运动辅助治疗胰岛素抵抗提供新思路。     

MG53 and disordered metabolism in striated muscle

MG53 is a member of tripartite motif family (TRIM) that expressed most abundantly in striated muscle. Using rodent models, many studies have demonstrated the MG53 not only facilitates membrane repair after ischemia reperfusion injury, but also contributes to the protective effects of both pre- and post-conditioning. Recently, however, it has been shown that MG53 participates in the regulation of many metabolic processes, especially insulin signaling pathway. Thus, sustained overexpression of MG53 may contribute to the development of various metabolic disorders in striated muscle. In this review, using cardiac muscle as an example, we will discuss muscle metabolic disturbances associated with diabetes and the current understanding of the underlying molecular mechanisms; in particular, the pathogenesis of diabetic cardiomyopathy. We will focus on the pathways that MG53 regulates and how the dysregulation of MG53 leads to metabolic disorders, thereby establishing a causal relationship between sustained upregulation of MG53 and the development of muscle insulin resistance and metabolic disorders. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

MG53 attenuates nitrogen mustard‐induced acute lung injury

Nitrogen mustard (NM) is an alkylating vesicant that causes severe pulmonary injury. Currently, there are no effective means to counteract vesicant‐induced lung injury. MG53 is a vital component of cell membrane repair and lung protection. Here, we show that mice with ablation of MG53 are more susceptible to NM‐induced lung injury than the wild‐type mice. Treatment of wild‐type mice with exogenous recombinant human MG53 (rhMG53) protein ameliorates NM‐induced lung injury by restoring arterial blood oxygen level, by improving dynamic lung compliance and by reducing airway resistance. Exposure of lung epithelial and endothelial cells to NM leads to intracellular oxidative stress that compromises the intrinsic cell membrane repair function of MG53. Exogenous rhMG53 protein applied to the culture medium protects lung epithelial and endothelial cells from NM‐induced membrane injury and oxidative stress, and enhances survival of the cells. Additionally, we show that loss of MG53 leads to increased vulnerability of macrophages to vesicant‐induced cell death. Overall, these findings support the therapeutic potential of rhMG53 to counteract vesicant‐induced lung injury.     

beta-catenin 基因在人骨肉瘤MG63 细胞系的表达变化

目的：观察分析beta-catenin 基因在人骨肉瘤MG63 细胞系的表达变化。方法：培养MG63 细胞株,用不同浓度的Wnt3a 蛋白或 相同浓度、不同时间段的Wnt3a 蛋白刺激液MG63细胞生长,用FQ-PCR在mRNA 水平检测beta-catenin 在MG63 细胞系的表达 变化。培养MG63 细胞株,用相同浓度的Wnt3a 蛋白刺激,比较刺激组与空白对照组的细胞数目的差别。结果：在细胞生长的第 0,1 天两组的MG63 细胞数量对比没有统计学差异（P＞0.05）;而在第2,3,4 天对照组细胞增殖速度较刺激组缓缓,经比较（t=4. 109,3.892,5.215,均P＜0.05）。beta-catenin 基因表达不会因为Wnt3a 的浓度增加而增加。100 ng/mL的Wnt3a 蛋白刺激beta-catenin 基 因表达最高,其他几个浓度的beta-catenin 基因表达对比没有统计学差异（P>0.05）。在100 ng/mL的Wnt3a蛋白刺激下,beta-catenin 基 因表达会随时间延长而增加,6 h时茁-catenin基因表达最高（P<0.05）,随后茁-catenin基因表达则没有统计学差异（P>0.005）。结 论：Wnt蛋白对MG63 细胞有正向增加增殖作用。激活Wnt/beta-catenin 信号通路能促进MG63 的细胞增殖。     

Proteasome inhibitor MG132 enhances TRAIL-induced apoptosis and inhibits invasion of human osteosarcoma OS732 cells

MG132 as a proteasome inhibitor could induce apoptosis in various cancer cells. This study aimed to discuss the effect of proteasome inhibitor MG132 on the TRAIL-induced apoptosis of human osteosarcoma OS732 cells. MG132 and TRAIL were applied on OS732 cells respectively or jointly. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di-phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope, flow cytometry, and transwell invasion chamber methods. The protein levels of DR5, caspase-3, caspase-8, p27^kip1 and MMP-9 were measured by Western blot analysis. The results indicated that combination of MG132 and TRAIL had the effect of up-regulating expression of DR5, caspase-3, caspase-8 and p27^kip1, down-regulating expression of MMP-9 and inducing apoptosis as well as suppressing the ability of invasion of OS732 cells. The survival rate of combined application of 10 μM MG132 and 100 ng/ml TRAIL on OS732 cells was significantly lower than that of the individual application (p < 0.01). Changes of cellular shape and apoptotic rates also indicated the apoptosis-inducing effect of combined application was much stronger than that of individual application. Cell cycle analysis showed combination of MG132 and TRAIL mostly caused OS732 cells arrested at G2–M-phase. The invasion ability of OS732 cells was restrained significantly in the combined group compared with the individual group and control group.     

Matrix Generator(MG):一个基于DNA片段的0/1矩阵生成程序

分子群体遗传学研究的特点是取样量大--存在于群体样本中的遗传变异必须要充分代表该群体和该物种的遗传变异量及分析的位点数多--位点样本必须恰当代表基因组.大样本的群体取样和位点取样产生大量的原始数据,使原始数据人工处理非常困难甚至不可能,从而迫切需要原始数据处理的自动化.目前一些大公司提供的凝胶图像收集仪器和配套的软件已经使原始数据的获取基本上自动化或半自动化.获得DNA片段分子量数据后,必须把这些分子量数据转变成可反映操作单位(样本)之间关系的数据矩阵,原来用于计算分子量的那些软件已不实用或派不上用场.目前,除了用于fAFLP的Binthere弥补了部分不足外,还没有此类软件.Binthere存在固定栏宽(Bin)的缺陷,也就是将分子量最大值与最小值之间等分的方法来归纳不同操作单位(OUT)之间的异同,使得分子量绝对值差很小的数据可能被归入不同的栏,导致结果不正确.为了解决这类问题,我们设计编写了一个新的软件,取名为Matrix Generator(MG).与同类软件相比,MG具有两个主要优点:(1)采用动态栏宽和智能归并算法,克服了固定栏宽可能造成的错误;(2)可用于非荧光标记的分子标记技术.MG的基本思路是:分子量差异越小的片段,越可能是同缘片段,越应该处于相同的栏内.为此,我们采用绝对对应的动态过程.也就是说,从最小分子量到最大分子量之间的栏目数不是事先确定,而是由所分析的所有样品的特点和所使用的凝胶的分辨率(用户根据凝胶的特点给出数值)决定的.当两片段的差异小于凝胶所能达到的分辨率时,两片段被认为是同缘片段而归入相同的栏内.归并的过程从差异最小值开始,直至任意两片段的差异都大于凝胶的分辨率为止.这样就排除了同缘片段被隔离或者非同缘片段被合并的错误,从而使最可能同缘的片段归结在同一位点.MG第一版(V1.0,DOS版)集中体现了实用和易用的优点而没有包含同类软件所具有的一些功能,所以MG必须与其他软件结合使用.对于非荧光标记的分子标记技术,如RAPD、RFLP、AFLP等,可用Quantity One等软件得到分子量,用Excel生成样品与(分子量数据代表的)DNA片段矩阵,然后用MG处理.对于荧光标记的分子标记技术,如fAFLP、fSSR等,除可以用Excel生成矩阵外,可直接用Binthere和Genotyper等生成分子量矩阵,然后用MG处理.MG输出的矩阵经过适当编辑后,就可用后续的软件如Paup、Ntsys、Philip等运算.为了检验MG的有效性,我们用六道木属(Abelia)的AFLP分析数据进行检验,14个样品的DNA片段分别用Binthere和MG进行处理.前者得到295个含信息的位点,后者得到210个含信息的位点.用Nei and Li(1979)的算法分别计算距离矩阵并对两距离矩阵作Mantel检验.结果,两矩阵之间存在一定的差别,但相似性系数高达0.941 63,说明两种方法总体上会得到相似的结果,但局部会有所不同.用Paup对两矩阵作进一步分析,生成两个Neighbor-joining(NJ)树.结果表明,MG生成的数据更符合实际情况,而且分辨率高.     

蛋白酶体抑制剂MG132诱导K562和HeLa细胞凋亡程度存在明显差异

蛋白酶体抑制剂MG132诱导人白血病细胞K562和宫颈癌细胞HeLa凋亡,用3个不同浓度的蛋白酶体抑制剂MG132处理人白血病细胞K562和宫颈癌细胞HeLa,通过MTT检测、annexin Ⅴ/ PI 双染法、流式细胞术、酶标仪和Western 印迹分别检测MG132对K562细胞和HeLa细胞的生长效应、细胞凋亡率、细胞内活性氧(ROS)水平和caspase-3活性变化的影响.蛋白酶体抑制剂MG132诱导K562细胞凋亡明显,对HeLa细胞诱导凋亡不明显.结果表明,蛋白酶体抑制剂MG132特异性诱导不同肿瘤细胞凋亡的程度存在明显差异.     

The value of MG7‐Ag and COX‐2 for predicting malignancy in gastric precancerous lesions

Here we aimed to first investigate the clinical value of combined detection of MG7‐Ag and COX‐2 (cyclo‐oxygenase 2) in prediction of advances in gastric precancerous lesions. Immunohistochemical analysis was used to examine the expression of MG7‐Ag and COX‐2 in 396 cases of patients with gastric precancerous lesions, including 66 cases of atrophic gastritis, 106 cases of intestinal metaplasia, 174 cases of low‐moderate‐grade dysplasia and 50 cases of high‐grade dysplasia. The relation of MG7‐Ag and COX‐2 staining with various clinicopathological features was analysed by follow‐up study. The positive rates of MG7‐Ag and COX‐2 were increased gradually from atrophic gastritis (21.2%, 28.8%), intestinal metaplasia (36.8%, 44.3%), low‐moderate‐grade dysplasia (51.4%, 58.6%) to high‐grade dysplasia (72%, 80%). Double positive staining of MG7‐Ag and COX‐2 in gastric precancerous lesions had an increased risk of precancerous progression over 22 times, compared with negative ones. However, the expression of MG7‐Ag and COX‐2 was not significantly correlated with age and gender of patients. MG7‐Ag and COX‐2 might play an important role in the process of carcinogenesis and progression of gastric cancer. Combined detection of MG7‐Ag and COX‐2 was of value of predicting early gastric cancer from precancerous lesions.     

Citrate permease gene expression in Lactococcus lactis subsp. lactis strains IL1403 and MG1363

Abstract Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis . Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.     

大肠杆菌MG1655菌株ERIC-PCR图谱主带序列组成分析

ERIC-PCR已经在细菌分类,鉴定及混合菌群分析中得到广泛应用,但对其产物形成规律的认识仍存在分歧,以大肠杆菌MG1655为对象,对其ERIC-PCR指纹图谱中1.1kb主带中的DNA片段进行了克隆,测序,基因组定位以及引物匹配分析。结果表明,这条1.1kb主带由分布在基因组中不同位置的3种序列不同的片段组成,各片段的丰度差异较大,最高为97.89%;3种片段中的2种所在的基因组区域仅一端含有ERIC序列,推测对含有ERIC序列的基因组DNA进行扩增时,ERIC-PCR是一种非随机扩增。     

粪肠球菌MG 2108对小鼠结肠炎症的缓解作用及机制研究

【目的】为了筛选能抑制鼠类柠檬酸杆菌(Citrobacter rodentium)诱发的小鼠结肠炎的益生菌,并研究其干预机制。【方法】对4株筛选的菌株进行人工模拟胃肠液耐受试验,并体外测试它们对鼠类柠檬酸杆菌的抑制能力,最终筛选出粪肠球菌(Enterococcus faecalis)MG 2108。72只雄性7周龄ICR小鼠经过适应性饲养7d后,被随机分为2组：正常对照组(MC组,24只,生理盐水)和炎症对照组(IC组,48只,1×10¹⁰CFU/mL灌胃鼠类柠檬酸杆菌),7d后各采12只小鼠,通过结肠组织HE染色和炎症因子检测实验,判断炎症模型建成。原MC组(剩下12只小鼠)更名为NC组,用以区别建模前后的正常对照组,IC组随机分成3组：自然恢复组(IR组,12只,生理盐水)、环丙沙星组(CF组,12只,4mg/mL环丙沙星)和粪肠球菌MG 2108组(EF组,12只,1×10⁸CFU/mL粪肠球菌MG 2108)。18d后结束灌胃,所有小鼠麻醉后眼球取血,解剖。【结果】粪肠球菌MG 2108可以缓解和修复鼠类柠檬酸杆菌引发的小鼠结肠和肝脏损伤,并且通过降低炎症细胞因子的表达水平和增加紧密连接蛋白的表达水平,促进了结肠炎症组织的修复。它改变了肠道微生物菌群结构,EF组的肠杆菌属(Enterorhabdus)和阿克曼菌属(Akkermansia)等有益菌群的丰度增加,同时短链脂肪酸也显著增加(P<0.05),并且优于CF组和IR组。【结论】粪肠球菌MG2108是一株有利于肠道健康的益生菌,治疗鼠类柠檬酸杆菌诱导的小鼠结肠炎效果优于环丙沙星,自然恢复组效果明显差于EF组。     

Escitalopram oxalate induces apoptosis in U‐87MG cells and autophagy in GBM8401 cells

Glioblastoma multiforme (GBM) is recognized as a most aggressive brain cancer with the worst prognosis and survival time. Owing to the anatomic location of gliomas, surgically removing the tumour is very difficult and avoiding damage to vital brain regions during radiotherapy is impossible. Therefore, therapeutic strategies for malignant glioma must urgently be improved. Recent studies have demonstrated that selective serotonin reuptake inhibitors (SSRIs) have cytotoxic effect on certain cancers. Considering as a more superior SSRI, escitalopram oxalate exhibits favourable tolerability and causes generally mild and temporary adverse events. However, limited information is revealed about the influence of escitalopram oxalate on GBM. Therefore, an attempt was made herein to explore the effects of escitalopram oxalate on GBM. The experimental results revealed that escitalopram oxalate significantly inhibits the proliferation and invasive ability of U‐87MG cells and significantly reduced the expressions of cell cycle inhibitors such as Skp2, P57, P21 and P27. Notably, escitalopram oxalate also induced significant apoptotic cascades in U‐87MG cells and autophagy in GBM8401 cells. An animal study indicated that escitalopram oxalate inhibits the proliferation of xenografted glioblastoma in BALB/c nude mice. These findings implied that escitalopram oxalate may have potential in treatment of glioblastomas.     

Offspring from intracytoplasmic sperm injection of aged mouse oocytes treated with caffeine or MG132

Postovulatory mammalian oocytes age significantly in culture. B6D2F1 or ICR strain mouse oocytes were collected 16 h after hCG injection and then cultured for up to 40 h post hCG at 37 °C under 5% CO(2) in air. After intracytoplasmic sperm injection (ICSI), B6D2F1 and ICR oocytes lost full-term developmental potential by 30 h and 26 h after hCG administration, respectively. However, using supplementation with 10 mM caffeine or 1-5 μM of MG132, we could obtain live offspring from oocytes at 34 h (BDF1, 5%-21%) or 28 h (ICR, 5%-18%), whereas none were obtained from untreated aged oocytes. Caffeine maintained normal meiotic spindle morphology, whereas MG132 maintained maturation-promoting factor activity. These treatments did not affect the potential of fresh oocytes for fertilization and subsequent development. Thus, it should be safe to use these chemicals in routine in vitro fertilization and offspring could be generated by ICSI of aged fertilization failed oocytes.     

A comparative study of proteasomal inhibition and apoptosis induced in N27 mesencephalic cells by dopamine and MG132

Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.     

MatriX Generator （MG）：一个基于DNA片段的0／1矩阵生成程序

分子群体遗传学研究的特点是取样量人——存在于群体样本中的遗传变异必须要充分代表该群体和该物种的遗传变异量及分析的位点数多——位点样本必须恰当代表基因组。大样本的群体取样和位点取样产生大量的原始数据,使原始数据人工处理非常困难甚至不可能,从而迫切需要原始数据处理的自动化。目前一些大公司提供的凝胶图像收集仪器和配套的软件已经使原始数据的获取基本上自动化或半自动化。获得DNA片段分子量数据后,必须把这些分子量数据转变成可反映操作单位(样本)之间关系的数据矩阵,原来用于计算分子量的那些软件已不实用或派不上用场。目前,除了用于fAFLP的Binthere弥补了部分不足外,还没有此类软件。Binthere存在固定栏宽(Bin)的缺陷,也就是将分子量最大值与最小值之间等分的方法来归纳不同操作单位(OUT)之间的异同,使得分子量绝对值差很小的数据可能被归入不同的栏,导致结果不正确。为了解决这类问题,我们设计编写了一个新的软件,取名为Matrix Generator (MG)。与同类软件相比,MG具有两个主要优点：(1)采用动态栏宽和智能归并算法,克服了固定栏宽可能造成的错误：(2)可用于非荧光标记的分子标记技术。MG的基本思路是：分子量差异越小的片段,越可能是同缘片段,越应该处于相同的栏内。为此,我们采用绝对对应的动态过程。也就是说,从最小分子量到最大分子量之间的栏目数不是事先确定,而是由所分析的所有样品的特点和所使用的凝胶的分辨率(用户根据凝胶的特点给出数值)决定的。当两片段的差异小于凝胶所能达到的分辨率时,两片段被认为是同缘片段而归入相同的栏内。归并的过程从差异最小值开始,直至任意两片段的差异都大于凝胶的分辨率为止。这样就排除了同缘片段被隔离或者非同缘片段被合并的错误,从而使最可能同缘的片段归结在同一位点。MG第一版(v1．0,DOS版)集中体现了实用和易用的优点而没有包含同类软件所具有的一些功能,所以MG必须与其他软件结合使用。对于非荧光标记的分子标记技术,如RAPD、RFLP、AFLP等,可用Quantity One等软件得到分子量,用Excel生成样品与(分子量数据代表的)DNA片段矩阵,然后用MG处理。对于荧光标记的分子标记技术,如fAFIP、fSSR等,除可以用Excel牛成矩阵外,可直接用Binthere和Genotyper等生成分子量矩阵,然后用MG处理。MG输出的矩阵经过适当编辑后,就可用后续的软件如Paup、Ntsys、Philip等运算。为了检验MG的有效性,我们用六道木属(Abelia)的AFIJP分析数据进行检验,14个样品的DNA片段分别用Binthere和MG进行处理。前者得到295个含信息的位点,后者得到210个含信息的位点。用Nei and Li (1979)的算法分别计算距离矩阵并对两距离矩阵作Mantel检验。结果,两矩阵之间存在一定的差别,但相似性系数高达0．941 63,说明两种方法总体上会得到相似的结果,但局部会有所不同。用Paup对两矩阵作进一步分析,生成两个Neighbor-joining (NJ)树。结果表明,MG生成的数据更符合实际情况,而且分辨率高。     

蛋白酶体抑制剂MG132对青扦花粉萌发和花粉管生长的影响

泛素/蛋白酶体系统(UPP)是真核细胞内蛋白质选择性降解的主要途径,而蛋白酶体是UPP中蛋白质降解的场所。本文应用细胞学、统计学方法以及FTIR技术研究了蛋白酶体抑制剂MG132对青扦(Peceawilsonii)花粉萌发、花粉管生长的影响。结果表明:MG132显著抑制青扦花粉萌发和花粉管生长,并导致花粉管形态异常,主要表现为花粉管亚顶端出现液泡化,并且液泡随着培养时间的延长而扩大到整个花粉管,花粉管濒临死亡;而DMSO以及非蛋白酶体抑制剂E-64不产生类似结果;半薄切片结果表明,MG132处理后不仅花粉管细胞质发生液泡化,生殖细胞也发生液泡化;FTIR分析进一步表明,MG132处理后,花粉管顶端的细胞壁蛋白和果胶质含量大幅度下降。上述结果表明:MG132通过抑制蛋白酶体活性显著影响青扦花粉萌发及花粉管生长;UPP在青扦花粉萌发、花粉管极性生长模式的建立和维持过程中起重要作用;抑制蛋白酶体活性将导致青扦花粉管的程序性死亡。     

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.