Novel Aspects of the SubA Subunit of the Subtilase Cytotoxin

The subtilase cytotoxin (SubAB) belongs to the family of AB₅ toxins and is produced together with Shiga toxin (Stx) by certain Stx-producing E. coli strains (STEC). For most AB-type toxins, it is assumed that cytotoxic effects can only be induced by a complete holotoxin complex consisting of SubA and SubB. However, it has been shown for SubAB that the enzymatically active subunit SubA, without its transport and binding domain SubB, induces cell death in different eukaryotic cell lines. Interestingly, the molecular structure of SubA resembles that of the SubAB complex. SubA alone is capable of binding to cells and then being taken up autonomously. Once inside the host cell, SubA is transported, similar to the SubAB holotoxin, via a retrograde transport into the endoplasmatic reticulum (ER). In the ER, it exhibits its enzymatic activity by cleaving the chaperone BiP/GRP78 and thereby triggering cell death. Therefore, the existence of toxic single SubA subunits that have not found a B-pentamer for holotoxin assembly might improve the pathogenic potential of subtilase-producing strains. Moreover, from a pharmacological aspect, SubA might be an interesting molecule for the targeted transport of therapeutic molecules into the ER, in order to investigate and specifically modulate processes in the context of ER stress-associated diseases. Since recent studies on bacterial AB₅ toxins contributed mainly to the understanding of the biology of AB-type holotoxins, this mini-review specifically focus on that recently observed single A-effect of the subtilase cytotoxin and addresses whether a fundamental shift of the traditional AB₅ paradigm might be required.     

High dietary methionine plus cholesterol stimulates early atherosclerosis and late fibrous cap development which is associated with a decrease in GRP78 positive plaque cells

The role of homocysteine, or its precursor methionine, in the formation of fibrous caps and its association with endoplasmic reticulum (ER) stress is unclear. Homocysteine can stimulate collagen accumulation and upregulate the ER stress chaperone glucose regulated protein 78 (GRP78). The aim of this study was to determine if high dietary methionine would increase fibrous caps, and that removal of an atherogenic diet would decrease the amount of ER stressed cells. New Zealand white rabbits were fed for 2, 4, or 12 weeks an atherogenic diet [1% methionine + 0.5% cholesterol (2MC, 4MC or 12MC)]; for 4 or 12 weeks a 0.5% cholesterol diet (4Ch, 12Ch); and to study plaque regression, an MC diet for 2 or 4 weeks accompanied by 10 weeks of a normal diet (2MCr, 4MCr). Endothelial function, atherosclerosis and GRP78 positive cells were studied. Endothelial function was abolished in 4MC and atherosclerosis increased 17-fold ( P  < 0.05) compared with 4Ch. Fibrous caps composed 48% of total plaque area in 12MC vs. 10% in 12Ch ( P  < 0.01), and 12MC expressed less GRP78 plaque cells vs. 12Ch ( P  < 0.01). Four MCr had less plaque GRP78 cells than 12MC ( P  < 0.05) and less endothelial GRP78 cells ( P  < 0.01). In addition, GRP78 positive cells were the highest in 4MC, but decreased in all other groups ( P  < 0.01). GRP78 positive cells within the fibrous cap inversely correlated with cap size ( r ² = 0.9). These studies suggest that high dietary methionine could be beneficial for plaque stabilisation, and a normal diet also stabilises plaque and decreases the number of stressed plaque cells.     

HMGB1和ENA-78在突发性耳聋患者治疗前后的变化

目的:探寻两种炎症介质血清高迁移率蛋白-1(HMGB1)以及中性粒细胞激活肽-78(ENA-78)在特发性突发性聋患者体内随病情变化的不同,及其这两种物质对该病患者的机体影响和所发挥的作用。方法:采用双抗夹心包板、免疫的方法(ELISA)来检测受试者体内中血清HMGB1和ENA-78的含量,受试者包括114例确诊的突发性耳聋患者(分为低度,中度和重度),38例其他疾病对照患者和36例正常健康的成年对照者。并观察这两种物质在患者治疗前后浓度上所产生的不同。结果:患有特发性突发性聋的患者按本文方案治疗后体内的HMGB1和ENA-78含量比治疗前降低显著,且患病程度越重的被测者血清中HMGB1和ENA-78的浓度越大,有正向关系;患有突发性耳聋的受试者两种被测物的含量高于患有其他疾病及健康的对照者,具有统计学意义(P<0.01)。结论:对于患有特发性突发性聋的患者,HMGB1和ENA-78在体内血清中的浓度可以作为诊断和患者患病程度的检测标准参考。     

Expression of glucose-regulated stress protein GRP78 is related to progression of melanoma

Aims:  Glucose-regulated protein 78 (GRP78) is a protein translated in response to endoplasmic reticulum (ER) stress that has been implicated in the pathogenesis and resistance to therapy of a variety of cancers. The aim of this study was to investigate its expression and role in the development and progression of human melanoma.
Methods and results:  The immunohistochemical expression of GRP78 in naevi, primary melanoma and melanoma metastases from 171 patients was correlated with clinicopathological factors and patient survival. The GRP78 immunoreactivity score (IRS) was 0.2 in compound naevi, 0.65 in dysplastic naevi, 4.65 in naevi adjacent to primary melanoma, 2.4 in melanoma in situ , 11.2 in thin (≤1.0 mm) and 18.1 in thick (>1.0 mm) primary melanoma. It was 18 and 17.3 in subcutaneous and lymph node metastases, respectively ( P  < 0.0001). GRP78 expression was positively correlated with increasing tumour thickness ( P  = 0.001) and with increasing dermal tumour mitotic index ( P  = 0.0004). Disease-free survival (χ² = 8.0703, P  = 0.0045) and overall survival (χ² = 6.2633, P  = 0.0123) in melanoma patients with IRS >25 were significantly lower than in melanoma patients with IRS <25.
Conclusions:  GRP78 expression appears to correlate with known correlates of melanoma progression and survival and requires further evaluation as a prognostic biomarker in melanoma.     

小檗碱对胰岛素抵抗大鼠氧化应激和内质网应激的影响

目的观察小檗碱对胰岛素抵抗大鼠氧化/抗氧化状态和内质网应激(endoplasmic reticulum stress,ERstress)的影响,并探讨其改善胰岛素抵抗的分子机制。方法采用高脂高热卡饮食饲养8wk制备胰岛素抵抗大鼠模型,成模后随机分为小檗碱组(BER组)、Alpha-硫辛酸组(ALA组)和模型组(MOD组),各组以相应的药物干预4wk。另设正常组(NOR组),予普通饲料喂养,不予药物干预。比较各组大鼠空腹血糖(FBG)、空腹胰岛素水平(Fins)和胰岛素敏感性(ISI)。血清中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性以观察药物对大鼠氧化和抗氧化的影响。Westernblot方法测定肝脏组织中的ER应激标志物c-Jun氨基端激酶(c-junNH2-terminal kinase,JNK)和Phospho-c-Jun(Ser73)(p-c-Jun)的含量变化。RT-PCR方法测定肝脏组织ER应激标志物葡萄糖调节蛋白GRP78(glucose regulated protein78,GRP78)mRNA表达水平,以观察小檗碱对胰岛素抵抗大鼠ER应激的影响。结果与NOR组比较,MOD组大鼠血清中MDA含量明显升高,SOD、GSH-Px活性明显降低(P均<0.05)。BER组大鼠SOD、GSH-Px的活性较MOD组明显升高(P<0.01,P<0.05),MDA含量较模型组明显下降。比较各组肝组织p-c-Jun/JNK的水平,MOD组较NOR组明显升高(P<0.01),BER组及ALA组较MOD组均明显下降(P均<0.01)。比较各组肝组织中GRP78mRNA水平,MOD组较NOR组明显升高(P<0.05),BER组及ALA组较MOD组均明显下降。结论小檗碱能提高胰岛素抵抗大鼠的胰岛素敏感性,增加其抗氧化能力,改善其ER应激状态,其作用机制可能与改善ER应激有关。     

宫颈癌组织中MHC-Ⅰ类抗原呈递相关蛋白CNX HSP90 GRP94的表达及意义

目的：探讨宫颈炎和宫颈癌组织中CNX、HSP90、GRP94蛋白和HPV16感染在维吾尔族和汉族妇女中的表达及相关关系。方法：采用免疫组化SP法和PCR技术检测宫颈炎组织66例（汉族39例，维吾尔族27例）以及宫颈癌组织92例（汉族30例，维吾尔族62例）中CNX、HSP90、GRP94蛋白的表达和HPV16的感染情况。结果：CNX蛋白在维、汉宫颈癌组织中的阳性率低于维、汉宫颈炎组织中阳性率表达（67．39％，86．36％）（P〈0．05）。并且，CNX蛋白的阳性率在宫颈炎组织中维吾尔族高于汉族（100％，76．92％）（P〈0．05）。HSP90和CRP94蛋白在维、汉宫颈癌组织中的阳性率表达高于维、汉宫颈炎组织（77．17％，56．06％；77．17％，60．60％）（P〈0．05）。宫颈癌组织中HSP90蛋白阳性率表达维吾尔族低于汉族（74．19％，83．33％）（P〈0．05）。HPV16感染在宫颈炎及宫颈癌中的阳性率均为维吾尔族高于汉族（P〈0．05）。通过相关性研究发现，在维吾尔族妇女宫颈癌组织中HSP90与GRP94呈正相关性（r=0．249．P〈0．05）。HSP90蛋白的阳性表达率与HPV16型感染在宫颈癌组织中呈显著正相关（r=0．361，P〈0．05）。结论：通过对CNX、HSP90、GRP94蛋白和HPV16联合检测能提高宫颈癌的检出率。     

滋补脾阴方药含药血清对内质网应激神经元损伤的保护作用及机制研究

目的:运用中药血清药理学方法研究滋补脾阴方药含药血清对内质网应激的保护作用并探讨其机制。方法:以N-糖链抑制剂衣霉素(tunicamycin,Tm)刺激小鼠神经瘤母细胞Neuro2a建立内质网应激模型,滋补脾阴方药含药血清为干预组,采用逆转录聚合酶链式反应方法观察葡萄糖调节蛋白78(glucose regulated protein78,GRP78)和凋亡促进因子CCAAT/增强子结合蛋白同源蛋白(CCAAT/EBP homologousprotein,CHOP)的mRNA表达;乳酸脱氢酶(lactate dehydrogenase,LDH)释放试验观察Tm、星形孢菌素(staurosporine,STS)刺激Neuro2a细胞后的LDH泄漏率。结果:各浓度滋补脾阴方药含药血清预处理组GRP78及CHOP mRNA表达与Tm(5μg/ml)组相比,表达水平明显下调,差异有统计学意义(P<0.05),因此滋补脾阴方药含药血清可以抑制Tm诱导内质网应激时GRP78及CHOP mRNA的表达。各浓度滋补脾阴方药含药血清预处理组与Tm组相比,LDH泄漏率明显下降(P<0.05),因此滋补脾阴方药含药血清可以降低Tm和STS刺激后的LDH泄漏率。结论:滋补脾阴方药具有神经保护作用,其机制可能是通过抑制内质网应激及线粒体损伤。     

Chemokines as novel therapeutic targets in inflammatory diseases

Chemokines and their receptors are a large family of inflammatory molecules responsible for a number of biological functions, including the accumulation of leukocytes at tissue sites. Over the past 10 years, a number of studies have indicated a role for chemokines and chemokine receptors in the pathophysiology of several inflammatory diseases, examples of which are multiple sclerosis, atherosclerosis, rheumatoid arthritis, and gastrointestinal diseases including hepatic disease. For this reason, it is not surprising that modulation of their pharmacology could be a prime target for drug discovery. This commentary provides a brief synopsis of our current knowledge of the role of chemokines and their receptors in the inflammatory process, and highlights the pros and possibly cons of chemokine and chemokine receptor antagonism in the therapeutic approach to several inflammatory diseases.