Congenital long QT syndrome caused by the F275S KCNQ1 mutation: Mechanism of impaired channel function

Congenital long QT syndrome is characterized by a prolongation of ventricular repolarization and recurrent episodes of life-threatening ventricular tachyarrhythmias, often leading to sudden death. We previously identified a missense mutation F275S located within the S5 transmembrane domain of the KCNQ1 ion channel in a Chinese family with long QT syndrome. We used oocyte expression of the KCNQ1 polypeptide to study the effects of the F275S mutation on channel properties. Expression of the F275 mutant, or co-expression with the wild-type S275 polypeptide, significantly decreased channel current amplitudes. Moreover, the F275S substitution decreased the rates of channel activation and deactivation. In transfected HEK293 cells fluorescence microscopy revealed that the F275S mutation perturbed the subcelluar localization of the ion channel. These results indicate that the F275S KCNQ1 mutation leads to impaired polypeptide trafficking that in turn leads to reduction of channel ion currents and altered gating kinetics.     

The localization of the ER retrieval sequence for the calcium pump SERCA1

Abstract

A number of studies using chimeric constructs made by fusing endoplasmic/sarcoplasmic reticulum calcium pump (SERCA) sequences with those of the plasma membrane located calcium pump (PMCA) have suggested that the retention/retrieval signal responsible for maintaining SERCA in the endoplasmic reticulum (ER) is located within the N-terminus of these pumps. Because of the difficulties in identifying the presence of constructs at the plasma membrane we have used a trans-Golgi network (TGN) marker to evaluate whether chimeric proteins are retained by the ER or have lost their retention/retrieval sequences and are able to enter the wider endomembrane system and reach the TGN. In this study, attempts to locate this retention/retrieval sequence demonstrate that the retention sequences are located not in the N-terminus, as previously suggested, but in the largely transmembranous C-terminal domain of SERCA. Further attempts to identify the precise retention/retrieval motif using SERCA1/PMCA3 chimeras were unsuccessful. This may be due to the fact that introducing SERCA1 sequences into the C-terminal PMCA3 sequence and vice versa disrupts the organization of the closely packed transmembrane helices leading to retention of such constructs by the quality control mechanisms of the ER. An alternative explanation is that SERCAs have targeting motifs that are non-linear, being made up of several segments of sequence to form a patch that interacts with the retrieval machinery.     

Localization of ribophorin II to the endoplasmic reticulum involves both its transmembrane and cytoplasmic domains

Proteins that are concentrated in specific compartments of the endomembrane system in order to exert their organelle-specific function must possess specific localization signals that prevent their transport to distal regions of the exocytic pathway. Some resident proteins of the endoplasmic reticulum (ER) that are known to escape with low efficiency from this organelle to a post ER compartment are recognized by a recycling receptor and brought back to their site of residence. Other ER proteins, however, appear to be retained in the ER by mechanisms that operate in the organelle itself. The mammalian oligosaccharyltransferase (OST) is a protein complex that effects the cotranslational N-glycosylation of newly synthesized polypeptides, and is composed of at least four rough ER-specific membrane proteins: ribophorins I and II (RI and RII), OST48, and Dadl. The mechanism(s) by which the subunits of this complex are retained in the ER are not well understood. In an effort to identify the domains within RII responsible for its ER localization we have studied the fate of chimeric proteins in which one or more RII domains were replaced by the corresponding ones of the Tac antigen, the latter being a well characterized plasma membrane protein that lacks intrinsic ER retention signals and serves to provide a neutral framework for the identification of retention signals in other proteins. We found that the luminal domain of RII by itself does not contain retention information, while the cytoplasmic and transmembrane domains contain independent ER localization signals. We also show that the retention function of the transmembrane domain is strengthened by the presence of a flanking luminal region consisting of 15 amino acids.     

CD2-associated protein is widely expressed and differentially regulated during embryonic development

CD2-associated protein (CD2AP) is an adapter protein that is involved in various signaling and vesicular trafficking processes and also functions as a linker between plasma membrane proteins and the actin cytoskeleton. The protein is known to have important functions in T cells and glomerular podocytes, but it is also expressed by many other adult-type tissues and cells. Here we analyzed the expression of the protein during early embryonic development and organogenesis of the mouse. The results showed differential tissue-specific regulation of CD2AP in developing and maturing organs. In oocytes and pre-implantation embryos, CD2AP was located diffusely in the cytoplasm, whereas in late blastocysts it was concentrated to the intercellular contacts. During organogenesis, CD2AP was distinctly upregulated upon, e.g., the pretubular aggregation of metanephric mesenchyme cells and the appearance of the osteoblastic rim around cartilages during endochondral ossification. High CD2AP expression was also observed during epithelial-like conversion of some highly specialized secretory cell types such as the odontoblasts, the cells of the choroid plexus and the decidualized cells of the endometrial stroma. In other instances, such as the development of the proximal tubuli of the kidney and the flat alveolar epithelium of the lung, the protein was downregulated upon differentiation and maturation of the cells. Finally, certain cells, e.g., glomerular podocytes, those forming the collecting ducts of the kidney, and the urothelium of the kidney pelvis, expressed CD2AP throughout their differentiation and maturation. Multiple molecules and complex pathways regulate embryogenesis, and scaffolding proteins apparently have pivotal roles in targeting and finetuning, e.g., growth factor- or hormone-induced processes. The cell-type specific spatio-temporal regulation of CD2AP during development suggests that this adapter protein is a key regulatory partner in many signaling pathways and cellular processes governing differentiation and morphogenesis.     

原发性及狼疮性肾病综合征患者血清PAI-1、Lp(a)水平变化及其临床价值探讨

目的:探讨原发性及狼疮性肾病综合征患者纤溶酶原激活物抑制因子1(PAI-1)和血清脂蛋白a[Lp(a)]的水平变化及其检测的临床应用价值。方法:选取病理类型明确,临床初诊为肾病综合征的患者138例。其中原发性肾病综合征70例,为PNS组;系统性红斑狼疮继发性肾病综合征患者68例,为LNS组。同期选取本院健康体检正常者64例,为正常对照NC组。全自动生化分析仪检测各组血清Lp(a)和血脂等指标;酶联免疫吸附(Elisa)法测定血清PAI-1水平。结果:1与NC组比较,血清Lp(a)和PAI-1水平在PNS和LNS两组中均显著升高(P0.05),PNS组比LNS组升高更为明显,差异有统计学意义(P0.05);2LP(a)与PAI-1秩相关系数(rs)分析,在PNS组中r_s=0.328,P=0.006,LNS组中r_s=0.439,P=0.006;3二元logistic回归分析表明,LP(a)和PAI-1均是PNS和LNS的危险因素;4ROC曲线分析表明,血清Lp(a)、PAI-1对PNS和LNS诊断的ROC曲线下面积(AUC~(ROC))分别为0.895、0.874和0.848、0.813,两者联合检测对PNS和LNS诊断的AUC~(ROC)分别为0.947和0.919。结论:血清Lp(a)与PAI-1水平在PNS和LNS患者体内均明显升高,PNS患者升高更为显著;Lp(a)与PAI-1水平在PNS和LNS患者中均显著正相关;LP(a)和PAI-1均是PNS和LNS的危险因素,两者水平的变化与PNS和LNS的发生相关。联合检测Lp(a)与PAI-1水平对PNS和LNS的诊治具有一定的临床应用价值。     

Genetic association and functional role of Crohn disease risk alleles involved in microbial sensing,autophagy, and endoplasmic reticulum (ER) stress

Genome-wide association studies have identified several genes implicated in autophagy (ATG16L1, IRGM, ULK1, LRRK2, and MTMR3), intracellular bacterial sensing (NOD2), and endoplasmic reticulum (ER) stress (XBP1 and ORMDL3) to be associated with Crohn disease (CD). We studied the known CD-associated variants in these genes in a large cohort of 3451 individuals (1744 CD patients, 793 ulcerative colitis (UC) patients and 914 healthy controls). We also investigated the functional phenotype linked to these genetic variants. Association with CD was confirmed for NOD2, ATG16L1, IRGM, MTMR3, and ORMDL3. The risk for developing CD increased with an increasing number of risk alleles for these genes (P < 0.001, OR 1.26 [1.20 to 1.32]). Three times as many (34.8%) CD patients carried a risk allele in all three pathways, in contrast to 13.3% of the controls (P < 0.0001, OR = 3.46 [2.77 to 4.32]). For UC, no significant association for one single nucleotide polymorphism (SNP) was found, but the risk for development of UC increased with an increasing total number of risk alleles (P = 0.001, OR = 1.10 [1.04 to 1.17]). We found a genetic interaction between reference SNP (rs)2241880 (ATG16L1) and rs10065172 (IRGM) in CD. Functional experiments hinted toward an association between an increased genetic risk and an augmented inflammatory status, highlighting the relevance of the genetic findings.     

Endoplasmic reticulum stress--a double edged sword for Z alpha-1 antitrypsin deficiency hepatoxicity

Several diverse disorders, including the liver disorder Z alpha-1 antitrypsin deficiency as well as cystic fibrosis, Alzheimer's, and Parkinson's disease arise from the same general disease mechanism and are now categorized under the term “conformational diseases”, characterized by abnormal folding and subsequent aggregation of an underlying protein. In recent years, several important research advances in the cell biology of aggregation-prone mutant proteins and pathobiological mechanisms of liver disease in general have proven paramount to our understanding of Z alpha-1 antitrypsin deficiency. This liver disease underlines the principle mechanisms of conformational disorders contained within the four pillars of endoplasmic reticulum stress: (1) protein degradation, (2) endoplasmic overload response, (3) unfolded protein response and (4) cellular death pathway. This four-stage model of Z alpha-1 antitrypsin hepatoxicity is elegant in its simplicity and helps explain the clinical manifestations of this condition. Endoplasmic reticulum stress responses have evolved to be protective, however when they are ineffective, toxic damage occurs demonstrating how these responses can be described as a double edged sword. In this context, one of the most perplexing problems in modern biology is to understand how the cell “chooses” between adaptation and demise in response to stress. When one pathway becomes predominant, a delicate balance is perturbed and either an adaptive or a lethal response ensues. Understanding how the endoplasmic reticulum stress signals potentially play a role in directing a clinical outcome may lead to better prospects of more rational approaches to investigation and therapy for this liver disease.     

Cisd2 mediates lifespan: is there an interconnection among Ca2+ homeostasis,autophagy, and lifespan?

Abstract

CISD2, an evolutionarily conserved novel gene, plays a crucial role in lifespan control and human disease. Mutations in human CISD2 cause type 2 Wolfram syndrome, a rare neurodegenerative and metabolic disorder associated with a shortened lifespan. Significantly, the CISD2 gene is located within a region on human chromosome 4q where a genetic component for human longevity has been mapped through a comparative genome analysis of centenarian siblings. We created Cisd2 knockout (loss-of-function) and transgenic (gain-of-function) mice to study the role of Cisd2 in development and pathophysiology, and demonstrated that Cisd2 expression affects lifespan in mammals. In the Cisd2 knockout mice, Cisd2 deficiency shortens lifespan and drives a panel of premature aging phenotypes. Additionally, an age-dependent decrease of Cisd2 expression has been detected during normal aging in mice. Interestingly, in the Cisd2 transgenic mice, we demonstrated that a persistent level of Cisd2 expression over the different stages of life gives the mice a long-lived phenotype that is linked to an extension in healthy lifespan and a delay in age-associated diseases. At the cellular level, Cisd2 deficiency leads to mitochondrial breakdown and dysfunction accompanied by cell death with autophagic features. Recent studies revealed that Cisd2 may function as an autophagy regulator involved in the Bcl-2 mediated regulation of autophagy. Furthermore, Cisd2 regulates Ca²⁺ homeostasis and Ca²⁺ has been proposed to have an important regulatory role in autophagy. Finally, it remains to be elucidated if and how the regulation in Ca²⁺ homeostasis, autophagy and lifespan are interconnected at the molecular, cellular and organism levels.     

来氟米特配伍雷公藤多甙治疗难治性肾病综合征的疗效观察

目的：观察来氟米特联合雷公藤多甙治疗难治性肾病综合征（RNS）的疗效。方法：选取52例RNS患者随机分为观察组（32例）与对照组（30例）,其中对照组给予来氟米特和激素联合治疗,观察组给予来氟米特配伍雷公藤多甙治疗。疗程均为6个月,治疗期间比较两组疗效,检测患者24h尿蛋白定量、血清总蛋白和白蛋白、血肌酐、血尿素氮、谷丙转氨酶变化情况,同时观察患者有无不良反应。结果：①治疗6个月后,观察组完全缓解率和总有效率分别为28.13%、78.13%,略高于对照组26.67%的完全缓解效率和76..67%的总有效率,但两组间差异无统计学意义（P〉0.05）。②经过治疗,RNS患者24h尿蛋白定量逐渐下降,而血清总蛋白和白蛋白水平逐渐增加,与治疗前比较差异均有显著性（P〈0.01）,其中观察组各指标变化比对照组明显（P〈0.05）③治疗后观察组与对照组患者尿素氮、血肌酐含量均较治疗前显著降低（P〈0.01或0.05）,而谷丙转氨酶水平无明显变化（P〉0..05）,两组间差异无显著性（P〉0..05）。④观察组不良反应发生率为12.50%,明显低于对照组23.33%的发生率,组间差异有显著性（P〈0.05）。结论：米氟米特联合雷公藤多甙治疗能明显缓解RNS患者的临床症状,改善肾功能,减少蛋白尿的产生,且短期内不良反应较少。     

成人原发肾病综合征患者血尿酸与血脂代谢的关系

目的:研究成人原发肾病综合征(PNS)患者高尿酸血症的患病率及其与血脂代谢的关系。方法:将109例成人PNS患者根据其尿酸水平分为高尿酸血症组与血尿酸正常组,检测患者的血脂、血清脂蛋白、ALB及24小时尿蛋白定量,分析成人PNS患者高尿酸血症的患病率,比较高尿酸血症组与血尿酸正常组PNS患者的一般情况及血脂水平,并分析PNS患者血尿酸水平的相关因素。结果:成人PNS患者高尿酸血症的发病率为24.77%;高尿酸血症组患者TG及24小时尿蛋白定量水平明显高于血尿酸正常组(P0.01),两组患者TC、LDL-C、HDL-C、Apo AI及Apo B比较差异无统计学意义(P0.05);成人PNS患者血尿酸水平与TG及24小时尿蛋白定量水平呈正相关(r=0.350,P=0.001;r=0.533,P=0.014),与TC、LDL-C、HDL-C及ALB无明显相关性(P0.05)。结论:成人PNS患者高尿酸血症的发生率较高,高尿酸血症患者具有更高的TG及尿蛋白水平,且成人PNS患者血尿酸水平与TG及24小时尿蛋白定量具有相关性,应高度重视成人PNS患者的血尿酸水平。     

Reduced Penetrance and Variable Expression of SCN5A Mutations and the Importance of Co-inherited Genetic Variants: Case Report and Review of the Literature

Mutations in the SCN5A gene are responsible for multiple phenotypical presentations including Brugada syndrome, long QT syndrome, progressive familial heart block, sick sinus syndrome, dilated cardiomyopathy, lone atrial fibrillation and multiple overlap syndromes. These different phenotypic expressions of a mutation in a single gene can be explained by variable expression and reduced penetrance. One of the possible explanations of these phenomena is the co-inheritance of genetic variants. We describe a family where the individuals exhibit a compound heterozygosity in the SCN5A gene including a mutation (R1632H) and a new variant (M858L). Individuals with both the mutation and new variant present with a more severe phenotype including spontaneous atrial tachyarrhythmia at young age. We give an overview of the different phenotypes of "SCN5A disease" and discuss the importance of co-inherited genetic variants in the expression of SCN5A disease.     

Production of biologically active GM-CSF in sugarcane: a secure biofactory

Over 300 transgenic sugarcane plants representing approx. 200 independent lines producing the human cytokine granulocyte macrophage colony stimulating factor (GM-CSF) were analyzed for recombinant protein accumulation and activity levels. Expression constructs differed in use of the maize polyubiquitin 1, Mubi-1, or the sugarcane polyubiquitin 9, SCubi9, promoters; presence or absence of a C-terminal HDEL tag for ER retention; and presence or absence of a 6X Histidine tag for metal ion affinity purification. Accumulation of GM-CSF protein ranged from undetectable to 0.02 of total soluble protein. No significant difference was observed between the two promoters; however, the ER retention tag was required for higher accumulation levels. Human bone marrow cells (TF-1), which require GM-CSF for cell division, proliferated when growth media was supplemented with transgenic sugarcane extracts. Comparison to purified commercially produced GM-CSF indicated the sugarcane-produced protein had essentially identical activity levels. In a 14-month field trial, accumulation levels remained stable. This is the first report of field production of GM-CSF. During the field trial, no flowering of the trial plants occurred; no pollen or seed was produced. Drying, burning, and burial of the test plants effectively blocked possible routes for the transgenic sugarcane to enter the environment or food supply. Sugarcane may provide a highly secure system for biofactory production of pharmaceutical proteins.This revised version was published online in May 2005 with corrections to the last authors name.     

苯磺酸左旋氨氯地平片联合脂必泰治疗糖尿病合并冠心病痰瘀交阻证的临床疗效观察

摘要目的：观察苯磺酸左旋氨氯地平片联合脂必泰胶囊治疗糖尿病合并冠心病痰瘀交阻证的临床疗效。方法：120例糖尿病性冠心病痰瘀交阻证患者随机分为两组,均给与注射胰岛素控制血糖。对照组（n=60）给与阿托伐他汀10mg,每日1次口服。治疗组（n=60）给与苯磺酸左旋氨氯地平片5mg,每天1次口服;脂必泰胶囊1粒,每天2次口服。两组均治疗3个月。分析比较治疗前后空腹血糖（FBG）、总胆固醇（TC）等指标,心电图（ECG）,并记录中医症状积分。结果：治疗组总有效率86．67％,对照组总有效率51．67％,两组相比较,差异有显著性（P〈0．01）。两组空腹血糖（VBO）、糖化血红蛋白（HbAlc）、甘油三酯（TO）等指标治疗后与治疗前比较,差异有统计学意义（P〈0．05或P〈0．01）。治疗后治疗组总胆固醇（TC）、高密度脂蛋白（HDL．C）与对照组相比较,变化显著（P〈0．05）。治疗组中医症状积分下降较对照组明显（P〈O．05或P〈0．01）。结论：苯磺酸左旋氨氯地平片联合脂必泰胶囊治疗糖尿病性冠心病痰瘀交阻证疗效确切。     

基于网络药理学探讨“黄芪-白术-熟地黄”组方防治肾病综合征的作用机制

本研究旨在通过网络药理学方法和分子对接技术探讨黄芪-白术-熟地黄组方(HBS)治疗肾病综合征的作用机制.通过多个数据库获取肾病综合征基因并进行功能模块分解,找出肾病综合征基因参与的主要生物学过程.通过文献以及数据库查找HBS活性成分和基因靶点,筛选出HBS治疗肾病综合征的有效靶点.通过有效靶点的KEGG和GO富集分析,...     

来氟米特配伍雷公藤多甙治疗难治性肾病综合征的疗效观察

目的:观察来氟米特联合雷公藤多甙治疗难治性肾病综合征(RNS)的疗效。方法:选取52例RNS患者随机分为观察组(32例)与对照组(30例),其中对照组给予来氟米特和激素联合治疗,观察组给予来氟米特配伍雷公藤多甙治疗。疗程均为6个月,治疗期间比较两组疗效,检测患者24h尿蛋白定量、血清总蛋白和白蛋白、血肌酐、血尿素氮、谷丙转氨酶变化情况,同时观察患者有无不良反应。结果:①治疗6个月后,观察组完全缓解率和总有效率分别为28.13%、78.13%,略高于对照组26.67%的完全缓解效率和76..67%的总有效率,但两组间差异无统计学意义(P>0.05)。②经过治疗,RNS患者24h尿蛋白定量逐渐下降,而血清总蛋白和白蛋白水平逐渐增加,与治疗前比较差异均有显著性(P<0.01),其中观察组各指标变化比对照组明显(P<0.05)③治疗后观察组与对照组患者尿素氮、血肌酐含量均较治疗前显著降低(P<0.01或0.05),而谷丙转氨酶水平无明显变化(P>0..05),两组间差异无显著性(P>0..05)。④观察组不良反应发生率为12.50%,明显低于对照组23.33%的发生率,组间差异有显著性(P<0.05)。结论:米氟米特联合雷公藤多甙治疗能明显缓解RNS患者的临床症状,改善肾功能,减少蛋白尿的产生,且短期内不良反应较少。