CYP2E1 and ALDH2 Genotypes and Alcohol Dependence in Japanese

The genotypes of the CYP2E1 and ALDH2 loci of alcoholic (alcohol dependence) and nonalcoholic (healthy) Japanese were investigated to examine the relationship between the polymorphism of CYP2E1 (C₁/C₂) and ALDH2 ( ALDH2*1/ALDH2*2 ), and the susceptibility to alcoholism. There was no significant difference in C₂ gene frequency between alcoholics (0.19) and nonalcoholics (controls) (0.20), whereas there was a significant difference in ALDH2 allele frequency, suggesting that, in Japanese, the C₂ genotype of CYP2E1 may have nothing to do with the risk of developing alcohol dependence. However, the ALDH2*1 allele may influence drinking behavior and the development of alcohol dependence. Furthermore, racial interethnic differences in the frequency of the mutated allele of the CYP2E1 gene (CJ were found, like the ALDH2 gene. Japanese healthy controls showed a significantly higher frequency of the C₂ allele than did Swedish healthy controls (0.05; reported by Persson et al., FEBS Lett. 319:207-211,1993).     

Detection of single nucleotide substitution by competitive allele-specific short oligonucleotide hybridization (CASSOH) with immunochromatographic strip

Recent advances in human genome research have revealed that genetic polymorphisms, such as single nucleotide polymorphisms (SNPs), are closely associated with susceptibility to various common diseases and adverse drug reactions. Also, numerous mutations responsible for a number of genetic diseases have been identified. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We have devised a novel low-tech method for the detection of a single nucleotide substitution using competitive allele-specific short oligonucleotide hybridization with immunochromatographic strip. The gene of interest is PCR-amplified, hybridized to an allele-specific short oligonucleotide probe in the presence of a competitive oligonucleotide, and subjected to chromatography using a DNA test strip at room temperature. The genotype is unambiguously determined by the presence or the absence of visible purple lines on a strip. Feasibility of the method was demonstrated by the detection of a prevalent disease-causing mutations in glycogen storage disease type Ia (G6PC), medium-chain acyl-CoA dehydrogenase deficiency (ACADM), non-ketotic hyperglycinemia (GLDC), and clinically important polymorphisms in the CYP2C19 gene and the aldehyde dehydrogenase 2 gene (ALDH2). The procedure does not demand either technical expertise or expensive instruments and is readily performed in local clinical laboratories. The result is obtained within 10 min after PCR. This rapid and simple method of SNP detection may be used for point-of-care genetic diagnosis with potentially diverse clinical applications. Hum Mutat 22:166-172, 2003.     

Molecular abnormality and cDNA cloning of human aldehyde dehydrogenases

Usual human livers contain two major ALDH isozymes, i.e., cytosolic ALDH1 and mitochondrial ALDH2, while approximately 50% of Orientals are "atypical" and have only the ALDH1 and are missing the ALDH2. Instead, the atypical livers contain an enzymatically inactive but immunologically cross-reactive material (CRM) corresponding to the ALDH2 component. Some Orientals are found to be atypical also in the ALDH1 locus, i.e., they are missing the enzymatically active ALDH1 but contain a large amount of CRM corresponding to the ALDH1 component. cDNA for ALDH1 and that for ALDH2 were cloned and their nucleotide sequences were determined. The amino acid sequences of ALDH1 and ALDH2 were deduced from their cDNA sequences. The molecular abnormality of the inactive ALDH2(2) is found to be the substitution of Glu at the 14th position from the COOH-terminal of the protein by Lys which resulted from G----A transition in the gene.     

Association of ALDH2 Genotypes and Alcohol Intake with Dietary Patterns: The Bunkyo Health Study

Dietary habits are associated with various diseases and assessed by dietary patterns (DPs). Since the ALDH2 genotype is correlated with alcohol and several food preferences, this genotype is probably associated with DPs. In this cross-sectional study of 1612 elderly adults, we investigated the effects of the ALDH2 genotype on DPs and the mediating role of alcohol intake. We identified the ALDH2 genotype and conducted a dietary history survey, then used principal component analysis to determine DPs for each gender. We performed multiple regression analysis to determine the independent contribution of the ALDH2 genotype and alcohol intake to DP scores. We identified three DPs: the “Japanese side dish type” (DP1), the “Japanese dish with alcohol type” (DP2), and the “Western dish with alcohol type” (DP3). In men, the single nucleotide polymorphism ALDH2 rs671 was significantly associated with all DP scores. When alcohol intake was added as a covariate, ALDH2 rs671 was still significantly correlated with the DP2 score but not with the DP1 or DP3 score, and alcohol intake was significantly correlated with all DP scores. In women, ALDH2 rs671 was significantly associated with the DP2 and DP3 scores; however, after adding alcohol intake as a covariate, these associations disappeared, and alcohol intake significantly correlated with all DP scores. In conclusion, the ALDH2 genotype was associated with several DPs in elderly adults, but most associations were mediated by alcohol intake.     

Clinicopathological prognostic factors for gallbladder carcinoma: a retrospective study

BackgroundGallbladder carcinoma (GBC) is an uncommon neoplasm with poor long-term survival. Worldwide the incidence rates vary according to geographic area. The multifactorial aetiology and the rarity of the disease limits the studies to improve outcomes in patients, since the treatment remains mostly surgical. The aim of this study was to identify clinicopathological prognostic factors for survival in patients with GBC submitted to surgery in our institution—a tertiary centre in Portugal. Also, to assess the expression of possible biomarkers (HER2, CD44 and ALDH1) in GBC, as well as the frequency of microsatellite instability (MSI) tumours.MethodsClinicopathological characteristics of 41 consecutive patients that underwent surgical resection for GBC (2008–2019) at our hospital were retrospectively reviewed. Clinicopathological factors were assessed and an immunohistochemical (IHC) analysis was done. Microsatellite stability (MSS) was considered if there was maintenance of nuclear expression of MLH1, MSH2, MSH6 and PMS2. Human epidermal growth factor receptor 2 (HER2) expression was evaluated according to the rules applied for gastric cancer and expression of CD44 and ALH1 was evaluated in order to detect cancer stem cells (CSC). Survival analysis was conducted using Kaplan-Meier and Cox regression was used to find prognostic factors.ResultsIncidence of GBC in our cohort of patients was 0.45%, most commonly affecting females. Median overall survival (OS) was 23 months with a 39.6% 5-year survival rate. Stage > II [hazard ratios (HR) =8.58; P=0.007], lymphovascular invasion (LVI) (HR =4.06; P=0.045) and hepatic resection (HR =0.288; P=0.034) independently influenced survival. HER2 positivity and high expression of CD44 or ADLH1 did not show significant influence in survival (P=0.649, P=0.868 and P=0.914, respectively), although HER2 and ALDH1 positive patients showed a tendency to a shorter OS, compared to negative patients. We found no relation between these biomarkers expression and disease stage. All analysed samples had MSS.ConclusionsGBC patients with a worse prognosis can be identified. The overexpression of HER2 could select patients for targeted therapy and prompt tissue sampling in unresectable patients.     

乳腺原发癌和淋巴结转移癌中ALDH1A1和TGF-β1的表达及预后价值

目的探讨乳腺原发癌和相应淋巴结转移癌中乙醛脱氢酶1(ALDH1A1)和转化生长因子-β1(TGF-β1)的表达情况及与患者预后的相关性,并探讨ALDH1A1与TGF-β1之间的相关性。方法收集有淋巴结转移的乳腺非特殊型浸润性癌197例,采用免疫组化方法检测ALDH1A1和TGF-β1在原发癌及相应淋巴结转移癌中的表达情况,并对临床病理参数及预后进行统计学分析。结果 ALDH1A1和TGF-β1在乳腺原发癌和相应淋巴结转移癌中表达的一致率分别为87.82%和86.80%,两者呈明显的正相关(P<0.05);Kaplan-Meier生存曲线结合Log-rank检验显示,在原发癌和淋巴结转移癌中,ALDH1A1和TGF-β1均为不良预后因素(P<0.05);单因素Cox比例风险回归模型分析显示,组织学分级、肿块大小、阳性淋巴结个数、原发癌和转移癌中ALDH1A1和TGF-β1均是患者预后的危险因素(P<0.05),多因素分析结果显示原发癌ALDH1A1、转移癌TGF-β1及阳性淋巴结个数是患者预后的独立危险因素(P<0.05);Spearman相关性分析显示,在乳腺原...     

脂肪变性供肝冷缺血损伤的研究进展

肝移植是治疗终末期肝病的重要手段,然而供者短缺限制了肝移植的发展,如何扩大供肝来源成为学术界的难题。近年来非酒精性脂肪性肝病（NAFLD）供者比例增加,合理利用脂肪变性供肝是扩大供者池的可行方案。肝移植前供肝保存过程的冷缺血损伤增加了术后器官功能不全的发生率。因此了解脂肪变性供肝冷缺血损伤机制和干预措施尤为重要。脂肪变性供肝冷缺血损伤在细胞器层面具体表现为线粒体、溶酶体、内质网的损伤,蛋白层面主要表现腺苷酸活化蛋白激酶（AMPK）、乙醛脱氢酶2（ALDH2）、血红素加氧酶（HO）-1的表达增加。本文就脂肪变性供肝冷缺血损伤的研究进展及相关干预措施做一综述。     

ALDH2基因导入对K562细胞增殖和抗氧化损伤的影响

目的 克隆人乙醛脱氢酶2(ALDH2)基因,研究ALDH2基因导入慢性粒细胞白血病细胞系K562细胞后对其增殖和抗氧化损伤的影响.方法 从肝细胞中克隆人ALDH2基因,构建真核表达载体,用脂质体法将其导入K562细胞中,用RT-PCR和Western blot法检测ALD-H2基因的表达,锥虫蓝拒染法和MTT法检测转基因组细胞的增殖水平及对氧自由基引起的氧化损伤的反应;在此基础上,利用RT-PCR以及荧光分光光度法进一步检测氧自由基诱导后转基因组细胞中血红素加氧酶-1(HO-1)的表达和细胞内活性氧类(ROS)的产生.结果 成功克隆人ALDH2基因并将构建的真核表达载体转染K562细胞,RT-PCR和Western blot法检测到ALDH2的高表达.锥虫蓝拒染法和MTT结果显示转基因组细胞增殖水平明显高于对照组(P＜0.05),经基因修饰的细胞对H2O2耐受性增高,H2O2的IC50值提高了7.8倍(IC50值分别为12.3μnol/L和1.4 μmol/L,P＜0.01).HO-1的表达和ROS的产生随H2O2浓度的增大而增加,而一定浓度H2O2诱导后HO-1的表达和ROS的产生在转基因组中显著低于对照组(P＜0.05).结论 ALDH2基因导人K562细胞后可增加对氧自由基引起的细胞损伤的耐受性,起到保护作用,该过程伴随着ROS水平以及HO-1表达的降低.     

逆转录病毒双顺反子策略有效转导和共表达ALDH1基因与mdr1基因的研究

目的研究含内在核糖体进入位点(IRES) 的逆转录病毒双顺反子载体介导的醛脱氢酶基因(ALDH1)与多药耐药基因(mdr1)转导和共表达.方法以携带ALDH1与mdr1基因的重组逆转录病毒双顺反子G1Na-ALDH1-IRES-mdr1(G1Na-AIM)为载体,电穿孔法转导双嗜型包装细胞PA317,长春新碱筛选;所得病毒生产细胞PA317/ AIM与单嗜型包装细胞GP+E86乒乓感染提高病毒滴度;以重组病毒上清转染K562细胞,应用聚合酶链反应(PCR)和Southern blot检测转移基因的整合,流式细胞术(FCM)及MTT分析基因表达,集落培养法测定转导效率.结果双嗜型病毒上清滴度达1.0×105cfu/ml.基因修饰细胞K562/ AIM经PCR 和Southern blot证实双基因稳定整合至基因组,对4-氢过氧化环磷酰胺(4-HC)及长春新碱(VCR)耐药 (提高3～10倍),FCM及集落法检测基因转导效率为62%～70%.结论逆转录病毒IRES双顺反子载体能引导不同类型的耐药基因有效共表达,可用于扩大耐药范围,进行体内显性选择.     

醛脱氢酶8A1融合蛋白的表达及其多克隆抗体的制备与鉴定

目的:重组表达醛脱氢酶8A1(aldehyde dehydrogenase 8 family member A1,ALDH8A1)蛋白,制备其多克隆抗体,并进行抗体的特异性鉴定,以及蛋白在组织和细胞内的定位。方法:以成人肝cDNA文库为模板,PCR扩增ALDH8A1目的片段,并构建重组表达载体pGEX-4T-1-ALDH8A1和pET-32a-ALDH8A1,经测序鉴定插入载体的DNA片段序列正确后,转化大肠杆菌BL21,诱导表达GST-ALDH8A1和His-AL-DH8A1融合蛋白。经Western blot确定其为目的蛋白后,纯化重组融合蛋白,并以GST-ALDH8A1免疫家兔制备抗人AL-DH8A1多克隆抗体。用His-ALDH8A1包板,ELISA法测定兔抗人ALDH8A1血清效价;Western blot鉴定兔抗人ALDH8A1血清在重组蛋白和天然蛋白中的反应特异性;免疫组化法确定ALDH8A1蛋白在组织和细胞中的定位。结果:成功构建重组表达载体pGEX-4T-1-ALDH8A1和pET-32a-ALDH8A1;获得包涵体形式表达的GST-ALDH8A1和可溶性形式表达的His-ALDH8A1融合蛋白;应用纯化的重组蛋白GST-AL-DH8A1免疫家兔,获得兔抗人ALDH8A1血清,ELISA测定抗血清的效价为1∶256000。Western blot结果显示,制备的AL-DH8A1兔多抗可特异地识别成人肝总蛋白以及293T、A549、HeLa、U937、HepG2细胞总蛋白中一个相对分子质量(Mr)约53000的ALDH8A1天然蛋白,与文献报道的ALDH8A1的Mr一致,表明ALDH8A1在正常肝组织与癌细胞中都有表达。免疫组化结果表明ALDH8A1蛋白定位于人肝癌细胞胞质中。结论:成功制备出兔抗人ALDH8A1多克隆抗体,此抗体可应用于ELISA,Western blot和免疫组化检测,为进一步研究ALDH8A1的功能奠定了基础。