基因操作技术在药物代谢酶系研究中的应用

采用基因敲除与转基因技术,可制备具有人类CYP450药物氧化代谢种属特性的“人源化”整体动物模型,在外源性化学物质与肿瘤发生的易感性研究等方面,具有重要的药理学和毒理学意义,也可为新药的研制与开发提供新的更为可靠的评价手段。     

肝靶向配体半乳糖基白蛋白和多聚谷氨酸

化学合成两类去唾液酸糖蛋白受体(ASGPR)的人工配体——半乳糖基白蛋白(Gal_nHSA)和半乳糖基多聚-L-谷氨酸(Gal_nPLGA), 并以 ¹²⁵I标记的去唾液酸胎球蛋白(ASF)为标准配体,测定了合成配体抑制 ¹²⁵I-ASF与大鼠肝细胞膜ASGPR结合的IC₅₀值. 结果表明,Gal₁₂HSA、Gal₁₅HSA、Gal₂₆HSA、Gal₃₀HSA和Gal₃₄PLGA均能够有效地抑制 ¹²⁵I-ASF与ASGPR的结合,且前者与ASGPR的亲和力随半乳糖基化程度的增加而增加. 这些合成配体来源丰富、制备简单,适合于作为药物或基因肝靶向运送的导向配体.     

鼻病毒非结构蛋白2B 诱导细胞发生内质网应激

为探讨鼻病毒非结构蛋白2B诱导内质网应激和细胞凋亡的机制,本研究构建了鼻病毒非结构蛋白2B的真核表达载体p2B‐GFP ,通过转染BHK‐21细胞检测相关标志蛋白的变化情况。结果显示,非结构蛋白2B定位表达于BHK‐21细胞内质网,诱导内质网应激标志蛋白Grp78、CHOP的表达增加,并使活化转录因子6（ATF6）的转录活性增加,还诱导BHK‐21细胞发生核浓缩而凋亡,使凋亡标志蛋白PARP发生降解而减少。结果提示,鼻病毒非结构蛋白2B可诱导细胞发生内质网应激,并经该途径诱导细胞凋亡。     

Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known function in catalyzing methylation. In situations of extremely low levels of S-adenosyl methionine (SAM), DNMT-3A and -3B might demethylate C-5 methyl cytosine (5mC) via deamination to thymine, which is subsequently replaced by an unmodified cytosine through the base excision repair (BER) pathway. Alternatively, 5mC when converted to 5- hydroxymethylcytosine (5hmC) by TET enzymes, might be further modified to an unmodified cytosine by DNMT-3A and -3B under oxidized redox conditions, although exact pathways are yet to be elucidated. Interestingly, even direct conversion of 5mC to cytosine might be catalyzed by DNMTs. Here, we summarize the evidence on the DNA dehydroxymethylase and demethylase activity of DNMT-3A and -3B. Although physiological relevance needs to be demonstrated, the current indications on the 5mC- and 5hmC-modifying activities of de novo DNA C-5 methyltransferases shed a new light on these enzymes. Despite the extreme circumstances required for such unexpected reactions to occur, we here put forward that the chromatin microenvironment can be locally exposed to extreme conditions, and hypothesize that such waves of extremes allow enzymes to act in differential ways.     

嗜热链球菌CGMCC 1.1864所产的一种新型细菌素ST9

本文利用琼脂扩散法测定嗜热链球菌(Streptocccus thermophilus) CGMCC 1.1864发酵上清液的抑菌效果, 结果表明此菌能够产生抑菌物质, 且在排除有机酸和过氧化氢的影响后上清液不但能抑制革兰氏阳性菌, 对革兰氏阴性菌也有抑制能力。此抑菌物质具有热稳定性, 于100°C处理 2 h及121°C处理20 min仍保留抑菌活性, 但若将其在100°C处理2 h的上清液立即置于?20°C保存, 其抑菌活性有较大损失。常温下(37°C), 该抑菌物质在pH 2.0?9.0范围内有很好的稳定性。发酵上清液经各种蛋白酶及?-淀粉酶处理后抑菌活性完全消失, 而对过氧化氢酶不敏感, 表明此抑菌物质为多肽, 属于细菌素, 本文初步将其命名为嗜热链球菌素ST9。由于ST9对其产生菌具有吸附作用, 选择pH吸附释放法对该嗜热链球菌素进行粗提, 然后经SephadexG-25凝胶层析柱除去杂蛋白, 最后冷冻干燥得纯品。通过Tricine-SDS-PAGE分析得到其分子量约为5.0 kD。     

吉富罗非鱼IGF2基因分离及其单核苷酸多态性与体型、增重相关性

胰岛素样生长因子2(insulin-like growth factor2,IGF2)是控制动物生长和脂肪沉积的重要基因之一。本文采用PCR方法分离了吉富罗非鱼(GIFT strain Nile tilapia Oreochromis niloticus)IGF2基因5475bp,包含由4个外显子组成的整个阅读框669bp以及3个内含子。通过比对吉富罗非鱼10个个体IGF2序列,共发现11处单核苷酸多态性(single nucleotide polymorphism,SNP)位点,本文检测了内含子1的621nt(C/T)和外显子3的161nt(A/G)两位点在192尾吉富罗非鱼中的基因型分布,并分析不同基因型与体型、增重的相关性。使用四引物扩增受阻体系PCR检测内含子1的621nt基因型,结果显示,CC、CT、TT基因型频率在雄鱼中分别为0.32、0.32、0.36,在雌鱼中分别为0.38、0.38、0.24;与体型、增重的相关性分析表明,此位点不同基因型只与雄鱼体型(体高/体长)显著相关(P0.05),CC型个体显著高于CT和TT型个体。外显子3的A/G转换导致了MSPⅠ酶切位点改变,使用PCR-RFLP法检测该位点基因型,结果显示整个群体中不存在AA基因型,在雄鱼中,GG、AG基因型频率分别为0.71、0.29,而雌鱼中则为0.75和0.25;与体型、增重的相关性分析表明,此位点不同基因型只与雄鱼增重极显著相关(P0.01),GG型的雄鱼明显较AG型增重快。     

Deficiency in a Glutamine-Specific Methyltransferase for Release Factor Causes Mouse Embryonic Lethality

Biological methylation is a fundamental enzymatic reaction for a variety of substrates in multiple cellular processes. Mammalian N6amt1 was thought to be a homologue of bacterial N⁶-adenine DNA methyltransferases, but its substrate specificity and physiological importance remain elusive. Here, we demonstrate that N6amt1 functions as a protein methyltransferase for the translation termination factor eRF1 in mammalian cells both in vitro and in vivo. Mass spectrometry analysis indicated that about 70% of the endogenous eRF1 is methylated at the glutamine residue of the conserved GGQ motif. To address the physiological significance of eRF1 methylation, we disrupted the N6amt1 gene in the mouse. Loss of N6amt1 led to early embryonic lethality. The postimplantation development of mutant embryos was impaired, resulting in degeneration around embryonic day 6.5. This is in contrast to what occurs in Escherichia coli and Saccharomyces cerevisiae, which can survive without the N6amt1 homologues. Thus, N6amt1 is the first glutamine-specific protein methyltransferase characterized in vivo in mammals and methylation of eRF1 by N6amt1 might be essential for the viability of early embryos.Nucleic acids, proteins, carbohydrates, and lipids, as well as a body of small molecules, are subject to methylation in a wide variety of biological contexts (3). The majority of methylation reactions are catalyzed by S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTases). These enzymes ubiquitously exist in species from all three domains of life.Methylation of DNA occurs on one of two bases: cytosine or adenine (19). In prokaryotes, adenine methylation is as widespread as cytosine methylation. In contrast, eukaryotic genomes are devoid of adenine methylation or this type of methylation is too rare to be detected (23, 26). Intriguingly, two putative N⁶-adenine DNA MTases, named N6amt1 and N6amt2, are encoded in the mouse and human genomes. In addition to the conserved AdoMet-binding signature motif GXGXG and other sequence elements, they possess the NPPY motif characteristic of the N⁶-adenine or N⁴-cytosine DNA MTases in bacteria (6, 14). N6amt1 was thus proposed as an AdoMet-dependent DNA MTase, although no evidence had been provided that N6amt1 could methylate DNA (23).No functional clue for N6amt1 existed until two groups independently identified Escherichia coli HemK, distantly related to N6amt1, as a protein MTase for polypeptide release factors RF1 and RF2 (8, 17). The HemK gene was initially discovered in a genetic screen for heme biosynthesis mutants (18), although subsequent studies revealed no direct involvement in heme metabolism. The presence of an NPPY motif, thought to be restricted to members of the adenine and cytosine amino methyltransferases, led to the suggestion that HemK could be an AdoMet-dependent DNA MTase (2). However, a series of genetic and biochemical experiments finally revealed that HemK methylates the side-chain amide group of a glutamine residue in the universally conserved tripeptide motif GGQ of the two release factors in E. coli (8, 17). Methylation of the release factors ensures efficient translation termination and release of newly synthesized peptide from the ribosome (16). Similarly, the yeast HemK homologue, YDR140w (Mtq2p), was confirmed to methylate the eukaryotic release factor eRF1 on a corresponding glutamine residue (9, 22). More recently, the human homologue N6amt1 (HemK2) was reported to methylate release factor 1 (eRF1) in vitro (5).We initially sought to characterize the function of N6amt1 as a potential DNA adenine MTase. Interestingly, the human N6amt1 gene is located on chromosome 21q21.3, a critical region for Down syndrome (1, 20). In this study, we report the identification of murine N6amt1 as a glutamine-specific MTase of eRF1 both in vitro and in vivo. Mammalian eRF1, the only mammalian release factor, is indeed methylated at the glutamine residue of the GGQ motif. Inactivation of the N6amt1 gene by targeted disruption led to embryonic lethality in the mouse. These data confirm that N6amt1 functions as a protein MTase in mammals and indicate that modulation of the eRF1 activity by N6amt1-mediated glutamine methylation might be essential for embryo viability.     

Reducing Pectobacterium virulence by expression of an N-acyl homoserine lactonase gene Plpp-aiiA in Lysobacter enzymogenes strain OH11

An N-acyl homoserine lactonase gene aiiA, transcribed by a strong and constitutive Escherichia coli promoter P_lpp (Accession No. EU723847), was transformed into Lysobacter enzymogenes strain OH11, creating strain OH11A. The N-acyl-homoserine lactone (AHL)-degradation assay showed that transformant OH11A acquired the ability to degrade AHL molecules produced by Agrobacterium tumefaciens, Pectobacterium carotovorum, Pseudomonas syringae pv. tomato strain DC3000 and Acidovorax avenae subsp. citrulli. Pathogenicity tests showed that while the parental strain OH11 did not reduce P. carotovorum infection, the transformant OH11A caused a strong reduction of Pectobacterium virulence on Chinese cabbage and cactus, whereas strain OH11A did not seem to interfere with the normal growth of this pathogen in cabbages. In antimicrobial activity assays, strain OH11A and OH11 showed similar antimicrobial activity against Phytophthora capsici and Sclerotinia sclerotiorum. This work provided a new strategy for developing genetically engineered multi-functional L. enzymogenes strains that possessed the ability to biologically control fungal pathogens and reduce bacterial pathogenicity.     

非体外循环与并行循环不停跳冠脉搭桥术炎性因子的比较

目的:通过对比非体外循环与并行循环下不停跳冠脉搭桥术炎性因子的变化,观察不同方法对机体全身炎性反应程度的影响.方法:选择2009年6月～2010年1月在我院行冠脉搭桥术患者30例,其中选取采用并行循环下不停跳冠脉搭桥术(OnP-BH CABG)和非体外循环冠脉搭桥术(OPCABG)患者各15例,即A组和B组.检测T1(术前)、T2(手术结束时)、T3(术后6小时)、T4(术后12小时)、T5(术后24小时)不同时间点血浆肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、白介素-8(IL-8)、肌钙蛋白I(cTnI)含量,并记录桥血管数量、精神状况、引流量及ICU入住时间.结果:两组血浆TNF-α、IL-6、IL-8、cTnI舍量T1时无统计学意义(P>0.05),T2、T3、T4、T5均呈升高趋势.A组较B组T2、T3、T4、T5各时间点TNF-α、IL-6、IL-8指标升高差异显著(P<0.01);cTnI升高幅度略缓,T4、T5差异无统计学意义.比较患者术后精神状况及ICU入住时间,B组均优于A组(P>0.05).术中搭桥血管支数无统计学差异、A组引流量较B组多,有统计学意义(p=0.02).结论:并行循环下不停跳冠脉搭桥术与非体外循环冠脉搭桥术相比,心肌损伤略重;体外循环可加重机体全身炎性反应程度.

Abstract：

Objective: The aim of this study was to compare the differences of inflammatory cytokines level and inflammatory reaction in patients between on-pump beating heart CABG and off-pump CABG. Methods: Select the patients from June 2009 to Jan 2010 who underwent the CABG in our hospital with on-pump beating heart CABG and off-pump CABG and each group was 15 patients. That was named Group A and group B. Serial blood samples were collected at the following intervals respectively: before operation and Oh, 6h, 12h, 24h after the operation to evaluate the concentration of TNF-α , IL-6, IL-8 and cTn I. Recorded and compared the grafts, mental status、bleeding and the time of intensive care unit. Results: There was no significant difference between two groups in plasma TNF -α , IL - 6, IL - 8 and cTn I level at T1. The level of TNF -α , IL - 6 and IL - 8 was significantly increased at T2 - T5 compared with that before operation (T1) in both groups. However, the plasma concentrations of TNF -α , IL - 6, IL - 8 were significantly lower in group B than group A. The level of cTnI was significantly increased at T2-T3 compared with T1, and no significantly difference at T4, T5. The general condition of the patients in group B was better than group A. The bleeding was significantly fewer in groups B. Conclusions: Myocardial injury was slightly heavier in patients with on pump beating heart CABG than off pump CABG. Cardiopulmonary bypass can aggravate inflammatory reaction.     

B19病毒XA株VP1独特区真核表达系统的建立及鉴定

目的:克隆B19病毒XA株VP1u基因,构建真核重组表达载体.方法:从已构建好的B19病毒XA株原核表达载体中获得VP1u基因,将其克隆入真核表达载体plRES2-EGFP中,经酶切鉴定并测序验证后,获得真核表达载体plRES2-EGFP-VP1u.将其转染至HeLa细胞,提取细胞总蛋白,用Western blot技术检测VP1u蛋白的表达.结果:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u,荧光显微镜下可见pIRES2-EGFP-VP1u转染HeLa细胞后表达EGFP蛋白而发出绿色荧光,Western blot证明VP1u蛋白在HeLa细胞中表达.结论:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u并在HeLa细胞中正确表达,为今后B19病毒VP1u基因疫苗的研究奠定基础.