Progress toward re‐engineering non‐ribosomal peptide synthetase proteins: a potential new source of pharmacological agents

Many important pharmaceutical agents, including vancomycin, bleomycin, cyclosporin, and several antibiotics, are produced by non‐ribosomal peptide synthetase (NRPS) enzymes in microorganisms. The NRPS pathway produces an extensive library of products using multienzyme complexes acting in an assembly‐line fashion. Engineering an NRPS system to produce an even greater variety of products, some of which may also have beneficial therapeutic value, would be an enormous advantage. Several approaches have been successful in generating novel NRPS products: mutational biosynthesis during which nonnatural substrates are fed to an organism; domain and module swapping between different species to generate hybrid enzymes; and rational site‐directed mutagenesis, based either on phylogeny or computational prediction, intended to switch substrate specificity and produce altered products. This review will highlight the progress in these areas and describe research in the future that will extend the capacity for re‐engineering NRPS systems. Drug Dev. Res. 66:9–18, 2006. © 2006 Wiley‐Liss, Inc.     

凝血栓蛋白1基因异常甲基化与大肠腺癌关联

目的 探讨凝血栓蛋白l（THBS1）基因启动子CpG岛异常甲基化与大肠腺癌及其临床病理特征的关联。方法 THBS1基因甲基化状态用甲基化特异性PCR检测。结果 大肠腺癌、癌旁组织中，THBSI基因启动子cpG岛甲基化率的差异有显著性（X^2=5．93，P=0．025）；老年患者肿瘤组织中THBS1基因甲基化率明显商于非老年患者（X^2=5.68，P=0．017），直径≥3cm的肿瘤组织中THBSl基因甲基化率显著高于直径〈3cm的肿瘤（X^2=4．16，P=0．041），C期和D期肿瘤组织中THBS1基因甲基化率显著高于A期或B期肿瘤（X^2=8．04，u=2，P=0．018）。结论 THBS1基因甲基化与大肠腺癌的发生有关，肿瘤以老年、晚期和直径较大的肿瘤多见。     

胃癌组织p16基因蛋白表达的意义

目的　检测 p1 6基因蛋白在胃癌组织、癌旁组织中的表达及其分布特点 ,分析其与胃癌临床病理学特征及预后的关系 .方法　采用 S- P免疫组织化学法对 53例胃癌组织及 35例癌旁组织进行 p1 6蛋白的定位观察 .结果　各病理类型胃癌组织、癌旁组织均有 p1 6基因蛋白表达 .阳性率分别为 62 .3% (33/53)和 88.6% (31 /35) ,阳性细胞的棕黄色颗粒主要位于细胞核 .胃癌 p1 6基因蛋白表达与性别、年龄、肿瘤部位在统计学上无差异 (P>0 .0 5) ;而与组织学类型、病理分级、淋巴结转移、临床病理分期在统计学上有差异 (P<0 .0 5) .p1 6蛋白阳性者 5年生存率 51 .0 %高于 p1 6蛋白阴性者 2 0 .0 % (P<0 .0 5) .结论　 p1 6基因缺失和表达水平的改变与胃癌发生、发展密切相关 ,检测 p1 6基因蛋白表达可作为辅助临床判断胃癌的生物学行为及推测预后的指标     

实验性心包炎心钠素与内皮素变化及其临床意义

目的　研究实验性心包炎心钠素 (ANP)与内皮素 (ET)的变化 ,为临床诊治小儿心包炎提供实验依据。方法　家兔 2 4只 ,随机分为实验组 1 3只 ,在心包腔内注入 30 %尿素 (2ml/kg) ;对照组 1 1只 ,在心包腔内注入生理盐水 (2ml/kg)作为对照。注射前与注射后 1、4、7、1 0、1 5与 2 1d分别测定血浆ANP与ET。注射后2 1d测定心肌中ANP与ET ,同时切取心脏作病理与电镜检查。结果　注射后 1～ 1 0d血浆ANP和ET较注射前与对照组明显增高 (P <0 .0 5) ,注射后 1 5dANP下降 ,注射后 2 1d血浆与心肌中的ANP均较注射前与对照组明显下降 (P <0 .0 1 ) ,注射后 2 1d血浆与心肌ET则显著增高 (P <0 .0 1 )。届时心包病理与电镜检查示心包增厚 ,心肌萎缩与损害。结论　心包炎早期血浆ANP与ET均升高 ,说明心功能不全处于代偿期 ,应早期切开引流 ,心包炎后期血浆ANP下降而ET持续升高表示心包已增厚 ,心肌发生萎缩与损害 ,心功能处于失偿期 ,应及时行心包大部剥脱手术 ,解除心肌束缚 ,有望心功能得到恢复     

人原发性肝癌中p16基因表达及CpG岛甲基化状态的研究

目的　进一步探讨人原发性肝癌中 p16基因 m RNA转录水平变化与其基因启动子区 Cp G岛甲基化的关系 ,及其在肝癌发生中的意义。方法　采用狭缝印迹杂交检测 2 0例人原发性肝癌及相应的癌旁、远癌组织及 2例正常人肝组织中 p16基因 m RNA表达水平 ,以甲基化特异性 PCR分析各组织中 p16基因启动子区 Cp G岛的甲基化状况 ,并进行统计分析。结果　 2 0例人原发性肝癌中 14例 (70 % ) p16 m RNA水平比远癌组织显著降低 ;13例 (6 5 % )显示 p16基因启动子区 Cp G岛甲基化 ,其中 84 .6 % (11例 )伴 p16 m RNA转录水平降低。结论　人原发性肝癌中存在高频率的 p16基因表达失活 ,其主要机制可能是启动子区 Cp G岛甲基化抑制了基因的转录 ,在人原发性肝癌的发生发展中有重要作用。其临床诊断和治疗意义有待进一步深入研究。     

C57小鼠源性淋巴瘤的基因枪途径抗肿瘤免疫研究

[目的]诱导C57小鼠建立有效的抗肿瘤免疫.[方法]用基因枪途径给C57小鼠腹部接种含有粒巨噬细胞集落刺激因子(GMCSF)基因的质粒,24h后在同一部位注射FBL3肿瘤细胞破碎后的上清液,15d后用3×106个FBL3肿瘤细胞皮下接种攻击.[结果]肿瘤的生长受到抑制,小鼠的生存时间明显延长.[结论]基因枪途径局部接种含有GMCSF基因的质粒,可增强机体的抗肿瘤免疫,抑制肿瘤细胞的生长.     

No Correlation Between Atrial Natriuretic Peptide Concentrations and Echocardiographic Measurements of Left Atrial Size or Left Ventricular Size and Function in Patients with Persistent Atrial Fibrillation

Atrial fibrillation (AF) may be associated with activation of atrial natriuretic peptide (ANP). The exact trigger for the release of ANP is still being debated. Atrial volume, pressure, and wall stretch are considered to be the main determinants of ANP activation. The aim of the study was to evaluate plasma ANP concentrations in patients with persistent AF and to analyze the echocardiographic determinants of ANP concentration in this group. The study population included 67 patients, 59 ± 7 years of age, with a median AF duration of 5.5 months (range 0.1–12). The relationship between plasma ANP concentrations and echocardiographic left atrial (LA) diameter and volume, and left ventricular (LV) diameter and ejection fraction (EF) was analyzed by logistic regression analysis. The median baseline plasma ANP concentration was 63 pg/mL (range 21–126) in the study group versus 34 pg/mL (range 16–73) in a control group. The mean left antero-posterior atrial dimension, LA volume, LV enddiastolic diameter, and LVEF were 48 mm, 104 mL, 52 mm, and 54%, respectively. A significant linear positive correlation was found between plasma ANP concentration and maximal LA volume (r = 0.62, P < 0.01). A negative correlation was found between LVEF and plasma ANP concentration (r =−0.42, P = 0.01). However, by multivariate regression analysis, no echocardiographic parameter was an independent predictor of plasma ANP concentration. Plasma ANP concentrations were independent of echocardiographic measurements of LA size or LV size and function in patients with persistent AF.     

Neurofilament M and calbindin D28k are present in mutually exclusive subpopulations of enteric neurons in the rat submucous plexus

Immunocytochemical methods have been used to examine the localisation of 3 neurofilament proteins and the calcium binding protein, calbindin D_28k, in whole mount preparations of the submucous plexus in the Wistar rat. Neurofilament-M (160 kDA protein) was present in 40% of the submucosal neurons, staining fine filaments in the soma and the axonal processes. Calbindin D_28k was present in 40% of the submucosal neurons staining both the soma and nerves within the plexus. The neurofilament proteins and calbindin D_28k were never observed within the same neurons. Neurofilament-M was co-localised with substance P and calcitonin gene-related peptide but not somatostatin or the other neuropeptides investigated. Calbindib D_28k was co-localised with vasoactive intestinal polypeptide and neuropeptide Y. Galanin- and somatostatin-immunoreactive neurons did not contain either the neurofilament proteins or calbindin D_28k. The results demonstrate the presence of subsets of submucosal neurons that can be distinguished by the presence of neurofilament-M or calbinsin D_28k.     

PTEN基因敲除降低辐射敏感性及其机制

目的 探讨PTEN基因敲除对辐射敏感性的影响及机制。方法 采用流式细胞术检测MEF1和MEF1/PTEN^－/－细胞内ROS水平;采用Western blot方法,检测H₂O₂和DPI预处理后AKT激酶的表达变化;采用细胞克隆形成率试验分析细胞对⁶⁰Co γ射线的敏感性。结果 PTEN基因敲除后细胞ROS水平增加,辐射敏感性降低。H₂O₂和DPI预处理后影响MEF1细胞AKT激酶活性,但对MEF1/Pten^－/－细胞无影响。 结论 PTEN基因敲除阻断了ROS对AKT的介导,AKT激酶持续活化,可能是辐射敏感性降低的重要原因。     

Molecular basis of severe myoclonic epilepsy in infancy

Severe myoclonic epilepsy (SMEI) or Dravet syndrome is caused by mutations of the SCN1A gene that encodes voltage-gated sodium channel alpha-1 subunit. Recently, we generated and characterized a knock-in (KI) mice with an SCN1A nonsense mutation that appeared in three independent SMEI patients. The SCN1A-KI mice well reproduced the SMEI disease phenotypes. Both homozygous and heterozygous knock-in mice developed epileptic seizures within the first postnatal month. In heterozygous knock-in mice, trains of evoked action potentials in inhibitory neurons exhibited pronounced spike amplitude decrement late in the burst but not in pyramidal neurons. We further showed that in wild-type mice the Nav1.1 protein is expressed dominantly in axons and moderately in somata of parbalbumin (PV) – positive inhibitory interneurons. Our immunohistochemical observations of the Nav1.1 are clearly distinct to the previous studies, and our findings has corrected the view of the Nav1.1 protein distribution. The data indicate that Nav1.1 plays critical roles in the spike output from PV interneurons and further, that the specifically altered function of these inhibitory circuits may contribute to epileptic seizures in the mice. These information should contribute to the understanding of molecular pathomechanism of SMEI and to develop its effective therapies.