人鸟氨酸脱羧酶抗酶1突变基因的克隆与原核表达

克隆人鸟氨酸脱羧酶抗酶1(Homo sapiensornithine decarboxylase antizyme 1,HOAZ1)开放性阅读框+1核糖体移码位点缺失的突变基因,构建突变基因的原核表达质粒,分离纯化其原核表达的重组蛋白。采用巢式-PCR和重叠延伸-PCR技术,从人非小细胞肺癌细胞株A549的cDNA中获得人类鸟氨酸脱羧酶抗酶1开放性阅读框+1核糖体移码(+1RF)位点缺失突变的基因序列(DM-HOAZ1)。将该序列克隆到原核表达载体pET-28a(+)后,转化表达菌Rosseta(DE3)感受态细胞。阳性克隆用IPTG诱导重组蛋白表达,然后在尿素变性条件下经Ni-NTA树脂亲和层析纯化重组HOAZ1。原核表达和纯化的HOAZ1重组蛋白用Western Blot鉴定。结果显示,成功获得HOAZ1开放阅读框中+1RF位点缺失的突变基因和该突变基因的原核表达质粒pET-28a(+)/DM-HOAZ1;用pET-28a(+)/DM-HOAZ1转化大肠杆菌后,HOAZ-1可被IPTG诱导性高表达,且表达量随诱导时间延长递增;原核表达的HOAZ1可用Ni-NTA树脂亲和层析有效纯化。建立了原核表达和分离纯化HOAZ1蛋白的试验方法,为进一步研究HOAZ1的功能和临床应用奠定了基础。     

实时无标记细胞分析系统 (RTCA) 在药物心肌毒性检测和生物活性研究中的应用

实时无标记细胞分析系统(Real time xCELLigence analysis system,RTCA)是一种新型细胞检测技术,能够连续监测、记录及分析细胞活动产生的各种信息,在药物研究中的心肌毒性评估和细胞生物活性考察方面都可以发挥重要作用.文中首先对RTCA的原理与特点进行了介绍,然后分别对RTCA在心肌毒性...     

基于实时无标记细胞分析系统的气道平滑肌细胞收缩舒张效应检测方法的建立

该研究利用实时无标记细胞分析系统(xCELLigence real-time cell analysis system,RTCA)建立气道平滑肌细胞(airway smooth muscle cells,ASMCs)收缩舒张效应检测方法.筛选适宜细胞接种密度,接种于RTCA检测板中监测细胞动态生长曲线;利用组胺刺激细胞...     

叠氮溴化丙锭-荧光定量PCR法实时快速检测5种乳杆菌活菌数方法的建立与应用

[背景]乳杆菌属是发酵食品中最常见的微生物之一,与食品的品质和安全密切相关,定量检测乳杆菌活菌数、解析乳杆菌群落组成对发酵乃至肠道微生物等具有重要意义.[目的]建立一种在种水平上定量检测5种乳杆菌活菌数的叠氮溴化丙锭-荧光定量PCR(propidium monoazide-quantitative PCR,PMA-qP...     

Inhibition of the rat adrenal ornithine decarboxylase activity by immobilization stress and/or dexamethasone

The effects of immobilization stress and/or dexamethasone (DEX) on the adrenal ornithine decarboxylase (ODC) activities of sham-operated and adrenal-medulloectomized (enucleated) male Sprague-Dawley rats were investigated. On day 11 after surgery, rats were injected with saline or DEX (1 mg/kg), 3 h before the time of sacrifice (0600 h or 1800 h). Four groups, from sham-operated and enucleated rats (ENU) treated with saline or DEX were subjected to immobilization stress for 1 h prior to sacrifice. Groups of rats from stress-sham-DEX, non stress-sham-DEX, stress-sham, non stress-sham, stress-ENU-DEX, non stress-ENU-DEX, stress-ENU, and non stress-ENU were sacrificed at 0600 h or 1800 h on day 11 after surgery. Adrenal glands were excised and later analyzed for ODC activities. Results indicated that DEX and/or immobilization stress inhibited ODC activities (p < 0.05) in normal and regenerating adrenal glands at 1800 h and ODC activity varies diurnally, the activity being greater at 1800 h than at 0600 hours (p < 0.001).     

Putrescine activates oxidative stress dependent apoptotic death in ornithine decarboxylase overproducing mouse myeloma cells

Accumulation of putrescine in ornithine decarboxylase overproducing cells provokes apoptotic death that is inhibited by 2-difluoromethylornithine, a specific inhibitor of ODC. The apoptotic process provoked by putrescine involves the release of cytochrome c from the mitochondria and activation of caspases cascades demonstrated by the cleavage of caspase-2, polyA-ribose polymerase (PARP), and proteolytic cleavage of the translation initiation factor 4G (eIF4G). The general caspases inhibitor BD-fmk inhibits PARP cleavage but not cell death. Aminoguanidine, an inhibitor of amine oxidases, inhibits the cleavage of PARP and cell death, whereas the antioxidant BHA inhibits PARP cleavage but not cell death. The intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) inhibits both PARP cleavage and cell death. Although the ability of BAPTA/AM to inhibit the induction of apoptosis may suggest that the accumulating putrescine stimulates the release of Ca2+, such a Ca2+ elevation was not observed. We suggest that the accumulation of putrescine leads to oxidative stress that causes cell death.     

K-FGF mediated transformation and induction of metastatic potential involves altered ornithine decarboxylase and S-adenosylmethionine decarboxylase expression – role in cellular invasion

Adult male Wistar rats were exposed to intermittent high altitude hypoxia of 7000 m simulated in a hypobaric chamber for 8 h/day, 5 days a week; the total number of exposures was 25. The concentration of individual phospholipids and their fatty acid (FA) profile was determined in right (RV) and left (LV) ventricles. Adaptation to hypoxia decreased the concentration of diphosphatidylglycerol (DPG) in hypertrophied RV by 19% and in non-hypertrophied LV by 12% in comparison with normoxic controls. Chronically hypoxic hearts exhibited lower phospholipid n-6 polyunsaturated FA (PUFA) content mainly due to decreased linoleic acid (18:2n-6), which was opposed by increased n-3 PUFA mainly due to docosahexaenoic acid (22:6n-3) in phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI). The content of arachidonic acid (20:4n-6) was unchanged in total phospholipids, but in PC it was increased in both ventricles (by 22%) and in PE decreased in LV only (by 20%). Chronic hypoxia increased the un-saturation index of PC and PE in both ventricles. The content of monounsaturated FA (MUFA) was increased and 18:2n-6 decreased in DPG. The proportion of saturated FA was increased in PC and PI of hypoxic RV but not LV. The FA composition of phosphatidylserine was not altered in hypoxic ventricles. It is concluded that chronic hypoxia led to only minor changes in individual phospholipid concentration in rat ventricular myocardium, but markedly altered their FA profile. These changes, in particular the greater incorporation of n-3 PUFA into phospholipids and increased un-saturation index, may lead to a better preservation of membrane integrity and thereby contribute to improved ischemic tolerance of chronically hypoxic hearts.     

抗酶的研究进展

抗酶（antizyme）是当细胞内多胺水平升高时刺激机体合成的一种小分子量调节蛋白,能特异性地与鸟氨酸脱羧酶（omithine decarboxylase,ODC）结合,经泛素非依赖途径被26S蛋白酶体降解,从而使多胺合成减少;抗酶还可以调节多胺转运,以稳定细胞内多胺水平。近年来随着生物技术的不断发展,对抗酶的认识也逐步深入,本文综述了抗酶家族、合成、作用及定位等方面的研究进展。     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC ornithine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO difluoromethylornithine     

Docking and molecular dynamics studies of peptide inhibitors of ornithine decarboxylase: a rate-limiting enzyme for the metabolism of Fusarium solani

Fusarium solani causes a wide variety of diseases in plants. Polyamine biosynthesis is responsible for the growth and pathogenicity of the fungus. The initial step of this pathway involves the decarboxylation of ornithine to putrescine, and is catalyzed by the enzyme ornithine decarboxylase (ODC). Inhibiting this process may be a promising approach for the management of fungal disease in various crops. Therefore, there is a need to develop inhibitors of ODC that have higher binding capacity than ornithine. Fifteen peptides were designed and modeled based on physicochemical properties of residues in the active site of ODC. The peptide GLIWGNGPF showed the highest dock score. It is assumed that the de novo design of peptides could be a potential approach to inhibit polyamine biosynthesis. Molecular dynamics studies make an important contribution to understanding the effect of the binding of peptides and the stability of an ODC-peptide complex system.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:8.