甲硫腺苷磷酸化酶基因和鸟氨酸脱羧酶在卵巢癌中的表达及意义

目的： 研究甲硫腺苷磷酸化酶（MTAP）基因和鸟氨酸脱羧酶（ODC）在卵巢癌中的表达，探讨两者与卵巢癌发病机制的关系。方法：收集60例新鲜的卵巢癌组织。采用逆转录聚合酶链反应测定癌组织中MTAP mRNA的表达,Western印迹法检测甲硫腺苷磷酸化酶蛋白质的表达情况; 同时采用高效液相色谱法测定卵巢癌组织中鸟氨酸脱羧酶的活性。20例正常卵巢组织为对照。结果：卵巢癌中MTAP mRNA的表达水平为0.42±0.11,低于正常卵巢的表达水平0.81±0.18,卵巢癌中MTAP mRNA的表达缺失率为15％ (9/60)。 MTAP蛋白表达结果与mRNA基本一致。 MTAP mRNA的表达与卵巢癌病理类型、肿瘤分期、组织分级无明显相关性(P>0.05)。卵巢癌组织中鸟氨酸脱羧酶ODC的活性为(3.82±1.03) U,比正常卵巢组织中ODC的活性(1.38±0.59)U高(P<0.01)。ODC的活性与卵巢癌组织分级呈正相关。9例MTAP表达阴性的卵巢癌ODC活性为(4.83±1.27) U,显著高于MTAP表达阳性的卵巢癌患者(P<0.05)。结论：卵巢癌存在MTAP基因表达减弱或缺失。MTAP基因表达缺失导致ODC的活化可能是卵巢癌的发病机制之一。     

鸟氨酸脱羧酶/多胺系统在大鼠缺血预适应心肌保护中的作用

目的：探讨鸟氨酸脱羧酶（ODC）/多胺系统在缺血预适应（IPC）心肌保护中的作用。方法:应用离体灌流大鼠心脏复制模拟心肌缺血/再灌注模型。心脏随机分为6组：对照组 (control)、缺血/再灌注组 (IR)、弱缺血预适应组 (IPCw)、强缺血预适应组 (IPCs)、IPCw+多胺抑制剂组 (DF-EG-IPCw)和IPCs+多胺抑制剂组(DF-EG-IPCs)。免疫印迹法定量分析多胺合成限速酶鸟氨酸脱羧酶 (ODC)的表达;高效液相色谱测定心肌组织中的多胺（腐胺、精脒、精胺）含量;Powerlab多导生理记录系统记录心脏功能;氯化三苯基四氮唑 (TTC) 染色检测心肌梗死面积;TUNEL方法检测细胞凋亡率,比较其中的差异性。结果:（1）与对照组比,IR组ODC表达下调,腐胺含量增加,精胺含量减少,总多胺池减少（P<0.05）,此时心功能下降（LVDP、HR、CF均低于对照组,P<0.05）,心肌梗死面积及心肌细胞凋亡率增加（P<0.05）;（2）与IR组比,弱及强缺血预处理组（IPCw、IPCs）心肌ODC表达上调,腐胺含量减少,精胺含量增加,总多胺池增加（P<0.05或P<0.01）,此时大鼠心功能有明显改善（LVDP、HR、CF与IR组比,P<0.05）,心肌梗死面积及心肌细胞凋亡率均明显降低（P<0.01）;（3）给予多胺抑制剂后,心肌ODC表达,腐胺、精脒、精胺及总多胺池含量均明显降低（DF-EG-IPCw组 vs IPCw组;DF-EG-IPCs组vs IPCs组,P<0.05或P<0.01）,心功能明显下降（P<0.05）,心肌梗死面积及细胞凋亡率均明显增加（P<0.05）。结论:缺血预适应能明显上调大鼠心肌ODC/多胺系统,减轻缺血/再灌注心肌损伤;多胺合成代谢抑制剂取消了预适应介导的心功能改善、缩小心肌梗死面积及减少心肌细胞凋亡的作用,提示ODC/多胺系统可能参与了缺血预适应介导的心肌保护作用。     

鸟氨酸脱羧酶抗酶-1过表达促进非小细胞肺癌细胞系A549凋亡

目的探讨上调鸟氨酸脱羧酶(ODC)抗酶-1(AZ-1)对非小细胞肺癌细胞系A549多胺代谢酶表达和细胞凋亡的影响。方法制备靶向提高人源性AZ-1表达的pcDNA3.1-HOAZ-1质粒,用其与空载质粒pcDNA3.1(-)分别转染A549细胞,并设立未经任何处理的空白对照组,48 h后分别收集总RNA和蛋白质,通过反转录聚合酶链反应和蛋白质印记分析分别检测AZ-1、ODC和鸟氨酸脱羧酶抗酶抑制剂-1(AZIN-1)的表达;同样的处理条件下应用流式细胞计量术检测细胞凋亡。结果在转染pcDNA3.1-HOAZ-1质粒后,ODC表达被明显抑制,AZIN-1的表达调节不明显;过表达AZ-1引起细胞凋亡增加(P0.05)。结论过表达AZ-1能抑制ODC表达和促进A549细胞凋亡。     

雄激素相关酶活性与BPH关系的研究

测定良性前列腺增生症(BPH)和正常人前列腺组织及不同细胞中5α-还原酶、鸟氨酸脱羧酶(ODC)及γ-谷氨酰转肽酶(r-GT)活性。结果显示:(1)5α-还原酶主要存在于前列腺间质细胞核中,BPH前列腺间质细胞中5α-还原酶活性约为正常前列腺的2倍;(2)BPH前列腺全匀浆和间质细胞中ODC活性分别为正常组织相应组分的2.4和2.6倍;(3)BPH前列腺全匀浆、上皮和间质细胞中r-GT活性分别为正常前列腺相应组分的4.5倍、3.2倍和4.1倍。提示5α-还原酶、ODC及r-GT活性升高与BPH的发生可能有密切关系。     

过表达ODC抑制氧化应激诱导大鼠心肌细胞凋亡的研究

目的构建过表达鸟氨酸脱羧酶（ODC）基因慢病毒载体,并研究其对氧化应激诱导大鼠H9C2心肌细胞凋亡的影响。方法构建大鼠ODC基因pHIV-ODC过表达质粒,与包装质粒psPAX2、pMD2G共转染293FT细胞,检测其侵染效率;包装慢病毒并侵染H9C2心肌细胞;72 h后观察其侵染效率,RT-PCR法检测细胞ODC mRNA的表达,利用CCK-8、Hochest33258染色检测ODC对H2O2诱导H9C2心肌细胞凋亡的影响。结果酶切及测序鉴定ODC序列正确插入慢病毒过表达载体。侵染H9C2细胞72 h后ODC mRNA水平较阴性慢病毒感染组显著升高。过表达ODC能抑制H2O2诱导的细胞活力下降,减轻H2O2诱导的心肌细胞凋亡,与H2O2组比较差异均有统计学意义（P〈0.001）。结论成功构建的过表达ODC慢病毒,上调H9C2细胞中ODC的表达,过表达ODC能显著抑制氧化应激诱导的H9C2心肌细胞凋亡。     

外源多胺对大鼠早期再生肝鸟氨酸脱羧酶蛋白水平的调节

目的 研究多胺(腐胺、精脒和精胺)对大鼠再生肝鸟氨酸脱羧酶(ODC)蛋白水平的调节,分析多胺在肝再生中的作用.方法 外源多胺(溶于0.9%NaCl)皮下注射雄性SD大鼠(180～220 g),部分肝切除(PH)诱导的大鼠再生肝中ODC蛋白水平的分析采用免疫印迹(Western blotting)方法.结果 高剂量的腐胺(20 ms/kg体重)、精脒(0.15 mg/kg体重)和精胺(6 mg/kg体重)处理后,ODC蛋白水平在PH后12 h内均低于对照组,且各组间变化趋势相近;而低剂量腐胺(0.02 mg/kg体重)、精脒(0.03 mg/kg体重)及精胺(0.06 ms/kg体重)处理组在PH后4h ODC水平迅速升高,分别比对照组高15.1%、29.5%和30.3%.结论 一定剂量的多胺对大鼠早期再生肝ODC蛋白水平有反馈调节作用,其中精脒、精胺的作用较强,腐胺较弱.     

皮质酮对大鼠肝再生过程中鸟氨酸脱羧酶的抑制

目的 研究大鼠体内主要的糖皮质激素--皮质酮对部分肝切除(PH)诱导的再生肝鸟氨酸脱羧酶(ODC)基因表达及酶活性的影响.方法 乙醚麻醉大鼠,于PH前3d行双侧肾上腺切除术及假手术,皮质酮悬浮于芝麻油中皮下注射玄肾上腺鼠.用RT-PCR、Western blotting及高效液相色谱法(HPLC)分别测定ODC mRNA、ODC蛋白及酶活性.结果 与再生肝相比,完整肝中ODC mRNA、蛋白及其酶活性水平较低.PH后所有组(每组n=6)中ODCmRNA水平均显著升高,5h达到峰值.7h前mRNA含量由高到低依次为去肾上腺组、去肾上腺假手术组(即对照组)、10和40ms/kg体重皮质酮处理组.去肾上腺组(n=13,下同)的ODC蛋白量高于对照组.10mg/kg体重皮质酮处理,在PH后0～24h下降,40mg/kg皮质酬处理组最低.PH后,所有组(每组n=6)ODC活性迅速升高,6h达到峰值;皮质酮处理后活性在6h下降显著,40ms/kg体重皮质酮处理组活性最低.结论 皮质酮处理后,大鼠完整肝及再生肝中ODC mRNA及蛋白水平呈剂量依赖性下降;ODC活性变化的部分原因是由于ODC mRNA及其蛋白量的改变.     

外源多胺对大鼠再生肝细胞鸟氨酸脱羧酶基因转录的调节

目的 研究天然多胺(腐胺、精脒和精胺)对大鼠再生肝细胞鸟氨酸脱羧酶(ODC)基因转录的调节,探讨多胺在肝再生中的作用.方法 大鼠部分肝切除术诱导再生肝,采用原位杂交技术分析ODC mRNA的表达量,外源多胺(溶于0.9%NaCl)的处理用皮下注射法.结果 外源多胺处理后,再生肝ODC mRNA水平的变化趋势与对照组相似,但不同种类、剂量的多胺,结果差异明显.在所观察到的整个肝再生期间,高剂量腐胺(20 mg/kg体重)处理组mRNA水平始终低于对照组,特别在部分肝切除后2、4和10 h与对照组相比具有显著性差异(P＜0.05);低剂量(0.02 mg/kg体重)的腐胺处理组,只在4 h和12 h显著高于对照组.高剂量精脒(0.15 mg/kg体重)处理组的ODC mRNA水平始终低于对照组(在10 h例外);而低剂量(0.03 mg/kg体重)处理组则不同,ODC mRNA水平表现出先抑制(在2 h)后促进(在4 h和6 h)的特点.精胺的作用与精脒相似.结论 不同浓度的多胺(主要是精脒和精胺)对大鼠再生肝细胞ODC基因转录存在着反馈调节作用.     

多胺在异丙肾上腺素所致大鼠心肌肥厚中的作用及意义

目的： 研究多胺、鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰基转移酶（SSAT）在异丙肾上腺素所致大鼠心肌肥厚中的作用及机制。方法: 异丙肾上腺素（ISO）皮下注射复制大鼠心肌肥厚模型,应用反向高效液相色谱（RP-HPLC）、RT-PCR和Western blotting结合图像分析系统,分别检测ISO作用不同时点大鼠心肌组织多胺含量、ODC和SSAT mRNA和蛋白的表达。结果: 与对照组比较,心脏重量参数在ISO注射后7 d时显著增加,ISO注射后1 d时腐胺含量增加（P<0.05）,5 d、7 d时显著增加（P<0.01）;精脒含量在ISO注射后3 d时开始增加,ISO注射后7 d时增加显著（P<0.01）,精胺含量略有增多（P<0.05）,总多胺池显著增加。心肌组织ODC和SSAT的 mRNA表达在ISO注射后1 d时升高(P<0.05或P<0.01）,并持续在较高水平。心肌组织ODC和SSAT的蛋白表达分别在ISO注射后1 d和ISO注射后5 d时升高,ISO注射后7 d时显著升高（P<0.01）。结论: 大鼠心肌组织多胺含量增加和ODC、SSAT的表达增强可能参与ISO所致心肌肥厚的病理过程。     

多胺与生物大分子

一、多胺概述 多胺是一类多阳离子的脂肪族胺。广泛存在于生物界。生理性多胺主要有精脒(spd),精胺(sp)及其前体腐胺(pu)。原核细胞一般只含pu和spd。眞核细胞还有sp。它们的化学结构如图1所示。多胺是L-鸟氨酸及L-蛋氨酸代谢的衍生物(图2)。哺乳动物细胞的pu是鸟氨酸脱羧酶(ODC)催化鸟氨酸脱羧而求。ODC以磷酸吡哆醛为辅基。除了前列腺外,多数成年休止的组织细胞ODC活性很低,它是多胺生物合成的限速酶,其半衰期是已知的眞核细胞酶中最短的,一般为20分钟右左。在生长快的器官和肿瘤组织中ODC活     

10-羟基喜树碱的提取方法及工艺优化研究

<正>10-羟基喜树碱是从我国特有植物喜树(Camptotheca acuminala Decne)中分离得到的20多个单体中抗肿瘤作用最强的生物碱,它不仅是临床效果较好的抗肿瘤药物,而且还是制备其他喜树碱类衍生物药品的重要中间体。由于喜树     

Effect of pyridoxine deficiency on aromatic L-amino acid decarboxylase in adult rat brain

Summary We have investigated the effect of pyridoxine deficiency on aromatic L-amino acid decarboxylase (AADC) using both dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5HTP) as substrates in the rat brain. The activity ratios of DOPA decarboxylase/5HTP decarboxylase measured under optimal substrate and cofactor concentrations were different in the cerebellum, cerebral cortex, corpus striatum and hypothalamus of the normal rat. In pyridoxine deficiency, there were no parallel decreases in DOPA and 5HTP decarboxylase activities in various brain regions. Dialysis of brain homogenates, in the presence and absence of hydroxylamine, resulted in a total or near total loss of 5HTP decarboxylase activity compared to DOPA decarboxylase activity, indicating that pyridoxal phosphate may be more tightly bound to DOPA decarboxylase than to 5HTP decarboxylase. These results, indicating that pyridoxine deficiency has differential effects on the activity of AADC, are consistent with our earlier observation of non-parallel changes in dopamine and serotonin content in various brain regions of the pyridoxine-deficient rat.     

Impaired development of cerebellar cortex in rats treated postnatally with alpha-difluoromethylornithine

alpha-Difluoromethylornithine specifically and irreversibly inhibits the enzyme ornithine decarboxylase. Ornithine decarboxylase catalyses the initial step in the synthesis of polyamines, which are thought to play an essential role in growth and development of mammalian tissues. The current study examined the effects of alpha-difluoromethylornithine on the ontogenic development of the rat cerebellar cortex. Animals injected daily with alpha-difluoromethylornithine on postnatal days 1-21 suffered a deficit in the number of granule cells and many of the remaining granule cells became trapped in the molecular layer during migration. Purkinje cells were also scattered throughout the molecular layer and their mean diameter was 38% smaller than in controls. In general, the cerebellar cortex of alpha-difluoromethylornithine-treated rats failed to progress much beyond the stage of development reached in control rats during the first postnatal week. These effects of alpha-difluoromethylornithine were already clearly visible at 10-15 days of age. The final size of the cerebellum as a whole and of individual folia was markedly subnormal. These data indicate that polyamines play an obligatory role in cerebellar neurogenesis and histogenesis.     

Induction of ornithine decarboxylase in Leptomonas seymouri

Ornithine decarboxylase, the initial enzyme of polyamine biosynthesis, was induced in vitro in Leptomonas seymouri, a parasite of Diptera, by resuspending stationary phase cells with fresh medium. Induction was biphasic with peaks at 2 and 8 h. Activity increased about 20-fold over 22 h under control conditions. Induction was completely blocked by cycloheximide and was suppressed by actinomycin D, alpha-amanitin, putrescine, spermidine and spermine. The enzyme half-life was 45 min in cells treated with cycloheximide 24 h post induction. These observations suggest the presence of a highly sensitive mechanism for regulation of ornithine decarboxylase as found in mammalian and other eukaryotic cells.     

鸟氨酸脱羧酶抗酶蛋白与细胞增殖抑制

鸟胺酸脱羧酶抗酶(ornithine decarboxylase antizyme,OAZ)基因通过特殊的阅读框移机制翻译全长有功能的抗酶蛋白,结合并降解鸟氨酸脱羧酶,使细胞内多聚胺浓度下降,抑制细胞增殖。抗酶还能结合cyclinD1,阻滞细胞周期。此外,OAZ还能够引起肿瘤细胞逆分化。它在多种肿瘤细胞中呈低表达,对肿瘤细胞的基因治疗有重要意义。     

Changes in cadaverine and putrescine metabolism in the mouse kidney induced to growth by an anabolic steroid

The formation of cadaverine and putrescine was studied in the kidneys of gonadectomized male mice stimulated to growth by nandrolone. an anbolic steroid with low androgenic activity. Administration of nandrolone resulted in an increased kidney weight and elevated activities of lysine and ornithine decarboxylase (assayed by measurement of the formation of ¹⁴CO₂ from the I-¹⁴C-labelled amino acids). The responses were dose and time dependent. The elevated enzyme activities were reflected by an increased endogenous kidney content of cadaverine and putrescine as well as in an increased urinary excretion of the diamines. Further, the kidney content and the urinary excretion of the polyamines sper-midine and spermine were elevated on nandrolone treatment. Fractionation of kidney extracts on pore gradient electrophoresis revealed an apparent molecular weight of about 95000 Daltons of the lysine decarboxylase as well as of the ornithine decarboxylase. On electrofocusing it was evident that both enzymes were present as more than one isoelectric form. However, the main form in both cases focused at a pH of about 5.0.     

Breaking through the acid barrier: An orchestrated response to proton stress by enteric bacteria

The ability of enteropathogens such as Salmonella and Escherichia coli to adapt and survive acid stress is fundamental to their pathogenesis. Once inside the host, these organisms encounter life-threatening levels of inorganic acid (H+) in the stomach and a combination of inorganic and organic acids (volatile fatty acids) in the small intestine. To combat these stresses, enteric bacteria have evolved elegant, overlapping strategies that involve both constitutive and inducible defense systems. This article reviews the recent progress made in understanding the pH 3 acid tolerance systems of Salmonella and the even more effective pH 2 acid resistance systems of E. coli. Focus is placed on how Salmonella orchestrates acid tolerance by modulating the activities or levels of diverse regulatory proteins in response to pH stress. The result is induction of overlapping arrays of acid shock proteins that protect the cell against acid and other environmental stresses. Most notable among these pH-response regulators are RpoS, Fur, PhoP and OmpR. In addition, we will review three dedicated acid resistance systems of E. coli, not present in Salmonella, that allow this organism to survive extreme (pH 2) acid challenge.