鸟氨酸脱羧酶高活性微生物菌株的筛选

根据鸟氨酸脱羧酶(ODC)催化底物L-鸟氨酸脱羧生成腐胺,从而引起培养基中pH升高的特点,设计了一种高效、经济的筛选方法,并以此作为初筛手段从土壤中快速分离可产生较高ODC活力微生物菌种。研究发现培养后pH的变化与微生物产酶能力存在着明显的正相关性,R2=0.914 2。对从土壤和活性污泥样品中分离得到的343株细菌进行有效初筛以及经过摇瓶发酵测定酶活的复筛试验后,筛选得到6株高酶活的菌株,其中菌株CJW07和CGW27的ODC活性分别达到了121.32 U/mL和109.25 U/mL。     

外源植物生长调节物质对烟草根中烟碱含量和烟碱合成酶活性变化的生理效应

水培法研究烟草打顶和喷施外源生长调节物质的结果表明:打顶的比不打顶的烟草根中鸟氨酸脱羧酶(ODC)、腐胺N-甲基转移酶(PMT)和N-甲基腐胺氧化酶(MPO)活性升高,烟叶中烟碱含量剧增;打顶喷施ABA和6-BA烟叶中烟碱含量升高,喷施IAA和GA3的下降,IAA的效果更明显.     

鸟氨酸脱羧酶抗酶抑制因子-1与细胞增殖

多胺(腐胺、精脒、精胺)参与细胞增殖、分化和凋亡等重要生命过程,细胞内多胺代谢紊乱也与包括肿瘤在内的多种疾病的发生发展密切相关。鸟氨酸脱羧酶抗酶抑制因子-1(antizyme inhibitor-1,AZIN1)是重要的多胺代谢调节蛋白,它通过多种途径调控细胞的生长。AZIN1能与鸟氨酸脱羧酶抗酶(antizyme,AZ)相互作用,解除后者对多胺合成限速酶鸟氨酸脱羧酶(ornithine decarboxylase,ODC)的抑制,由此上调细胞内多胺的含量。AZIN1还能通过调节细胞周期蛋白cyclin D1的降解速度和干扰细胞中心体复制而影响细胞增殖。AZIN1基因转录后修饰和某些特定mi RNA也对AZIN1的细胞增殖调节功能有重要影响。现对该研究领域的研究进展做简要综述。     

三穗鸭ODC1基因多态性与屠体性状关联性分析

[目的]寻找与三穗鸭屠体性状相关的遗传标记。[方法]以139只贵州三穗鸭为研究对象,采用PCR-SSCP技术和DNA测序方法,对鸟氨酸脱羧酶1(Ornithine decarboxylase,ODC1)基因进行遗传多态性研究。[结果]在ODC1基因中检测到1个碱基变异,位于第4外显子109 bp处,为A→G突变,且在此位点的优势等位基因型为AA基因型,A为优势等位基因。ODC1基因Ex4处多态性对三穗鸭的腿肌重、半净膛重等屠体性状存在显著影响(P0.05)。[结论]研究初步推测ODC1基因多态性与屠体性状具有显著的影响,A109G可作为穗鸭屠体性状的遗传标记,为建立三穗鸭的分子辅助标记选择方法提供初步依据,推进选育进程。     

鸟氨酸脱羧酶与肿瘤

多胺是广泛存在于哺乳类组织细胞中的小分子有机化合物,参与细胞的生长和分化等重要的生理过程,也是肿瘤细胞的快速生长所必需。鸟氨酸脱羧酶（omithine decarboxylase,ODC）是多胺合成代谢途径中的第一个限速酶,ODC活性的异常会引起包括肿瘤在内的一系列疾病的发生,由此该酶成为近年来研究的热点。简要综述了ODC与肿瘤关系的研究进展。     

花生叶片衰老中多胺代谢酶活性及游离多胺含量变化（简报）

花生叶片衰老过程中,多胺代谢酶精氨酸脱羧酶（ADC）、鸟氨酸脱羧酶（ODC）和多胺氧化酶（PAO）活性逐渐下降,而腐胺（Put）含量迅速上升,精胺（SPm）、亚精胶（Spd）含量下降,致使衰老期间Put／（Spd＋Spm）迅速上升。     

鸟氨酸脱羧酶与肿瘤

研究鸟氨酸脱羧酶(ODC)在肿瘤细胞增殖中的调节作用是多年来一直探索的课题,近年来有了一些新的进展。ODC可从核酸和蛋白质生物合成中的多个环节促进肿瘤细胞增殖;而ODC的活性又可从多个方面受诱导剂和抑制剂的影响。ODC的这些生物学性质为研究肿瘤病因和研制防治肿瘤药物提供了新的线索。本文就近年来对ODC活性的诱导作用和影响因素以及ODC促使肿瘤生成的作用机制的研究方面的某些新进展作一概要介绍。     

鸟氨酸脱羧酶与肿瘤

研究鸟氨酸脱羧酶(ODC)在肿瘤细胞增殖中的调节作用是多年来一直探索的课题,近年来有了一些新的进展。ODC可从核酸和蛋白质生物合成中的多个环节促进肿瘤细胞增殖;而ODC的活性又可从多个方面受诱导剂和抑制剂的影响。ODC的这些生物学性质为研究肿瘤病因和研制防治肿瘤药物提供了新的线索。本文就近年来对ODC活性的诱导作用和影响因素以及ODC促使肿瘤生成的作用机制的研究方面的某些新进展作一概要介绍。     

异柠檬酸裂解酶肽类抑制剂筛选及活性检测

[目的]通过噬菌体的肽库筛选并合成具有抑制ICL活性的多肽。[方法]以ICL为基础,利用Discovery Studio 2.1中ligentfit模块,将筛选出多肽与优化后ICL进行分子对接。合成并进行生物活性检测。[结果]通过噬菌体肽库筛选得到6条的七肽成功和ICL对接。合成后的6条七肽质谱检测结果均正确。对6条七肽进行体外生物活性检测,对ICL酶的活性均明显具有抑制的作用。其中3条七肽抑制率超过了70%。[结论]利用虚拟筛选优化了ICL抑制剂的筛选过程,筛选并合成ICL抑制剂,抑制率为75%、64%、66%、77%、78%、67%,为抗结核多肽药物研发提供基础。     

γ-氨基丁酸对低氧胁迫下甜瓜幼苗多胺代谢的影响

以‘西域一号’甜瓜为试验材料,采用营养液水培法,研究了低氧胁迫下外源添加γ-氨基丁酸(GABA)对甜瓜幼苗多胺代谢的影响.结果表明：与通气对照相比,低氧胁迫处理的甜瓜幼苗谷氨酸脱羧酶(GAD)活性和GABA含量显著提高,同时多胺合成酶活性提高诱导多胺含量显著增加,但二胺氧化酶(DAO)和多胺氧化酶(PAO)活性也显著提高;根系精氨酸脱羧酶(ADC)活性提高幅度较大,导致根系游离态腐胺含量较高,而叶片鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(SAMDC)活性提高幅度较大,导致叶片游离态亚精胺(Spd)含量较高;根系游离态DAO和PAO活性显著低于叶片,其细胞壁结合态PAO活性显著高于叶片.与低氧胁迫处理相比,低氧胁迫下外源添加GABA处理的甜瓜幼苗叶片和根系中GABA和谷氨酸含量均显著提高,而GAD活性显著降低;精氨酸、鸟氨酸、甲硫氨酸含量的提高促使多胺合成酶活性显著提高,从而诱导多胺含量显著增加,DAO和PAO活性显著降低.     

Correlation between Ornithine Decarboxylase and Putrescine in Tomato Plants Infected by Citrus Exocortis Viroid or Treated with Ethephon

We have investigated the arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities and the levels of conjugated polyamines to explain the decrease of free putrescine level caused by citrus exocortis viroid (CEVd) and ethephon treatment in tomato (Lycopersicon esculentum Mill. cv Rutgers) plants (J.M. Belles, J. Carbonell, V. Conejero [1991] Plant Physiol 96: 1053-1059). This decrease correlates with a decrease in ODC activity in CEVd-infected or ethephon-treated plants; ADC activity was not altered. CEVd infection had no effect on polyamine conjugates, and ethephon produced a decrease in putrescine conjugates. Interference with ethylene action by silver ions prevented the decrease in ODC activity and in free and conjugated putrescine. It is suggested that changes in putrescine level after CEVd infection and ethephon treatment are regulated via ODC activity and that conjugation is not involved.     

Ornithine decarboxylase activity in human endometrium and endometrial cancer cells

Summary Ornithine decarboxylase (ODC) activities were significantly higher in proliferative endometrium during the estrogen-dominated follicular phase of the menstrual cycle than in secretory endometrium after the formation of the progesterone-secreting corpus luteum. The enzymatic activity was increased about fivefold by renewal of the medium during incubations of endometrial fragments or isolated endometrial glands. Endometrial adenocarcinoma cells (HEC-1, HEG-50), both in monolayers and suspension, also responded to medium renewal by increasing ODC activity about 10-fold after 4 h, with subsequent reduction to control levels after 7 h. These effects were blocked by actinomycin D and cycloheximide. Endometrial stromal cells exhibited highly variable ODC activities at different passages. Difluoromethylornithine (DFMO) and sodium molybdate had marked antiproliferative effects in HEC-50 cultures, reducing cell numbers to 10 to 20% of control values 11 d after plating and inhibiting ODC activity by approximately 80% on Day 7. The antiproliferative effect of DFMO, but not that of molybdate, was reversed by 10 μM putrescine, the product of ODC activity. In contrast to DFMO, molybdate had no effect on ODC activity of cell homogenates. Molybdate did not elicit antizyme formation in HEC-50 cells under conditions in which putrescine did. These results indicate that ODC activity, present in both epithelial and stromal cells, as shown analytically and also by autoradiography after labeling with [³H]DFMO, may be related to cell proliferation in vivo and that proliferation of human endometrial cancer cells in culture can be arrested by DFMO and by molybdate. This investigation was supported by PHS grant HD 07197, awarded by the National Institute of Child Health and Human Development and PHS grant CA 15648, awarded by the National Cancer Institute.     

Increased Putrescine Biosynthesis through Transfer of Mouse Ornithine Decarboxylase cDNA in Carrot Promotes Somatic Embryogenesis

Carrot (Daucus carota L.) cells were transformed with Agrobacterium tumefaciens strains containing 3[prime]-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of a cauliflower mosaic virus 35S promoter. A neomycin phosphotransferase gene linked with a nopaline synthase promoter was used to select transformed cell lines on kanamycin. Although the nontransformed cells contained no ODC, high amounts of mouse-specific ODC activity were observed in the transformed cells. Transgenic cells showed a significant increase in the cellular content of putrescine compared to control cells. Spermidine, however, remained unaffected. Not only did the transformed cells exhibit improved somatic embryogenesis in the auxin-free medium, they also regenerated some embryos in the presence of inhibitory concentrations of 2,4-dichlorophenoxyacetic acid. These cells acquired tolerance to [alpha]-difluoromethylarginine (a potent inhibitor of arginine decarboxylase) at concentrations that inhibit growth as well as embryogenesis in nontransformed carrot cells, showing that the mouse ODC can replace the carrot arginine decarboxylase for putrescine biosynthesis in the transgenic cells.     

Modulation of insulin induced ornithine decarboxylase by putrescine and methylputrescines in H-35 hepatoma cells

Summary The effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min.1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with K_i values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a K_i = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 µM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 µM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.Abbreviations ODC Ornithine Decarboxylase - TLC Thin Layer Chromatography - DNEM Dulbecco's Modified Essential Medium - PBS Phosphate Buffered saline - PEG Polyethyleneglycol     

Apparent ornithine decarboxylase activity, measured by 14CO2 trapping, after frozen storage of rat tissue and rat tissue supernatants

Ornithine decarboxylase (ODC) activity of rat tissues was measured by the standard 14CO2 trapping method after frozen storage (-60 or -70 degrees C) of the tissues or their 105,000g supernatants. True ODC activity was determined by two methods: (a) addition of the inhibitors alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, or aminooxyacetate (AOA), an inhibitor that blocks the decarboxylation of ornithine by mitochondrial enzymes; and (b) chromatographic analysis of the reaction products. In the frozen supernatants of liver and spleen, ODC activity changed only slightly after 1 day but increased 29 and 14%, respectively, by 30 days; activity in kidney supernatant decreased 17% after 1 day and remained near that level at 30 days. Kidney and spleen ODC activity was inhibited 90-100% by DFMO, but apparent liver ODC activity was inhibited only 60-75%. In the supernatant prepared from tissue stored frozen for 1 day, apparent ODC activity in liver increased 500% over that activity in the freshly prepared supernatant; at 23 days, apparent activity increased 755% for liver and 121% for kidney. After 23 days, DFMO did not inhibit apparent ODC activity in supernatants from frozen liver and inhibited ODC in frozen kidney by only 49%. With AOA, the ODC activities of the fresh and frozen supernatants were similar, indicating that the large increase in apparent ODC activity in frozen tissue was due to artifacts from the metabolism of ornithine via the mitochondrial pathway. HPLC analysis of the reaction products resulting from the incubation of uniformly labeled [14C]ornithine with the fresh and frozen preparations indicated no increase in putrescine with the frozen preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell cycle phase-dependent induction of ornithine decarboxylase-antizyme

The activities of ornithine decarboxylase (ODC) and ODC inhibitory protein (ODC-antizyme) were studied in Ehrlich ascites tumor cells, separated according to their position in the cell cycle by centrifugal elutriation. Release and/or synthesis of ODC-antizyme was induced by putrescine treatment. Each mouse received an intraperitoneal injection of 25 mumoles of putrescine at 0, 1, 2, and 3 hr after tumor transplantation. Tumor cells obtained from putrescine-treated and control mice at 4 hr after transplantation were separated into fractions representing all phases of the cell cycle. The cell cycle distribution of the tumor cells in each fraction was determined by flow cytometry. In control tumor cells the ODC activity exhibited two maxima; in late-G1/early-S and in late-S/G2. A marked decrease in ODC activity was observed in mid-S phase. This decrease coincided with maximum ODC-antizyme activity (revealed by putrescine treatment), suggesting that ODC-antizyme is involved in the regulation of ODC activity during the cell cycle.     

Spermidine mediates degradation of ornithine decarboxylase by a non-lysosomal, ubiquitin-independent mechanism

The mechanism of spermidine-induced ornithine decarboxylase (ODC, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum-free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine (Glass and Gerner: Biochem. J., 236:351-357, 1986; Sertich et al.: J. Cell Physiol., 127:114-120, 1986). Treatment of cells with 10 microM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [35S]methionine-labeled ODC and separation by two-dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine-mediated inactivation of ODC occurred in a temperature-dependent manner exhibiting pseudo-first-order kinetics over a temperature range of 22-37 degrees C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22 degrees C for 3 hours with 10 microM spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents (NH4Cl, chloroquine, antipain, leupeptin, chymostatin) had no effect on ODC degradation. ODC turnover was not dependent on ubiquitin-dependent proteolysis. Shift of ts85 cells, a temperature-sensitive mutant for ubiquitin conjugation, to 39 degrees C (nonpermissive for ubiquitin-dependent proteolysis) followed by addition of spermidine led to a rapid decline in ODC activity, with a rate similar to that seen at 32 degrees C (the permissive temperature). In contrast, spermidine-mediated ODC degradation was substantially decreased by inhibitors of protein synthesis (cycloheximide, emetine, and puromycin). These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non-lysosomal, ubiquitin-independent pathway.     

Initial characterization of a HTC cell variant partially resistant to the anti-proliferative effect of ornithine decarboxylase inhibitors

Under the selective pressure of -α-methylornithine (α-MeOrn), a competitive inhibitor of ornithine decarboxylase (ODC) (EC 4.1.1.17) a clone of rat hepatoma tissue culture (HTC) cells has been isolated and designated HMO_A. The growth of this clone is affected by the drug only after a lag period of three generations. The same partial resistance was observed to -α-difluoromethyl ornithine (α-DFMeOrn), an irreversible inhibitor of ODC. HMO_A cells showed elevated ODC activity with a concomitant increase of the putrescine content but no change in S-adenosyl- -methionine decarboxylase (SAM-DC) (EC 4.1.1.50) activity. Evidence is given that this overproduction of putrescine may be responsible for the partial resistance of HMO_A cells to the anti-proliferative effect of the ODC inhibitors. α-MeOrn increases ODC and SAM-DC activities and α-DFMeOrn raises SAM-DC activity in a time and cell line-dependent manner. These findings may support the concept that intracellular putrescine and spermidine play a direct or indirect regulatory role in the expression of ODC and SAM-DC. Thus, the variant cell should be useful for studies on the genetic regulation of polyamine metabolism in eukaryotic cell systems.     

Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.