嘧啶核苷的研究进展

嘧啶核苷包括尿嘧啶核苷和胞嘧啶核苷,其在食品工业和医药行业上应用广泛。介绍了嘧啶核苷的用途、测定方法、生产方法等;根据代谢控制发酵原理,以尿苷生产菌的选育为例,详细介绍了嘧啶核苷生产菌的育种策略,并对嘧啶核苷生产菌的育种实例、育种展望进行了综述。     

超氧自由基与嘧啶碱基及其核苷反应的ESR研究

用自旋捕集技术和ＥＳＲ方法,以ＭＮＰ为捕集剂,研究了紫外线辐照核黄素产生超氧自由基等活性氧与嘧啶碱基及其核苷的反应,确定了尿嘧啶,胞嘧啶和胸腺嘧啶及其核苷所产生的自旋加合物自由基的类别,讨论了自由基的形成机制,揭示了超氧自由基与嘧啶碱基及其核苷的反应是不是直接进行的,而是通过羟基自由基来实现的。     

添加核苷对肝素黄杆菌发酵产肝素酶的影响

研究了添加核苷对肝素黄杆菌发酵产肝素酶的影响,结果发现,单种核苷的添加会抑制产酶,而复合核苷的添加则促进产酶,当4种核苷的添加比例与肝素酶mRNA中4种相应核苷酸的比例一致时,促进作用最强。通过HPLC检测,证实添加后核苷很快进入了菌体内。HPLC的结果还表明,菌体内嘧啶核苷酸和嘌呤核苷酸的合成代谢可能不平衡,这对产酶是不利的。为此,还研究了通过添加天冬氨酸以增强嘧啶核酸合成代谢的调节方式,使产酶得到了提高。     

高聚物高效液相色谱柱分离分析嘧啶和核苷

建立了使用高聚物型色谱柱,反相高效液相色谱法(RP-HPLC)测定5种嘧啶和核苷的方法,样品经甲醇溶解,以p H为4.4的0.01 mol/L KH2PO4缓冲盐溶液和甲醇为流动相进行梯度洗脱,流速1.0 m L/min,温度20℃,紫外检测波长251 nm。考察了流动相中不同因素对分离分析的影响,实现了5种嘧啶和核苷混合物的有效分离。柱效可达37 400 N/m,优于常规的C18-硅胶色谱柱(柱效为21 400 N/m),为该高聚物填料在嘧啶和核苷化合物检测方面的广泛应用提供了有关依据。     

嘧啶结构骨架类抗疟药研究进展

嘧啶结构骨架是抗疟史上最为重要的药效团之一,因其具有多种药理活性且较易于合成与改造而受到广泛的关注。近年来,部分传统的抗疟药物已出现了耐药性,治疗效果明显降低,因此对传统抗疟药物进行结构修饰已势在必行。对近10年来发现的新型嘧啶结构骨架化合物(苯并嘧啶、胺基嘧啶、喹啉并嘧啶和噻吩并嘧啶)在结构特征、抗疟活性和构效关系等方面进行了综述,以期为新型抗疟药的设计提供一些参考。     

疾病干预的新靶点:嘌呤与嘧啶受体

内源性核苷、核苷酸通过嘌呤与嘧啶受体(P受体),参与机体组织器官多种功能的调节。在肿瘤、细胞凋亡、局部缺血、伤口愈合、骨质疏松、药物毒性、炎症及痛觉等病理状态下,P受体的内源性配体核苷与核苷酸发挥保护作用,P1、P2受体及其受体亚型的选择性激动剂和桔抗剂具有宽广的临床应用前景。     

Cobetia marina CICC10367渗透压冲击下羟基四氢嘧啶的合成及释放

摘要:【目的】筛选获得耐受渗透压冲击的羟基四氢嘧啶合成菌株,利用“细菌挤奶”工艺提高羟基四氢嘧啶的产率。【方法】从盐池中分离羟基四氢嘧啶合成菌株,并对其进行形态、生理生化及16S rDNA鉴定。考察了培养基及其NaCl浓度对羟基四氢嘧啶合成的影响,在优化的条件下利用“细菌挤奶”工艺制备羟基四氢嘧啶。【结果】筛选获得的一株羟基四氢嘧啶合成菌株,鉴定为Cobetia marina CICC10367（C. marina CICC10367）。NaCl浓度为90 g/L的、谷氨酸单钠为唯一碳氮源的培养基有利于羟     

友菌素核苷转移酶amiE基因的克隆、表达和功能鉴定

【目的】克隆和表达二糖核苷类抗生素友菌素生物合成基因簇中的核苷转移酶基因amiE,并研究AmiE的体外催化功能。【方法】采用PCR技术将编码257个氨基酸的葡萄糖-1-磷酸核苷转移酶基因amiE克隆到表达载体pET28a上,构建质粒pCSG4001,转化入大肠杆菌E.coli BL21(DE3)中诱导表达;利用亲和层析分离纯化蛋白AmiE,以葡萄糖-1-磷酸和胸腺嘧啶三磷酸(TTP)或尿嘧啶三磷酸(UTP)为底物,利用高效液相检测AmiE的体外酶活;以甘露糖-1-磷酸、半乳糖胺-1-磷酸和半乳糖-1-磷酸和TTP作为底物,进一步研究AmiE对底物的选择性。【结果】N-末端融合组氨酸标签的AmiE蛋白在大肠杆菌中获得了可溶性表达,通过亲和层析纯化出的AmiE能够以TTP(或UTP)和葡萄糖-1-磷酸作为底物,催化形成胸腺嘧啶二磷酸葡萄糖(TDP-glucose)或者尿嘧啶二磷酸葡萄糖(UDP-glucose),但对其他三种底物,无明显催化活性。【结论】大肠杆菌中表达纯化的核苷转移酶AmiE能够体外催化形成TDP-葡萄糖(或UDP-葡萄糖),确证了AmiE作为核苷转移酶的催化功能,同时表明AmiE对底物具有一定的选择性。     

嗜盐细菌中四氢嘧啶和羟基四氢嘧啶的生物合成及其生物学功能

在嗜盐细菌盐适应中,四氢嘧啶(1,4,5,6-四氢-2-甲基-4-嘧啶羧酸)和羟基四氢嘧啶(1,4,5,6-四氢-2-甲基-5-羟基-4-嘧啶羧酸)发挥着十分重要的作用。四氢嘧啶的生物合成以L-天冬氨酸-β-半醛(ASA)为底物,依次由2,4-二氨基丁酸转氨酶(EctB),2,4-二氨基丁酸乙酰基转移酶(EctA)和四氢嘧啶合成酶(EctC)催化反应,分别生成L-2,4-二氨基丁酸(DABA),N-乙酰-L-2,4-二氨基丁酸(ADABA)和四氢嘧啶。羟基四氢嘧啶则由四氢嘧啶羟化酶(EctD)将四氢嘧啶羟基化产生。通常,四氢嘧啶合成基因以ectABC基因簇的形式存在,而羟基四氢嘧啶合成基因ectD单独存在。四氢嘧啶生物合成基因在微生物菌株和转基因经济作物中的表达可以提高其耐盐碱旱等抗逆能力,羟基四氢嘧啶合成基因的表达可以增强宿主耐热和耐干燥的能力。四氢嘧啶类相容性溶质的生物学功能及其潜在应用已成为前沿性研究热点。     

8-氧化咖啡因和嘧啶类生物碱在普洱熟茶中的存在

应用柱层析分离技术,从普洱熟茶中首次分离到8-氧化咖啡因,嘧啶类生物碱(胸腺嘧啶脱氧核苷、胸腺嘧啶和尿嘧啶) ,黄酮类配糖体(黄杞甙) ,以及简单酚类化合物(1 ,2 ,4-苯三酚、1 ,3-苯二酚和4-甲基-1 ,2-二苯酚)。由于普洱熟茶是由大叶茶经微生物后发酵生产的, 8-氧化咖啡因显然是茶叶中的咖啡因在微生物作用下形成的转化产物。胸腺嘧啶脱氧核苷亦可能是茶叶中的嘧啶类生物碱与微生物中的核苷类化合物在后发酵过程中缩合形成的。二者均为新发现的普洱熟茶的特征性成分。     

Scheduling of anticancer drugs

Many cancer patients are treated with a combination of anticancer drugs. Here, we discuss the importance of drug scheduling and the need for studies that investigate the optimal timing of the various anticancer drugs. Positron emission tomography (PET) using radiolabeled anticancer drugs could be an important tool for those studies.     

Biotransformation of the antitumor intermediate 5-fluorouridine by recombinant Escherichia coli with high pyrimidine nucleoside phosporylase activity

Abstract

5-Fluorouridine (5-FUrd) is a precursor of the widely used antitumor drug doxifluridine. We have produced 5-FUrd by biotransformation by cloning the gene encoding pyrimidine nucleoside phosphorylase (PyNPase) from Enterobacter aero-genes CMCC (B) 45103 and expression in Escherichia coli BL21 (DE3), resulting in recombinant E. coli BL21 (DE3)/ pET28a-PyNPase. After medium optimization, the PyNPase activity in the fermentation broth was 1613 U mg^–1, which was 54-fold that of E. aerogenes. Under optimal conditions (cell concentration, 0.5 g L^–1; uridine, 10 mM; 5-fluorouracil, 45 mM; temperature, 50°C; pH, 7.8), more than 90% of uridine was converted to 5-FUrd, suggesting that this is a valuable tool for application in the preparation of antiviral and antitumor drugs.     

纳米金在抗肿瘤研究中的应用

肿瘤的早期诊断依然是目前需要攻克的难关,现有的抗肿瘤药物因具有较大毒性和缺乏特异性而存在很大的局限性。纳米金能够被多种基团修饰从而获得对肿瘤细胞的靶向性,已成为当前抗肿瘤研究的热点。研究发现纳米金可以通过抑制血管生成、携带抗肿瘤药物以及光热效应等达到肿瘤治疗的目的,同时由于修饰后的纳米金对肿瘤细胞具有靶向性,在肿瘤的早期诊断方面也具有重要的意义。纳米金在体内的分布和代谢与其大小、形态及所带电荷有关,有关纳米金毒性和生物相容性性的评价,还有待于进一步研究。     

Correlation between expression of oncogene products and resistance to anticancer drugs in cultured ovarian cancer cell lines

Despite recent advances in the application of chemotherapy to ovarian cancer, the development of alternative therapies that retain activity against drug-resistant-tumors remains a high priority. We analyzed a number of cultured ovarian cancer cell lines of different tissue types for the presence or absence of sensitivity to various anticancer drugs as well as expression patterns of oncogene products (erbB-2, EGFR, bcl-2). As a result, we identified oncogene products that were related to resistance. Using 9 cultured cell lines of ovarian cancers (serous, mucinous, endometrioid, clear, undifferentiated), sensitivities to anticancer drugs were investigated using the MTT assay. The phenotypes of oncogene products expressed by the above cultured cell lines were analyzed by Western blotting. The oncogene products involved in resistance to anticancer drugs were identified by multivariate analysis. Positive correlation between the resistance to anticancer drugs and the oncogene products was obtained by multivariate analysis for (a) CDDP and erbB-2 (b) x p-16 and erbB-2, and (c) MMC and EGFR. Correlation between resistance to anticancer drugs and expression of certain oncogene products was obtained in ovarian cancers, suggesting that sensitivity to anticancer drugs could be predicated prior to chemotherapy.     

Antineoplastic potential of medicinal plants

Cancer is one of the most dreaded diseases worldwide and the incidence is on the rise in both developing and developed countries. It is treated by chemotherapy, radiotherapy and surgery. In spite of advances in treatment strategies, cancer still remains a major cause of death. Research is on for development of better drugs which are more effective and simultaneously cause fewer side effects. Plants have been used for the treatment of various ailments of man and animals since ages. They are being screened extensively to explore the possibility of development of economically viable anticancer drugs. Natural products of plant origin currently constitute a considerable proportion of commercially available antineoplastic drugs. This review gives an insight into commercially available anticancer drugs of plant origin and also patents granted to plant derived components, extracts and polyherbal formulations possessing anticancer activity. The exhaustive work reviewed here on antineoplastic activity of various plants both in vitro and experimental models throughout the world will help design further research in this field.     

L-histidinol in experimental cancer chemotherapy: improving the selectivity and efficacy of anticancer drugs, eliminating metastatic disease and reversing the multidrug-resistant phenotype.

Human cancer chemotherapy is limited by two major problems: the failure of commonly used anticancer drugs to act against tumor cells in a specific manner and the ability of malignant cells to resist killing by antineoplastic agents. Experimentally, both of these problems can be solved by using L-histidinol in combination with conventional anticancer drugs. A structural analogue of the essential amino acid L-histidine and an inhibitor of protein biosynthesis. L-histidinol improves the selectivity and the efficacy of a variety of cancer drugs in several transplantable murine tumors. Furthermore, L-histidinol circumvents the drug-resistant traits of a variety of cancer cells, including those showing multidrug resistance. This review will summarize these properties of L-histidinol, present new evidence on its ability to increase the vulnerability of both drug-sensitive and drug-resistant human leukemia cells to various anticancer drugs, and show that, in addition to inhibiting protein synthesis, L-histidinol acts as an intracellular histamine antagonist. The establishment of a connection between the latter mechanism and the capacity to modulate anticancer drug action has resulted in a clinical trial in the treatment of human cancer.     

Current understanding of synergistic interplay of chitosan nanoparticles and anticancer drugs: merits and challenges

Recent advances have been made in cancer chemotherapy through the development of conjugates for anticancer drugs. Many drugs have problems of poor stability, water insolubility, low selectivity, high toxicity, and side effects. Most of the chitosan nanoparticles showed to be good drug carriers because of their biocompatibility, biodegradability, and it can be readily modified. The anticancer drug with chitosan nanoparticles displays efficient anticancer effects with a decrease in the adverse effects of the original drug due to the predominant distribution into the tumor site and a gradual release of free drug from the conjugate which enhances drug solubility, stability, and efficiency. In this review, we discuss wider applications of numerous modified chitosan nanoparticles against different tumors and also focusing on the administration of anticancer drugs through various routes. We propose the interaction between nanosized drug carrier and tumor tissue to understand the synergistic interplay. Finally, we elaborate merits of drug delivery system at the tumor site, with emphasizing future challenges in cancer chemotherapy.     

Synthesis of pheophorbide-a conjugates with anticancer drugs as potential cancer diagnostic and therapeutic agents

Pheophorbide-a, a chlorine based photosensitizer known to be selectively accumulated in cancer cells, was conjugated with anticancer drugs, doxorubicin and paclitaxel in the purpose of selective cancer diagnosis and therapy. Pheophorbide-a was conjugated with anticancer drugs via directly and by the use of selective cleavage linkers in cancer cell. The fluorescence of pheophorbide-a and doxorubicin conjugate by excitation at 420 or 440 nm was greatly diminished possibly by the energy transfer mechanism between two fluorescent groups. However, upon treatment in cancer cells, the conjugate showed to be cleaved to restore each fluorescence of pheophorbide-a and doxorubicin after 48 h of incubation. Also, pheophorbide-a conjugates either with doxorubicin and paclitaxel inhibited the growth of various cancer cells more potently than pheophorbide-a, which displayed very weak inhibitory activity. The results indicated that the pheophorbide-a conjugates with anticancer drugs could be utilized for selective cancer therapy as well as for the fluorescence detection of cancer.     

Polyether ionophores—promising bioactive molecules for cancer therapy

The natural polyether ionophore antibiotics might be important chemotherapeutic agents for the treatment of cancer. In this article, the pharmacology and anticancer activity of the polyether ionophores undergoing pre-clinical evaluation are reviewed. Most of polyether ionophores have shown potent activity against the proliferation of various cancer cells, including those that display multidrug resistance (MDR) and cancer stem cells (CSC). The mechanism underlying the anticancer activity of ionophore agents can be related to their ability to form complexes with metal cations and transport them across cellular and subcellular membranes. Increasing evidence shows that the anticancer activity of polyether ionophores may be a consequence of the induction of apoptosis leading to apoptotic cell death, arresting cell cycle progression, induction of the cell oxidative stress, loss of mitochondrial membrane potential, reversion of MDR, synergistic anticancer effect with other anticancer drugs, etc. Continued investigation of the mechanisms of action and development of new polyether ionophores and their derivatives may provide more effective therapeutic drugs for cancer treatments.     

AlgiMatrix? Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

Background

Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.

Methods

Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids.

Results

IC₅₀ values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.

Conclusion

The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.