Newer human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2) inhibitors as potential anticancer agents

Human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2), being an age-old target, has attracted attention recently for anticancer drug development. Mycophenolic acid (MPA), a well-known immunosuppressant drug, was used a lead structure to design and develop modestly potent and selective analogues. The steep structure–activity relationship (SAR) requirements of the lead molecule left little scope to synthesise newer analogues. Here, newer MPA amides were designed, synthesised and evaluated for hIMPDH2 inhibition and cellular efficacy in breast, prostate and glioblastoma cell lines. Few title compounds exhibited cellular activity profile better than MPA itself. The observed differences in the overall biological profile could be attributed to improved structural and physicochemical properties of the analogues over MPA. This is the first report of the activity of MPA derivatives in glioblastoma, the most aggressive brain cancer.     

谷氨酰胺磷酸核糖焦磷酸转酰胺酶研究进展

谷氨酰胺磷酸核糖焦磷酸转酰胺酶是生物．体内嘌呤产物合成途径的关键酶,负责催化全合成途径的第一步反应。肌苷属于嘌呤核苷,是食品和医药行业广泛应用的重要产品。谷氨酰胺磷酸核糖焦磷酸转酰胺酶在肌苷的生物合成途径中起重要调节作用,对其深入研究将有助于提高肌苷的产量,对工业化生产有重大意义。本从谷氨酰胺磷酸核糖焦磷酸转酰胺酶的属性、功能、结构和基因表达与调控方面对其做了介绍,为肌苷产量的提高工作奠定了基础。     

Patterns of adenine metabolism and caffeine biosynthesis in different parts of tea seedlings

Contents of purine alkaloids in different parts of tea ( Camellia sinensis L. cv. Yabukita ) seedlings, seeds and tissue cultures were determined with high-performance liquid chromatography. More than 99% of the caffeine detected was in the leaves of the 4-month-old seedlings. The amount expressed per g fresh weight was higher in older leaves. Theobromine, a precursor of caffeine biosynthesis, was found only in younger leaves. Zero or only trace amounts of theophylline, a degradation product of caffeine, were found in the seedlings. Almost all the caffeine in tea seeds was found in the seed coats. Theobromine and theophilline could not be detected in any part of the seeds.
Tracer experiments using [8-¹⁴C]-adenine indicate that (i) caffeine biosynthesis from [8-¹⁴C]-adenine occurs only in younger leaves,(ii) salvage of [8-¹⁴C]-adenine for nucleic acid synthesis takes place in all parts of the seedlings, (iii) considerable degradation of [8-¹⁴C]-adenine by conventional purine degradation pathway via uric acid takes place in roots and lower parts of stem tissue.
The results strongly suggest that caffeine is synthesized in younger leaves and accumulated within the leaves. Both caffeine contents and its synthetic activity from adenine were extremely low in tissue culture of tea.     

DNA合成技术及应用

DNA从头合成技术是指以寡核苷酸链为起始的合成DNA片段的技术,其不断进步是合成生物学快速发展的基石之一。常规使用的连接介导的DNA合成技术和PCR介导的DNA合成技术日益成熟,精确合成长度已经达到0．5—1kb。微阵列介导的DNA合成技术不断发展,其低成本、高通量的特点吸引了人们的注意;而酵母体内DNA合成技术的成功探索也为体外DNA合成提供了一种补偿方法。DNA合成在优化密码子用于异源表达、构建异源代谢途径、合成人工基因组以及合成减毒病毒用于疫苗研制等方面有广泛应用。综述了DNA从头合成技术的研究进展,并介绍了DNA合成的前沿应用。     

Synthesis and Mass Spectrometric Fragmentation Characteristics of Imidazole Ribosides–Analogs of Intermediates of Purine De Novo Synthetic Pathway

Two inherited deficiencies have been described in purine de novo synthesis pathway. Both the defects are diagnosed by detecting ribosides—dephosphorylated substrates of the enzymes—in patient's urine. We describe here a synthesis and mass spectrometric fragmentation of ribosides potentially of diagnostic importance for defects in the second part of the pathway. All the species, except 5-amino-4-imidazolesuccinocarboxamideriboside can be synthesized from the commercially available 5-amino-4-imidazolecarboxamideriboside by chemical methods. Fragmentation spectra of the compounds were obtained by the ion trap mass spectrometry. During fragmentation an opening of the imidazole ring was not observed for any of the compounds but loss of its substituents in the form of small molecules (NH₃, CO₂, CO) is the major route of fragmentation. The ribose moiety cleaves off molecule(s) of water, undergoes a cross-ring cleavage or breaks away as a whole.     

Purine composition of the crystalline cytoplasmic inclusions of Paramecium tetraurelia

Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.     

A novel class of unstable 6-thioguanine-resistant cells from dog and human kidneys

Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion. In medium containing a relatively high concentration (10g/ ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10^–4 to 10^–5. A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in thioguanine medium, consistent with a reduction in hypoxanthine phosphoribosyltransferase activity. When these thioguanine-resistant primary clones were subsequently grown in the absence of thioguanine and assayed for the thioguanine-resistant phenotype and hypoxanthine phosphoribosyltransferase activity, it was found that most were now thioguanine-sensitive and yielded cell free extracts with substantial amounts of hypoxanthine phosphoribosyltransferase activity. In contrast, thioguanine-resistant human clones grown continuously in the presence of thioguanine yielded cell free extracts with little or no detectable hypoxanthine phosphoribosyltransferase activity. Southern blot analysis demonstrated no structural alterations in the hypoxanthine phosphoribosyltransferase gene in thioguanine-resistant primary human kidney clones. These results suggest that a novel mechanism(s) for thioguanine resistance and the control of hypoxanth phosphoribosyltransferase expression may occur in dog and human kidney cells.Abbreviations AG 8-azaguanine - APRT adenine phosphoribosyltransferase - DAPI 4-6 diamino-2-phenylindole - DV Dulbecco-Vogt - HAT hypoxanthine, aminopterin, thymidine - HPRT hypoxanthine phosphoribosyltransferase - PRPP 5-phosphoribosyl 1-pyrophosphate - TG 6-thioguanine - TG^r thioguanine-resistant - TG^s thioguanine-sensitive - TIP thymidine triphosphate     

枯草芽孢杆菌嘌呤合成途径的修饰对腺苷积累的影响

【目的】研究嘌呤操纵子中purF、purM、purN、purH和purD基因的共同过表达对枯草芽孢杆菌发酵生产腺苷的影响。【方法】利用温敏质粒pKS1,以单交换的形式增加了purF基因在基因组上的拷贝数,同时将强启动子P43插入嘌呤操纵子中,使嘌呤合成途径中purF基因及其下游purM、purN、purH和purD基因的表达水平得到加强,通过实时定量PCR(Realtime Quantitative PCR,RT-qPCR)测定相关基因(purF、purM、purN、purH和purD)的转录水平;通过酶活性检测分析关键酶基因扩增对PRPP转酰胺酶活性的影响;通过发酵实验考察出发菌株与工程菌株的生长、耗糖和腺苷积累情况。【结果】实时定量PCR结果表明,purF、purM、purN、purH和purD基因的表达水平均有不同程度的提高,嘌呤合成途径中关键酶PRPP转酰胺酶的活性是出发菌株的2.4倍。摇瓶发酵实验发现工程菌腺苷产量较出发菌提高17.5%,糖苷转化率增加26.1%。5 L罐发酵实验表明,虽然工程菌的菌体生长受到一定的影响,但在相同发酵周期内腺苷产量比出发菌提高了9.7%。【结论】嘌呤操纵子中purF、purM、purN、purH和purD基因转录水平的增强能够提高腺苷的产量,为通过代谢工程技术改造腺苷生产菌提供了理论依据和研究思路。     

Adenine salvage activity during callus induction and plant growth

Adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) activity was monitored in crude extracts of Arabidopsis thaliana tissues and callus. Changes in APRT activity during germination were determined within different organs of the mature plant and during callus induction. APRT activity was constitutively expressed in all organs examined. There was an increase in APRT activity detected in seeds beginning 3 days following imbibition, after which the level decreased to that found in leaf tissue of mature plants. There was also an increase in APRT activity early during callus induction. A mutant that lacks APRT activity had a diminished capacity for callus induction in both the presence and absence of exogenous cytokinin. The results are consistent with the hypothesis that an increase in APRT activity is associated with actively dividing cells. The significance of these observations is discussed relative to the role of APRT in adenylate and cytokinin metabolism during plant development.