N 6 -( 2 -isopentenyl) adenosine: biosynthesis in vitro in transfer RNA by an enzyme purified from Escherichia coli

An enzyme has been partially purified from Escherichia coli which catalyzes in vitro the transfer of the Δ²-isopentenyl group from Δ²-isopentenyl pyrophosphate to an adenosine residue in Mycoplasma sp. (Kid) tRNA. The product of the reaction is N⁶-(Δ²-isopentenyl) adenosine, which is known to be absent in this Mycoplasma tRNA. The enzyme has an approximate molecular weight of 55,000 daltons, requires reduced sulfhydryl groups and a divalent metal ion for full activity, and is specific for tRNA.     

广义虫草类真菌来源的N~6‐(2‐羟乙基)腺苷的研究开发现状与思考

N~6-(2-羟乙基)腺苷(简称HEA)是虫草菌丝体中得到的第一个生物来源的钙离子拮抗剂,具有抑制癌细胞生长、降低炎症反应、保护肾脏、镇静、镇痛和抗惊厥等多种功效,已经成为一个研究热点。本文对HEA的菌株来源、生理功能、生物合成、提取纯化、化学合成的研究现状等方面进行了总结,对有关专利进行了评价和分析,提出从种质资源入手拓宽HEA来源,优化HEA的提取纯化方法,并深入探究其生物合成途径,为HEA的深入研究和开发应用提供指导。     

Loss of myocardial protection from ischemic preconditioning following chronic exposure to R(-)-N6-(2-phenylisopropyl)adenosine is related to defect at the adenosine A1 receptor

Exogenously administered adenosine agonist will protect myocardium against infarction during ischemia. However, long-term exposure to adenosine agonists is associated with loss of this protection. To determine why this protection is lost, isolated, perfused rabbit hearts were studied after administration of R(-)-N⁶-(2-phenylisopropyl)adenosine (PIA), 0.25 mg/h IP, for 3-4 days to intact animals. All hearts experienced 30 min of regional ischemia and 120 min of reperfusion. Control groups 1 and 2 were untreated. In group 1 this ischemia/reperfusion was the only intervention, whereas group 2 hearts were preconditioned with a cycle of 5 min global ischemia/10 min reperfusion preceding the 30 min regional ischemia. Groups 3-5 had been chronically exposed to PIA. Group 3 hearts had 1 preconditioning ischemia/reperfusion cycle before the prolonged ischemia. Group 4 received a 5 min infusion of 0.1 mol/L phenylephrine in lieu of global ischemia, whereas group 5 was instead treated with 1 mol/L carbachol. Infarct size averaged 32% of the risk zone in group 1, whereas ischemic preconditioning limited infarction to 8.2 in group 2. Prolonged exposure of group 3 hearts to PIA resulted in the inability of preconditioning with 5 min global ischemia to protect (28.7 ± 4.4% infarction). However, protection was restored by either phenylephrine, an agonist of ₁-adrenergic receptors which couple to G_q and stimulate PKC, or carbachol, an agonist of M₂-muscarinic receptors which couple instead to G_i as do adenosine A₁ receptors (5.2 ± 1.7% and 9.2 ± 2.1% infarction, resp.). Therefore, cross tolerance to ischemic preconditioning develops after chronic PIA infusion. Since both the G_i and the PKC components of the preconditioning pathway were shown to be intact, tolerance must have been related to downregulation or desensitization of the A₁ adenosine receptor.     

Putative role of the adenosine A3 receptor in the antiproliferative action of N 6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N ⁶-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A₃ receptor (hA₃R) ligands with affinities in the high nanomolar range (K _i values of 159 and 649 nM, respectively). These values were comparable to the observed K _i value of adenosine on hA₃R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A₃R. In a functional assay in Chinese hamster ovary cells transfected with hA₃R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A₃R antagonist VUF5574. Both IPA and reference A₃R agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A₃R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A₃R-independent mechanism, as was previously reported for other A₃R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A₃R.     

一株蝉拟青霉产N~6-(2-羟乙基)腺苷静置发酵条件的研究

为了提高蝉拟青霉122菌株中N~6‐(2‐羟乙基)腺苷(HEA)的产量。采用静置液体培养方法,以HEA产量为指标,筛选得到122菌株在查氏培养基上的HEA产量优于沙氏和PD培养基;以查氏培养基为基础培养基,在26℃培养40d时,HEA产量达到最大;在黑暗条件培养优于光照条件培养;最优碳源是蔗糖和葡萄糖;最优氮源是磷酸氢二铵;最优无机盐是KH_2PO_4;最优前体物为次黄嘌呤;最适氨基酸为L‐谷氨酸。正交实验结果表明蔗糖是影响122菌株产HEA的最主要的因子,在筛选出的优化培养基上得到122菌株HEA的最大产量为(130.22±0.60)mg/L,较优化前HEA的产量提高9.73倍。     

Immunospecific binding of tRNA precursors containing N6-(delta 2-isopentenyl) adenosine

Antibodies specific for N6-(delta 2-isopentenyl) adenosine (i6A) were immobilized on Sepharose and this adsorbent (Sepharose-anti-i6A) was used to selectively isolate bacteriophage T4 tRNA precursors containing i6A/ms2i6A from an unfractionated population of 32P-labeled T4 RNAs. The results showed that antibodies to i6A selectively bound only those tRNA precursors containing i6A/ms2i6A. Binding of tRNA precursors by antibody and specificity of the binding was assessed by membrane binding using 32P-labeled tRNA precursor. Binding was highly specific for i6A/ms2i6A residues in the tRNA precursors. This binding can be used to separate modified from unmodified precursor RNAs and to study the biosynthetic pathways of tRNA precursors.     

蝉花虫草提取物N~6-(2-羟乙基)腺苷对小鼠肾脏缺血再灌注损伤的保护作用

研究蝉花虫草提取物N~6-(2-羟乙基)腺苷[N~6-(2-hydroxyethyl)-adenosine,HEA]对小鼠肾脏缺血再灌注损伤(ischemia reperfusion,IR)的影响。选择20–25g雄性C57BL/6小鼠,随机分成5组。假手术组小鼠仅接受腹中线开腹、游离双侧肾蒂及缝合腹部操作;IR组小鼠制成肾脏IR模型,不给药;HEA低剂量组、HEA中剂量组、HEA高剂量组分别在建立肾脏缺血再灌注模型前15min腹腔注射HEA(2.5mg/kg、5mg/kg和7.5mg/kg)。再灌注24h后检测各组肾功能指标血清肌酐(serum creatinine,Scr)和尿素氮(blood urea nitrogen,BUN)水平。苏木精-伊红染色后用光学显微镜观察肾脏组织形态学改变。电镜和TUNEL分析法观察肾小管上皮细胞凋亡状况。实时荧光定量RT-PCR检测细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)、白细胞介素(interleukin-1β,IL-1β)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)等基因的m RNA的表达情况。结果显示,与假手术组比较,IR组BUN和Scr水平明显升高(P0.01,P0.05);肾脏病理表现为上皮细胞碎片和管型,且最为严重;与IR组比较,HEA 7.5mg/kg组血清BUN和Scr水平显著降低(P0.05);HEA 5mg/kg组和HEA 7.5mg/kg组肾损伤情况明显改善(P0.05,P0.01);电镜观察可知HEA 7.5mg/kg组明显改善肾小管上皮细胞的损伤程度,并且保护线粒体和细胞核膜的完整性。原位末端标记法[terminal dexynucleotidyl transferase(Td T)-mediated d UTP nick end labeling,TUNEL]显示HEA治疗组凋亡细胞数量明显低于IR组(P0.01)。HEA治疗组ICAM-1、IL-1β和TNF-αm RNA的水平均低于IR组(P0.01)。所以预处理HEA 7.5mg/kg对肾脏缺血再灌注有保护作用。     

Amperometric immunosensor based on polypyrrole/poly(m-pheylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(Delta2-isopentenyl) adenosine assay

A novel immunosensor based on a multilayer-coated glassy carbon electrode was designed to determine isopentenyl adenosine (iPA) in plants. The multilayer consists of polypyrrole and poly(m-phenylenediamine) with K4Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The ferrocyanide doped in polypyrrole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iPA catalyzed the oxidation of the added glucose with the formation of H2O2, which is in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine). The current of the oxidized production of ferrocyanide reduced at -50 mV is inversely proportional to the concentration of iPA in the competitive immunoreaction. This immunosensor is able to be used about 40 times; after that its surface can be regenerated for a new immunosensor assembly by washing with 0.1M citrate-phosphate buffer (pH 4.6). The percentage of current response reduction (CR%) (y) is linearly related to the logarithm of the concentration of iPA (x) in the 5-300 microg/ml range, with a regression equation of the form y = 42.13x - 27.79 and a correlation coefficient of 0.9861. Five hybrid rice grain samples were analyzed with results in satisfactory agreement to those obtained by high-performance liquid chromatography.     

(E)-2-(2-Hydroxyethylidene)-6-methyl-5-heptenal (alpha-acariolal) and (E)-2-(2-hydroxyethyl)-6-methyl-2,5-heptadienal (beta-acariolal), two new types of isomeric monoterpenes from Caloglyphus polyphyllae (Acari: Acaridae)

The opisthonotal gland secretion of the acarid mite, Caloglyphus polyphyllae, contained two new monoterpenes, (E)-2-(2-hydroxyethylidene)-6-methyl-5-heptenal (1) and (E)-2-(2-hydroxyethyl)-6-methyl-2,5-heptadienal (2), to which we have given the trivial names alpha- and beta-acariolal in relation to alpha- and beta-acaridial (3 and 4), respectively. Elucidation of the structure of 1 was established mainly from 1H-NMR and GC/MS spectral data after partial purification, together with the fact that 1 was recovered in the more-polar fraction from a silica gel column than alpha- and beta-acaridial (3 and 4) present in the secretion. Compound 2 was obtained in the same fraction as a mixture with 1. Based on the facts that 2 had the same molecular weight by GC/MS and the same polarity as that of 1, compound 2 was assumed to be a structural analog of 1. The structures of compounds 1 and 2 were confirmed by their synthesis in nine and ten respective steps starting from alpha-bromo-gamma-butyrolactone.