Induction control of purine catabolism in Schizosaccharomyces pombe

Summary The purine catabolic enzymes uricase, allantoinase, allantoicase and ureidoglycollase are highly induced by purines in wild-type Schizosaccharomyces pombe cells. In contrast, urease activity is constitutive. Attempts have been made to identify the inducer or inducers involved. There appears to be a sequential form of induction, uricase being induced by uric acid, allantoinase by allantoic acid (and possibly allantoin), allantoicase by allantoic acid, ureidoglycollase by ureidoglycollic acid. This sequential control of purine breakdown is compared with that found in other fungi.     

Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (P_i) from the breakdown of extracellular ATP. Enzyme activity involved Ca²⁺/Mg²⁺-dependent and Ca²⁺/Mg²⁺-independent dephosphorylation of ATP. Ca²⁺/Mg²⁺-dependent ecto-ATPase was active over a limited range of 0.01–1.0 mM ATP, while Ca²⁺/Mg²⁺-independent ATPase activity was active over a range of 0.1–30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0–9.0), but increased in a relatively linear manner with: (1) time of reaction (0–90 min), (2) number of cells (1–20 oocytes), and (3) temperature (10–37°C). Ecto-ATPase activity was unaffected by ouabain (100 M), sodium azide (100 M), and oligomycin (5 g/ml) (as inhibitors of endo-ATPases) and -glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP,-methylene ATP, ,-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P₂-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido) napthalene-1,3,5-trisulphonic acid), 100 M] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca²⁺/Mg²⁺-dependent enzyme. This striking property of suramin may point to a structural similarity between the ATP-binding sites of ecto-ATPase and purinoceptors, a potentially complicating factor where purinoceptors expressed in oocytes are used to test the potency of agonists and the efficacy of receptor antagonists and enzyme inhibitors.     

A study of dose-response relationships of allpurinol in the presence of low or high purine turnover

Summary Effects of allopurinol (125–500 mg/m² body surface) were studied in normal subjects during periods of 18 days both during a purine-free, isoenergetic liquid formula diet and additional intake of ribonucleic acid, 4 g/day. Plasma uric acid and renal excretion of uric acid, oxypurines (hypoxanthine plus xanthine) and orotic acid were measured and total purine excretion calculated. Effects of allopurinol were evaluated by comparison of the results obtained in the steady state during diet alone (average of days 7–10) with those during allopurinol administration (days 16–18).During the purine-free diet, plasma uric acid was lowered more than urinary uric acid by allopurinol on doses of 250–500 mg/m² (44%–54% of control values on 500 mg/m²), demonstrating an increase in renal clearance. At the same dose, the uric acid lowering effect of allopurinol was more pronounced with than without purine loads (plasma 41%, urine 32% of control on 500 mg/m² during purine intake), while renal uric acid clearance was decreased. The more pronounced reduction of uric acid excretion during purine administration was balanced to the greater part by a more pronounced increased in oxypurine excretion. Total purine excretion was reduced by about 20% during the purine-free diet irrespective of dose. The size of this purine deficit was doubled, but was also independent of dose during addition of purines. Orotic acid excretion increased with dose during allopurinol treatment and was reduced by addition of purines.With respect to uric acid lowering effects, these results are in accordance with findings in patients overproducing uric acid endogenously and suggest that the uric acid lowering effect of allopurinol is enhanced with increasing concentrations of purine bases, presumably due to the tight binding of oxipurinol to xanthine oxidase. The small uricosuric effect of allopurinol seen during ingestion of the purine-free diet possibly is attributable to drug-induced orotic aciduria. The increase in size during purine intake of the purine deficit may result from reduced absorption of dietary purines during allopurinol treatment. Apparently, maximum effects of allopurinol on endogenous synthesis and/or absorption of purines from the gut are exerted by low doses.Abbreviations HPRT Hypoxanthine phosphoribosyl transferase - PRPP Phosphoribosyl pyrophosphate - RNA Ribonucleic acid This study was supported in part by grants of the Deutsche Forschungsgemeinschaft and Deutsche Wellcome GmbHDedicated to Prof. Nepomuk Zöllner on the occasion of his 65th birthday     

The hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus has unique properties

Tritrichomonas foetus, an anaerobic, flagellated protozoan parasite, is incapable of de novo purine nucleotide synthesis, and depends primarily on the salvage of purine bases from the host. The hypoxanthine-guanine-xanthine phosphoribosyl-transferase (HGXPRTase) from this organism has been purified to homogeneity by ammonium sulfate precipitation and Sephacryl-HR100 gel filtration, followed by anion exchange FPLC. Hypoxanthine, guanine and xanthine phosphoribosyl-transferase activities co-eluted in all the purification steps, suggesting that they are associated with the same enzyme protein. The molecular mass of the native protein, as estimated by gel filtration, is 24 kDa. The molecular mass estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is also 24 kDa. Non-denaturing polyacrylamide gel electrophoresis of the purified protein, followed by activity staining with either [¹⁴C]hypoxanthine, [¹⁴C]guanine or [¹⁴C]xanthine, also demonstrates that the enzyme is a monomer of 24 kDa. This monomeric structure is distinctive from all the other reported PRTases which are either dimers or tetramers. Furthermore, unlike the mammalian HGPRTase, which is heat stable, the T. foetus enzyme is heat labile. Kinetic studies with the purified T. foetus HGXPRTase showed that the apparent Kms for hypoxanthine, guanine and xanthine were 4.1 μM, 3.8 μM and 52.4 μM respectively. This recognition of xanthine as a substrate by the parasite enzyme with only about a 10-fold higher Km value than those for hypoxanthine and guanine distinguishes it from the mammalian HGPRTase, which cannot use xanthine as a substrate, as well as the HGXPRTases of Eimeria tenella and Plasmodiumfalciparum, which are dimers, with xanthine about 100-times less proficient as a substrate. T. foetus HGXPRTase is thus a unique enzyme with opportunity for specific inhibitor design.     

Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [³H]adenosine 1.8-fold within 30 s, approx. half of the [³H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [¹⁴C]Inosine, [¹⁴C]hypoxanthine and [³H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC₅₀ approx. 0.7 μM), but not nitrobenzylthioinosine (15 μM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 μM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [³H]adenosine and [¹⁴C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purimes may also be present.     

Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity

From a mutagenized population of wildtype Leishmania donovani promastigotes, a clonal cell line, TUBA2, was isolated by virtue of its ability to survive and grow in 20 microM tubercidin (7-deazaadenosine). The TUBA2 clone was also 1000-fold less sensitive than the parental line to growth inhibition by formycin A, another cytotoxic adenosine analog. Parental and mutant cells, however, were equally sensitive to growth inhibition by formycin B, allopurinol riboside, and 6-thioguanosine. Mutant cell extracts, unlike those prepared from wildtype cells, did not phosphorylate radiolabelled adenosine, tubercidin, or formycin A. Intact adenosine kinase-deficient cells did not accumulate exogenous tubercidin or formycin A but incorporated [14C]adenosine at rates 25% of those found for parental cells. The uptake data suggest that adenosine kinase plays an important role in the metabolism of adenosine but indicate alternative metabolic pathways for this nucleoside. The metabolism of adenosine to the nucleotide level in TUBA2 cells appears to be initiated via deribosylation to adenine. Significant amounts of both adenosine hydrolytic and adenosine phosphorylytic activities have been detected in L. donovani promastigotes. Furthermore, L. donovani extracts could slowly catalyze the deamination of formycin A. The isolation and characterization of adenosine kinase-deficient cells has provided considerable insight into the function of the purine pathway in L. donovani.     

糖皮质激素及嘌呤类似物在炎症性肠病治疗中的应用与评价

目的：合理应用糖皮质激素及嘌呤类似物治疗炎症性肠病。方法：复习文献,总结糖皮质激素及嘌呤类似物治疗炎症性肠病的适应证、用药时机、疗程、疗效及不良反应。结果及结论：糖皮质激素适用于氨基水杨酸制剂疗效不佳的轻、中型患者,尤其适用于重型活动期及暴发型患者;应根据患者炎症性肠病病变的程度和范围选择合适的剂量、给药方式,注意药品不良反应。嘌呤类似物主要适用于对糖皮质激素依赖的UC病例及维持缓解用药以及激素疗效不佳的轻、中度炎症、瘘管性病变以及激素依赖的CD病例。起效慢,不单独用于急性期的治疗;不良反应存在个体差异,用于维持缓解,宜长期用药。     

病证结合诊治原发性痛风36例

目的：对３６例原发性痛风的诊治思路、方法和经验作回顾性总结。方法：以病证并辨为前提,紧扣基本病机组拟基本方,并随证及突出表现灵活加味。结果：３６例经治疗１～５个疗程,治愈２２例（占６１．１１％）,好转１２例（占３３．３３％）,无效２例（占５．５６％）,总有效率为９４．４４％。结论：早期诊断和鉴别诊断,查明有无肾功能不全及其合并疾病,抓住湿、热、痰、瘀、虚五端组方并坚持治疗,避免各种诱发和加重因素,为提高本病诊治水平的关键。     

炎性痛大鼠背根神经节中p38丝裂原活化蛋白激酶激活调节嘌呤2X3受体对痛觉过敏的影响

目的 研究完全弗氏佐剂(complete freund's adjuvant,CFA)导致的炎性痛大鼠的背根神经节(dorsal root ganglia,DRG)中,磷酸化p38丝裂原活化蛋白激酶(phospho-p38 mitogen-activated protein kinase,p-p38MAPK)与嘌呤2X3(purine 2X3,P2X3)受体的相互关系,及其对于痛觉过敏的影响. 方法 选择雄性SD大鼠72只,体重200 g～250 g.采用随机数字表法分为6组,每组12只:Sham+二甲基亚砜(dimethyl sulfoxide,DMSO) (Sham+DMSO)组、CFA+DMSO组、Sham+P2X3抑制剂A-317491 (Sham+A)组、CFA+A-317491(CFA+A)组、Sham+p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38MAPK)抑制剂SB203580(Sham+SB)组和CFA+SB203580(CFA+SB)组,6组大鼠每组又分为两个亚组,每组6只,即行为学组和Western blot组.行为学组在造模前及造模后1、2、4d检测热缩足潜伏期(thermal withdrawal latency,TWL),观察各组大鼠行为学变化.Western blot组在造模后2d处死,取DRG检测p-p38MAPK及P2X3的表达. 结果 与造模前及Sham+DMSO组比较,CFA+DMSO组、CFA+A组、CFA+SB组大鼠TWL均明显降低[造模后2 dTWL分别为(16.3±1.1)、(5.0±0.7)、(12.6±1.5)、(10.7±1.6)s](P＜0.05),而CFA+A组及CFA+SB组大鼠TWL均较CFA+DMSO组升高(P＜0.05).CFA致炎2d后p-p38MAPK以及P2X3表达均显著增加(P＜0.05);鞘内注射SB203580后,p-p38MAPK与P2X3的表达水平都有明显降低(P＜0.05);鞘内注射A-317491后,P2X3的表达水平显著降低(P＜0.05),而p-p38MAPK表达并未有明显变化(P＞0.05). 结论 炎性痛时p38MAPK的激活可介导P2X3受体的上调.     

苦茶的化学成分

目的：对苦茶（Camellia assamica vat．kucha Chang et Wang）叶的化学成分进行研究。方法：采用水提取,聚酰胺、Sephadex LH-20、ODS柱和HPLC等手段分离纯化,通过理化常数和谱学数据鉴定化合物的结构。结果：分离鉴定了10个化合物,其中3个为嘌呤生物碱类化合物,分别为theacfine（1）,caffeine（2）,theobromine（3）;7个为茶多酚类,分别为（＋）-catechin（4）,（-）-epigallocatechin（5）,（-）-gallocatechin（6）,（-）-epigaUocatechin 3-O—gallate（7）,（-）-gallocatechin 3-O—gallate（8）,1,2,6-tri—O—galloyl-β—D—glucose（9）,gallicacid（10）。结论：化合物4-10为首次从该植物中分离得到,并首次对化合物1的核磁数据进行了全归属。