Progress toward re‐engineering non‐ribosomal peptide synthetase proteins: a potential new source of pharmacological agents

Many important pharmaceutical agents, including vancomycin, bleomycin, cyclosporin, and several antibiotics, are produced by non‐ribosomal peptide synthetase (NRPS) enzymes in microorganisms. The NRPS pathway produces an extensive library of products using multienzyme complexes acting in an assembly‐line fashion. Engineering an NRPS system to produce an even greater variety of products, some of which may also have beneficial therapeutic value, would be an enormous advantage. Several approaches have been successful in generating novel NRPS products: mutational biosynthesis during which nonnatural substrates are fed to an organism; domain and module swapping between different species to generate hybrid enzymes; and rational site‐directed mutagenesis, based either on phylogeny or computational prediction, intended to switch substrate specificity and produce altered products. This review will highlight the progress in these areas and describe research in the future that will extend the capacity for re‐engineering NRPS systems. Drug Dev. Res. 66:9–18, 2006. © 2006 Wiley‐Liss, Inc.     

补益脾肾方佐治小儿哮喘的临床研究

目的比较小儿支气管哮喘中、西医治疗疗效.方法将64例支气管哮喘患儿随机分为两组治疗组32例,给予常规西药治疗及对症处理;中药组32例,在常规西药治疗的基础上加服中药(补益脾肾方),监测两组的ET-1、NO水平,比较两组的临床疗效.结果急性期ET-1、NO显著增高,入院时两组比较,差异无显著性(P＞0.05).治疗2w后,中药组ET-1、NO明显下降,两组比较,差异有显著性(P＜0.05).1年后随访患儿中,中药组(23例)轻度哮喘2例,中度哮喘3例,重度哮喘2例;治疗组(20例)依次分别为5例、4例、4例;1年内中药组与治疗组患儿哮喘发作次数比较未发作(例)61,发作＞3次(例)15,两组比较差异有显著性(P＜0.01).结论补益脾肾中药可降低支气管哮喘患儿的血ET-1、NO水平,减轻哮喘发作时的症状;随访1年患儿哮喘发作次数明显减少.我们认为补益脾肾中药可提高支气管哮喘患儿的机体抵抗力,减轻气管炎症损伤,对防治小儿支气管哮喘有效.     

填髓益脑法对大鼠脑缺血再灌注损伤的保护作用

目的观察填髓益脑法对局灶性脑缺血再灌注损伤后脑组织的保护作用。方法雄性成年SD大鼠40只随机分为假手术组、造模组、通络组、填髓益脑组。线栓法制作大鼠局灶性脑缺血模型。填髓益脑组、通络组均采用相应药物灌胃，假手术组和造模组以等量生理盐水灌胃。观察再灌注24h时神经行为学评分，检测NO、GLU及SOD。结果造模组神经行为学评分明显高于填髓益脑组与通络组，填髓益脑组与通络组相近：填髓益脑组与通络组NO、GLU、SOD较之造模组有明显差异。结论填髓益脑法对局灶性脑缺血再灌注损伤有明显的保护作用，其效果与通络法相同。其作用机制可能是通过阻止脑组织SOD活性降低、减少GLU的释放、降低NO含量而发挥神经保护作用。     

Evidence for a NO synthase in porcine platelets which is stimulated during activation/aggregation

Abstract: We tried to characterize the porcine platelet nitric oxide (NO) synthase and its L-arginine (L-arg)/NO metabolism. Using RT-PCR we could show a constitutive endothelial NOS (ecNOS) and an inducible NOS (iNOS) similar mRNA in platelets. The NOS protein could be evidenced by an ecNOS specific antibody which also bound in platelets. This finding could be confirmed by Western blot showing an ecNOS in the membrane but not the cytosolic fraction; iNOS protein could not be detected. Using NADPH-diaphorase staining we could show NO synthase in preactivated platelets but not in resting platelets, indicating that the platelet NOS may be activated during platelet activation/aggregation. Porcine L-arg plasma levels (9.31 × 10^–5 mol/l ± 10%) could be shown to be in the same range as human plasma levels. Moreover, we could show that the NO precursor L-arg and hydroxy-L-arginine (OHarg) concentration dependently inhibited collagen induced platelet aggregation. Summarizing these results confirm the existence of and further characterize porcine platelet NO synthases.     

萘普生对大鼠着床期宫内PGF_(2α)含量及宫内节育器抗生育作用的影响

本文测定了对照组及两个萘普生(前列腺素合成酶抑制剂)处理组大鼠着床期宫内PGF2α的含量。结果表明,对照组、萘普生低剂量组(2mg/kg体重)和高剂量组(20mg/kg体重)的PGF2α含量分别为6.71±0.93、2.19±0.34和1.01±0.18ng/100g组织(三组间P<0.01)。同时,还观察到在本实验条件下,萘普生无明显干扰节育器抗生育作用,药物本身亦未呈现显著抗生育活性。     

Functional role and histological demonstration of nitric-oxide-mediated inhibitory nerves in dog sphincter of Oddi

Abstract Nitric oxide (NO) plays an important physiological role in regulating gastrointestinal motility. Involvement of endogenous NO was evaluated in the response to non-adrenergic, non-cholinergic (NANC) nerve stimulation of the dog sphincter muscle of Oddi. Transmural electrical stimulation (TES), nicotine (10^?5M) and K⁺ (10 mM) produced only a relaxation in the sphincter muscle strips contracted with substance P, which was not potentiated by atropine. The TES-induced relaxation was abolished by tetrodotoxin (3 times 10^?7 M) and oxyhaemoglobin (1.6 times 10^?5 M), but not affected by atropine (10^?7 M), propranolol (10^?7 M), phentolamine (10^?7 M), indomethacin (10^?6 M), chole-cystokinin (CCK, 10^?8 M) and vasoactive intestinal polypeptide (VIP, 10^?8 M). The relaxation was also abolished by treatment with N^G-nitro-L-arginine (L-NA, 10^?5 M), an NO synthase inhibitor. Nicotine produced a transient relaxation, which was abolished by tetrodotoxin, hexamethonium (10^?5 M) and L-NA, but not affected by atropine and N^G-nitro-D-arginine (D-NA, 10^?5 M). The addition of K⁺ elicited a transient relaxation, which was abolished by tetrodotoxin and L-NA. The inhibitory effects of L-NA were antagonized by L-arginine (10^?3 M). The presence of neurons containing nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase was histochemically demonstrated in the sphincter of Oddi. These findings may indicate that TES, nicotine and K⁺ liberate NO from NANC inhibitory nerve which is involved in the relaxation of the dog sphincter of Oddi. The muscular tone does not seem to be regulated by cholinergic nerves under the experimental conditions used.     

急性脑梗死后血浆NO、NOS、ET含量的动态变化及尼莫地平对其影响

目的　观察急性脑梗死 (ACI)后血浆一氧化氮 (NO)、一氧化氮合成酶 (NOS)、内皮素 (ET)含量的动态变化 ,以及尼莫地平治疗后对其影响。方法　ACI患者 110例 ,随机分成尼莫地平组 (5 0例 ) (在常规治疗基础上用尼莫地平 )和常规治疗组 (6 0例 )。在发病后不同时点动态观察血浆NO、NOS、ET含量 ,并设 5 0例脑动脉硬化患者为对照组。结果　脑梗死后血浆ET含量显著升高 ,直至恢复期 ;NO、NOS先增高后下降 ;尼莫地平组和常规组比较ET有显著差异 (P <0 .0 1) ,NO、NOS差别不显著 (P >0 .0 5 )。结论　NO、NOS、ET参与并影响了ACI后复杂的病理生理过程 ;尼莫地平部分通过对ET含量的影响发挥其对脑梗死的治疗作用     

脑梗死患者血浆氧化低密度脂蛋白与一氧化氮、假性血友病因子及血浆颗粒膜蛋白的关系研究

目的探讨动脉粥样硬化性脑梗死患者血浆氧化低密度脂蛋白与血管内皮损伤及血小板活化程度的关系。方法用酶联免疫吸附测定（ELISA）的方法检测49例脑梗死患者和50例相匹配的对照组血浆氧化低密度脂蛋白（OX-LDL）、血管性假血友病因子（VWF）、血浆颗粒膜蛋白（GMP-140）水平，同时用硝酸还原酶比色法测定血清一氧化氮（NO）水平，并把、VWF、GMP-140、NO与OX-LDL作相关分析。结果脑梗死组血浆OX-LDL、、VWF、GMP-140明显高于对照组（t=2．91，P〈0．01；t=3．94，P〈0．001；t=2．08，P〈0．05），而脑梗死组血清NO水平明显低于对照组（t=4．02，P〈0．001）；相关分析表明血浆OX-LDL水平与血清NO水平呈负相关（r=-0．204，P〈0．05），与血浆懈呈正相关（r=0．60，P〈0．01），与血浆GMP-140呈正相关（r=0．430，P〈0．01）。结论脑梗死患者的血浆OX-LDL明显增高，而OX-LDL增高可能是脑梗死的危险因素。     

再生障碍性贫血患者血清一氧化氮与细胞因子水平分析

生物体内的一氧化氮 (NO)作为一种反应极强的效应分子 ,不仅参与免疫调控 ,而且也是造血祖细胞生长和分化不可缺少的调节因子 [1 ]。本文通过对再生障碍性贫血 (AA)患者血清 NO与白细胞介素 - 2 (IL- 2 )、肿瘤坏死子 (TNF)、血小板生成素 (TPO)、红细胞生成素 (EPO)、GM- CSF、5种细胞因子水平测定 ,分析其与外周血象及各细胞因子间的相互关系 ,并探讨 NO及 IL- 2等细胞因子在 AA发生发展过程中的作用。1　材料和方法1.1　材料1.1.1　标本来源　 AA患者 5 0例 ,以 2 0 0 2年 3月至 2 0 0 4年4月我院确诊为 AA的患者为研究对象…