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.     

谷氨酰胺肠外营养对创伤后多器官衰竭患者的营养作用

目的探讨补充谷氨酰胺(Gln)的肠外营养在严重创伤后多器官衰竭(MOF)患者治疗中的作用。方法采用随机对照方法,将60例MOF患者分为Gln组和对照组,每组30例。Gln组给予Gln1.5ml/kg联合常规治疗[热量104～125kJ/(kg·d),其中脂肪供热40%,氮供给量为0.2～0.25g/(kg·d)的完全胃肠外营养(TPN)支持];对照组仅给予常规治疗,连续7d。对照分析营养指标、免疫指标、并发症及临床结果。结果所有患者营养指标(血清白蛋白、前清蛋白、转铁蛋白)和免疫指标(IgG、IgA、IgM)明显减低,处于负氮平衡。第8天时,Gln组营养指标明显高于治疗前及对照组(P<0.05),负氮平衡得到纠正(P<0.05),而对照组仅血清白蛋白、转铁蛋白较治疗前增高(P<0.05),仍处于负氮平衡(P>0.05)。Gln组免疫指标明显高于治疗前及对照组(P<0.01),而对照组仅IgG高于治疗前(P<0.01)。对照组14例(46.7%)出现了血清胆红素、丙氨酸转氨酶和血糖的升高,而Gln组仅4例(13.3%)增高(P<0.05),至14d时对照组病死率为36.7%,而Gln组仅为10%,两者相比差异有显著性(P<0.05)。结论应用Gln的肠外营养能够明显纠正MOF患者的营养代谢障碍,纠正负氮平衡,增强免疫功能,降低并发症及病死率。     

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。     

The effect of bacterial melanin on electrical activity of neurons of the substantia nigra under conditions of GABA generation

The changes in spike activity of single neurons of the compact part of the substantia nigra, evoked by nucleus caudatus stimulation under conditions of long-term registration of the single and multiple, isolated and combined actions of GABA, GABA-amide, glutamine, and ethanolamine-O-sulfate (EOS) were studied in albino rats. Inhibition of poststimulus activity under GABA action was recorded and the inhibitory effect of GABA-amide was revealed. Primary excitatory and subsequent inhibitory effects of glutamine in combination with EOS were shown. The subsequent administration of bacterial melanin, synthesized by a mutant culture of Bacillus thuringiensis (BT-M) evoked a clear-cut and prolonged excitatory reaction during all the combined actions of GABA, GABA-amide, glutamine, and EOS. Preliminary administration of BT-M abolished the inhibitory poststimulus effects of GABA, GABA-amide, and EOS, as well as glutamine-induced excitation.     

Effects of endotoxin on intestinal hemodynamics, glutamine metabolism, and function

The purpose of this study was to investigate the intestinal hemodynamics and gut glutamine metabolism during endotoxemia, and their correlation with altered intestinal absorptive capacity and permeability. Seventeen Sprague-Dawley rats were used in the study. The endotoxin group (ENDO) recieved endotoxin (10 mg/kg intraperitoneally,n=9), while the control group (CONT,n=8) received saline injection. Twelve hours later, D-xylose (0.5 g/kg) and fluorescein isothiocyanate-dextran (FITC-dextran, 750 mg/kg) were given by oral gavage. One hour later abdominal aortic (AA) blood flow, superior mesenteric venous (SMV) flow, mean arterial pressure (MAP), central venous pressure (CVP), and SMV pressure (SMVP) were also measured. The MAP, AA, and SMV blood flow decreased (P<0.05), while the CVP and SMVP increased (P<0.05) in the ENDO group as compared with the CONT group. The ENDO group showed significant decreases for both intestinal glutaminase activity and net intestinal glutamine uptake (P<0.05). The D-xylose concentration in SMV decreased significantly (P<0.05) in the ENDO group as compared with the CONT group. However, the plasma FITC-dextran concentration showed no significant difference between the groups. Endotoxin produced a hypodynamic effect in rats 12h after intraperitoneal administration in association with both a decreased intestinal glutamine metabolism and an absorptive capacity.     

经口补充谷氨酰胺对烧伤大鼠小肠功能的影响

大鼠２０％ＢＳＡⅢ°烧伤后１４天，门静脉、后腔静脉和腹主动脉血浆谷氨酰胺（Ｌ一Ｇｌｕｔａｍｉｎｅ，Ｇｌｎ）含量均明显降低，小肠粘膜的蛋白质、ＤＮＡ、ＡＴＰ含量及乳糖酶活性均明显低于正常。烧伤后在饲料中补充２％或５％Ｇｌｎ，大鼠血浆Ｇｌｎ水平，小肠粘膜的蛋白质、ＤＮＡ、ＡＴＰ含量，以及乳糖酶活性均明显高于烧伤对照和补充甘氨酸（Ｇｌｙ）组，其中ＡＴＰ含量和乳糖酶活性接近正常组。补充５％Ｇｌｎ的大鼠血浆Ｇｌｎ水平高于２％Ｇｌｎ组，其他指标则并不优于补充２％Ｇｌｎ的动物。上述结果表明，烧伤后经口补充Ｇｌｎ，有助于支持肠道的物质和能量代谢，防治或减轻肠道功能的损伤。     

Gln与rhGh对肝硬化病人肠黏膜屏障的影响

目的观察Gln与rhGh各单独应用及联合应用对肝硬化病人小肠黏膜屏障形态学和功能学的影响。方法20个肝硬化病人随机分成四组,给予等氮等热量营养支持7d,Gln+rhGh组口服Gln[0.3g/(Kg·d)]和皮下注射rhGh(10U/d);Gln组口服Gln;rhGh组应用皮下注射rhGh;CONT对照组应用相应安慰剂。观察治疗前后患者小肠黏膜通透性和十二指肠黏膜绒毛高度和隐窝深度的变化,分别比较。结果Gln+rhGh组治疗后小肠黏膜通透性有显著性的降低(P<0.05),且显著低于其他三组(P<0.05);十二指肠黏膜绒毛高度和隐窝深度有显著性的增高(P<0.05),且显著高于其他三组(P<0.05)。结论Gln与rhGh联合应用能减低肠道黏膜的通透性和维持其形态学的完整。     

Immunocytochemistry of Amino-Acids in the Rodent Pituitary Using Extremely Specific, Very High Titre Antisera

The compartmental interrelationships of the metabolically related amino-acids glutamate, GABA and glutamine and the metabolically unrelated amino-acids taurine and glycine in the rodent pituitary, were investigated by light microscopic immunocytochemistry using highly specific antisera. Glutamate-like immunoreactivity was abundant in astrocytes in the posterior pituitary. Glutamine immunoreactivity was present only at low levels in the posterior pituitary, but was abundant in astrocytes within the intermediate lobe. Other glia-like ceils in the anterior pituitary were also glutamine-immunoreactive. GABA immunoreactivity was abundant in the intermediate lobe but absent from anterior and posterior lobes. The GABA immunoreactivity mainly took the form of small punctata, the majority of which were in intimate apposition to the glutamine-immunoreactive glia. Strong taurine immunoreactivity was present in astrocytes in the posterior pituitary but only weak labelling was present in intermediate and anterior lobes of the pituitary. Specific glycine immunoreactivity was not detected in the pituitary. These results suggest that glutamate-immunoreactive astrocytes in the posterior pituitary, unlike glia in loci such as the retina, do not convert much, if any, of their glutamate content into glutamine (or if they do, it is rapidly further metabolized to another compound), whereas those astrocytes in the intermediate lobe do contain glutamine. The spatial association of GABAergic fibres with glutamine-positive astrocytes raises the possibility that astrocytes in the intermediate lobe receive a GABAergic innervation. Glutamate, glutamine and taurine (or their metabolites) may have roles as neuroactive substances regulating pituitary secretion.     

谷氨酰胺对内毒素血症大鼠小肠营养作用的实验研究

35只大鼠随机分为四组 ,A组为正常对照组 ,B组为谷氨酰胺肠外营养组 (Gln TPN) ,C组为不含Gln的常规TPN组和经肠道营养的D组。内毒素以每天每公斤体重 2mg的剂量 ,混入营养液或等渗盐水中 ,持续滴注 5d。结果显示 ,B组肠壁蛋白质和DNA含量高于C组 ,其中回肠蛋白质含量有显著性差异 (P <0 .0 5)。在绒毛高度、小肠粘膜及全层厚度等指标B组也明显优于C组 (P <0 .0 5) ,经肠道营养的D组在上述组织学观察数值上均与正常组相近 ,且粘膜厚度、绒毛高度均超过正常值 (P <0 .0 5)。B、D两组肠道细菌易位率均低于C组 (P <0 .0 5)。研究结果表明 ,加入Gln的TPN有助于防止内毒素血症大鼠肠粘膜萎缩及细菌易位。早期应用经肠道营养更具有积极的生理意义