Glutamine-dependent asparagine synthetase from Lupinus luteus

Asparagine synthetase (glutamine-hydrolyzing [l-aspartate: l-glutamine amido-ligase (AMP-forming), E.C. 6.3.5.4] was purified over 500-fold from cotyledon extracts of 1-week-old yellow lupin seedlings. The enzyme was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. The reaction products were shown to be asparagine, AMP, PPi and glutamate. The limiting K_m values were for aspartate 1·3 mM, for MgATP 0·14 mM and for glutamine 0·16 mM. Positive homotropic cooperativity was observed for MgATP only, and gel filtration studies indicated that the substrate-free enzyme (MW 160 000) associated to a dimer (MW 320 000 in the presence of MgCl₂ and ATP. The purified enzyme, which had some glutaminase activity, catalyzed an aspartate- and glutamine-independent ATP-PPi exchange reaction at a rate 5–7-fold higher than the rate of asparagine synthesis. Initial velocity studies and exchange data indicated an overall ping-pong mechanism. Compared to similar enzymes isolated from mammalian tumor cells, the lupin enzyme appears to be unique with respect to MW, reaction mechanism and regulatory properties. The allosteric properties observed suggest an important role for this enzyme in the regulation of asparagine biosynthesis.     

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.     

A Novel Sesquiterpene,Tuberonone, from Solanum tuberosum L.

The effects of the ovarian hormones progesterone and estrone on the conversion of tryptophan to nicotinamide in rats were investigated. Female rats were fed for 35 days with a 20% casein diet, or with a 20% casein diet containing 0.1% progesterone, or 0.001% estrone, or 0.1% progestrone and 0.001% estrone. The conversion ratio of tryptophan to nicotinamide on the last day of the experiment was 2% in the groups fed with the 20% casein diet and the diet containing 0.1% progesterone, but around 1.2% in the group fed with 0.001% estrone, and 0.7% in the group fed with 0.1% progesterone and 0.001% estrone. These results demonstrated that administration of ovarian hormones significantly decreased the conversion of tryptophan to nicotinamide.     

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD_260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.     

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.     

Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes

Summary Genetically defined alkaline phosphatase (ALP) isozymes from the larval midgut tissues ofBombyx mori were purified and characterized. The membrane-bound form (m-ALP) was solubilized with 1% Triton X-100, then purified by DEAE-Sephacel, Con A-Sepharose 4B and Ultrogel AcA 34 column chromatography. The soluble form (s-ALP) was purified by DEAE-Sephacel, epoxy Toyopearl coupled with phosphonic acid and Ultrogel AcA 34 column chromatography. About 840- and 650-fold purification were achieved for m-ALP and s-ALP, respectively, and both ALPs were homogeneous as judged by poly-acrylamide gel electrophoresis. Both forms were found to be similar (MW=68 000 in gel permeation chromatography, and a single subunit as a monomer in denaturing SDS-polyacrylamide gels with MW=58 000 for m-ALP and MW=61 000 for s-ALP). The pH optima of ALPs were shown to lie at 10.9 (m-ALP) and 9.8 (s-ALP), the former being extremely stable even in pH 10–12 which accords with the physiological milieu inBombyx midgut lumen. The K_m values of the m-ALP and s-ALP forp-nitrophenyl phosphate were 1.99 and 1.49 mM, respectively. Both ALPs had a similar substrate specificity.l-Cysteine strongly inhibited both ALPs, but inhibitory effects ofl-phenylalanine,l-homoarginine andl-leucine were undetectable for s-ALP and very weak for m-ALP. A comparison of enzymatic properties on two ALPs suggested that each isozyme plays different roles.Abbreviations m-ALP membrane-bound alkaline phosphatase - s-ALP soluble alkaline phosphatase     

Isolation and characterization of catechol oxidase from Solanum melongena

Catechol oxidase was distributed in soluble and particulate fractions of Solanum melongena. The purified preparation appears to be homogeneous by polyacrylamide gel electrophoresis. The enzyme shows two pH maxima—with catechol, 6.5 and 7.5; and with dopa, 6.5 and 7.9. The latent form of the enzyme does not occur in S. melongena. The preparation resembles the enzyme from other sources in substrate specificity towards various mono- and diphenols, having a higher affinity for catechol than dopa; this tendency increases on purification. The cresolase activity decreases with purification and a lag period with p-cresol is observed. The oxidation of mono- and diphenols is inhibited by ascorbic acid, sulphydryl compounds and chelating agents.     

长裙竹荪抑菌活性物质的分离纯化及其抑菌效果

长裙竹荪Dictyophora indusiata是珍贵的食药用真菌,具有很强的抑菌作用,在天然防腐剂开发方面具有广阔的应用前景。本研究以长裙竹荪的抑菌活性为指标,通过萃取、3次不同流动相的硅胶柱层析、1次反相柱层析和薄层层析法对竹荪提取物进行分离纯化,得到一个抗菌活性强的单体化合物。根据核磁共振波谱等数据分析,推断该化合物为间苯三酚。以巨大芽孢杆菌和肠炎沙门氏菌为供试菌,用平板打孔法及原位抑菌法测定该化合物的抑菌效果,结果表明：该化合物对这两种菌有很强的抑制作用,半抑制浓度分别为83.06μg/mL和51.58μg/mL。本研究首次从长裙竹荪中获得具有抗菌活性的单体化合物间苯三酚,为竹荪天然抗菌物质的开发提供理论依据。     

桔黄赛多孢菌胞外胰蛋白酶的酶学特性

桔黄赛多孢菌Scedosporium aurantiacum是慢性肺病患者的常见呼吸道定植菌,在免疫缺陷人群中可引起侵袭性感染,致死率高,但由于致病机理不明,目前仍然缺乏有效的防控手段。我们在前期研究中,通过差异蛋白组学及酶工程技术发现分泌胰蛋白酶是桔黄赛多孢菌的潜在毒力因子,目前对这种蛋白酶的遗传信息、结构及致病机制并不清楚。本研究用Superdex S-200分子筛和DEAE-Sepharose离子交换两种填料将这种蛋白酶分离纯化出来,通过酶谱验证了纯化效果。进一步研究发现,这种胰蛋白酶对bFSR、bLSTR和bEKK 3种底物的水解性能最佳,对zFR和bzLR的水解性能最差。酶解最快的反应所对应的K_m为6.09μmol/L,V_max为13.01μmol/L/s,K_cat为23.65/s;酶解最慢的反应所对应的K_m为29.94μmol/L,V_max为11.35μmol/L/s,K_cat为20.63/s。研究结果对于填补赛多孢菌毒力因子研究的空白、针对毒力因子开发新型的抗真菌药物和治疗方法都具有重要意义。     

阿加曲班抗凝对连续血液净化患儿凝血功能及单核细胞TLR2rMnX、TLR4rMnX表达水平的影响

目的:探讨阿加曲班抗凝治疗连续血液净化患儿的疗效及对凝血功能及单核细胞TLR2rMnX、TLR4rMnX表达水平的影响。方法:选取从2017年3月至2018年10月于我院儿童重症医学科接受连续血液净化治疗的患儿86例进行研究,将其按照随机抽签法分成研究组与对照组。对照组予以普通肝素抗凝治疗,研究组予以阿加曲班抗凝治疗。分别比较两组的28 d死亡率、治疗前后凝血功能血小板计数(PLT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)和单核细胞TLR2rMnX、TLR4rMnX表达水平、治疗过程中滤器与管路凝血程度、使用寿命以及穿刺部位出血情况的差异。结果:研究组28d死亡率(2.33%)比对照组(9.30%)低,但差异无统计学意义(x2=0.849,P=0.357)。治疗后研究组APTT(31.61±1.26)s、FIB水平(6.61±1.80)g/L较对照组的(27.92±1.44)s、(5.58±1.72)g/L明显更高(t=12.646、2.713,P=0.000、0.008)。研究组治疗过程中滤器与管路凝血程度0级人数占比(93.02%)相比对照组(76.74%)较高,而Ⅱ级人数占比(0.00%)相比对照组(9.30%)较低(x2=4.440、4.195,P=0.035,0.041)。研究组穿刺部位出血等级为0级人数占比(93.02%)高于对照组(74.42%),而Ⅱ级人数占比(0.00%)低于对照组(9.30%)(x2=5.460,4.195;P=0.019,0.041)。研究组管路、滤器使用寿命(18.73±7.74)h、(20.84±8.01)h相比对照组的(14.57±6.88)h、(16.20±7.15)h均较长(t=2.634、2.834,P=0.010,0.006)。治疗后研究组单核细胞TLR2rMnX、TLR4rMnX表达水平为(4.72±1.39)、(3.22±0.82),均低于对照组的(8.30±1.44)、(5.11±0.94)(t=11.729、9.936,P=0.000、0.000)。结论:阿加曲班抗凝应用于连续血液净化患儿中的疗效相比普通肝素抗凝更佳,且有利于改善凝血功能和单核细胞TLR2rMnX、TLR4rMnX表达水平,能够降低滤器或管路凝血发生风险,同时有效降低穿刺部位出血风险,增加管路、滤器使用寿命。