Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates

The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35 g l⁻¹ and 1.40 g l⁻¹ from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals.     

Screening and isolation of potential lactate dehydrogenase inhibitors from five Chinese medicinal herbs: Soybean,Radix pueraria,Flos pueraria,Rhizoma belamcandae,and Radix astragali

Stroke is among the leading causes of death and severe disability worldwide. Flavonoids have been extensively used in the treatment of ischemic stroke by reducing lactate dehydrogenase levels and thereby enhancing blood perfusion to the ischemic region. Here, we used ultrafiltration high‐performance liquid chromatography coupled with diode array detection and mass spectrometry for the rapid screening and identification of flavonoids from five Chinese medicinal herbs: soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali. Using PC12 cells as a suitable in vitro model of toxicity, cell viability was quantitated using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. The results showed that the extracts of soybean and the six major components, namely, acetyldaidzin, malonylgenistin, daidiain, glycitin, genistin, and acetylcitin; the extract of R. pueraria and its main component daidzein; the extract of F. pueraria and its three major components, tectorigenin, tectoridin, and tectorigenin‐7‐O‐xylosylglucosid; and the extract of R. belamcandae and its main component, tectoridin, were strong lactate dehydrogenase inhibitors. Also, the components of R. astragali showed no bioactivity. These findings indicate that the ultrafltration high‐performance liquid chromatography coupled with diode array detection and mass spectrometry method could be utilized in rapid screening and separation of bioactive compounds from a complex matrix.     

An HPLC method for the determination of the free cortisol/cortisone ratio in human urine

The measurement of the urinary free cortisol-cortisone ratio has been reported to be a sensitive indicator of renal 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD 2) activity. This converts biologically active cortisol to inactive cortisone. A decrease in its activity (e.g. through disease or inhibition caused by a therapeutic agent or a foodstuff) may increase cortisol levels and susceptibility towards hypertension. The method presented here uses a simple isocratic tandem column HPLC system. The method has been validated and found to be robust and reproducible. The lower limit of quantification (LLOQ) was found to be 10 ng/mL for both cortisol and cortisone. Samples of urine (n = 99) from patients, most of whom were on complex combinations of drugs, were analyzed and 92% of samples were found to give successful results with this method (cortisol and cortisone above LLOQ). The ratio ranged from 0.07 to 5.61. No interferences were noted from the drugs that the patients were taking. It was also found that a morning spot urine sample gave comparable results to 24 h collection samples, thus making sample collection much easier.     

Microchip-based capillary electrophoresis for determination of lactate dehydrogenase isoenzymes

This article describes a novel microchip-based capillary electrophoresis and oncolumn enzymatic reaction analysis protocol for lactate dehydrogenase (LDH) isoenzymes with a home-made xenon lamp-induced fluorescence detection system. A microchip integrated with a temperature-control unit is designed and fabricated for low-temperature electrophoretic separation of LDH isoenzymes, optimal enzyme reaction temperature control, and product detection. A four-step operation and temperature control are employed for the determination of LDH activity by on-chip monitoring of the amount of incubation product of NADH during the fixed incubation period and at a fixed temperature. Experiments on the determination of LDH standard sample and serum LDH isoenzymes from a healthy adult donor are carried out. The results are comparable with those obtained by conventional CE. Shorter analysis times and a more stable and lower background baseline can be achieved. The efficient separation of different LDH forms indicates the potential of microfluidic devices for isoenzyme assay.     

Design,Synthesis, Biological Evaluation and In Silico Study of Benzyloxybenzaldehyde Derivatives as Selective ALDH1A3 Inhibitors

Aldehyde dehydrogenase 1A3 (ALDH1A3) has recently gained attention from researchers in the cancer field. Several studies have reported ALDH1A3 overexpression in different cancer types, which has been found to correlate with poor treatment recovery. Therefore, finding selective inhibitors against ALDH1A3 could result in new treatment options for cancer treatment. In this study, ALDH1A3-selective candidates were designed based on the physiological substrate resemblance, synthesized and investigated for ALDH1A1, ALDH1A3 and ALDH3A1 selectivity and cytotoxicity using ALDH-positive A549 and ALDH-negative H1299 cells. Two compounds (ABMM-15 and ABMM-16), with a benzyloxybenzaldehyde scaffold, were found to be the most potent and selective inhibitors for ALDH1A3, with IC₅₀ values of 0.23 and 1.29 µM, respectively. The results also show no significant cytotoxicity for ABMM-15 and ABMM-16 on either cell line. However, a few other candidates (ABMM-6, ABMM-24, ABMM-32) showed considerable cytotoxicity on H1299 cells, when compared to A549 cells, with IC₅₀ values of 14.0, 13.7 and 13.0 µM, respectively. The computational study supported the experimental results and suggested a good binding for ABMM-15 and ABMM-16 to the ALDH1A3 isoform. From the obtained results, it can be concluded that benzyloxybenzaldehyde might be considered a promising scaffold for further drug discovery aimed at exploiting ALDH1A3 for therapeutic intervention.     

Determination of breath alcohol using a differential-type amperometric biosensor based on alcohol dehydrogenase

A differential-type amperometric biosensor based on conventional thick-film technology has been developed for breath alcohol measurement. The amperometric breath alcohol biosensor utilizes the alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) cofactor which produce reduced NADH as a product of the oxidation of alcohol. The biosensor was designed in a differential format consisting of a common Ag/AgCl reference electrode, an active working electrode containing the ADH, and the inactive working electrode containing only bovine serum albumin instead of the ADH. The differential signal between the active working electrode and the inactive working electrode minimized the interference from a large number of oxidizable species present in a person's breath. Prior to the amperometric measurement the biosensor was hydrated simply by dipping it into a phosphate buffer solution at pH 7.4. The NADH produced from the enzymatic reaction was oxidized at the working electrode biased at a potential of 470 mV vs. an on-board Ag/AgCl reference electrode. The biosensor can measure a person's breath alcohol over the concentration range 20–800 ppm routinely required in a test of drunken driving.     

北美海蓬子甜菜碱醛脱氢酶基因的克隆及表达载体构建

以北美海蓬子种子为材料, 采用RACE技术获得北美海蓬子BADH基因的cDNA全长序列. 结果表明: BADH基因cDNA全长为1836bp, 其中开放阅读框(ORF)为1503bp, 编码500个氨基酸, 预测蛋白的分子量为54.5kDa, 理论等电点为5.31. 推测出BADH氨基酸中含有甜菜碱醛脱氢酶家族高度保守的十肽序列(VTLELGGKSP)及与酶功能相关的半胱氨酸残基(Cys). 序列比对和系统进化树显示, 北美海蓬子与盐节木和盐穗木的亲缘关系最近, 同源性达94%. 并构建了植物表达载体pCAMBIA3301-BADH, 将其成功导入到农杆菌EHA105中, 为进一步研究该基因的功能奠定了基础.     

Real‐time measurement of H/D exchange in a microdialysis Raman quartz cell

We present here a novel quartz cell for monitoring H/D exchange in biomolecules using Raman spectroscopy. This cell is combined with a syringe to pump heavy water through a hollow microdialysis fibre, which is inserted into the cell. The deuterium efflux into the sample has been studied as a function of the molecular weight cut‐off of the microdialysis fibre and compared with other microcell systems comprising conventional glass capillaries. The fastest D₂O efflux that we have obtained (k_d = 0.38 ± 0.008 min⁻¹) permits to measure exchange rates of 2.5 min⁻¹ or less. Application of this cell to deuterium exchange in glyceraldehyde‐3‐phosphate dehydrogenase reveals a class of H‐atoms highly resistant to deuteration, which is consistent with a previous infrared study on this protein. Copyright © 2009 John Wiley & Sons, Ltd.     

计时电流法研究4-(N-亚硝基甲氨基)-1-(3-吡啶基)-1-丁酮对谷氨酸脱氢酶活性的影响

用计时电流法研究了烟草特有亚硝胺之一4 (N 亚硝基甲氨基) 1 (3 吡啶基) 1 丁酮(NNK)对谷氨酸脱氢酶(GLDH)催化活性的影响,测定了NNK存在与否时GLDH酶促反应的最大反应速率及米氏常数或表观米氏常数。实验结果表明,NNK对GLDH的催化活性有明显抑制作用,而且属于可逆竞争性抑制,测得在25℃,pH 8.0时抑制常数Ki为176μmol·L-1。