Environmentally Sensitive Color-Shifting Fluorophores for Bioimaging

We introduce color-shifting fluorophores that reversibly switch between a green and red fluorescent form through intramolecular spirocyclization. The equilibrium of the spirocyclization is environmentally sensitive and can be directly measured by determining the ratio of red to green fluorescence, thereby enabling the generation of ratiometric fluorescent probes and biosensors. Specifically, we developed a ratiometric biosensor for imaging calcium ions (Ca²⁺) in living cells, ratiometric probes for different proteins, and a bioassay for the quantification of nicotinamide adenine dinucleotide phosphate.     

基于SERS纳米探针的细胞内硝基还原酶检测

在缺氧的肿瘤细胞内, 硝基还原酶(NTR)通常过表达且其含量高低与缺氧程度呈正相关, 因此开发高选择性检测NTR的方法对早期肿瘤诊断至关重要. 本文通过修饰对硝基苯硫酚(p?NTP)到金纳米粒子(Au NPs)表面构建了一种表面增强拉曼散射(SERS)探针. 在缺氧条件下, 以还原型烟酰胺腺嘌呤二核苷酸(NADH)作为电子供体, NTR可催化还原芳香硝基为芳香胺, 导致纳米探针的SERS光谱发生变化, 从而实现NTR的高选择性检测, 检出限低至18 ng/mL. 该探针毒性低、 生物兼容性好, 可用于缺氧条件下A549细胞内的NTR分析, 为肿瘤细胞的缺氧现象评估提供了一种有效的策略.     

Kinetic approach for the purification of nucleotides with magnetic separation

The isolation of β‐nicotinamide adenine dinucleotide is of great importance since it is widely used in different scientific and technologic fields such as biofuel cells, sensor technology, and hydrogen production. In order to isolate β‐nicotinamide adenine dinucleotide, first 3‐aminophenyboronic acid functionalized magnetic nanoparticles were prepared to serve as a magnetic solid support and subsequently they were used for reversible adsorption/desorption of β‐nicotinamide adenine dinucleotide in a batch fashion. The loading capacity of the 3‐aminophenyboronic acid functionalized nanoparticles for β‐nicotinamide adenine dinucleotide adsorption was 13.0 μmol/g. Adsorption kinetic and isotherm studies showed that the adsorption process followed a pseudo‐second‐order kinetic model and the experimental data can be represented using Langmuir isotherm model. The 3‐aminophenyboronic acid functionalized magnetic nanoparticles were proposed as an alternative support for the β‐nicotinamide adenine dinucleotide purification. The results elucidated the significance of magnetic separation as a fast, relatively simple, and low‐cost technique. Furthermore, the magnetic supports can be reused at least five times for purification processes.     

Multiphoton imaging of actin filament formation and mitochondrial energetics of human ACBT gliomas

We studied the three-dimensional (3D) distribution of actin filaments and mitochondria in relation to ACBT glioblastoma cells migration. We embedded the cells in the spheroid form within collagen hydrogels and imaged them by in situ multiphoton microscopy (MPM). The static 3D overlay of the distribution of actin filaments and mitochondria provided a greater understanding of cell-to-cell and cell-to-substrate interactions and morphology. While imaging mitochondria to obtain ratiometric redox index based on cellular fluorescence from reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide we observed differential sensitivity of the migrating ACBT glioblastoma cells to femtosecond laser irradiation employed in MPM. We imaged actin-green fluorescent protein fluorescence in live ACBT glioma cells and for the first time observed dynamic modulation of the pools of actin during migration in 3D. The MPM imaging, which probes cells directly within the 3D cancer models, could potentially aid in working out a link between the functional performance of mitochondria, actin distribution and cancer invasiveness.     

Cathodic reduction of nicotinamide adenine dinucleotide and other adenine-containing compounds in acidic media

Both nicotinamide adenine dinucleotide (NAD⁺) and acid-hydrated NADH, as well as adenine, adenosine, adenosine mono-, di-, and tri-phosphate and adenosine diphosphoribose, undergo four-electron reductions of the protonated adenine ring in acidic media. The values of αn_a (transfer coefficient times the number of electrons involved in the rate-determining step), n (total number of electron transferred), and p (number of protons involved in the rate-determining step) agree well with values previously reported for adenine. Cathodic stripping voltammetry of an adsorbed film can be applied to these compounds. Rapid scan rates are required to eliminate the slow desorption step at ?1.1 V vs. SCE for some of these compounds. Hydration of the nicotinamide ring of NADH appears to inhibit this desorption step, but does not appear to be related directly to the electroactivity of the hydration product.     

A label-free fluorescence DNA probe based on ligation reaction with quadruplex formation for highly sensitive and selective detection of nicotinamide adenine dinucleotide

A simple label-free fluorescent sensing scheme for sensitive and selective detection of nicotinamide adenine dinucleotide (NAD(+)) has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation. This approach can detect 0.5 nM NAD(+) with high selectivity against other NAD(+) analogs.     

Novel nicotinamide adenine dinucleotide analogues as selective inhibitors of NAD-dependent enzymes

Three novel dinucleotide analogues of nicotinamide adenine dinucleotide (NAD⁺) have been synthesised from d-ribonolactone. These compounds incorporate a thiophene moiety in place of nicotinamide and are hydrolytically stable. They have been evaluated as inhibitors of adenosine diphosphate ribosyl cyclase, glutamate dehydrogenase and Sir2 acyltransferase activities. Enzyme specificity and a high level of inhibition was observed for the dehydrogenase.     

一些生物活性分子的合成进展

对一些生物活性分子的合成研究进展，如NADH（nicotinamide adenine dinucleotide hydrogen)模型、类胡萝卡素相关分子、卟啉衍生物的合成研究作 了简要的概述。     

酶法测定乳酸

利用乳酸在氧化型烟酰胺腺嘌呤二核苷酸（NAD）存在的条件下,由乳酸脱氢酶（L-LDH）催化生成丙酮酸和还原型烟酰胺腺嘌呤二核苷酸（NADH）。通过测定NADH吸光度的变化率可得出乳酸的酶促反应速度,并制得标准曲线。样品中的乳酸可由标准曲线求得。讨论了试剂用量、pH、温度和共存离子对测定的影响。     

Separation of flavins and nicotinamide cofactors in Chinese hamster ovary cells by capillary electrophoresis

Simultaneous extraction, separation and quantitation of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in Chinese Hamster Ovary (CHO) cells were investigated. The separation of flavins and nicotinamide cofactors was performed by capillary electrophoresis with laser-induced fluorescence detection at the excitation wavelength of 325 nm. The separation protocol was established by investigating the excitation wavelength, high voltage and effects of buffer nature, pH and concentration. All endogenous fluorophores riboflavin, FAD, FMN, NADH and NADPH show wide linear range of quantitation. The limits of detection for the five compounds ranged from 4.5 to 23 nM. Extraction conditions were optimized for high-efficiency recovery of all endogenous fluorophores from CHO cells. To account for the complex matrix of cell extracts, a standard addition method was used to quantify FAD, FMN, NADH and NADPH in CHO cells. The quantitative results should be useful to reveal the metabolic status of cells. The protocols for extraction, separation and quantitation are readily adaptable to normal and cancer cell lines for the analysis of endogenous fluorophores.     

生化教材中存在44年的瑕疵——吡啶环上氮原子的化合价

Understanding redox reactions in reaction of nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺) requires a clear grasp of the textbook content. The reactions in nicotinamide coenzymes with reduced and oxidized forms have been compared in various biochemistry textbooks. Incorrect interpretations usually emphasize the valence changes that at nitrogen in the pyridine ring of a nicotinamide from +5 to +3. Actually, the valence of nitrogen in pyridine ring is -3. We have gathered shreds of evidences and provide here possible suggestions and caution for readers and instructors.     

Reductive metabolism of nitro-p-phenylenediamine by rat liver

Reductive metabolism of the hair dye constituent, nitro-p-phenylenediamine (2-nitro-1,4-diaminobenzene, NPDA), and its acetylated metabolite, NPDA N4-acetate, was investigated with rat liver subcellular fractions, microsomes and cytosol. Under anaerobic conditions, these compounds were reduced to their corresponding amines by these fractions. The microsomal nitro-reducing activity was retarded completely by air and strongly by carbon monoxide. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) functioned more effectively than reduced nicotinamide adenine dinucleotide (NADH) as an electron donor in the microsomal reduction of the nitro compounds, and flavin mononucleotide (FMN) gave rise to a marked enhancement in the microsomal activity, especially when added to an anaerobic incubation mixture containing both NADH and NADPH. The cytosolic nitro-reducing activity was attributed to xanthine oxidase, aldehyde oxidase and other unknown enzyme(s), based on the results of cofactor requirements and inhibition experiments.     

Facile synthesis of near infrared fluorescent trypsin-stabilized Ag nanoclusters with tunable emission for 1,4-dihydronicotinamide adenine dinucleotide and ethanol sensing

A facile chemical synthetic route was developed to prepare near-infrared fluorescent trypsin-stabilized Ag nanoclusters (Try-Ag NCs). The fluorescence emission wavelength of the produced Try-Ag NCs is tunable by simple adjusting pH value of the synthesis system, and the Try-Ag NCs offer a symmetric fluorescent excitation and emission peak. The fluorescence of Try-Ag NCs remains constant in the presence of various ions and molecules, and it can be effectively quenched by 1,4-dihydronicotinamide adenine dinucleotide (NADH) instead of its oxidized forms nicotinamide adenine dinucleotide (NAD⁺). This property enables the Try-Ag NCs to be a novel analytical platform to monitor biological reaction involved with NADH. In this work, the Try-Ag NCs was also applied to analyze ethanol based on the generation of NADH which was the product of NAD⁺ and ethanol in the catalysis of alcohol dehydrogenase. And the proposed platform allowed ethanol to be determined in the range from 10 to 300 μmol/L with 5 μmol/L detection limit.     

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast†

The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed‐phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose‐6‐phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.     

Indirect electrochemical reduction of nicotinamide coenzymes

Nicotinamide coenzymes nicotinamide adenine dinucleotide (NAD(+)) and nicotinamide adenine dinucleotide phosphate (NADP(+)) were electrochemically reduced to NADH and NADPH, respectively. As direct reduction of nicotinamide coenzymes leads to inactive by-products, an indirect method using (pentamethylcyclopentadienyl-2,2'-bipyridine aqua) rhodium (III) as the mediator, was applied. A phosphate buffer solution, pH 8, with 1-10 mM NAD(P)(+) and 2.5-200 microM mediator, was pumped through a glassy carbon packed bed cathode. Virtually all the NAD(P)(+) was reduced to NAD(P)H in the cell. No sign of mediator loss due to side-reactions was detected though the mediator molecules shuttled hundreds of times between the oxidised and the reduced form. Adsorption of mediator molecules on the surface of the carbon cathode was found to be important for the reduction process. Due to strong adsorption, only minute amounts of mediator were consumed.     

Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in‐capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β‐nicotinamide adenine dinucleotide 2′‐phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β‐nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver–Burk equation, the Michaelis–Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.     

Amperometric measurement of hexacyanoferrate(III)-coupled dehydrogenase reactions.

The electrochemical oxidations of hexacyanoferrate II ion and reduced nicotinamide adenine dinucleotide at carbon electrodes are described. Amperometric methods for nicotinamide adenine dinucleotide oxidoreductase analyses by amperometric monitoring of hexacyanoferrate II are reported for lactic dehydrogenase in serum.     

Synthesis of Nicotinamide Adenine Dinucleotide (NAD) Analogues with a Sugar Modified Nicotinamide Moiety

The synthesis of nicotinamide adenine dinucleotide (NAD) analogues in which the ribose unit of the nicotinamide moiety is replaced by a hexitol, altritol, and cyclohexenyl sugar mimic is described.     

一种香豆素荧光标记多肽的合成及应用

Sirtuin蛋白是一类称为依赖烟酰胺腺嘌呤二核苷酸（NAD）的组蛋白去乙酰化酶,共有7个成员,均是潜在的疾病治疗靶点。 然而,目前的荧光筛选方法,只适用于SIRT1~SIRT3。 因此,根据SIRT5的新酶活,设计、合成了针对SIRT5的荧光标记多肽（ISGASE(SuK) AMC）,并通过LC-MS和荧光检测证明了该荧光标记多肽能应用于SIRT5的活性筛选。     

脱氢酶电化学生物传感器的研究进展

自然界中超过400种脱氢酶使用辅酶-烟酰胺腺嘌呤二核苷酸(NAD+)或烟酰胺腺嘌呤二核苷酸磷酸(NADP+)作为生物催化反应中氢和电子的传递体,因此烟酰胺型辅酶的电化学氧化对构筑此类脱氢酶电化学生物传感器具有重要的意义.本文介绍了还原型辅酶在人工电子媒介体存在下的电化学氧化,以及脱氢酶电化学生物传感器的设计和应用.