A continual spectrophotometric assay for amino acid decarboxylases

A spectrophotometric method for assaying the activity of three amino acid decarboxylases is reported. This method makes use of the coupled reaction of the decarboxylase with phosphoenolpyruvate carboxylase and malate dehydrogenase. The assay is simple and rapid and allows continuous monitoring of the reaction progress. The kinetic parameters obtained using this method for diaminopimelate decarboxylase, lysine decarboxylase, and arginine decarboxylase are comparable to values obtained by radiochemical methods.     

A combination method of chemical with enzyme reactions for identification of membrane proteins

A simple method for effective analysis of various proteins has been developed, including membrane proteins, with LC-MS/MS, using CNBr and acetic acid cleavage in one reaction for the digestion of both the M/ and /D/ positions within the target proteins. This dual chemical reaction has been compared with traditional CNBr or an acid cleavage method using a rat kidney membrane fraction and it showed an advantage of the dual reaction with respect to a high number of peptides detected and a high protein recovery. Furthermore, when this dual chemical reaction was combined with trypsin digestion, the number of proteins surprisingly increased approximately 3.0 times more than in the cases with the trypsin digestion only. It was also 1.9 times more than in cases dealing with Tube-Gel trypsin digestion, which is one of the most efficient digestion methods. In addition, it was shown that this dual chemical reaction could be applied to an in-gel digestion. Using the combination of the chemical and enzyme reaction, 172 proteins including 95 membrane proteins were identified. This indicated that this method is one of the efficient systems in single MS/MS analysis. In particular, many membrane proteins identified in this study were detected by a new combination, but not by a traditional trypsin digestion method.     

Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I

A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5'-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5'-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5'-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.     

Fermentation data analysis and state estimation in the presence of incomplete mass balance

A method is developed for identifying measurement errors and estimating fermentation states in the presence of unidentified reactant or product. Unlike conventional approaches using elemental balances, this method employs an empirically determined basis, which can tolerate unidentified reaction species. The essence of this approach is derived from the concept of reaction subspace and the technique of singular value decomposition. It is shown that the subspace determined via singular value decomposition of multiple experimental data provides an empirical basis for identifying measurement errors. The same approach is applied to fermentation state estimation. Via the formulation of the reaction subspace, the sensitivity of state estimates to measurement errors is quantified in terms of a dimensionless quantity, maximum error gain (MEG). It is shown that using the empirically determined subspace, one can circumvent the problem of unidentified reaction species, meanwhile reducing the sensitivity of the estimates.     

A global optimization method for prediction of muscle forces of human musculoskeletal system

Inverse dynamic optimization is a popular method for predicting muscle and joint reaction forces within human musculoskeletal joints. However, the traditional formulation of the optimization method does not include the joint reaction moment in the moment equilibrium equation, potentially violating the equilibrium conditions of the joint. Consequently, the predicted muscle and joint reaction forces are coordinate system-dependent. This paper presents an improved optimization method for the prediction of muscle forces and joint reaction forces. In this method, the location of the rotation center of the joint is used as an optimization variable, and the moment equilibrium equation is formulated with respect to the joint rotation center to represent an accurate moment constraint condition. The predicted muscle and joint reaction forces are independent of the joint coordinate system. The new optimization method was used to predict muscle forces of an elbow joint. The results demonstrated that the joint rotation center location varied with applied loading conditions. The predicted muscle and joint reaction forces were different from those predicted by using the traditional optimization method. The results further demonstrated that the improved optimization method converged to a minimum for the objective function that is smaller than that reached by using the traditional optimization method. Therefore, the joint rotation center location should be involved as a variable in an inverse dynamic optimization method for predicting muscle and joint reaction forces within human musculoskeletal joints.     

利用酶催化反应过程曲线确定动力学参数的新方法

本文提出了一个利用过程曲线确定酶催化反应动力学参数的新方法.利用这一方法,仅仅根据两条实验曲线就可以确定单底物酶催化反应的全部动力学参数,并且所有的图形都是(?)     

Convenient method for studying enzyme kinetics.

A convenient method for enzyme kinetic studies is introduced. The method includes identification of reaction mechanism and estimation of the associated kinetic constants with a minimum number of experiments. The application of the method is illustrated by using literature data. Factors limiting the application of this method are also discussed.     

An enzymatic colorimetric method for measuring naringin using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) in the presence of peroxidase

A sensitive colorimetric method for naringin estimation using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as peroxidase substrate is described. The method is based on the coupling reaction of an ABTS radical cation with an oxidation product of naringin formed by peroxidase. This coupling reaction leads to the formation of a purple-colored compound with a maximum absorption at 560 nm. A molar absorption coefficient at this wavelength was calculated to be 13,286 +/- 200 M-1 cm-1. The lowest amount of naringin that can be detected is 1 nmol. The application of this method to the quantification of naringin in grapefruit tissues is presented.     

Computational study of the Sonogashira cross-coupling reaction in the gas phase and in dichloromethane solution

The Sonogashira cross-couplig reaction, consisting of oxidative addition, cis-trans isomerization, transmetalation, and reductive elimination, was computationally modeled using the DFT B3LYP/cc-pVDZ method for reaction between bromobenzene and phenylacetylene. Palladium diphosphane was used as a catalyst, copper(I) bromide as a co-catalyst and trimethylamine as a base. The reaction mechanism was studied both in the gas phase and in dichloromethane solution using PCM method. The complete catalytic cycle is thermodynamically strongly shifted toward products (diphenylacetylene and regenerated palladium catalyst) and is exothermic being in accordance with experimental data. The rate-determining step is the oxidative addition, since the highest point on the Gibbs energy graph of the complete reaction is the transition state of this step. This conclusion is also supported by recent experimental data. The computed energy profile suggests that the transmetalation step is initiated by the dissociation of neutral ligand, while the activation Gibbs energy of this step is 0.1 kcal mol(-1) in the gas phase.     

Modular decomposition of metabolic systems via null-space analysis

We describe a method by which the reactions in a metabolic system may be grouped hierarchically into sets of modules to form a metabolic reaction tree. In contrast to previous approaches, the method described here takes into account the fact that, in a viable network, reactions must be capable of sustaining a steady-state flux. In order to achieve this decomposition we introduce a new concept--the reaction correlation coefficient, phi, and show that this is a logical extension of the concept of enzyme (or reaction) subsets. In addition to their application to modular decomposition, reaction correlation coefficients have a number of other interesting properties, including a convenient means for identifying disconnected subnetworks in a system and potential applications to metabolic engineering. The method computes reaction correlation coefficients from an orthonormal basis of the null-space of the stoichiometry matrix. We show that reaction correlation coefficients are uniquely defined, even though the basis of the null-space is not. Once a complete set of reaction correlation coefficients is calculated, a metabolic reaction tree can be determined through the application of standard programming techniques. Computation of the reaction correlation coefficients, and the subsequent construction of the metabolic reaction tree is readily achievable for genome-scale models using a commodity desk-top PC.     

Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase.

The polymerase chain reaction (PCR) is a technique to amplify a specific DNA sequence millions of times. The thermostable enzyme Taq polymerase allows this procedure to take place under conditions of high specificity and automatization. By combining the techniques of PCR and dideoxy sequencing, it is possible to perform DNA sequencing independently of their structures. The cyclic sequencing reaction is carried out in the presence of an excess amount of sequencing primer and a radioactive nucleotide ([alpha-35S]dATP) using a DNA thermal cycler. Different reaction conditions were investigated and optimized including nucleotide ratios in each termination mix, primer/template ratios, amount of a radioactive nucleotide, and the program of the reaction. This method allows the detection of single base substitutions in heterozygous alleles, and the detection of homozygous deletions. A new RFLP of the human porphobilinogen deaminase (PBGD) gene was identified using this technique. This RFLP is created by one base difference (cytosine or adenine) that changes the restriction site for Apa LI. The alternative sequencing method described in this study is a simple and time-saving procedure that can also be used for large sequencing projects.     

An Adiabatic Molecular Dynamics Method for the Calculation of Free Energy Profiles

A new molecular dynamics method for calculating free energy profiles for rare events is presented. The new method is based on the creation of an adiabatic separation between the reaction coordinate subspace and the remaining degrees of freedom within a molecular dynamics run. This is achieved by associating with the reaction coordinate(s) a high temperature and large mass, thereby allowing the activated process to occur while permitting the remaining degrees of freedom to respond adiabatically. In this limit, by applying a formal multiple time scale Liouville operator factorization, it can be rigorously shown that the free energy profiles are obtained directly from the probability distribution of the reaction coordinate subspace and, therefore, require no postprocessing of the output data. The new method is applied to a variety of model problems and its performance tested against free energy calculations using the "bluemoon ensemble" approach. The comparison shows that free energy profiles can be calculated with greater ease and efficiency using the new method.     

A new substrate for the measurement of N-acetyltransferase activity

A sensitive colorimetric method for the measurement of N-acetyltransferase (NAT) is described. It is based on the high rate of acetylation of 2-(p-aminobenzamido)pyridine by the liver enzyme and the lack of it by the blood NAT. A linear relationship was found between enzyme concentration and reaction rate. The reaction rate was also proportional to the substrate concentration. Inhibition of the reaction was observed at high substrate concentrations. The NAT levels in the liver and kidney of rat, rabbit, mouse and man were measured using this procedure. The tissues of dog failed to acetylate this substrate. The method is applicable to kinetic studies such as the analysis of inhibition reactions with o-phenanthroline and p-chloromercuribenzoate.     

基于退火缓冲液的SLiCE无缝克隆方法的改良

旨在建立一种在采用低感受态细胞转化的条件下完成Seamless ligation cloning extract(SLiCE)无缝克隆的方法。通过在SLiCE反应中引入变性和复性步骤来提高无缝克隆的效率。结果显示,当DNA片段经过SLiCE处理后再进行变性和复性处理,其产生的克隆子比对照多7倍左右,经过菌落PCR验证SLiCE无缝克隆的重组效率高达100%,在此条件下,采用1.7×10^6 CFU/μg转化效率的感受态细胞转化SLiCE反应产物,不仅能够得到重组成功的克隆子,且经验证重组效率仍高达100%。采用引入变性和复性步骤的SLiCE方法,使用常用的CaCl2方法制备的普通感受态细胞,就能得到阳性克隆子,实现无缝克隆,表明本方法能提升SLiCE技术的克隆效率和实用性。     

Rapid method for detection of urea hydrolysis by yeasts.

A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.     

Rapid method for detection of urea hydrolysis by yeasts.

A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.     

One-pot synthesis of 3,4-dihydropyrimidin-2(1H)-ones using chloroacetic acid as catalyst

A simple and effective synthesis of 3,4-dihydropyrimidin-2(1H)-one derivatives from aldehydes, 1,3-dicarbonyl compounds and urea or thiourea using chloroacetic acid as catalyst under solvent-free conditions is described. Compared with the classical Biginelli reaction conditions, this new method has the advantage of good to excellent yields and short reaction time.     

Kinetics of amino acid production from bean dregs by hydrolysis in sub-critical water

Amino acids play an important physiological role in all life-forms and can be recovered from bean dregs waste using sub-critical water hydrolysis. This work deals with the hydrolysis kinetics of bean dregs. Kinetics was conducted in a temperature range of 200–240°C using a 300-ml stainless steel batch reactor. Since the reaction kinetics in sub-critical water is very complicated, a simplified kinetic model to describe the hydrolysis of bean dregs is proposed: a single consecutive reaction. The differential equations resulting from the model were fit to experimental data to obtain kinetic rate constants. By means of the Arrhenius plot, the activation energy as well as the pre-exponential factor was determined. A good agreement between the simplified model and the experimental data was obtained. The kinetic parameters provided useful information for understanding the hydrolysis reaction of bean dregs. The experimental results show that the best hydrolysis technology is: reaction temperature 200°C, reaction time 20 min. Under this condition, the total amino acid yield reaches 52.9%. Based on the results, this method could become an efficient method for bean dregs liquefaction, producing valuable amino acid.     

Cationic heme undecapeptide as stain for detecting glycosaminoglycans on cellulose acetate strips

A rapid and sensitive method has been developed for detecting glycosaminoglycans on cellulose acetate strips. The method is based on the electrostatic interactions between glycosaminoglycans and cationic heme undecapeptide. The complexes are identified by peroxidatic reaction using 3,3'-diaminobenzidine as H2 donor. The detection limit is 2.5-10 ng for sulfated glycosaminoglycans and 60 ng for hyaluronic acid. The sensitivity of this method is higher than of those using Alcian blue or toluidine blue (500 ng).     

Optimizing the spectrofluorimetric determination of cefdinir through a Taguchi experimental design approach

The aim of this work is to optimize a spectrofluorimetric method for the determination of cefdinir (CFN) using the Taguchi method. The proposed method is based on the oxidative coupling reaction of CFN and cerium(IV) sulfate. The quenching effect of CFN on the fluorescence of the produced cerous ions is measured at an emission wavelength (λ_em) of 358 nm after excitation (λ_ex) at 301 nm. The Taguchi orthogonal array L₉ (3⁴) was designed to determine the optimum reaction conditions. The results were analyzed using the signal‐to‐noise (S/N) ratio and analysis of variance (ANOVA). The optimal experimental conditions obtained from this study were 1 mL of 0.2% MBTH, 0.4 mL of 0.25% Ce(IV), a reaction time of 10 min and methanol as the diluting solvent. The calibration plot displayed a good linear relationship over a range of 0.5–10.0 µg/mL. The proposed method was successfully applied to the determination of CFN in bulk powder and pharmaceutical dosage forms. The results are in good agreement with those obtained using the comparison method. Finally, the Taguchi method provided a systematic and efficient methodology for this optimization, with considerably less effort than would be required for other optimizations techniques. Copyright © 2015 John Wiley & Sons, Ltd.