Modelling of Lipase Catalysed Transesterification Reactions

Transesterification of substituted ethanols with ethyl acetate is studied in the presence of two different lipases. The steric bulk of the substituent group plays a more important role than the electronic factors in the reaction. The reversible reaction is modelled mathematically, and the rate parameters in the model estimated by a transient parameter estimation procedure. The effect of lipase quantity, reaction temperature and substrate concentration on the reaction is studied independently. Product quantity exhibits a maxima for increasing substrate concentration which is modelled successfully with a substrate product inhibition model, and compared with the experimental data     

Kinetic properties of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> bifunctional GlmU

The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (d-N-GlcNAc-l-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg²⁺, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmU enzyme activities and the kinetic properties will facilitate high-throughput screening of GlmU inhibitors.     

Kinetic Modelling for the Assay of Nortriptyline Hydrochloride Using Potassium Permanganate as Oxidant

Kinetic methods for accurate determination of nortriptyline hydrochloride have been described. The methods are based on the oxidation of nortriptyline hydrochloride with KMnO₄ in acidic and basic media. In acidic medium, the decrease in absorbance at 525.5 nm and in basic medium, the increase in absorbance at 608.5 nm were measured as a function of time. The variables affecting the reactions were carefully investigated and optimised. Kinetic models such as initial rate, rate constant, variable time and fixed time were employed to construct the calibration curves. The initial rate and fixed time methods were selected for quantification of nortriptyline hydrochloride. In acidic medium, the calibration curves showed a linear response over the concentration range 10–50 μg mL⁻¹ for initial rate and 10–60 μg mL⁻¹ for fixed time method (2 min). In basic medium, the calibration graphs were linear over the concentration range 10–100 μg mL⁻¹ for initial rate and fixed time methods (4 min). In acidic medium, the limits of detection for initial rate and fixed time methods (2 min) were 1.02 and 3.26 μg mL⁻¹, respectively. In basic medium, the limits of detection were found to be 1.67 and 1.55 μg mL⁻¹ for initial rate and fixed time methods (4 min), respectively. The initial rate and fixed time methods have been successfully applied to the determination of nortriptyline hydrochloride in commercial dosage form. Statistical comparison of the results of the proposed methods with those of reference method exhibited excellent agreement and there is no significant difference between the compared methods in terms of accuracy and precision.KEY WORDS: dosage forms, fixed time method, initial rate method, nortriptyline, potassium permanganate     

Stepwise Kinetic Equilibrium Models of Quantitative Polymerase Chain Reaction

ABSTRACT: BACKGROUND: Numerous models for use in interpreting quantitative PCR (qPCR) data are present in recent literature. The most commonly used models assume the amplification in qPCR is exponential and fit an exponential model with a constant rate of increase to a select part of the curve. Kinetic theory may be used to model the annealing phase and does not assume constant efficiency of amplification. Mechanistic models describing the annealing phase with kinetic theory offer the most potential for accurate interpretation of qPCR data. Even so, they have not been thoroughly investigated and are rarely used for interpretation of qPCR data. New results for kinetic modeling of qPCR are presented. RESULTS: Two models are presented in which the efficiency of amplification is based on equilibrium solutions for the annealing phase of the qPCR process. Model 1 assumes annealing of complementary targets strands and annealing of target and primers are both reversible reactions and reach a dynamic equilibrium. Model 2 assumes all annealing reactions are nonreversible and equilibrium is static. Both models include the effect of primer concentration during the annealing phase. Analytic formulae are given for the equilibrium values of all single and double stranded molecules at the end of the annealing step. The equilibrium values are then used in a stepwise method to describe the whole qPCR process. Rate constants of kinetic models are the same for solutions that are identical except for possibly having different initial target concentrations. Analysis of qPCR curves from such solutions are thus analyzed by simultaneous non-linear curve fitting with the same rate constant values applying to all curves and each curve having a unique value for initial target concentration. The models were fit to two data sets for which the true initial target concentrations are known. Both models give better fit to observed qPCR data than other kinetic models present in the literature. They also give better estimates of initial target concentration. Model 1 was found to be slightly more robust than model 2 giving better estimates of initial target concentration when estimation of parameters was done for qPCR curves with very different initial target concentration. Both models may be used to estimate the initial absolute concentration of target sequence when a standard curve is not available. CONCLUSIONS: It is argued that the kinetic approach to modeling and interpreting quantitative PCR data has the potential to give more precise estimates of the true initial target concentrations than other methods currently used for analysis of qPCR data. The two models presented here give a unified model of the qPCR process in that they explain the shape of the qPCR curve for a wide variety of initial target concentrations.     

Kinetic Modelling of in Vitro Lipid Peroxidation Experiments - 'Low Level' Validation of a Model of in Vivo Lipid Peroxidation

Kinetic modelling overcomes some of the drawbacks of purely intuitive thinking in integrating information accumulated on chemical reactions involved in oxidative stress. However, it is important to assess if current knowledge about the reactions that mediate lipid peroxidation already allows satisfactory modelling of this process in near-to-physiological conditions. In this paper, a set of increasingly complex in vitro experiments on antioxidants (a-tocopherol and ascorbate) and lipid peroxidation in heterogeneous systems is simulated. Quantitative to semiquantitative agreement is found between experimental and simulation results. In addition, this theoretical analysis provided useful insights, suggested new hypotheses and experiments and pointed out relevant aspects needing further research. The results encourage and serve as partial validation for the formulation of relatively detailed mathematical models of in vivo lipid peroxidation. Some important aspects of the formulation and analysis of such models are discussed.     

A Kinetic Analysis of Coupled (or Auxiliary) Enzyme Reactions

As a case study, we consider a coupled (or auxiliary) enzyme assay of two reactions obeying the Michaelis–Menten mechanism. The coupled reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction is the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the coupled reaction is described by a pair of interacting Michaelis–Menten equations. Moreover, we show that when the indicator reaction is fast, the quasi-steady-state dynamics are governed by three fast variables and one slow variable. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis–Menten equations, are derived. The theory can be extended to deal with more complex sequences of enzyme-catalyzed reactions.     

Pathway-Dependent Effectiveness of Network Algorithms for Gene Prioritization

A network-based approach has proven useful for the identification of novel genes associated with complex phenotypes, including human diseases. Because network-based gene prioritization algorithms are based on propagating information of known phenotype-associated genes through networks, the pathway structure of each phenotype might significantly affect the effectiveness of algorithms. We systematically compared two popular network algorithms with distinct mechanisms – direct neighborhood which propagates information to only direct network neighbors, and network diffusion which diffuses information throughout the entire network – in prioritization of genes for worm and human phenotypes. Previous studies reported that network diffusion generally outperforms direct neighborhood for human diseases. Although prioritization power is generally measured for all ranked genes, only the top candidates are significant for subsequent functional analysis. We found that high prioritizing power of a network algorithm for all genes cannot guarantee successful prioritization of top ranked candidates for a given phenotype. Indeed, the majority of the phenotypes that were more efficiently prioritized by network diffusion showed higher prioritizing power for top candidates by direct neighborhood. We also found that connectivity among pathway genes for each phenotype largely determines which network algorithm is more effective, suggesting that the network algorithm used for each phenotype should be chosen with consideration of pathway gene connectivity.     

Prioritization of liver MRI for distinguishing focal lesions

Liver cancer is one of the leading causes of cancer-related mortality worldwide.Magnetic resonance imaging(MRI) is a non-invasive imaging technique that is often used by radiologists for diagnosis and surgical planning.Analysis of a large amount of liver MRI data for each patient limits the radiologist's efficiency and may lead to misdiagnoses.The redundant MRI data,especially from dynamic contrast enhanced(DCE) sequences,is also a bottleneck in transmitting the images via the internet or PACS for remote consultancy in a reasonable amount of time.This study included 25 patients(aged between 20 and 70years) with liver cysts(seven cases),hemangiomas(eight cases),or hepatic cell carcinomas(10 cases).DCE T1 WI MRI was performed for all the patients.The diagnosis reference included typical MRI findings and post-surgery pathology.The methods were as follows:(i) MRI sequence pre-processing based on large vessels variation level set method to remove non-liver parts from MRI images;(ii) human visual model features(luminance,motion,and contour) extraction and fusion;(iii) anomaly-based MRI ranking;and(iv) methods assessment with the 25 patients' DCE MRI data.The prioritization methods applied to the DCE images could automatically assimilate and determine the content of the medical images,identifying the liver cysts,hemangiomas,and carcinomas.The average uniformity between radiologists and prioritization with the proposed method was 0.805,0.838,and0.818 for cysts,hemangiomas,and carcinomas,respectively,which indicates that the proposed method is an efficient method for liver DCE image prioritization.     

Production of Hexenol in a Two-Enzyme System: Kinetic Study and Modelling

In a two-enzyme system, successive action of hydroperoxide lyase from mint and yeast alcohol-dehydrogenase catalyses the conversion of hydroperoxy linolenic acid to hexenol. Kinetic behaviour was investigated separately for each enzyme: a lumped model based on the Michaelis-Menten approach shows the fate of the reactants in the system.     

Kinetic and Vibrational Isotope Effects of Proton Transfer Reactions in Channelrhodopsin-2

Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H₂O and in D₂O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm⁻¹ to 1738 cm⁻¹ in D₂O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm⁻¹ in D₂O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm⁻¹ and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.     

An Integrative Computational Approach for Prioritization of Genomic Variants

An essential step in the discovery of molecular mechanisms contributing to disease phenotypes and efficient experimental planning is the development of weighted hypotheses that estimate the functional effects of sequence variants discovered by high-throughput genomics. With the increasing specialization of the bioinformatics resources, creating analytical workflows that seamlessly integrate data and bioinformatics tools developed by multiple groups becomes inevitable. Here we present a case study of a use of the distributed analytical environment integrating four complementary specialized resources, namely the Lynx platform, VISTA RViewer, the Developmental Brain Disorders Database (DBDB), and the RaptorX server, for the identification of high-confidence candidate genes contributing to pathogenesis of spina bifida. The analysis resulted in prediction and validation of deleterious mutations in the SLC19A placental transporter in mothers of the affected children that causes narrowing of the outlet channel and therefore leads to the reduced folate permeation rate. The described approach also enabled correct identification of several genes, previously shown to contribute to pathogenesis of spina bifida, and suggestion of additional genes for experimental validations. The study demonstrates that the seamless integration of bioinformatics resources enables fast and efficient prioritization and characterization of genomic factors and molecular networks contributing to the phenotypes of interest.     

柱前衍生-RP-HPLC法测定青蒿中青蒿素的含量

采用柱前衍生-RP-HPLC法测定10个不同产地的青蒿药材中青蒿素的含量.采用Lichrospher 100 RP-18e(250 mm×4.6 mm,5μm,Merck KgaA,Germany)色谱柱,甲醇-0.01 mol/L醋酸钠-醋酸缓冲液(pH 5.8)(体积比62:38)为流动相;检测波长:260 nm;流速:0.5 mL/min;柱温:25℃.结果表明该法准确重现性好,可以为青蒿质量标准的制订提供科学依据.     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

嗜酸氧化亚铁硫杆菌生长动力学方程的应用

基于Monod模型推导出了A．f的生长动力学方程模型,采用Gauss-Newton算法确定了在不同初始条件下细菌生长的动力学参数,即最大比生长速率‰、Monod常数K及R0。通过在不同初始条件下细菌生长特性的研究,得到了相应初始生长条件下以限制性底物亚铁离子浓度为表征的生长动力学方程,理论上揭示了动力学参数变化对细菌生长的影响规律,其中生长动力学方程的数值模拟与实验数据相吻合。     

Assignment of EC Numbers to Enzymatic Reactions with Reaction Difference Fingerprints

The EC numbers represent enzymes and enzyme genes (genomic information), but they are also utilized as identifiers of enzymatic reactions (chemical information). In the present work (ECAssigner), our newly proposed reaction difference fingerprints (RDF) are applied to assign EC numbers to enzymatic reactions. The fingerprints of reactant molecules minus the fingerprints of product molecules will generate reaction difference fingerprints, which are then used to calculate reaction Euclidean distance, a reaction similarity measurement, of two reactions. The EC number of the most similar training reaction will be assigned to an input reaction. For 5120 balanced enzymatic reactions, the RDF with a fingerprint length at 3 obtained at the sub-subclass, subclass, and main class level with cross-validation accuracies of 83.1%, 86.7%, and 92.6% respectively. Compared with three published methods, ECAssigner is the first fully automatic server for EC number assignment. The EC assignment system (ECAssigner) is freely available via: http://cadd.whu.edu.cn/ecassigner/.     

The Kinetic Reaction Mechanism of the Vibrio cholerae Sodium-dependent NADH Dehydrogenase

The sodium-dependent NADH dehydrogenase (Na⁺-NQR) is the main ion transporter in Vibrio cholerae. Its activity is linked to the operation of the respiratory chain and is essential for the development of the pathogenic phenotype. Previous studies have described different aspects of the enzyme, including the electron transfer pathways, sodium pumping structures, cofactor and subunit composition, among others. However, the mechanism of the enzyme remains to be completely elucidated. In this work, we have studied the kinetic mechanism of Na⁺-NQR with the use of steady state kinetics and stopped flow analysis. Na⁺-NQR follows a hexa-uni ping-pong mechanism, in which NADH acts as the first substrate, reacts with the enzyme, and the oxidized NAD leaves the catalytic site. In this conformation, the enzyme is able to capture two sodium ions and transport them to the external side of the membrane. In the last step, ubiquinone is bound and reduced, and ubiquinol is released. Our data also demonstrate that the catalytic cycle involves two redox states, the three- and five-electron reduced forms. A model that gathers all available information is proposed to explain the kinetic mechanism of Na⁺-NQR. This model provides a background to understand the current structural and functional information.     

Reactions of the amino groups in ribonuclease A: I. Kinetic studies

A kinetic analysis has been made of the reaction of the amino groups of ribonuclease A with trinitrobenzene-sulfonic acid. The number of reactive groups and the number of subsets were markedly dependent on the nature and concentrations of the buffer and the pH. Apparent values of pK_a, calculated from the variation of the velocity constants with pH, could, in general, be obtained only for pH values above 7.4. Below this pH the velocity constants were greater than the values calculated from the intrinsic constants. The values of pK_a were in the range of 7.9 – 9.0, which are somewhat smaller than those derived from titration data.The change of behavior of the amino groups with pH is confirmed by a study of the effects of ionic strength on the reactions.The velocity constants generally appear to decrease with increasing concentration of protein.It is shown that there is a close correlation between the pH region in which large changes occur of the reactivities of the amino groups of RNase and the kinetics of the enzyme reaction.     

Prioritization of landscape connectivity for the conservation of Peary caribou

Adequate connectivity between discontinuous habitat patches is crucial for the persistence of metapopulations across space and time. Loss of landscape connectivity is often a direct result of fragmentation caused by human activities but also can be caused indirectly through anthropogenic climate change. Peary caribou (Rangifer tarandus pearyi) are widely dispersed across the islands of the Canadian Arctic Archipelago and rely on sea ice to move seasonally between island habitats throughout their range. Seasonal connectivity provided by sea ice is necessary to maintain genetic diversity and to facilitate dispersal and recolonization of areas from which caribou have been extirpated. We used least‐cost path analysis and circuit theory to model connectivity across Peary caribou range, and future climate projections to investigate how this connectivity might be affected by a warming climate. Further, we used measures of current flow centrality to estimate the role of High Arctic islands in maintaining connectivity between Peary caribou populations and to identify and prioritize those islands and linkages most important for conservation. Our results suggest that the Bathurst Island complex plays a critical role in facilitating connectivity between Peary caribou populations. Large islands, including Banks, Victoria, and Ellesmere have limited roles in connecting Peary caribou. Without rigorous greenhouse gas emission reductions our projections indicate that by 2100 all connectivity between the more southern Peary caribou populations will be lost for important spring and early‐winter movement periods. Continued connectivity across the Canadian Arctic Archipelago, and possibly Peary caribou persistence, ultimately hinges on global commitments to limit climate change. Our research highlights priority areas where, in addition to emission reductions, conservation efforts to maintain connectivity would be most effective.