Tarso B. L. Kist

A review of DNA sequencing techniques

The four best known DNA sequencing techniques are reviewed. Important practical issues covered are read-length, speed, accuracy, throughput, cost, as well as the automation of sample handling and preparation. The methods reviewed are: (i) the Sanger method and its most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other chemical methods; (iii) the Pyrosequencing method--DNA sequencing in real time by the detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with exonuclease (exonuclease digestion of a single molecule composed of a single strand of fluorescently labelled deoxynucleotides). Each method is briefly described, the current literature is covered, advantages, disadvantages, and the most suitable applications of each method are discussed.

Performance of an ultraviolet light‐emitting diode‐induced fluorescence detector in capillary electrophoresis

The performance of a fluorescence detector in capillary electrophoresis (CE) using a light‐emitting diode (LED) as excitation source is reported. An ultraviolet LED pulsed at a repetition rate of 500 Hz, combined with a time‐discrimination and averaging acquisition system, was used. Limits of detection of 3 and 18 fmoles (at a signal‐to‐noise ratio equal to 3) were achieved for fluorescamine‐derivatized bradykinin and lysine, respectively. This system exhibited a linear response for a concentration range between 54 and 417 νM for derivatized lysine, and between 1.81 and 23.58 νM for derivatized bradykinin. This detection system showed to be very convenient for routine analytical applications.

A liquid-liquid extraction procedure followed by a low temperature purification step for the analysis of macrocyclic lactones in milk by liquid chromatography-tandem mass spectrometry and fluorescence detection.

In this work a method is proposed and demonstrated for the analysis of the macrocyclic lactones abamectin, doramectin, eprinomectin, ivermectin and moxidectin in bovine milk by liquid chromatography coupled to mass spectrometry (LC-MS/MS) and liquid chromatography with fluorescence detection (LC-FL). The method is based on liquid-liquid extraction followed by a low temperature purification (LLE-LTP) step. Moreover, the proposed method was validated according to the Commission Decision 2002/657/EC, using LC-MS/MS and LC-FL for confirmatory and quantitative analysis, respectively. For LC-MS/MS the recovery rates observed ranged from 101.2 to 141.6% with coefficient of variation from 2.6 to 19.8%. For LC-FL the recovery rates observed ranged from 100.2 to 105% and coefficient of variations from 2.9 to 8.8%. Matrix effects were negligible due to the low temperature purification step. The quantification limits were far below the maximum limits established by regulations of all countries consulted. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.

Use of capillary electrophoresis with laser-induced fluorescence detection to screen and liquid chromatography-tandem mass spectrometry to confirm sulfonamide residues: validation according to European Union 2002/657/EC.

A multiresidue method is described for determining six sulfonamides (SAs) (sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole, sulfaquinoxaline and sulfadimethoxine) in liver by a capillary electrophoresis screening method and a liquid chromatography coupled to tandem mass spectrometry confirmatory assay. Samples were prepared by homogenizing the tissue, with sodium hydroxide and acetonitrile. After evaporation, extracts were injected in the capillary electrophoresis system or mass spectrometry system for confirmatory analysis. The detection of analytes was achieved by laser-induced fluorescence in capillary electrophoresis. Procedures were validated according to the European Union regulation 2002/657/EC determining specificity, selectivity and detection capability for screening method and decision limit, detection capability, specificity, selectivity, trueness and precision for confirmation method. The results of validation process demonstrate that the method is suitable for application in Brazilian statutory veterinary drug residue surveillance programs. Capillary electrophoresis was proved to be a fast, robust method with low time and reagents consumption.

Analysis of sulfonamides by capillary electrophoresis

The methods of analysis of sulfonamides (SFAs) using CE are reviewed. Sulfonamides were the first antimicrobial group of drugs used in medical treatment. These compounds are still used today in medicine and widely are employed in veterinary medicine, also for their growth promoter effects. Improved methods of analysis of sulfonamides are a constant challenge for researchers. CE is a new trend in the fields of pharmaceutical and food analyses. Several methods for the determination of SFAs by CE have been published in recent years, and the present review considers applications in quality control of pharmaceutical dosage forms, food analysis, determinations in serum, and other biological fluids as well as in electrophoresis experiments which examine the behavior of this class of compounds for theoretical studies of the technique. This review covers studies ranging from the pioneering works on sulfonamide analyses using classical electrophoresis to the more recent CE methods coupled to tandem mass spectrometers. The sections are divided following the EC modes like CZE, MEKC, and hyphenated methods (CE-MS, CE-MS/MS). A brief compilation of theoretical findings of sulfonamides electrophoretic behavior is also included. Parameters such as recoveries, LOD and LOQ, among others, are examined, covering works published until August 2008. This review can contribute to further research aimed to improve the analysis of SFAs by CE.

Cattle tick Boophilus microplus salivary gland contains a thiol-activated metalloendopeptidase displaying kininase activity

This work reports on the characterization of a metalloendopeptidase kininase present in Boophilus microplus salivary glands. Using the guinea pig ileum assay, salivary gland whole extracts (SGE) were found to have a potent kininase activity. Ion-exchange chromatography separated two kininase activities from SGE. The major enzymatic component, eluted at lower ionic strength, was named BooKase (Boophilus Kininase). Analysis of the hydrolysis products by capillary electrophoresis identified Phe5-Ser6 as the only hydrolyzable peptide bond in bradykinin after BooKase treatment. This is the same specificity as the mammalian thimet oligoendopeptidase (EC 3.4.24.15). Like this enzyme, BooKase is also a metallo-peptidase (requires Mn2+) and is activated by-SH protecting reagents. In addition, BooKase was partially inhibited by cFP-AAF-pAB, a specific inhibitor of thimet oligopeptidase. Contrary to other kininases, BooKase had no activity upon angiontensin I. Our results show that BooKase behaves as a typical peptidase with kinase activity.

DNA damage in brain cells and behavioral deficits in mice after treatment with high doses of amantadine

Amantadine (AMA) is an uncompetitive antagonist of the N‐methyl‐d‐aspartate receptor, with clinical application, acting on treatment of influenza A virus and Parkinsons disease. It has been proposed that AMA can indirectly modulate dopaminergic transmission. In high doses, the central nervous system is its primary site of toxicity. To examine deleterious effects on CNS induced by AMA, this study evaluated possible neurobehavioral alterations induced by AMA such as stereotyped behavior, the effects on locomotion and memory and its possible genotoxic/mutagenic activities. Adult male CF‐1 mice were treated with a systemic injection of AMA (15, 30 or 60 mg kg−1) 20 min before behavioral tasks on open field and inhibitory avoidance. Higher AMA doses increased the latency to step‐down inhibitory avoidance test in the training session in the inhibitory avoidance task. At 60 mg kg−1 AMA induced impairing effects on locomotion and exploration and hence impaired habituation to a novel environment. Stereotyped behavior after each administration in a 3‐day trial was observed, suggesting effects on dopaminergic system. Amantadine was not able to induce chromosomal mutagenesis or toxicity on bone marrow, as evaluated by the micronucleus assay. At the lowest dose tested, AMA did not induce DNA damage and it was unable to impair memory, locomotion, exploration or motivation in mice. However, higher AMA doses increased DNA damage in brain tissue, produced locomotor disturbances severe enough to preclude testing for learning and memory effects, and induced stereotypy, suggesting neurotoxicity.

Sample stacking in CZE using dynamic thermal junctions I. Analytes with low dpKa/dT crossing a single thermally induced pH junction in a BGE with high dpH/dT

The possibility to compress analyte bands at the beginning of CE runs has many advantages. Analytes at low concentration can be analyzed with high signal‐to‐noise ratios by using the so‐called sample stacking methods. Moreover, sample injections with very narrow initial band widths (small initial standard deviations) are sometimes useful, especially if high resolutions among the bands are required in the shortest run time. In the present work, a method of sample stacking is proposed and demonstrated. It is based on BGEs with high thermal sensitive pHs (high dpH/dT) and analytes with low dpKa/dT. High thermal sensitivity means that the working pKa of the BGE has a high dpKa/dT in modulus. For instance, Tris and Ethanolamine have dpH/dT=−0.028/°C and −0.029/°C, respectively, whereas carboxylic acids have low dpKa/dT values, i.e. in the −0.002/°C to+0.002/°C range. The action of cooling and heating sections along the capillary during the runs affects also the local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band compression is theoretically calculated using a simple model. Finally, this stacking method was demonstrated for amino acids derivatized with naphthalene‐2,3‐dicarboxaldehyde and fluorescamine using a temperature difference of 70°C between two neighbor sections and Tris as separation buffer. In this case, the BGE has a high pH thermal coefficient whereas the carboxylic groups of the analytes have low pKa thermal coefficients. The application of these dynamic thermal gradients increased peak height by a factor of two (and decreased the standard deviations of peaks by a factor of two) of aspartic acid and glutamic acid derivatized with naphthalene‐2,3‐dicarboxaldehyde and serine derivatized with fluorescamine. The effect of thermal compression of bands was not observed when runs were accomplished using phosphate buffer at pH 7 (negative control). Phosphate has a low dpH/dT in this pH range, similar to the dKa/dT of analytes. It is shown that ∣dKa/dT−dpH/dT∣≫0 is one determinant factor to have significant stacking produced by dynamic thermal junctions.

Stochastic Schrödinger equations in cavity QED: physical interpretation and localization

We propose physical interpretations, also valid for temperatures different from zero, for stochastic methods which have been developed recently to describe the evolution of a quantum system interacting with a reservoir. As opposed to the usual reduced density operator approach, which refers to ensemble averages, these methods deal with the dynamics of single realizations, and involve the solution of stochastic Schr?dinger equations. These procedures have been shown to be completely equivalent to the master equation approach when ensemble averages are taken over many realizations. We show that these techniques are not only convenient mathematical tools for dissipative systems, but may actually correspond to concrete physical processes, for any temperature of the reservoir. We consider a mode of the electromagnetic field in a cavity interacting with a beam of two- or three-level atoms, the field mode playing the role of a small system and the atomic beam standing for a reservoir at finite temperature, the interaction between them being given by the Jaynes-Cummings model. We show that the evolution of the field states, under continuous monitoring of the state of the atoms which leave the cavity, can be described in terms of either the Monte Carlo wavefunction (quantum jump) method or a stochastic Schr?dinger equation, depending on the system configuration. We also show that the Monte Carlo wavefunction approach leads, for finite temperatures, to localization into jumping Fock states, while the diffusion equation method leads to localization into states with a diffusing average photon number, which for sufficiently small temperatures are close approximations to mildly squeezed states. We prove analytically that, in the quantum jump situation, the system evolves in the mean towards a Fock state, even if an infinite number of photon-number amplitudes is present in the initial state.

Sample stacking in CZE using dynamic thermal junctions II: Analytes with high dpKa/dT crossing a single thermal junction in a BGE with low dpH/dT

In a previous work [M. Mandaji, et al., this issue] a sample stacking method was theoretically modeled and experimentally demonstrated for analytes with low dpKa/dT (analytes carrying carboxylic groups) and BGEs with high dpH/dT (high pH–temperature‐coefficients). In that work, buffer pH was modulated with temperature, inducing electrophoretic mobility changes in the analytes. In the present work, the opposite conditions are studied and tested, i.e. analytes with high dpKa/dT and BGEs that exhibit low dpH/dT. It is well known that organic bases such as amines, imidazoles, and benzimidazoles exhibit high dpKa/dT. Temperature variations induce instantaneous changes on the basicity of these and other basic groups. Therefore, the electrophoretic velocity of some analytes changes abruptly when temperature variations are applied along the capillary. This is true only if BGE pH remains constant or if it changes in the opposite direction of pKa of the analyte. The presence of hot and cold sections along the capillary also affects local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band stacking efficacy was also taken into account in the theoretical model presented. Finally, this stacking method is demonstrated for lysine partially derivatized with naphthalene‐2,3‐dicarboxaldehyde. In this case, the amino group of the lateral chain was left underivatized and only the alpha amino group was derivatized. Therefore, the basicity of the lateral amino group, and consequently the electrophoretic mobility, was modulated with temperature while the pH of the buffer used remained unchanged.