Journal of biochemical and biophysical methods最新文献

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3 cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G (∼ 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.

The growth thermogenic curves of Escherichia coli (E. coli) affected by berberine, coptisine and palmatine were determined quantitatively by microcalorimetry. The power–time curves of E. coli with and without the three berberines alkaloids (BA) were acquired, meanwhile the extent and duration of inhibitory effects on the metabolism were evaluated by growth rate constant (k), half-inhibitory ratio (IC₅₀), peak time of maximum heat-output power (t_p), total heat-production (Q_t) and so on. The inhibitory effects of BA on E. coli revealed that the sequence of their antimicrobial activity was berberine > coptisine > palmatine. The functional groups methylenedioxy at C2 and C3 on phenyl ring improve antimicrobial activity more remarkably than methoxyl at C2 and C3 on phenyl ring. However, the antimicrobial activity does not vary significantly with methylenedioxy or methoxyl at C9 and C10 on phenyl ring.

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.

Identification of problematic protein classes (domain types, protein families) that are difficult to predict from sequence is a key issue in genome annotation. ROC (Receiver Operating Characteristic) analysis is routinely used for the evaluation of protein similarities, however its results – the area under curve (AUC) values – are differentially biased for the various protein classes that are highly different in size. We show the bias can be compensated for by adjusting the length of the top list in a class-dependent fashion, so that the number of negatives within the top list will be equal to (or proportional with) the size of the positive class. Using this balanced protocol the problematic classes can be identified by their AUC values, or by a scatter diagram in which the AUC values are plotted against positive/negative ratio of the top list. The use of likelihood-ratio scoring (Kaján et al, Bioinformatics, 22, 2865–2869, 2007) the bias caused by class imbalance can be further decreased.

Cell complexes composed of two different cells labeled with different fluorophores can be detected as double positive events in the flow cytometer. Double positivity can originate not only from real complexes but from non-interacting coinciding cells as well. Coincidence has a high impact on the determination of the amount of platelet–granulocyte complexes since platelet concentration is in the orders of magnitude higher than that of the granulocytes. A computer model has been developed to simulate coincidence in the flow cytometer to reveal the contribution of coincidence to the overestimation of the total amount of platelet–granulocyte complexes. Mixtures of non-interacting fluorescent beads as well as EDTA-anticoagulated blood samples were analyzed in the flow cytometer.

An excellent fit was found between computer simulated and measured data pairs. Bead mixture in the flow cytometer and simulation of that resulted in 37.3 ± 1.3 and 35.7 ± 0.6% double positivity, respectively. 30.2 ± 4.3% double positivity was measured for EDTA-anticoagulated blood samples while simulation of that resulted in 28.3 ± 0.6%. Double positivity attributed to platelet–granulocyte complexes in slightly diluted blood samples might originate in coincidence and not from true complexes.

HPLC method was developed for determination of bisphenol A diglycidyl methacrylate (bis-GMA), bisphenol A diglycidyl acrylate (bis-GA), bisphenol A dimethacrylate (bis-DMA), glycidylmethacrylate (GMA) and triethylenglycol dimethacrylate (TEGDMA). Separation was carried out on a reversed phase Omnisphere 5 C18 column with a gradient mobile phase of CH₃CN/H₂O. UV detection was set at 205 nm and 275 nm parallel. The limits of quantification were found. The method has been applied for quantification of unreacted monomers trapped in polymer network of fillings.

Nowadays, very diverse human activities generate urgent demands for fast, sensitive reliable innovative tools capable of detecting major industrial, military, and other dangerous products. An important part of these compounds are free radicals. Capillary electrophoresis (CE), especially in its miniaturized format (lab-on-a-chip), and other electromigration methods offer special possibilities to resolve this problem. These measurements have a great opportuness because of very wide chemical and biological role of free radicals. Several compounds, e.g. monomers and some biologically important groups (as are nitrones) oppose oxidative challenges by virtue of their trap very rapidly oxygen- or carbon-centered radicals and generating other radical species which are stable and biochemically less harmful than the original ones. In many cases, conventionally, the relative trap capacity is measured against tert.-butylhydroperoxide (TBH). In this lecture are presented numerous important free radical species (active oxygen–, nitrogen- and carbon-centered ones, as HO, NO etc) and their adequate in vitro and in vivo applied bioanalytical methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence analysis. A simple and highly sensitive method is the capillary zone electrophoresis with amperometric detection (CZE-AD); It was introduced to determine indirectly OH by analysing its reaction products with salicylic and dihydroxybenzoic acids. Hydroxylated radical products of these acids are often used as a relative measurement in free radical research. Accurate determination of pK(a) values is important for proper characterization of newly synthesized molecules. CZE method was used for determination of their values. Are initiated new research fields as Fenton-, electro-Fenton and photoelectro-Fenton chemistry and foreseen their perspectives.

Nitric oxide is an important cell signaling molecule in physiology and pathophysiology. An indirect method for monitoring nitric oxide (NO) by determining nitrate and nitrite by microchip capillary electrophoresis (CE) with electrochemical (EC) detection has been developed. The amount of nitrite formed in this reaction (analyzed by capillary electrophoresis) was compared with the amount of oxygen consumed (measured by polarography). Were observed a linear relationship between the amount of consumed oxygen and the amount of nitrite formed in the measured range. These results demonstrate that polarographic measurements of the amount of oxygen consumed in the reaction with NO could be used to estimate the concentration of dissolved NO in authentic media. Polarography is an adequate method also to quantitative kinetic study of the free radical activity and of the trapping capacity of different compounds. This method is based on measure of the catalytic polarografic curr