Purification methods of mammalian catechol-O-methyltransferases

The protein purification strategies used for obtaining homogeneous rat and human soluble catechol-O-methyltransferase (S-COTM) polypeptides are reviewed. Expression and purification of recombinant rat and human S-COMT in Escherichia coli and for human S-COMT in baculovirus-infected insect cells made it possible to elucidate the S-COMT polypeptides in more detail. The application of these purification methods has allowed the crystallization of the rat S-COMT protein and the analysis of the kinetic properties of the enzyme in great detail. The availability of the pure S-COMT protein together with the structural data has also greatly enhanced the development of more potent COMT inhibitors.     

Tyrosine protein kinase assays

Protein kinases form a large family of enzymes that play a major role in a number of live processes. The study of their action is important for the understanding of the transformation mechanisms and of the normal and pathological growth events. The quality of an enzyme assay is often the key point of an enzymatic study. It must be flexible and compatible with various experimental conditions, such as those for the purification process, the screening of inhibitors and the substrate specificity studies. As will be shown in the present review, two categories of substrates, peptidic and proteic, should be distinguished. The use of peptide substrates facilitates the determination of the recognition requirements of the enzyme and of the kinetic effects of even minute variations in their sequence. These linear peptide structures are assumed to mimic a complex interaction between the enzyme and a proteic substrate in which distant amino acids in the sequence are vicinal in the folded substrate. Less amenable to a systematic study, but probably more adequate to investigate the natural substrate of a given kinase, are the proteic substrates. Obviously the tools to measure protein kinase activities are not the same in these two cases. The main difficulty in assaying protein kinases is the use of labelles γ-ATP, mostly at large excess concentration, since the final product of the reaction has to be separated from the non-reacted labelled ATP. In the case of peptide substrates, the difficulty is to separate them from ATP basing on differences of molecular mass. Despite the efforts of many investigators to rely upon differences in solubility, in charges or in “affinity”, this separation, which is crucial for the assay, is still an unsolved experimental problem. Chromatographic, as well as electrophoretic assays appeared relatively late in this domain, and more work in assessing new methodologies might bring new breakthroughs in the next few years. Specific, simple and reliable kinase assays are still a major challenge. Their improvement will help to conduct specificity studies, to elucidate complex growth mechanisms in which they are involved and to discover more selective potent inihibitors.     

Chromatographic methods for amylases

This review surveys recent developments in chromatographic methods for the separation of amylases from complex extracts, including the separation of isozymes. It contains two tables with the properties and molecular characteristics of α- and β-amylases from different sources as well as an updated review of methods for the determination of amylase activity. The main subject of this review is a detailed evaluation of the application of newly developed chromatographic methods for the purification of amylases.     

Association of the macrophage migration inhibitory factor gene − 173G/C polymorphism with inflammatory bowel disease: A meta-analysis of 4296 subjects

A variety of epidemiologic studies have focused on the association between macrophage migration inhibitory factor (MIF) gene − 173G/C polymorphism and inflammatory bowel disease (IBD). However, results in different studies have been inconsistent. In order to derive a more precise estimation of the associations, we performed this meta-analysis and systematic searches of electronic databases PubMed and Web of Science (up to April 30, 2013). Based on our search criteria, a total of seven eligible studies concerning the MIF − 173G/C polymorphism and IBD risk were included in the final meta-analysis, comprising 2162 IBD cases and 2134 controls. Significant association was found between MIF − 173G/C polymorphism and the risk of IBD when all studies were pooled into the meta-analysis (for C allele vs. G allele: OR = 1.25, 95% CI = 1.12–1.41, p = 0.000; for C/C vs. G/G: OR = 1.71, 95% CI = 1.23–2.39, p = 0.002; for C/C + G/C vs. G/G: OR = 1.24, 95% CI = 1.09–1.42, p = 0.002; for C/C vs. G/C + G/G: OR = 1.67, 95% CI = 1.20–2.33, p = 0.002). Heterogeneity and publication bias did not exist in the overall comparisons. The present meta-analysis suggests an association between the MIF − 173G/C polymorphism and IBD risk. However, due to few studies and the selection bias existed in some studies, the results should be interpreted with caution.     

Use of antioxidants to minimize the human health risk associated to mutagenic/carcinogenic heterocyclic amines in food

Heterocyclic amines (HAs) are mutagenic/carcinogenic compounds formed in meat during cooking. Several efforts have been made to minimize the risk associated to HA human exposure. Supplementation with antioxidants is considered a promising measure to reduce HA exposure because of their ability as inhibitors of HA formation or as blocking/suppressing agents on HA biotransformation/metabolism. The aim of this review is to present the current knowledge on the capability of synthetic and natural antioxidants to modulate HA-induced mutagenicity/carcinogenicity. Data show a general trend towards a reduction of HA formation both in model systems and in real foods as well as an effective modulation of biotransformation and metabolism. Phenolic compounds, particularly those from tea and olive oil, seem to be the most effective, although a great variability is observed because of the concentration-dependent pro- and antioxidant effects.     

Exposure to beta-carbolines norharman and harman

The aromatic beta-carbolines norharman and harman have been implicated in a number of human diseases including Parkinson's disease, tremor, addiction and cancer. It has been shown that these compounds are normal body constituents formed endogenously but external sources have been identified. Here, we summarise literature data on levels of norharman and harman in fried meat and fish, meat extracts, alcoholic drinks, and coffee brews. Other sources include edible and medicinal plants but tobacco smoke has been identified as a major source. Exposure levels from these different dietary sources are estimated to a maximum of 4 microg norharman per kg body weight (bw) per day and 1 microg harman per kg bw per day. Exposure via tobacco smoke depends on smoking habits and type of cigarettes but can be estimated to 1.1 microg/kg bw for norharman and 0.6 microg/kg bw for harman per package of cigarettes smoked. Studies on toxicokinetics indicate that inhalative exposure leads to a rapid increase in plasma levels and high bioavailability of norharman and harman. Oral bioavailability is lower but there are indications that sublingual absorption may increase dietary uptake of beta-carbolines. Endogenous formation can be estimated to be 50-100 ng/kg bw per day for norharman and about 20 ng/kg bw per day for harman but these rates may increase with high intake of precursors. Biomarker studies on plasma levels of beta-carbolines reported on elevated levels of norharman, harman or both in diseased patients, alcoholics and following tobacco smoking or consumption of beta-carboline-containing food. Cigarette smoking has been identified as major influence but dietary exposure may contribute to exposure.     

Quantitative structure-retention relationships in affinity high-performance liquid chromatography

In this report the affinity high-performance liquid chromatography data, which were determined on silica-based human serum albumin, alpha1-acid glycoprotein, keratin, collagen, melanin, amylose tris(3,5-dimethylphenylcarbamate), and basic fatty acid binding protein columns, are discussed. Using a quantitative structure-retention relationship (QSRR) approach the affinity data were interpreted in terms of structural requirements of specific binding sites on biomacromolecules. The unique chromatographic properties of immobilized artificial membrane and cholesterol stationary phases were also analyzed from the point of view of mimicking biological processes. It has been demonstrated that chemometric processing of appropriately designed sets of chromatographic data derived in systems comprising biomolecules provides information of relevance for molecular pharmacology and rational drug design.     

Protein database of Caenorhabditis elegans

Whole genome sequencing of the free-living nematode Caenorhabditis elegans is a prominent achievement in genomics and uncovers the existence of enormous known and unknown gene products. Characterization and linking of all gene products are the next challenging theme of biology. Genome-wide researches are already progressing on C. elegans and the fruits of these efforts are accessible through the internet. To link the sequence-function relationship, proteomic research has been applied to provide comprehensive information of the worm proteins. In addition to 2-dimensional gel electrophoresis for visualization of the proteome, recent advances in liquid chromatography (LC)-based technologies have allowed the large-scale analysis of proteins and are at cutting-edge of high-throughput analysis of focused proteome.     

Capillary electrophoresis for pharmacokinetic studies

Different analytical techniques involving capillary electrophoresis for the determination of drugs and metabolites in biological fluids are described. Pharmacokinetic studies carried out using capillary electrophoresis are presented, as well as the in vitro metabolism investigations. The advantages and the limitations of capillary electrophoresis for pharmacokinetic studies are discussed.