Screening for recombinant glutathione transferases active with monochlorobimane

A rapid and facile colony assay has been developed for catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferases (GST), expressed in Escherichia coli. The basis of the method is the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB). This screening method makes it possible to isolate and characterize one recombinant clone that is active with MCB among thousands of inactive variants. Colonies containing GSTs that catalyze the conjugation of GSH with MCB display fluorescence under long-wavelength UV light. The fluorescence is visible instantly. One rat and 11 human GSTs representing four distinct enzyme classes were studied, and all except human GST T1-1 gave rise to fluorescent colonies. The colony assay based on MCB can consequently be broadly applied for identifying active GSTs both after subcloning of wild-type enzymes and in the screening of mutant libraries. Populations of bacteria expressing GSTs can also be analyzed by flow cytometry.     

Prediction of partial membrane protein topologies using a consensus approach

We have developed a method to reliably identify partial membrane protein topologies using the consensus of five topology prediction methods. When evaluated on a test set of experimentally characterized proteins, we find that approximately 90% of the partial consensus topologies are correctly predicted in membrane proteins from prokaryotic as well as eukaryotic organisms. Whole-genome analysis reveals that a reliable partial consensus topology can be predicted for approximately 70% of all membrane proteins in a typical bacterial genome and for approximately 55% of all membrane proteins in a typical eukaryotic genome. The average fraction of sequence length covered by a partial consensus topology is 44% for the prokaryotic proteins and 17% for the eukaryotic proteins in our test set, and similar numbers are found when the algorithm is applied to whole genomes. Reliably predicted partial topologies may simplify experimental determinations of membrane protein topology.     

AZ 242, a novel PPARalpha/gamma agonist with beneficial effects on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats

Abnormalities in fatty acid (FA) metabolism underlie the development of insulin resistance and alterations in glucose metabolism, features characteristic of the metabolic syndrome and type 2 diabetes that can result in an increased risk of cardiovascular disease. We present pharmacodynamic effects of AZ 242, a novel peroxisome proliferator activated receptor (PPAR)alpha/gamma agonist. AZ 242 dose-dependently reduced the hypertriglyceridemia, hyperinsulinemia, and hyperglycemia of ob/ob diabetic mice. Euglycemic hyperinsulinemic clamp studies showed that treatment with AZ 242 (1 micromol/kg/d) restored insulin sensitivity of obese Zucker rats and decreased insulin secretion. In vitro, in reporter gene assays, AZ 242 activated human PPARalpha and PPARgamma with EC(50) in the micro molar range. It also induced differentiation in 3T3-L1 cells, an established PPARgamma effect, and caused up-regulation of liver fatty acid binding protein in HepG-2 cells, a PPARalpha-mediated effect. PPARalpha-mediated effects of AZ 242 in vivo were documented by induction of hepatic cytochrome P 450-4A in mice. The results indicate that the dual PPARalpha/gamma agonism of AZ 242 reduces insulin resistance and has beneficial effects on FA and glucose metabolism. This effect profile could provide a suitable therapeutic approach to the treatment of type 2 diabetes, metabolic syndrome, and associated vascular risk factors.     

Patterned gene programs and target remodeling following axotomy at a major site for sensory innervation

The genetic mechanisms that a target uses to reestablish the connections of regenerating axons were explored using oligonucleotide microarrays and real-time PCR. In normal and denervated mouse vibrissa follicles, patterns of genetic regulation were assessed in adjacent targets that normally receive different types of sensory and autonomic innervation. We show that a target remodeling occurs following axotomy involving reduced hair growth, altered hair follicle integrity and remodeling of the extracellular matrix. Also, we found two orphan receptors putatively involved in hair growth. Our data further demonstrate region-specific regulation of genes putatively involved in target-axon interactions. Thus, this study shows for the first time that major target remodeling occurs following denervation and suggests putative functions for several novel genes.     

Restricted dispersal in a long-distance migrant bird with patchy distribution,the great reed warbler

In patchily distributed species dispersal connects local populations into metapopulations. Reliable quantifications of dispersal are therefore crucial to understanding the population dynamics and genetic structure of such metapopulation systems. The great reed warbler (Acrocephalus arundinaceus) inhabits eutrophic lakes and has a patchy breeding distribution. In this study we investigated the dispersal pattern of the great reed warbler based on an extensive capture-recapture effort covering a large census area (22,500 km²). At two adjacent breeding sites (10 km apart) in southern Central Sweden, the "main study area", we ringed the majority of adult and nestling great reed warblers between 1992 and 1999. In 1998 and 1999, we opportunistically searched for territorial males at the majority of the Swedish breeding sites, and were able to examine about 56% of all males in the region. Analyses of recaptured males demonstrated that philopatry predominated. Sixty-nine percent of the recruiting nestlings returned to breed in the main study area (their natal area), and 92% of the resighted adults were found at the same breeding locality in both study years. Breeding dispersal was significantly more restricted than natal dispersal. Additional data on natal and breeding dispersal within the main study area in 1992-1999 suggested that females were as philopatric as males. The overall high level of philopatry, with only occasional longer dispersal distances documented, yielded a root-mean-square dispersal distance of 33 km per generation. High philopatry, short dispersal distances and similar dispersal patterns of male and female great reed warblers contrast the findings among birds in general, but conform to data of species having patchy breeding habitat and isolated populations. Restricted dispersal suggests limited gene flow even among several Swedish populations, which is in line with some previous findings of the population ecology of the great reed warbler: (1) structured mtDNA lineages among European populations; (2) small-scale population differences in song patterns; and (3) low genetic variation and occurrence of inbreeding depression in our main study population.     

Gold-coated capillary based 2,4-dichlorophenoxyacetic acid chemi-luminescent assays: possibilities towards multianalysis

The application of gold-coated glass capillaries for the design of a sensitive chemiluminescent immunoassay for 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The gold coating on the glass capillaries has been partially characterized and its effect on enhancing the signal intensity has been measured. A simple photo-multiplier tube-based photon detector is used for this purpose. At least three times improvement in the signal intensity is observed compared to uncoated glass capillaries, with a consequent improvement in the sensitivity of detection. Using such gold-coated glass capillaries, 2,4-D in the range 10(-9) to 10(-13) mol/l is detectable at a precision of +/-15% (CV%) and a limit of detection of 10(-15) mol/l is achievable. The possibility of using such gold-coated capillaries with a portable multianalytical set-up for field studies is also demonstrated.     

Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension

Streaking is a severe problem when narrow range basic immobilized pH gradient strips are used as the first dimension of two-dimensional (2-D) electrophoresis. It is demonstrated that this cysteinyl related streaking is eliminated when focusing is done in the presence of hydroxyethyl disulfide (DeStreak). Use of DeStreak also results in 2-D maps with simplified spot patterns and improved reproducibility.     

Dissociation of AMPK activity and ACCbeta phosphorylation in human muscle during prolonged exercise

During prolonged, low intensity exercise, the type of substrate utilized varies with time. If 5' AMP-activated protein kinase (AMPK) regulates muscle metabolism during exercise, signaling through AMPK would be expected to change in concordance with changes in substrate utilization. Six healthy, young males cycled (approximately 45% VO(2peak)) until exhaustion (approximately 3.5h). During exercise, leg glucose uptake and rate of glycogenolysis gradually decreased whereas free fatty acid uptake gradually increased. In the thigh muscle, the alpha AMPK subunits became progressively more phosphorylated on Thr(172) during exercise eliciting a parallel increase in alpha2 but not alpha1 AMPK activity. In contrast, after 1h of exercise, Ser(221) phosphorylation of acetyl-CoA carboxylase-beta (ACCbeta) peaked at 1h of exercise and returned to resting levels at exhaustion. Protein expression of alpha2 AMPK, alpha1 AMPK or ACCbeta did not change with time. These data suggest that AMPK signaling is not a key regulatory system of muscle substrate combustion during prolonged exercise and that marked activation of AMPK via phosphorylation is not sufficient to maintain an elevated ACCbeta Ser(221) phosphorylation during prolonged exercise.     

Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Determination of ximelagatran,an oral direct thrombin inhibitor,its active metabolite melagatran,and the intermediate metabolites,in biological samples by liquid chromatography-mass spectrometry

Analytical methods for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and intermediate metabolites, melagatran hydroxyamidine and melagatran ethyl ester, in biological samples by liquid chromatography (LC) positive electrospray ionization mass spectrometry (MS) using selected reaction monitoring are described. Isolation from human plasma was achieved by solid-phase extraction on octylsilica. Analytes and isotope-labelled internal standards were separated by LC utilising a C(18) analytical column and a mobile phase comprising acetonitrile-4 mmol/l ammonium acetate (35:65, v/v) containing 0.1% formic acid, at a flow-rate of 0.75 ml/min. Absolute recovery was approximately 80% for ximelagatran, approximately 60% for melagatran ethyl ester and >90% for melagatran and melagatran hydroxyamidine. Limit of quantification was 10 nmol/l, with a relative standard deviation <20% for each analyte and <5% above 100 nmol/l. Procedures for determination of these analytes in human urine and breast milk, plus whole blood from rat and mouse are also described.