Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage

We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[(2)H(4)]Tyrosine and o,o'-[(2)H(6)]dityrosine were used as internal standards. Calibration curves of the method were linear (r(2)=0.990-0.999) over a concentration range of 0.03-10 microM for o-tyrosine; 0.04-10 microM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05-10 microM for o,o'-dityrosine; and for m-tyrosine; 1.0-100 microM for p-tyrosine and for phenylalanine; and 0.01-10 microM for 8-OHdG. The detection limits were from 0.025 to 0.05 microM for the tyrosine derivatives; 0.01 microM for 8-OHdG; and 0.5 microM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%).The background levels of p-tyrosine, phenylalanine, o,o'-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890+/-590, 3420+/-730, 5.8+/-0.3, and 9.2+/-1.5 micromol/mol creatinine, respectively. Using the present HPLC-APCI-MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.     

Trypsin inhibition activity of heat-denatured ovomucoid: a kinetic study

A kinetic study was conducted on the effect of heating in the temperature range of 75-110 degrees C on the trypsin inhibition activity of ovomucoid. Heat treatment of isolated ovomucoid resulted in a time-dependent decrease in trypsin inhibition activity that could accurately be described by a first-order kinetic model. The magnitude and the temperature dependence of the rate constants was affected by the pH during heat treatment. The heat stability of ovomucoid was the lowest at pH 7.6. Heat treatments intended to decrease the trypsin inhibition activity should therefore be carried out as soon as possible after laying, because the ovomucoid was inactivated faster at the pH of fresh egg white (pH 7.6). The presence of the other egg white constituents decreased the heat stability of ovomucoid compared to that of the model system of ovomucoid in buffer, presumably by the formation of ovomucoid-lysozyme complexes in the former.     

Noninvasive visualisation of coronary atherosclerosis with multislice computed tomography

Computed Tomography (Electron Beam Tomography: EBCT and multislice computed tomography: MSCT) have recently emerged as non-invasive diagnostic modalities that can quantify coronary calcium which is not only as indicator of coronary risk, but also permits assessment of the coronary lumen. The 16-slice MS-CT, the most recent CT-scanner has a very high resolution, which allows non-invasive assessment of coronary plaques. This has led to a stimulus for further research to assess the role of MSCT coronary plaque imaging in the identification of high-risk coronary plaques.     

The solution structure of human mitochondria fission protein Fis1 reveals a novel TPR-like helix bundle

Fis1 in yeast localizes to the outer mitochondrial membrane and facilitates mitochondrial fission by forming protein complexes with Dnm1 and Mdv1. Fis1 orthologs exist in higher eukaryotes, suggesting that they are functionally conserved. In the present study, we cloned the human Fis1 ortholog that was predicted in a database, and determined the protein structure using NMR spectroscopy. Following a flexible N-terminal tail, six alpha-helices connected with short loops construct a single core domain. The C-terminal tail containing a transmembrane segment appears to be disordered. In the core domain, each of two sequentially adjacent helices forms a hairpin-like conformation, resulting in a six helix assembly forming a slightly twisted slab similar to that of a tandem array of tetratrico-peptide repeat (TPR) motif folds. Within this TPR-like core domain, no significant sequence similarity to the typical TPR motif is found. The structural analogy to the TPR-containing proteins suggests that Fis1 binds to other proteins at its concave hydrophobic surface. A simple composition of Fis1 comprised of a binding domain and a transmembrane segment indicates that the protein may function as a molecular adaptor on the mitochondrial outer membrane. In HeLa cells, however, increased levels in mitochondria-associated Fis1 did not result in mitochondrial translocation of Drp1, a potential binding partner of Fis1 implicated in the regulation of mitochondrial fission, suggesting that the interaction between Drp1 and Fis1 is regulated.     

Highly biased type 1 immune responses in mice deficient in LFA-1 in Listeria monocytogenes infection are caused by elevated IL-12 production by granulocytes

LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN-gamma producers in the liver and spleen of LFA-1(-/-) mice were markedly increased compared with heterozygous littermates and Valpha14(+)NKT cell-deficient mice, and NK cells were major IFN-gamma producers. Numbers of IL-12 producers were also markedly elevated in LFA-1(-/-) mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-gamma production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN-gamma-secreting NK cells in the liver of LFA-1(-/-) mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1(-/-) mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1(-/-) mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-gamma secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.     

Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.     

Adaptive reproductive variation along a pollution gradient in a wolf spider

When populations are exposed to environmental pollutants, growth and reproduction might be strongly reduced due to an increased detoxification effort. Sublethal metal pollution is therefore to be expected to cause the same selection pressure as a low resource habitat and might alter the reproductive strategy. Optimality models of life history theory predict that when resource availability is reduced, growth and reproductive output are reduced and that the release of fewer but larger propagules will be favoured. This was tested by applying a life history model to reproductive trait measurements in six populations of the wolf spider Pirata piraticus in which the assumptions of the model are satisfied. Internal Cd, Cu and Zn body burden were strongly correlated with each other, and differed strongly between the populations, indicating consistently differing metal exposure at the different sites. Pb levels were extremely variable within each population and did not differ between the populations. Females from populations with high concentrations of the first three heavy metals showed a strongly reduced reproductive output and fecundity, indicating a high reduction in resource availability due to detoxification processes. Egg size in contrast was negatively correlated with fecundity and reproductive output and as a consequence positively related with internal metal burden. Our results are thus in strong agreement with the predictions of the optimality models and confirm the benefits of a larger propagule size when resource availability is reduced.     

Coronary perfusion and muscle lengthening increase cardiac contraction: different stretch-triggered mechanisms

An increase in coronary perfusion, transversal stretch of the myocardium, increases developed force (F(dev)) (Gregg effect) through activation of stretch-activated ion channels (SACs). Lengthening of the muscle, longitudinal stretch of the myocardium, causes an immediate increase in F(dev) followed by a slow F(dev) increase (Anrep effect). In isometrically contracting perfused papillary muscles of Wistar rats, we investigated whether both effects were based on similar stretch-induced mechanisms by measuring F(dev) and intracellular Ca(2+) concentration ([Ca(2+)](i)) after a muscle length increase from 85% to 95% L(max) (length at which maximal isometric force develops) at low and high coronary perfusion before and after inhibition of SACs with gadolinium (10 micromol/l Gd(3+)). The increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect was of similar magnitude as the Anrep effect (from 3.5 +/- 0.8 to 3.9 +/- 1.2 mN/mm(2) and from 3.0 +/- 0.7% to 3.8 +/- 0.9% normalized [Ca(2+)](i), means +/- SE). SAC blockade completely blunted the increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect; however, it did not affect the Anrep effect. The slow force response, but not the calcium response, was augmented by an increase in coronary perfusion. Therefore, increased coronary perfusion, transversal stretch of the myocardium, and muscle lengthening, longitudinal stretch of the myocardium, increase myocardial contraction in the rat through different stretch-triggered mechanisms.