Metabolic profile of NO-flurbiprofen (HCT1026) in rat brain and plasma: a LC-MS study

Male rats were given the nitrooxybutyl ester of flurbiprofen HCT1026 (15 mg/Kg) by oral administration and plasma and brain levels of the parent drug and its potential metabolites (HCT1027 and flurbiprofen) were determined at different times post-administration by a validated HPLC method (UV-DAD detection; LOQ: 0.13 nmoles/ml and 0.3 nmoles/g respectively in plasma and brain tissue). Structural confirmation of the analytes was achieved by MS monitoring of their de-protonated (negative ion mode) or cationized/protonated (positive ion mode) molecular ions and of the relative fragment ions obtained by collision-induced dissociation (CID) experiments. The results indicate that flurbiprofen is the only metabolite found at measurable levels in both plasma and brain, while HCT1026 or its de-nitrated metabolite HCT1027 were always below the limit of detection at all the observation times. The same was observed after administration of the higher dose of HCT1026 (100 mg/Kg, i.p.). In orally-treated animals the time-course of flurbiprofen formation strictly parallels that of NOx (nitrite/nitrate) in plasma but not in brain, where the levels were always in the range of the controls. These data indicate that the NO molecule is not released from the parent drug within the brain.     

A hydrogen biosensor made of clay,poly(butylviologen), and hydrogenase sandwiched on a glass carbon electrode

A hydrogen gas (H(2)) biosensor was developed in which hydrogenase (H(2)ase) was immobilized and sandwiched between two layers of a montmorillonite clay and poly(butylviologen) (PBV) mixture on a glass carbon electrode. The immobilized PBV efficiently enhanced the electron transfer among the electrode, H(2)ase, and methyl viologen in solution. Both PBV and methyl viologen acted as the electron carrier in the clay-PBV-H(2)ase modified electrode. The clay-PBV-H(2)ase electrode catalyzed the oxidation of H(2) to protons (H(+)) with the electrons being transferred by viologen groups to the electrode. The activation energy of this process was 38+/-2 kJ/mol at pH 7. The catalytic current of the clay-PBV-H(2)ase electrode increased linearly when exposed to increasing concentrations of H(2) gas. In contrast, this electrode showed no activity when exposed to three combustible compounds, namely, carbon monoxide, methane and methanol. The optimum pH range for the oxidation of H(2) by the clay-PBV-H(2)ase electrode was from 7 to 10. Electron transfer process in the clay-PBV-H(2)ase electrode is discussed.     

Geometric algorithms for the analysis of 2D-electrophoresis gels.

In proteomics, two-dimensional gel electrophoresis (2-DE) is a separation technique for proteins. The resulting protein spots can be identified either by using picking robots and subsequent mass spectrometry or by visual cross inspection of a new gel image with an already analyzed master gel. Difficulties especially arise from inherent noise and irregular geometric distortions in 2-DE images. Aiming at the automated analysis of large series of 2-DE images, or at the even more difficult interlaboratory gel comparisons, the bottleneck is to solve the two most basic algorithmic problems with high quality: Identifying protein spots and computing a matching between two images. For the development of the analysis software CAROl at Freie Universit?t Berlin, we have reconsidered these two problems and obtained new solutions which rely on methods from computational geometry. Their novelties are: 1. Spot detection is also possible for complex regions formed by several "merged" (usually saturated) spots; 2. User-defined landmarks are not necessary for the matching. Furthermore, images for comparison are allowed to represent different parts of the entire protein pattern, which only partially "overlap." The implementation is done in a client server architecture to allow queries via the internet. We also discuss and point at related theoretical questions in computational geometry.     

Towards a framework for the evolutionary genomics of Kinetoplastids: what kind of data and how much?

The current status of kinetoplastids phylogeny and evolution is discussed in view of the recent progresses on genomics. Some ideas on a potential framework for the evolutionary genomics of kinetoplastids are presented.     

Dual interaction of synaptotagmin with mu2- and alpha-adaptin facilitates clathrin-coated pit nucleation

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds mu2- and alpha-adaptin, two of the four subunits of the endocytic adaptor complex AP-2. mu2 represents the major interacting subunit of AP-2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine-based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP-2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP-2 by synprint, the cytoplasmic synaptotagmin-binding domain of N-type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP-2 plays a key physiological role in the nucleation of endocytic clathrin-coated pits.     

The role of intravascular ultrasound imaging in vascular brachytherapy

Intracoronary brachytherapy has recently emerged as a new therapy to prevent restenosis. Initial experimental work was achieved in animal models and the results were assessed by histomorphometry. Initial clinical trials used angiography to guide dosimetry and to assess efficacy. Intravascular ultrasound (IVUS) permits tomographic examination of the vessel wall, elucidating the true morphology of the lumen and transmural components, which cannot be investigated on the lumenogram obtained by angiography. This paper reviews the use of IVUS in the clinical studies of brachytherapy conducted to date. IVUS allows clinicians to make a thorough assessment of the remodeling of the vessel and appears to have a major role to play in facilitating understanding of the underlying mechanisms of action in this emerging field. The authors propose that state-of-the-art IVUS techniques should be employed to further knowledge of the mechanisms of action of brachytherapy in atherosclerotic human coronary arteries.     

Gamma S-crystallin of bovine and human eye lens: solution structure, stability and folding of the intact two-domain protein and its separate domains

Human and bovine gammaS-crystallin (HgammaS and BgammaS) and their isolated N- and C-terminal domains were cloned and expressed as recombinant proteins in E. coli. HgammaS and BgammaS are found to be authentic according to their spectral and hydrodynamic properties. Both full-length proteins and isolated domains are monomeric and exhibit high thermal and pH stabilities. The thermodynamic characterization made use of chemically and thermally-induced equilibrium unfolding transitions at varying pH. In spite of its exemplary two-domain structure, gammaS-crystallin does not show bimodal unfolding characteristics. In the case of BgammaS, at pH 7.0, the C-terminal domain is less stable than the N-terminal one, whereas for HgammaS the opposite holds true. Differential scanning calorimetry confirms the results of chemically-induced equilibrium unfolding transitions. Over the whole pH range between 2.0 and 11.5, HgammaS-crystallin and its isolated domains (HgammaS-N and HgammaS-C) follow the two-state model. The two-state unfolding of the intact two-domain protein points to the close similarity of the stabilities of the constituent domains. Obviously, interactions between the domains do not contribute significantly to the overall stability of gammaS-crystallin. In contrast, the structurally closely related gammaB-crystallin owes much of its extreme stability to domain interactions.     

Functional characterization of a mammalian Sac1 and mutants exhibiting substrate-specific defects in phosphoinositide phosphatase activity

The Saccharomyces cerevisiae SAC1 gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane phosphoinositide phosphatases, the synaptojanins, and these Sac1 domains define novel phosphoinositide phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic phosphoinositide phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Deltasac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3, 5-bisphosphate phosphatase activities, is essential for the heterologous complementation of Sac1p defects in vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.     

The identification of antioxidants in dark soy sauce

Soy sauce is a traditional fermented seasoning in Asian countries, that has high antioxidant activity in vitro and some antioxidant activity in vivo. We attempted to identify the major antioxidants present, using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay as a guide. 3-Hydroxy-2-methyl-4H-pyran-4-one (maltol) was one of several active compounds found in an ethyl acetate extract of dark soy sauce (DSS) and was present at millimolar concentrations in DSS. However, most of the antioxidant activity was present in colored fractions, two of which (CP1 and CP2) were obtained by gel filtration chromatography. Their structural characteristics based on nuclear magnetic resonance (NMR) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) analysis suggest that carbohydrate-containing pigments such as melanoidins are the major contributors to the high antioxidant capacity of DSS.     

Improving model construction of profile HMMs for remote homology detection through structural alignment

Background  

Remote homology detection is a challenging problem in Bioinformatics. Arguably, profile Hidden Markov Models (pHMMs) are one of the most successful approaches in addressing this important problem. pHMM packages present a relatively small computational cost, and perform particularly well at recognizing remote homologies. This raises the question of whether structural alignments could impact the performance of pHMMs trained from proteins in the Twilight Zone, as structural alignments are often more accurate than sequence alignments at identifying motifs and functional residues. Next, we assess the impact of using structural alignments in pHMM performance.