Positional cloning and pathway analysis of the asthma susceptibility gene, NPSR1

In the present study, we identified a novel asthma susceptibility gene, NPSR1 (neuropeptide S receptor 1) on chromosome 7p14.3 by the positional cloning strategy. An earlier significant linkage mapping result among Finnish Kainuu asthma families was confirmed in two independent cohorts: in asthma families from Quebec, Canada and in allergy families from North Karelia, Finland. The linkage region was narrowed down to a 133-kb segment by a hierarchial genotyping method. The observed 77-kb haplotype block showed 7 haplotypes and a similar risk and nonrisk pattern in all three populations studied. All seven haplotypes occur in all three populations at frequences > 2%. Significant elevated relative risks were detected for elevated total IgE (immunoglobulin E) or asthma. Risk effects of the gene variants varied from 1.4 to 2.5. NPSR1 belongs to the G protein-coupled receptor (GPCR) family with a topology of seven transmembrane domains. NPSR1 has 9 exons, with the two main transcripts, A and B, encoding proteins of 371 and 377 amino acids, respectively. We detected a low but ubiquitous expression level of NPSR1-B in various tissues and endogenous cell lines while NPSR1-A has a more restricted expression pattern. Both isoforms were expressed in the lung epithelium. We observed aberrant expression levels of NPSR1-B in smooth muscle in asthmatic bronchi as compared to healthy. In an experimental mouse model, the induced lung inflammation resulted in elevated Npsr1 levels. Furthermore, we demonstrated that the activation of NPSR1 with its endogenous agonist, neuropeptide S (NPS), resulted in a significant inhibition of the growth of NPSR1-A overexpressing stable cell lines (NPSR1-A cells). To determine which target genes were regulated by the NPS-NPSR1 pathway, NPSR1-A cells were stimulated with NPS, and differentially expressed genes were identified using the Affymetrix HGU133Plus2 GeneChip. A total of 104 genes were found significantly up-regulated and 42 down-regulated 6 h after NPS administration. The up-regulated genes included many neuronal genes and some putative susceptibility genes for respiratory disorders. By Gene Ontology enrichment analysis, the biological process terms, cell proliferation, morphogenesis and immune response were among the most altered. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), were verified and confirmed by quantitative reverse-transcriptase-PCR. In conclusion, we identified a novel asthma susceptibility gene, NPSR1, on chromosome 7p14.3. NPS-NPSR1 represents a novel pathway that regulates cell proliferation and immune responses, and thus may have functional relevance in the pathogenesis of asthma.

Individual variation in in vitro efficacy of antiplatelet medication - aspirin and clopidogrel

Antiplatelet medication is known to decrease adverse effects in patients with atherothrombotic disease. However, despite ongoing antiplatelet medication considerable number of patients suffer from atherothrombotic events. The aims of the study were 1) to evaluate the individual variability in platelet functions and compare the usability of different methods in detecting it, 2) to assess variability in efficacy of antiplatelet medication with aspirin (acetylsalicylic acid) or the combination of aspirin and clopidogrel and 3) to investigate the main genetic and clinical variables as well as potential underlying mechanisms of variability in efficacy of antiplatelet medication. In comparisons of different platelet function tests in 19 healthy individuals PFA-100® correlated with traditional methods of measuring platelet function and was thus considered appropriate for testing individual variability in platelet activity. Efficacy of ongoing 100mg aspirin daily was studied in 101 patients with coronary artery disease (CAD). Aspirin response was measured with arachidonic acid (AA)-induced platelet aggregation, which reflects cyclo-oxygenase (COX)-1 dependent thromboxane (Tx) A2 formation, and PFA-100®, which evaluates platelet activation under high shear stress in the presence of collagen and epinephrine. Five percent of patients failed to show inhibition of AA-aggregation and 21% of patients had normal PFA-100® results despite aspirin and were thus considered non-responders to aspirin. Interestingly, the two methods of assessing aspirin efficacy, platelet aggregation and PFA-100®, detected different populations as being aspirin non-responders. It could be postulated that PFA-100® actually measures enhanced platelet function, which is not directly associated with TxA2 inhibition exerted by aspirin. Clopidogrel efficacy was assessed in 50 patients who received a 300mg loading dose of clopidogrel 2.5 h prior to percutaneous coronary intervention (PCI) and in 51 patients who were given a loading dose of 300mg combined with a five day treatment of 75mg clopidogrel daily mimicking ongoing treatment. Clopidogrel response was assessed with ADP-induced aggregations, due to its mechanism of action as an inhibitor of ADP-induced activation. When patients received only a loading dose of clopidogrel prior to PCI, 40% did not gain measurable inhibition of their ADP-induced platelet activity (inhibition of 10% or less). Prolongation of treatment so that all patients had reached a plateau of inhibition exerted by clopidogrel, decreased the incidence of non-responders to 20%. Polymorphisms of COX-1 and GP VI, as well as diabetes and female gender, were associated with decreased in vitro aspirin efficacy. Diabetes also impaired the in vitro efficacy of short-term clopidogrel. Decreased response to clopidogrel was associated with limited inhibition by ARMX, an antagonist of P2Y12-receptor, suggesting the reason for clopidogrel resistance to be receptor-dependent. Conclusions: Considerable numbers of CAD patients were non-responders either to aspirin, clopidogrel or both. In the future, platelet function tests may be helpful to individually select effective and safe antiplatelet medication for these patients.

Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana

Lysophosphatidic acid (LPA) acts as a signaling molecule that regulates diverse cellular processes and it can rapidly be metabolized by phosphatase and acyltransferase LPA phosphatase gene has not been identified and characterized in plants so far The BLAST search revealed that the At3g03520 is similar to phospholipase family. and distantly related to bacterial phosphatases The conserved motif. (J)4XXXNXSFD, was identified in both At3g03520 like phospholipases and acid phosphatases In silico expression analysis of At3g03520 revealed a high expression during phosphate starvation and abiotic stresses. This gene was overexpressed in Escherichia coli and shown to posses LPA specific phosphatase activity These results Suggest that this gene possibly plays a role in signal transduction and storage lipid synthesis.

Structures and molecular-dynamics studies of three active-site mutants of bovine pancreatic phospholipase A(2)

Phospholipase A(2) hydrolyzes phospholipids at the sn-2 position to cleave the fatty-acid ester bond of L-glycerophospholipids. The catalytic dyad (Asp99 and His48) along with a nucleophilic water molecule is responsible for enzyme hydrolysis. Furthermore, the residue Asp49 in the calcium-binding loop is essential for controlling the binding of the calcium ion and the catalytic action of phospholipase A2. To elucidate the structural role of His48 and Asp49, the crystal structures of three active-site single mutants H48N, D49N and D49K have been determined at 1.9 angstrom resolution. Although the catalytically important calcium ion is present in the H48N mutant, the crystal structure shows that proton transfer is not possible from the catalytic water to the mutated residue. In the case of the Asp49 mutants, no calcium ion was found in the active site. However, the tertiary structures of the three active-site mutants are similar to that of the trigonal recombinant enzyme. Molecular-dynamics simulation studies provide a good explanation for the crystallographic results.

Carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus)

The carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus) was investigated by quantitative precipitin analysis using blood group A, B, H, Le and I substances and by precipitation inhibition with various mono- and oligosaccharides. The lectin precipitated best with A1 substances and moderately with B and A2 substances, but not with H or Le substances. Inhibition assays of lectin-blood group A1 precipitation demonstration that A substance-derived oligosaccharides having the common structure: d-Ga1NAcα(1 → 3)d-Gal-(β1 → Image ) to a d-Glc, were the best inhibitors and about 8 and 4 times more active than d-Ga1NAc and d-Ga1NAcα(1 → 3)d-Ga1, respectively. A difucosyl A-specific oligosaccharide (A-penta), a monofucosyl (A-tetra) and a non-fucosyl containing (A5 II) oligosaccharide, d-Ga1NAcα(1 → 3)d-Ga1β(1 → 3)d-G1cNAc, had almost the same reactivity, suggesting that the fucose linked to the sub-terminal d-Ga1 or to the third sugar, d-GlcNAc, from the non-reducing end made no contribution to the carbohydrate binding. Although a terminal non-reducing d-Ga1NAc or d-Ga1 residue was indispensible for binding, the lectin bound not only to these terminal non-reducing galactopyranosyl residues, but also showed increased binding to oligosaccharides in which it was bonded to a sub-terminal d-Ga1 joined to a d-GlcNAc residue, as in blood group A or B substances. This defines the site, thus far, as complementary to a disaccharide plus the β linkage to the third sugar (d-Glc or d-GlcNAc) from the non-reducing end. The role of the β(1 → 3) or β(1 → 4) linkage of the sub-terminal non-reducing d-Gal to the d-GlcNAc requires further study.

Photocytotoxic Lanthanum(III) and Gadolinium(III) Complexes of Phenanthroline Bases Showing Light-Induced DNA Cleavage Activity

Lanthanide complexes of formulation [La(B)(2)(NO3)(3)] (1-3) and [Gd(B)(2)(NO3)(3)] (4-6), where B is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 4),dipyrido[3,2-d2',3'-f]quinoxaline (dpq in 2,5) and dipyrido[3,2-a2',3'-c]phenazine (dppz in 3, 6), have been prepared, characterized from physicochemical data, and their photoinduced DNA and protein cleavage activity studied The photocytotoxicity of the dppz complexes 3 and 6 has been studied using HeLa cancer cells. The complexes exhibitligand centered bands in the UV region The dppz complexes show thelowest energy band at 380 nm in N,N-dimethylformamide (DMF) The La(III)complexes are diamagnetic. The Gd(III) complexes (4-6) have magneticmoments that correspond to seven unpaired electrons The complexes are1(.)1 electrolytic in aqueous DMF The dpq and dppz complexes in DMFshow ligand-based reductions. The complexes display moderate binding propensity to calf thymus DNA giving binding constant values in the range of 5.7 x 10(4)-5.8 x 10(5) M-1 with a relative order. 3, 6 (dppz)> 2, 5 (dpq) > 1, 4 (phen) The binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes do not show any hydrolytic cleavage of plasmid supercoiled pUC19 DNA. The dpq and dppz complexes efficiently cleave SC DNA to its nicked circular form onexposure to UV-A light of 365 nm at nanomolar complex concentration. Mechanistic studies reveal the involvement of singlet oxygen (O-1(2)) and hydroxyl radical (HO center dot) as the cleavage active species.The complexes show binding propensity to bovine serum albumin (BSA)protein giving K-BSA values of similar to 10(5) M-1. The dppz complexes 3 and 6 show BSA protein cleavage activity in UV-A light of 365 nm The dppz complexes 3 and 6 exhibit significant photocytotoxicity in HeLa cells giving respective IC50 values of 341 nM and 573 nM in UV-A light of 365 nm for an exposure time of 15 min (IC50 > 100 mu M in dark for both the complexes) Control experiments show significant dark and phototoxicity of the dppz base alone (IC50 = 413 nM in light with 4 h incubation in dark and 116 mu M in dark with 24 h incubation). A significant decrease in the dark toxicity of the dppz base is observedon binding to the lanthanide ions while retaining similar phototoxicity.

Studies in the polarography of metal-amino acid complexes -Part II. Lead

1. The polarographic behaviour of glycine, α-alanine, β-alanine, valine, aspartic acid, glutamic acid and asparagine complexes of lead has been studied at various pH values and in presence of (1) NaOH, (2) Na2CO3 and (3) NH4 NO3+NH4OH. All the polarographic waves have been found to be reversible. 2. Experiments conducted on the effect of variation of pH, i.e., 7A2. The data have been analysed employing the equations developed by DeFord and Hume as modified by McKenzie and Mellor. 3. Only pure complexes are produced below pH 11·2 in the case of aspartic acid, glutamic acid and glycine, while mono hydroxy complexes are produced in α-alanine, valine and asparagine systems. 4. It has been found that no mixed hydroxy and mixed ammonia complexes are produced in presence of sodium hydroxide and ammonia-ammonium nitrate, respectively. However evidence is obtained for the formation of mixed carbonate complexes in glycine and aspartic acid systems in presence of sodium carbonate. 5. Thermodynamic data have been calculated from polarographic measurements for 18 complexes. 6. The suitability of incorporating amino acids in base solutions for the polarographic estimation of lead has been tested.

Vibrational analysis of the (B2P{cyrillic}→X2P{cyrillic}) system of NS

A few red degraded bands attributable to NS have been reported earlier by Fowler and Barker, Dressler and Barrow et al, and they occur in the same region (2300 to 2700 Å) as the bands of the known systems (C2∑+-X2P{cyrillic}) and (A2Δ-X2P{cyrillic}). Measurements made on the heads of some of these weak bands led Barrow et al. to believe that these bands may form a system analogous to the β-system of NO and be due to a2P{cyrillic}-2P{cyrillic} transition. The spectrum of NS has now been studied in a little more detail by means of an uncondensed discharge through dry nitrogen and sulphur vapour in the presence of argon and thirty three bands belonging to this system have been recorded in the region 2280 to 2760 Å. It has been found possible to represent the band heads by means of the equation {Mathematical expression}. Taking the lower state doublet interval as 223 cm-1, it is shown that the separation in the upper state is 94 cm-1. The ratio of the force constants in the upper and the ground states is found to be 0·39 and is nearly the same as that in the β-system of NO (0·30). The present vibrational analysis therefore supports the view that these new red degraded bands of NS arise from a (B2P{cyrillic}→X2P{cyrillic}) transition and the observed intensity distribution in the form of a wide parabola is also in qualitative agreement with what is expected from the moderately large Δ re (∼0·12Å) value.

Assessing the mitigation potential of forestry activities in a changing climate: A case study for Karnataka

The Clean Development Mechanism (CDM), Article 12 of the Kyoto Protocol allows Afforestation and Reforestation (A/R) projects as mitigation activities to offset the CO2 in the atmosphere whilst simultaneously seeking to ensure sustainable development for the host country. The Kyoto Protocol was ratified by the Government of India in August 2002 and one of India's objectives in acceding to the Protocol was to fulfil the prerequisites for implementation of projects under the CDM in accordance with national sustainable priorities. The objective of this paper is to assess the effectiveness of using large-scale forestry projects under the CDM in achieving its twin goals using Karnataka State as a case study. The Generalized Comprehensive Mitigation Assessment Process (GCOMAP) Model is used to observe the effect of varying carbon prices on the land available for A/R projects. The model is coupled with outputs from the Lund-Potsdam-Jena (LPJ) Dynamic Global Vegetation Model to incorporate the impacts of temperature rise due to climate change under the Intergovernmental Panel on Climate Change (IPCC) Special Report on Emissions Scenarios (SRES) A2, A1B and B1. With rising temperatures and CO2, vegetation productivity is increased under A2 and A1B scenarios and reduced under B1. Results indicate that higher carbon price paths produce higher gains in carbon credits and accelerate the rate at which available land hits maximum capacity thus acting as either an incentive or disincentive for landowners to commit their lands to forestry mitigation projects. (C) 2009 Elsevier B.V. All rights reserved.

Distribution of products around a burning droplet: an analytical approach

A knowledge of the concentration distribution around a burning droplet is essential if accurate estimates are to be made of the transport coefficients in that region which influence the burning rate. There are two aspects of this paper; (1) determination of the concentration profiles, using the simple assumption of constant binary diffusion coefficients for all species, and comparison with experiments; and (2) postulation of a new relation for the therinal conductivity, which takes into account the variations of both temperature and concentrations of various species. First, the theoretical concentration profiles are evaluated and compared with experimental results reported elsewhere [5]. It is found that the agreement between the theory and experiment is fairly satisfactory. Then, by the use of these profiles and the relations proposed in the literature for the thermal conductivity of a mixture of nonpolar gases, a new relation for thermal conductivity: K = (A1 + B1 T) + (A2 + B2 T) xr (21). is suggested for analytical solutions of droplet combustion problems. Equations are presented to evaluate A1, A2, B1, and B2, and values of these terms for a few hydrocarbons are tabulated.

Ultra-violet bands of mercury iodide

The ultraviolet bands of mercury iodide have been excited in uncondensed discharge and photographed with a quartz Littrow spectrograph. The bands in the region λ 2658 to 2530 Å have been analysed into two systems which may form the two components of a2Π-2∑ electronic transition with a2Π interval equal to 858·9 cm-1. These systems and the systems in the region λ 3095 to 2647 Å have the same lower state.

Structural, dielectric and ferroelectric properties of four-layer Aurivillius phase Na0.5La0.5Bi4Ti4O15

Monophasic Na0.5La0.5Bi4Ti4O15 powders were synthesized via the conventional solid-state reaction route. The X-ray powder diffraction (XRD), selected area electron diffraction (SAED) and high resolution transmission electron microscopy (HRTEM) studies carried out on the as synthesized powdered samples confirmed the phase to be a four-layer Aurivillius that crystallizes in an orthorhombic A2(1)am space group. The microstructure and the chemical composition of the sintered sample were examined by scanning electron microscope (SEM) equipped with an energy dispersive X-ray analyzer (EDX). The dielectric properties of the ceramics have been studied in the 27-700 degrees C temperature range at various frequencies (100 Hz to 1 MHz). A sharp dielectric anomaly was observed at 580 degrees C for all the frequencies corresponding to the ferroelectric to paraelectric phase transition. Saturated ferroelectric hysteresis loops were observed at 200 degrees C and the associated remnant polarization (P-r) and coercive field (E-c) were found to be 7.4 mu C/cm(2) and 34.8 kV/cm, respectively. AC conductivity analysis confirmed the existence of two different conduction mechanisms in the ferroelectric region. Activation energies calculated from the Arrhenius plots were similar to 0.24 eV and similar to 0.84 eV in the 300-450 degrees C and 450-580 degrees C temperature ranges, respectively. (C) 2010 Elsevier B.V. All rights reserved.

Methods and tools for mass spectrometric lipidome analysis

The analysis of lipid compositions from biological samples has become increasingly important. Lipids have a role in cardiovascular disease, metabolic syndrome and diabetes. They also participate in cellular processes such as signalling, inflammatory response, aging and apoptosis. Also, the mechanisms of regulation of cell membrane lipid compositions are poorly understood, partially because a lack of good analytical methods. Mass spectrometry has opened up new possibilities for lipid analysis due to its high resolving power, sensitivity and the possibility to do structural identification by fragment analysis. The introduction of Electrospray ionization (ESI) and the advances in instrumentation revolutionized the analysis of lipid compositions. ESI is a soft ionization method, i.e. it avoids unwanted fragmentation the lipids. Mass spectrometric analysis of lipid compositions is complicated by incomplete separation of the signals, the differences in the instrument response of different lipids and the large amount of data generated by the measurements. These factors necessitate the use of computer software for the analysis of the data. The topic of the thesis is the development of methods for mass spectrometric analysis of lipids. The work includes both computational and experimental aspects of lipid analysis. The first article explores the practical aspects of quantitative mass spectrometric analysis of complex lipid samples and describes how the properties of phospholipids and their concentration affect the response of the mass spectrometer. The second article describes a new algorithm for computing the theoretical mass spectrometric peak distribution, given the elemental isotope composition and the molecular formula of a compound. The third article introduces programs aimed specifically for the analysis of complex lipid samples and discusses different computational methods for separating the overlapping mass spectrometric peaks of closely related lipids. The fourth article applies the methods developed by simultaneously measuring the progress curve of enzymatic hydrolysis for a large number of phospholipids, which are used to determine the substrate specificity of various A-type phospholipases. The data provides evidence that the substrate efflux from bilayer is the key determining factor for the rate of hydrolysis.