Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the metzincin superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

The interaction between heparin and Lys49 phospholipase A(2) reveals the natural binding of heparin on the enzyme

We have studied at a molecular level the interaction of heparins on bothropstoxin-1 (BthTx-1), a phospholipase A(2) toxin. The protein was monitored using gel filtration chromatography, dynamic light scattering (DLS), circular dichroism (CD), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. The elution profile of the protein presents a displacement of the protein peak to larger complexes when interacting with higher concentration of heparin. The DLS results shows two R-h at a molar ratio of 1, one to the distribution of the protein and the second for the action of heparin on BthTx-I structures, and a large distribution with the increase of protein. The interaction is accompanied by significant changes in the CD spectra, showing two common features: a decrease in signal at 208 nm (3 and 6 kDa heparins) and an isodichroic point near 226 nm (3 kDa heparin). FTIR spectra indicate that only a few amino acid residues are involved in this interaction. Alterations in the ITFE by binding heparins suggest that the initial binding occurs on the ventral face of BthTx-1. Together, these results add an experimental and structural basis on the action mechanism of the heparins over the phospholipases A(2) and provide a molecular model to elucidate the interaction of the enzyme-heparin complex at a molecular level. (c) 2005 Elsevier B.V. All rights reserved.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Purification, biochemical and functional characterization of miliin, a new thiol-dependent serine protease isolated from the latex of Euphorbia milii

Miliin, a new thiol-dependent serine protease purified from the latex of Euphorbia milii possesses a molecular weight of 79 kDa, an isoelectric point of 4.3 and is optimally active at 60 degrees C in the pH range of and 7.5-11.0. Activity tests indicate that milliin is a thiol-dependent serine protease.

Search for B-s(0)->mu(+)mu(-) decays at D0

We report results from a search for the decay B-s(0)->mu(+)mu(-) using 1.3 fb(-1) of p (p) over bar collisions at root s = 1.96 TeV collected by the D0 experiment at the Fermilab Tevatron Collider. We find two candidate events, consistent with the expected background of 1.24 +/- 0.99, and set an upper limit on the branching fraction of B(B-s(0)->mu(+)mu(-))< 1.2x10(-7) at the 95% C.L.

Measurement of the p(p)over-bar -> WZ+X cross section at root s=1.96 TeV and limits on WWZ trilinear gauge couplings

We present measurements of the process p (P) over bar -> WZ + X -> l 'nu(l ')l (l) over bar at root s = 1:96 TeV,where l and l ' are electrons or muons. Using 1 fb(-1) of data from the D0 experiment, we observe 13 candidates with an expected background of 4.5 +/- 0.6 events and measure a cross section sigma(WZ) = 2.7(-1.3)(+1.7) pb. From the number of observed events and the Z boson transverse momentum distribution, we limit the trilinear WWZ gauge couplings to -0: 17 <= lambda(Z) <= 0.21 (Delta k(Z) <= 0.29(lambda(Z) = 0) at the 95% C.L. for a form factor scale Lambda = 2 TeV. Further, assuming that Delta g(1)(Z) = Delta k(Z), we find -0.12 <= Delta k(Z) <= 0.29(lambda(Z) = 0) at the 95% C. L. These are the most restrictive limits on the WWZ couplings available to date.

Measurement of inclusive differential cross sections for Upsilon(1S) production in p(p)over-bar collisions at root s=1.96 TeV

We present measurements of the inclusive production cross sections of the Upsilon(1S) bottomonium state in p (p) over bar collisions at root s=1.96 TeV. Using the Upsilon(1S)->mu(+)mu(-) decay mode for a data sample of 159 +/- 10 pb(-1) collected by the D0 detector at the Fermilab Tevatron collider, we determine the differential cross sections as a function of the Upsilon(1S) transverse momentum for three ranges of the Upsilon(1S) rapidity: 0 <\y(Upsilon)\<= 0.6, 0.6 <\y(Upsilon)\<= 1.2, and 1.2 <\y(Upsilon)\<= 1.8.

Measurement of the ratio of B+ and B-0 meson lifetimes

The ratio of the B+ and B-0 meson lifetimes was measured using data collected in 2002-2004 by the D0 experiment in Run II of the Fermilab Tevatron Collider. These mesons were reconstructed in B&RARR;μ(+)ν D*-X decays, which are dominated by B-0 and B&RARR;μ(+)ν(D) over bar X-0 decays, which are dominated by B+. The ratio of lifetimes is measured to be τ(+)/τ(0)=1.080&PLUSMN; 0.016(stat)&PLUSMN; 0.014(syst).

Search for right-handed W bosons in top quark decay

We present a measurement of the fraction f(+) of right-handed W bosons produced in top quark decays, based on a candidate sample of t (t) over bar events in the lepton+jets decay mode. These data correspond to an integrated luminosity of 230 pb(-1), collected by the D0 detector at the Fermilab Tevatron p (p) over bar Collider at root s=1.96 TeV. We use a constrained fit to reconstruct the kinematics of the t (t) over bar and decay products, which allows for the measurement of the leptonic decay angle theta(*) for each event. By comparing the cos theta(*) distribution from the data with those for the expected background and signal for various values of f(+), we find f(+)=0.00 +/- 0.13(stat)+/- 0.07(syst). This measurement is consistent with the standard model prediction of f(+)=3.6 x 10(-4).

Search for resonant second generation slepton production at the Fermilab Tevatron

We present a search for supersymmetry in the R-parity violating resonant production and decay of smuons and muon sneutrinos in the channels mu ->chi(0)(1)mu, mu ->chi(0)(2,3,4)mu, and nu(mu)->chi(+/-)(1,2)mu. We analyzed 0.38 fb(-1) of integrated luminosity collected between April 2002 and August 2004 with the D0 detector at the Fermilab Tevatron Collider. The observed number of events is in agreement with the standard model expectation, and we calculate 95% C.L. limits on the slepton production cross section times branching fraction to gaugino plus muon, as a function of slepton and gaugino masses. In the framework of minimal supergravity, we set limits on the coupling parameter lambda(')(211), extending significantly previous results obtained in Run I of the Tevatron and at the CERN LEP collider.

Search for production of single top quarks via tcg and tug flavor-changing-neutral-current couplings

We search for the production of single top quarks via flavor-changing-neutral-current couplings of a gluon to the top quark and a charm (c) or up (u) quark. We analyze 230 pb(-1) of lepton+jets data from p (p) over tilde collisions at a center of mass energy of 1.96 TeV collected by the D0 detector at the Fermilab Tevatron Collider. We observe no significant deviation from standard model predictions, and hence set upper limits on the anomalous coupling parameters kappa(c)(g)/Lambda and kappa(u)(g)/Lambda, where kappa(g) define the strength of tcg and tug couplings, and Lambda defines the scale of new physics. The limits at 95% C.L. are kappa(c)(g)/Lambda < 0.15 TeV-1 and kappa(u)(g)/Lambda < 0.037 TeV-1.

Measurement of the t(t)over-bar production cross section in p(p)over-bar collisions at root s=1.96 TeV using kinematic characteristics of lepton plus jets events

We present a measurement of the top quark pair production cross section in p (p) over bar collisions at root s=1.96 TeV utilizing 425 pb(-1) of data collected with the D0 detector at the Fermilab Tevatron Collider. We consider the final state of the top quark pair containing one high-p(T) electron or muon and at least four jets. We exploit specific kinematic features of t (t) over bar events to extract the cross section. For a top quark mass of 175 GeV, we measure sigma(t (t) over bar)=6.4(-1.2)(+1.3)(stat)+/- 0.7(syst)+/- 0.4(lum) pb, in good agreement with the standard model prediction.

Search for the flavour-changing neutral current B-s(0)->mu+mu- in p(p)over-bar collisions at root s=1.96 TeV with the D0 dectector

We present the results of a search for the flavor-changing neutral current decay B-s(0)-->mu(+)mu(-) using a data set with integrated luminosity of 240 pb(-1) of p (p) over bar collisions at roots=1.96 TeV collected with the D0 detector in run II of the Fermilab Tevatron collider. We find the upper limit on the branching fraction to be B(B-s(0)-->mu(+)mu(-))less than or equal to5.0x10(-7) at the 95% C.L. assuming no contributions from the decay B-d(0)-->mu(+)mu(-) in the signal region. This limit is the most stringent upper bound on the branching fraction B-s(0)-->mu(+)mu(-) to date.

Search for first-generation scalar leptoquarks in p(p)over-bar collisions at root s=1.96 TeV

We report on a search for pair production of first-generation scalar leptoquarks (LQ) in p (p) over bar collisions at root s=1.96 TeV using an integrated luminosity of 252 pb(-1) collected at the Fermilab Tevatron collider by the D0 detector. We observe no evidence for LQ production in the topologies arising from LQ(LQ) over bar -> eqeq and LQ(LQ) over bar -> eq nu q, and derive 95% C.L. lower limits on the LQ mass as a function of beta, where beta is the branching fraction for LQ -> eq. The limits are 241 and 218 GeV/c(2) for beta=1 and 0.5, respectively. These results are combined with those obtained by D0 at root s=1.8 TeV, which increases these LQ mass limits to 256 and 234 GeV/c(2).

Measurement of the t(t)over-bar production cross section in p(p)over-bar collisions at root s=1.96 TeV using lepton plus jets events with lifetime b-tagging

We present a measurement of the top quark pair (t (t) over bar) production cross section (sigma(t (t) over bar)) in pp collisions at root s = 1.96 TeV using 230 pb(-1) of data collected by the DO experiment at the Fermilab Tevatron Collider. We select events with one charged lepton (electron or muon), missing transverse energy, and jets in the final state. We employ lifetime-based b-jet identification techniques to further enhance the t F purity of the selected sample. For a top quark mass of 175 GeV, we measure sigma(t (t) over bar) 8.6(-1.5)(+1.6) (stat. + syst.) +/- 0.6(lumi.) pb, in agreement with the standard model expectation. (c) 2005 Published by Elsevier B.V.