Ornithine <em>d</em>ecarboxylase (ODC) is a pyri<em>d</em>oxal 5'-phosphate (PLP)-<em>d</em>epen<em>d</em>ent enzyme that catalyzes the rate-<em>d</em>etermining step in the biosynthesis of polyamines. ODC is a proven <em>d</em>rug target to treat African sleeping sickness. The x-ray crystal structure of Trypanosoma brucei ODC in complex with <em>d</em>-ornithine (<em>d</em>-Orn), a substrate analog, an<em>d</em> G418 (Geneticin), a weak non-competitive inhibitor, was <em>d</em>etermine<em>d</em> to <em>2</em>.5-A resolution. <em>d</em>-Orn forms a Schiff base with PLP, an<em>d</em> the si<em>d</em>e chain is in a similar position to that observe<em>d</em> for putrescine an<em>d</em> alpha-<em>d</em>ifluoromethylornithine in previous T. brucei ODC structures. The <em>d</em>-Orn carboxylate is positione<em>d</em> on the solvent-expose<em>d</em> si<em>d</em>e of the active site (si face of PLP), an<em>d</em> Gly-199, Gly-36<em>2</em>, an<em>d</em> His-197 are the only resi<em>d</em>ues within 4.<em>2</em> A of this moiety. This structure confirms pre<em>d</em>ictions that the carboxylate of <em>d</em>-Orn bin<em>d</em>s on the si face of PLP, an<em>d</em> it supports a mo<em>d</em>el in which the carboxyl group of the substrate l-Orn woul<em>d</em> be burie<em>d</em> on the re face of the cofactor in a pocket that inclu<em>d</em>es Phe-397, Tyr-389, Lys-69 (methylene carbons), an<em>d</em> Asp-361. Electron <em>d</em>ensity for G418 was observe<em>d</em> at the boun<em>d</em>ary between the two <em>d</em>omains within each ODC monomer. A ten-amino aci<em>d</em> loop region (39<em>2</em>-401) near the <em>2</em>-fol<em>d</em> axis of the <em>dimer</em> interface, which contributes several resi<em>d</em>ues that form the active site, is <em>d</em>isor<em>d</em>ere<em>d</em> in this structure. The <em>d</em>isor<em>d</em>ering of resi<em>d</em>ues in the active site provi<em>d</em>es a potential mechanism for inhibition by G418 an<em>d</em> suggests that allosteric inhibition from this site is feasible.

OBJECTIVE

Oral anticoagulation with vitamin K antagonists (VKAs) for stroke prevention in atrial fibrillation (AF) is effective but has significant limitations. AZD0837, a new oral anticoagulant, is a prodrug converted to a selective and reversible direct thrombin inhibitor (AR-H067637). We report from a Phase II randomized, dose-guiding study (NCT00684307) to assess safety, tolerability, pharmacokinetics, and pharmacodynamics of extended-release AZD0837 in patients with AF.

RESULTS

Atrial fibrillation patients (n = 955) with>> or =1 a<em>d</em><em>d</em>itional risk factor for stroke were ran<em>d</em>omize<em>d</em> to receive AZD0837 (150, 300, or 450 mg once <em>d</em>aily or <em>2</em>00 mg twice <em>d</em>aily) or VKA (international normalize<em>d</em> ratio <em>2</em>-3, target <em>2</em>.5) for 3-9 months. Approximately 30% of patients were naïve to VKA treatment. Total blee<em>d</em>ing events were similar or lower in all AZD0837 groups (5.3-14.7%, mean exposure 138-145 <em>d</em>ays) vs. VKA (14.5%, mean exposure 161 <em>d</em>ays), with fewer clinically relevant blee<em>d</em>ing events on AZD0837 150 an<em>d</em> 300 mg once <em>d</em>aily. A<em>d</em>verse events were similar between treatment groups; with AZD0837, the most common were gastrointestinal <em>d</em>isor<em>d</em>ers (e.g. <em>d</em>iarrhoea, flatulence, or nausea). <em>d</em>-<em>Dimer</em>, use<em>d</em> as a biomarker of thrombogenesis, <em>d</em>ecrease<em>d</em> in all groups in VKA-naïve subjects with treatment, whereas in VKA pre-treate<em>d</em> patients, <em>d</em>-<em>dimer</em> levels starte<em>d</em> low an<em>d</em> remaine<em>d</em> low in all groups. As expecte<em>d</em>, only a few strokes or systemic embolic events occurre<em>d</em>. In the AZD0837 groups, mean S-creatinine increase<em>d</em> by approximately 10% from baseline an<em>d</em> returne<em>d</em> to baseline following treatment cessation. The frequency of serum alanine aminotransferase>> or =3x upper limit of normal was similar for AZD0837 an<em>d</em> VKA.

CONCLUSIONS

AZD0837 was generally well tolerated at all doses tested. AZD0837 treatment at an exposure corresponding to the 300 mg od dose in this study provides similar suppression of thrombogenesis at a potentially lower bleeding risk compared with dose-adjusted VKA. This study is registered with ClinicalTrials.gov, number NCT00684307.

OBJECTIVE

An accelerated progression of atherosclerosis may contribute to the increased mortality due to cardiovascular disease reported in rheumatoid arthritis (RA). The aim of this study was to identify variables, related to disease onset as well as to disease progression, of importance for the presence of atherosclerosis, as diagnosed by B-mode ultrasonography, in patients with medium-term RA. The results are based on the co-analysis of retrospective data as well as cross-sectional data. The impact of RA per se on atherosclerosis was evaluated relative to age- and sex-matched controls.

METHODS

Thirty-nine RA patients, with a maximum age of 65 years, who had previously been included in a large retrospective cohort study, were assessed by duplex scanning after a disease duration of 19-<em>2</em>3 years. In the present study, factors identified in the two earlier studies were assessed for their potential relationship with intima-media wall thickness (IMT) of the common carotid artery (CCA), and the presence and grade of atherosclerotic plaques of the CCA and the common femoral artery, in regression models. The candidate co-variates were: variables reflecting inflammatory activity at disease onset and at the time of ultrasound assessment, established cardiovascular risk factors, pharmacological treatment [corticosteroids, disease-modifying anti-rheumatic drugs (<em>D</em>MAR<em>D</em>s)], and the presence of complications and co-morbidity identified during disease progression, as well as lipid levels, anti-lipid antibodies, haemostatic factors, and markers of immune activation measured at ultrasound assessment.

RESULTS

In patients with RA, analysis of simple linear regression models revealed those variables significantly associated with IMT-CCA to be age, tissue plasminogen activator (tPA) antigen, cholesterol, low density lipoprotein (L<em>D</em>L)-cholesterol, triglycerides, and atherosclerotic plaques while neither inflammatory status at disease onset, traditional cardiovascular risk factors, or pharmacological treatment during disease had any significant impact on IMT. In an estimated multiple linear regression model, variables associated with increasing log of IMT-CCA were the log of cholesterol and of soluble intracellular adhesion molecule 1 (sICAM-1), while methotrexate treatment tended to have a decreasing effect. In simple binary logistic regression, atherosclerotic plaques were associated with age, IMT-CCA, smoking, and the levels of sICAM-1, sE-selectin, interleukin-<em>2</em> soluble receptor alpha (IL-<em>2</em>sRalpha), plasminogen activator inhibitor-1 (PAI-1) mass, cholesterol, L<em>D</em>L-cholesterol, and the L<em>D</em>L/high density lipoprotein (H<em>D</em>L) ratio. A multiple approach indicated that plaques were associated with age, cholesterol, and sE-selectin. Severe plaques were associated with L<em>D</em>L-cholesterol and disease duration. Logistic regression in the age- and sex-matched case-control study revealed that IMT-CCA was, together with the <em>D</em>-<em>dimer</em>, associated with RA per se.

CONCLUSIONS

Levels of lipids and adhesion molecules were associated with the presence of atherosclerosis in RA. IMT-CCA was associated with RA per se. Disease duration could predict severe atherosclerotic plaques. Treatment with methotrexate seemed to decrease the IMT-CCA.

Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH <or= 4.0) hot springs >>or=70 degrees C) around the world. SSV1 contains a 15.5-kb double-stranded <em>D</em>NA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a <em>D</em>naA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP<em>2</em>, and VP3). The functions of the remaining <em>2</em>9 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 <em>D</em>-63. The structure reveals a helix-turn-helix motif that <em>dimer</em>izes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the <em>dimer</em> axis and thus exhibits <em>2</em>-fold symmetry. Two smaller conserved patches, also related by <em>2</em>-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that <em>D</em>-63 may function as an adaptor protein in macromolecular assembly.

The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta <em>2</em> interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, <em>D</em>. (1977) FEBS Lett. 8<em>2</em>, <em>2</em>43-<em>2</em>46]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of <em>2</em>.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (<em>2</em>) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta <em>2</em> interface. The tertiary changes are confined to residues at the alpha 1 beta <em>2</em> interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta <em>2</em> interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the <em>dimer</em>-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.

In physiological conditions, collagen degradation by fibroblasts occurs primarily via phagocytosis, an intracellular pathway that is thought to require collagen receptors and actin assembly for fibril internalization and degradation. Currently it is unclear which specific steps of collagen phagocytosis in fibroblasts involve actin filament assembly. As studies of phagocytosis in fibroblasts are complicated by the relatively slow rate of particle internalization compared to professional phagocytes, we have examined the role of collagen receptors and actin only in the initial collagen binding step. Prior to the binding of collagen-coated fluorescent beads by human gingival fibroblasts, a cell type that is avidly phagocytic in vitro, cells were treated with cytochalasin <em>D</em> (actin filament barbed-end capping) or swinholide A (actin <em>dimer</em> sequestering and severing) or latrunculin B (actin monomer sequestering). Bead binding and immunostaining of (alpha)(<em>2</em>)(beta)(1) and (alpha)(3)(beta)(1) integrin collagen receptors were measured by flow cytometry. After 1-3 hours of coincubation with beads, cytochalasin <em>D</em> or swinholide A eliminated actin filaments stained by rhodamine-phalloidin and inhibited collagen bead binding (reductions of <em>2</em>5% and 50%, respectively), possibly because of cell rounding and restricted interactions with beads. In contrast, latrunculin enhanced binding dose-dependently over controls (twofold at 1 microM) and induced the formation of brightly staining aggregates of actin and the retention of long cytoplasmic extensions. Latrunculin also reduced surface (beta)(1), (alpha)(<em>2</em>) and (alpha)(3) integrin staining up to 40% in bead-free and bead-loaded cells, indicating that latrunculin enhanced collagen receptor internalization. As determined by fluorescence recovery after photobleaching, latrunculin increased the mobility of surface-bound (beta)(1) integrin. The stimulatory effect of latrunculin on collagen bead binding was reduced to control levels by treatment with a (beta)(1) integrin inactivating antibody while a (beta)(1) integrin blocking antibody abrogated both bead binding and the latrunculin-induced stimulation. Immunoblotting of bead-associated proteins showed that latrunculin completely eliminated binding of (beta)-actin to collagen beads but did not affect (beta)(1) integrin binding. These data indicate that latrunculin-induced sequestration of actin monomers facilitates the disengagement of actin from (beta)(1) integrin receptors, increases collagen bead binding and enhances collagen receptor mobility. We suggest that these alterations increase the probability of adhesive bead-to-cell interactions.

The state of oligomerization of macrophage migration inhibitory factor (MIF, also known as glycosylation inhibiting factor, GIF) in solution has been variously reported as monomer, <em>dimer</em>, trimer, or mixtures of all three. Several crystal structures show MIF to be a trimer. Sedimentation velocity shows a recombinant human MIF sample is quite homogeneous, with 98% as a species with s(<em>2</em>0,w)=3.07 S and <em>D</em>(<em>2</em>0,w)=8.<em>2</em>9 x 10(-7) cm(<em>2</em>)/s. Using the partial specific volume calculated from the amino acid composition these values imply a mass of 33.56 k<em>D</em>a, well above that of <em>dimer</em>, but also 9% below the trimer mass of 37.035 k<em>D</em>a. Sedimentation equilibrium data at loading concentrations from 0.01 to 1 mg/ml show unequivocally that the self-association is extremely tight. However, the apparent mass is 33.53 k<em>D</em>a [95% confidence 33.<em>2</em>5-33.8<em>2</em>], again 9% below that expected for 100% trimer. To examine the possibility this protein has an unusual partial specific volume, sedimentation equilibrium was also done in H(<em>2</em>)O/<em>D</em>(<em>2</em>)O mixtures, giving 0.765+/-0.017 ml/g rather than the calculated 0.735 ml/g. With this revised partial specific volume, the equilibrium and velocity data each give M=37.9+/-<em>2</em>.8 k<em>D</em>a, fully consistent with a strongly-associated trimeric quaternary structure.

The overt <em>D</em>IC score of the <em>D</em>IC subcommittee of the ISTH includes a fibrin-related marker (FRM) as indicator of intravascular fibrin formation. The type of marker to be used has not been specified, but <em>D</em>-<em>dimer</em> antigen, or fibrin degradation products are used by most investigators. Soluble fibrin complexes have been suggested as more specific indicators of acute intravascular fibrin formation. The aim of the present study was to compare the predictive value of the overt <em>D</em>IC score concerning clinical outcome in a surgical intensive care cohort, using either <em>D</em>-<em>dimer</em> antigen, or soluble fibrin antigen as FRM. The cutoff values for <em>2</em> and 3 score points for the FRM were assigned on the basis of the <em>2</em>5% and 75% quartiles of 1870 plasma samples obtained from 359 ICU patients during a period of 6 months. For 331 patients with complete diagnostic workup and day 1 blood samples, the Iatro SF as FRM component of the overt <em>D</em>IC score displayed the highest prognostic power concerning clinical outcome. The <em>2</em>8-day mortality of patients with overt <em>D</em>IC at day 1, using Iatro SF as FRM assay was 50.0%, whereas <em>2</em>8-day mortality of patients without overt <em>D</em>IC was 14.0% (p <0.0001). Using M<em>D</em>A <em>D</em>-<em>dimer</em>, and TINAquant <em>D</em>-<em>dimer</em>, <em>2</em>8-day mortality was between 35.5% and 39.3% in patients with overt <em>D</em>IC, and 15.5% to 15.6% in patients without overt <em>D</em>IC. Selection of the FRM as component of the <em>D</em>IC score has a small, but relevant impact on the prognostic performance of the overt <em>D</em>IC score. The present data on the distribution of values may provide a basis for the selection of appropriate cutoff points for assigning <em>2</em>, and 3 points in the score.

3<em>D</em> domain swapping of proteins involves the interconversion of a monomer containing a single domain-domain interface and a <em>2</em>-fold symmetrical <em>dimer</em> containing two equivalent intermolecular interfaces. Human glyoxalase I has the structure of a domain-swapped <em>dimer</em> [Cameron, A. <em>D</em>., Olin, B., Ridderström, M., Mannervik, B., and Jones, T. A. (1997) EMBO J. 16, 3386-3395] but Pseudomonas putida glyoxalase I has been reported to be monomeric [Rhee, H.-I., Murata, K., and Kimura, A. (1986) Biochem. Biophys. Res. Commun. 141, 993-999]. We show here that recombinant P. putida glyoxalase I is an active <em>dimer</em> (kcat approximately 500 +/- 100 s-1; KM approximately 0.4 +/- 0.<em>2</em> mM) with two zinc ions per <em>dimer</em>. The zinc is required for structure and function. However, treatment of the <em>dimer</em> with glutathione yields an active monomer (kcat approximately 115 +/- 40 s-1; KM approximately 1.4 +/- 0.4 mM) containing a single zinc ion. The monomer is metastable and slowly reverts to the active <em>dimer</em> in the absence of glutathione. Thus, glyoxalase I appears to be a novel example of a single protein able to exist in two alternative domain-swapped forms. It is unique among domain-swapped proteins in that the active site and an essential metal binding site are apparently disassembled and reassembled by the process of domain swapping. Furthermore, it is the only example to date in which 3<em>D</em> domain swapping can be regulated by a small organic ligand.

Although the hepatitis <em>delta</em> virus genome contains multiple open reading frames, only one of these reading frames is known to be expressed during replication of the virus. This open reading frame encodes two distinct molecular species of hepatitis <em>delta</em> antigen (HDAg), p<em>2</em>4 <em>delta</em> and p<em>2</em>7 <em>delta</em>, depending on the location of the stop codon which terminates translation. We found antibody specific for p<em>2</em>7 <em>delta</em> to be capable of precipitating p<em>2</em>4 <em>delta</em> in extracts of infected liver, indicating that p<em>2</em>7 <em>delta</em> and p<em>2</em>4 <em>delta</em> form heterologous complexes in vivo. After cross-linking with 0.05% glutaraldehyde, specific HDAg <em>dimers</em> were detected in antigen prepared from both the liver and serum of an HDV-infected woodchuck carrier of woodchuck hepatitis virus. Guanidine HCl-denatured HDAg extracted from liver and dialyzed against phosphate-buffered saline sedimented in rate-zonal sucrose density gradients as 15S multimeric complexes. These 15S multimers were stable in the presence of 1.<em>2</em>% Nonidet P-40. After RNase digestion, the 15S complex was reduced to a 1<em>2</em>S complex without associated RNA, while boiling for 3 min in 1% sodium dodecyl sulfate-0.5% <em>2</em>-mercaptoethanol further reduced the 15S complex to 3S HDAg monomers. In the absence of glutaraldehyde cross-linking, HDAg extracted from liver migrated as monomer species in reducing and nonreducing gels, suggesting that the conserved cysteine residue present in p<em>2</em>7 <em>delta</em> does not play a role in the formation of either <em>dimers</em> or multimers. On the other hand, an amino-terminal chymotrypsin-digested HDAg fragment, with a predicted length of 81 or less amino acids, retained the ability to form <em>dimers</em>, consistent with the hypothesis that a coiled-coil motif present between residues <em>2</em>7 and 58 may play a role in HDAg protein interactions in vivo.

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homo<em>dimer</em>ic N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular <em>D</em>NA in the presence of Mn(<em>2</em>+), Mg(<em>2</em>+) or Ca(<em>2</em>+) ions, unlike human MutLalpha which shows endonuclease activity only in the presence of Mn(<em>2</em>+). We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CT<em>D</em>) consisting of amino acids 460-658 exhibits Mn(<em>2</em>+)-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CT<em>D</em> to be a <em>dimer</em>. The probable endonucleolytic active site is localized to a metal-binding motif, <em>D</em>MHAX<em>2</em>EX4E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and a mutant NgoL-CT<em>D</em> protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in <em>D</em>NA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the <em>D</em>[M/Q]HAX<em>2</em>EX4E motif.

A theoretical framework is presente<em>d</em> for analysis of all three "multiline" EPR spectra (MLS) arising from the tetramanganese (Mn(4)) cluster in the S(<em>2</em>) oxi<em>d</em>ation state of the photosynthetic water oxi<em>d</em>izing complex (WOC). Accurate simulations are presente<em>d</em> which inclu<em>d</em>e anisotropy of the g an<em>d</em> (four) (55)Mn hyperfine tensors, chosen accor<em>d</em>ing to a <em>d</em>atabase of (55)Mn(III) an<em>d</em> (55)Mn(IV) hyperfine tensors obtaine<em>d</em> previously using unbiase<em>d</em> least-squares spectral fitting routines. In view of the large (30%) anisotropy common to Mn(III) hyperfine tensors in all complexes, previous MLS simulations which have assume<em>d</em> isotropic hyperfine constants have require<em>d</em> physically unrealistic parameters. A simple mo<em>d</em>el is foun<em>d</em> which offers goo<em>d</em> simulations of both the native "19-<em>2</em>1-line" MLS an<em>d</em> the "<em>2</em>6-line" NH(3)-boun<em>d</em> form of the MLS. Both a <em>dimer</em>-of-<em>dimers</em> an<em>d</em> <em>d</em>istorte<em>d</em>-trigonal magnetic mo<em>d</em>els are examine<em>d</em> to <em>d</em>escribe the symmetry of the Heisenberg exchange interactions within the Mn(4) cluster an<em>d</em> thus <em>d</em>efine the initial electronic basis states of the cluster. The effect of rhombic symmetry <em>d</em>istortions is explicitly consi<em>d</em>ere<em>d</em>. Both magnetic mo<em>d</em>els correspon<em>d</em> to one of several possible structural mo<em>d</em>els for the Mn(4) cluster propose<em>d</em> in<em>d</em>epen<em>d</em>ently from Mn EXAFS stu<em>d</em>ies. Simulate<em>d</em> MLS were constructe<em>d</em> for each of the eight (or seven) <em>d</em>oublet states of the Mn(4) cluster in the WOC for the two viable oxi<em>d</em>ation mo<em>d</em>els (3Mn(III)-1Mn(IV) or 3Mn(IV)-1Mn(III)), an<em>d</em> using a wi<em>d</em>e range of axial Mn hyperfine tensors, with either coaxial or orthogonal tensor alignments. We fin<em>d</em> accurate simulations using the 3Mn(III)-1Mn(IV) oxi<em>d</em>ation mo<em>d</em>el. In the <em>dimer</em>-of-<em>dimers</em> coupling mo<em>d</em>el, the spin state conversion between two <em>d</em>oublet states |S(1<em>2</em>),S(34),S(T)|(7)/(<em>2</em>),4,(1)/(<em>2</em>>> an<em>d</em> |(7)/(<em>2</em>),3,(1)/(<em>2</em>>> is foun<em>d</em> to explain the large (<em>2</em>5%) contraction in the hyperfine splitting observe<em>d</em> upon conversion from the native MLS to the NH(3)-boun<em>d</em> MLS. Stabilization of this excite<em>d</em> state as the new groun<em>d</em> state is cause<em>d</em> by change in the intermanganese exchange coupling, without appreciable change in the intrinsic hyperfine tensors. The lack of goo<em>d</em> simulations of the Ca(<em>2</em>+)-<em>d</em>eplete<em>d</em> MLS suggests that Ca(<em>2</em>+)-<em>d</em>epletion changes both Mn ligation an<em>d</em> intermanganese exchange coupling. The 3Mn(IV)-1Mn(III) oxi<em>d</em>ation mo<em>d</em>el is <em>d</em>isfavore<em>d</em> because only approximate simulations coul<em>d</em> be foun<em>d</em> for the native MLS an<em>d</em> no agreement with the NH(3)-boun<em>d</em> MLS was obtaine<em>d</em>. The scalar part of the hyperfine tensors for both Mn(III) an<em>d</em> Mn(IV) ions were foun<em>d</em> to approximate (+/-5%) the values for the <em>d</em>imanganese(III,IV) catalase enzyme, suggesting similar overall ligan<em>d</em> types. However, the large (30%) anisotropic part of the Mn(III) hyperfine interaction is opposite in sign to that foun<em>d</em> in all tetragonally exten<em>d</em>e<em>d</em> six-coor<em>d</em>inate Mn(III) ions (i.e., the usual Jahn-Teller splitting). The <em>d</em>istribution of spin <em>d</em>ensity from the high-spin <em>d</em>(4) electron configuration of each Mn(III) ion correspon<em>d</em>s to a flattene<em>d</em> (oblate) ellipsoi<em>d</em>. This electronic <em>d</em>istribution is favore<em>d</em> in five-coor<em>d</em>inate ligan<em>d</em> fiel<em>d</em>s having trigonally compresse<em>d</em> bipyrami<em>d</em>al geometry, but it coul<em>d</em> also arise, in principle, in straine<em>d</em> six-coor<em>d</em>inate ligan<em>d</em> fiel<em>d</em>s having tetragonally compresse<em>d</em> geometry, i.e. [Mn(<em>2</em>)(&mgr;-O)](4+) (reverse Jahn-Teller <em>d</em>istortion). The resulting valence electronic configurations are <em>d</em>escribe<em>d</em> as e'(<em>2</em>)e"(<em>2</em>) an<em>d</em> (<em>d</em>(pi))(3)(<em>d</em>(x)()()<em>2</em>(-)(y)()()<em>2</em>)(1), respectively, in contrast to the (<em>d</em>(pi))(3)(<em>d</em>(z)()()<em>2</em>)(1) configuration common to unstraine<em>d</em> six-coor<em>d</em>inate tetragonally-exten<em>d</em>e<em>d</em> Mn(III) ions, such as foun<em>d</em> in the [Mn(<em>2</em>)(&mgr;-O)(<em>2</em>)](3+) core in several synthetic <em>dimers</em> an<em>d</em> catalase. Both of the former geometries pre<em>d</em>ict strongly oxi<em>d</em>izing Mn(III) ions, thereby suggesting a structural basis for the oxi<em>d</em>ative reactivity of the Mn(4) cluster in the WOC. The magnetic mo<em>d</em>el nee<em>d</em>e<em>d</em> to explain the MLS is not rea<em>d</em>ily reconcile<em>d</em> with the simplest structural an<em>d</em> electronic mo<em>d</em>els <em>d</em>e<em>d</em>uce<em>d</em> from EXAFS stu<em>d</em>ies of the WOC.

This clinical policy from the American College of Emergency Physicians is the revision of a <em>2</em>003 clinical policy on the evaluation and management of adult patients presenting with suspected pulmonary embolism (PE).(1) A writing subcommittee reviewed the literature to derive evidence-based recommendations to help clinicians answer the following critical questions: (1) <em>D</em>o objective criteria provide improved risk stratification over gestalt clinical assessment in the evaluation of patients with possible PE? (<em>2</em>) What is the utility of the Pulmonary Embolism Rule-out Criteria (PERC) in the evaluation of patients with suspected PE? (3)What is the role of quantitative <em>D</em>-<em>dimer</em> testing in the exclusion of PE? (4) What is the role of computed tomography pulmonary angiogram of the chest as the sole diagnostic test in the exclusion of PE? (5) What is the role of venous imaging in the evaluation of patients with suspected PE? (6) What are the indications for thrombolytic therapy in patients with PE? Evidence was graded and recommendations were given based on the strength of the available data in the medical literature.

BACKGROUND

Helical computed tomography (CT) is commonly used to diagnose pulmonary embolism, although its operating characteristics have been insufficiently evaluated.

OBJECTIVE

To assess the sensitivity and specificity of helical CT in suspected pulmonary embolism.

METHODS

Observational study.

METHODS

Emergency department of a teaching and community hospital.

METHODS

299 patients with clinically suspected pulmonary embolism and a plasma D -dimer level greater than 500 microgram/L.

METHODS

Pulmonary embolism was established by using a validated algorithm that included clinical assessment, lower-limb compression ultrasonography, lung scanning, and pulmonary angiography.

METHODS

Sensitivity, specificity, and likelihood ratios of helical CT and interobserver agreement. Helical CT scans were withheld from clinicians and were read 3 months after acquisition by radiologists blinded to all clinical data.

RESULTS

118 patients (39%) had pulmonary embolism. In 12 patients (4%), 2 of whom had pulmonary embolism, results of helical CT were inconclusive. For patients with conclusive results, sensitivity of helical CT was 70% (95% CI, 62% to 78%) and specificity was 91% (CI, 86% to 95%). Interobserver agreement was high (kappa = 0.823 to 0.902). The false-negative rate was lower for helical CT used after initial negative results on ultrasonography than for helical CT alone (21% vs. 30%). Use of helical CT after normal results on initial ultrasonography and nondiagnostic results on lung scanning had a false-negative rate of only 5% and a false-positive rate of only 7%.

CONCLUSIONS

Helical CT should not be used alone for suspected pulmonary embolism but could replace angiography in combined strategies that include ultrasonography and lung scanning.

The structures of the native fructose-1,6-bisphosphatase (Fru-1,6-Pase), from pig kidney cortex, and its fructose <em>2</em>,6-bisphosphate (Fru-<em>2</em>,6-P<em>2</em>) complexes have been refined to <em>2</em>.8 A resolution to R-factors of 0.194 and 0.188, respectively. The root-mean-square deviations from the standard geometry are 0.0<em>2</em>1 A and 0.016 A for the bond length, and 4.4 degrees and 3.8 degrees for the bond angle. Four sites for Fru-<em>2</em>,6-P<em>2</em> binding per tetramer have been identified by difference Fourier techniques. The Fru-<em>2</em>,6-P<em>2</em> site has the shape of an oval cave about 10 A deep, and with other dimensions about 18 A by 1<em>2</em> A. The two Fru-<em>2</em>,6-P<em>2</em> binding caves of the <em>dimer</em> in the crystallographically asymmetric unit sit next to one another and open in opposite directions. These two binding sites mutually exchange their Arg<em>2</em>43 side-chains, indicating the potential for communication between the two sites. The beta, <em>D</em>-fructose <em>2</em>,6-bisphosphate has been built into the density and refined well. The oxygen atoms of the 6-phosphate group of Fru-<em>2</em>,6-P<em>2</em> interact with Arg<em>2</em>43 from the adjacent monomer and the residues of Lys<em>2</em>74, Asn<em>2</em>1<em>2</em>, Tyr<em>2</em>64, Tyr<em>2</em>15 and Tyr<em>2</em>44 in the same monomer. The sugar ring primarily contacts with the backbone atoms from Gly<em>2</em>46 to Met<em>2</em>48, as well as the side-chain atoms, Asp1<em>2</em>1, Glu<em>2</em>80 and Lys<em>2</em>74. The <em>2</em>-phosphate group interacts with the side-chain atoms of Ser1<em>2</em>4 and Lys<em>2</em>74. A negatively charged pocket near the <em>2</em>-phosphate group includes Asp118, Asp1<em>2</em>1 and Glu<em>2</em>80, as well as Glu97 and Glu98. The <em>2</em>-phosphate group showed a disordered binding perhaps because of the disturbance from the negatively charged pocket. In addition, Asn1<em>2</em>5 and Lys<em>2</em>69 are located within a 5 A radius of Fru-<em>2</em>,6-P<em>2</em>. We argue that Fru-<em>2</em>,6-P<em>2</em> binds to the active site of the enzyme on the basis of the following observations: (1) the structure similarity between Fru-<em>2</em>,6-P<em>2</em> and the substrate; (<em>2</em>) sequence conservation of the residues directly interacting with Fru-<em>2</em>,6-P<em>2</em> or located at the negatively charged pocket; (3) a divalent metal site next to the <em>2</em>-phosphate group of Fru-<em>2</em>,6-P<em>2</em>; and (4) identification of some active site residues in our structure, e.g. tyrosine and Lys<em>2</em>74, consistent with the results of the ultraviolet spectra and the chemical modification. The structures are described in detail including interactions of interchain surfaces, and the chemically modifiable residues are discussed on the basis of the refined structures.(ABSTRACT TRUNCATE<em>D</em> AT 400 WOR<em>D</em>S)

The synthesis of four oligonucleoti<em>d</em>es containing alternating phosphorothioate groups, (Rp)-an<em>d</em> (Sp)-<em>d</em>[G(p(S)CpG)3p(S)C] an<em>d</em> (Rp)- an<em>d</em> (Sp)-<em>d</em>[C(p(S)GpC)p(S)G], by the phosphite approach is <em>d</em>escribe<em>d</em>. Silica gel to which <em>2</em>'(3')-O-acetyluri<em>d</em>ine an<em>d</em> 5'-succinyl groups were boun<em>d</em> serve<em>d</em> as support for oligomer synthesis. The syntheses were carrie<em>d</em> out by <em>dimer</em> a<em>d</em><em>d</em>ition with presynthesize<em>d</em> <em>d</em>iastereomerically pure <em>d</em>inucleosi<em>d</em>e phosphorothioates as buil<em>d</em>ing blocks. The pro<em>d</em>ucts were characterize<em>d</em> by 31P NMR, nuclease P1 <em>d</em>igestion, an<em>d</em> oxi<em>d</em>ation to the correspon<em>d</em>ing all-phosphate-containing oligomers. The ability of each oligomer to a<em>d</em>opt the Z conformation un<em>d</em>er high-salt con<em>d</em>itions was screene<em>d</em> for by circular <em>d</em>ichroism spectroscopy. Both (Rp)-<em>d</em>[G(p(S)CpG)3p(S)C] an<em>d</em> (Sp)-<em>d</em>[C(p(S)GpC)3p(S)G] are capable of forming Z-type structures at high NaCl concentrations. In the case of (Rp)-<em>d</em>[G(p(S)CpG)3p(S)C] where a phosphorothioate of the Rp configuration occurs 5' to a <em>d</em>eoxycyti<em>d</em>ine resi<em>d</em>ue, the B----Z transition is potentiate<em>d</em> in comparison to the unmo<em>d</em>ifie<em>d</em> oligomer. (Sp)-<em>d</em>[G(p(S)CpG)3p(S)C] an<em>d</em> (Rp)-<em>d</em>[C(p(S)GpC)3p(S)G] retain the B conformation even at high NaCl concentration.

Extracellular regulate<em>d</em> protein kinase <em>2</em> (ERK<em>2</em>) is a eukaryotic protein kinase whose activity is regulate<em>d</em> by mitogenic stimuli. To gain insight into the catalytic properties of ERK<em>2</em> an<em>d</em> to complement structure-function stu<em>d</em>ies, we un<em>d</em>ertook a pre-stea<em>d</em>y state kinetic analysis of the enzyme. To <em>d</em>o this, ERK<em>2</em> was quantitatively activate<em>d</em> by MAPKK1 in vitro by monitoring the stoichiometry an<em>d</em> site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic pepti<em>d</em>e analysis, an<em>d</em> (3<em>2</em>)P ra<em>d</em>iolabeling. Using a quench-flow apparatus, MgATP(<em>2</em>-) was rapi<em>d</em>ly mixe<em>d</em> (<1 ms) with both ERK<em>2</em> an<em>d</em> the protein substrate EtsDelta138 in the presence of a saturating total concentration (<em>2</em>0 mM) of magnesium ion at <em>2</em>7 <em>d</em>egrees C an<em>d</em> pH 7.5. An exponential burst of pro<em>d</em>uct was observe<em>d</em> over the first few millisecon<em>d</em>s that followe<em>d</em> mixing. This burst ha<em>d</em> an amplitu<em>d</em>e alpha of 0.44 an<em>d</em> was followe<em>d</em> by a slower linear phase. The pre-stea<em>d</em>y state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(<em>2</em>) = 109 +/- 9 s(-1) an<em>d</em> k(3) = 56 +/- 4 s(-1). These are attribute<em>d</em> to rapi<em>d</em> phosphorylation of EtsDelta138 an<em>d</em> the process of pro<em>d</em>uct release, respectively. Single-turnover experiments provi<em>d</em>e<em>d</em> an in<em>d</em>epen<em>d</em>ent <em>d</em>etermination of k(<em>2</em>) (106 +/- <em>2</em>5 s(-1)). The observe<em>d</em> catalytic constant (k(cat)(obs)) was foun<em>d</em> to be sensitive to the concentration of ERK<em>2</em>. The <em>d</em>ata fit a mo<em>d</em>el in which ERK<em>2</em> monomers form <em>dimers</em> an<em>d</em> suggest that both the monomeric an<em>d</em> <em>d</em>imeric forms of ERK<em>2</em> are active with catalytic constants (k(cat)) of <em>2</em>5 an<em>d</em> 37 s(-1), respectively. In a<em>d</em><em>d</em>ition, the mo<em>d</em>el suggests that in the presence of saturating concentrations of both magnesium an<em>d</em> substrates ERK<em>2</em> subunits <em>d</em>issociate with a <em>d</em>issociation constant (K(<em>d</em>)) of 3<em>2</em> +/- 16 nM.

Telomeric C-stran<em>d</em> sequences form non-Watson-Crick base-paire<em>d</em> structures in supercoile<em>d</em> plasmi<em>d</em>s an<em>d</em> in oligonucleoti<em>d</em>es at low pH. Here we examine oligonucleoti<em>d</em>es compose<em>d</em> of <em>2</em> or 4 repeats of the human telomeric C-stran<em>d</em> sequence <em>d</em>(CCCTAA)n. At low pH, the <em>2</em>-repeat molecule forms a <em>dimer</em> which exhibits H1'-H1' nuclear Overhauser effects (NOEs) between stacke<em>d</em> CC+ base pairs. These NOEs are characteristic of the i-motif, which is a tetraplex compose<em>d</em> of two intercalate<em>d</em> CC+ <em>d</em>uplexes. The 4-repeat molecule forms an intramolecular monomeric structure at low pH, suggesting that four contiguous cytosine tracts fol<em>d</em> into a CC+ intercalate<em>d</em> tetraplex. These unusual structures may be relevant to the formation of guanine tetraplexes by complementary G-rich sequences. They may also provi<em>d</em>e a general mechanism for self-recognition by nucleic aci<em>d</em>s.

BACKGROUND

The purpose of this study was to evaluate the relative importance of systemic hypercoagulability, preexisting and acquired risk factors, and specific injury patterns in the development of venous thromboembolism (VTE) after injury.

METHODS

Injured patients with an Injury Severity Score>> or = 15 were followed with lower extremity venous duplex ultrasonography, prothrombin fragment 1 + <em>2</em>, and quantitative <em>D</em>-<em>dimer</em> levels at 1 and 3 days and then weekly until discharge.

RESULTS

Among 101 patients with a mean Injury Severity Score of <em>2</em>7.3 +/- 10.5 followed for 1<em>2</em>.4 +/- 8.7 days, <em>2</em>8 (<em>2</em>7.7%) developed a lower extremity thrombosis, <em>2</em> (1.9%) sustained a pulmonary embolism, and 1 (0.9%) had a symptomatic upper extremity thrombosis. Although admission fragment 1 + <em>2</em> and <em>D</em>-<em>dimer</em> levels were elevated in 81.4% and 100% of patients, respectively, mean levels were not significantly different in those with or without VTE. VTE was more common (p < 0.05) among those with obesity, age>> 40 years, immobilization for>> 3 days, spine fractures, and lower extremity fractures. However, only obesity (p = 0.004) and immobilization>> 3 days (p = 0.05) were independent predictors of VTE in a multivariate analysis.

CONCLUSIONS

Although elevated in seriously injured patients, neither markers of activated coagulation nor specific injury patterns are predictive of VTE. Associations with immobilization and obesity suggest that VTE after injury is a systemic hypercoagulable disorder with local manifestations of thrombosis related to lower extremity stasis.

Background: Abnormal coagulation parameters have been reported in COVID-19 patients. Although the underlying mechanism of COVID-19 coagulopathy remains unknown, it has been suggested to be a form of disseminated intravascular coagulation (DIC).

Objectives: The aim of our study was to analyze the coagulation parameters of patients with COVID-19, determine if coagulation factors consumption occurs and identify potential prognostic biomarkers of the disease.

Patients/methods: Blood samples from hospitalized patients with COVID-19 pneumonia were collected. We performed basic coagulation tests and quantification of coagulation factors and physiological inhibitor proteins. Laboratory data were compared with clinical data and outcomes.

Results: The study involved 206 patients (63.6% male). D-dimer was particularly elevated (median 450 ng/ml; IQR 222.5-957.3). Free protein S levels were below the normal range (median 56.6%; IQR: 43.6-68.9) and factor VIII showed an increasing trend (median 173.4%; IQR: 144.1-214.9). However, all coagulation factors were within normal limits. We found no correlation between abnormal coagulation parameters and thrombosis, except for higher D-dimer (HR 1.99; 95% CI 1.3-3.1; p=0.002).

Conclusions: COVID-19 is associated with coagulopathy that correlates with poor prognosis. However, we did not demonstrate a consumption of coagulation factors, as seen in DIC.

Keywords: COVID-19; D-dimer; SARS-CoV-2 infection; coagulation factors; coagulopathy.

BACKGROUND

It has been demonstrated that there is an increased risk of venous thromboembolism (VTE) during air travel on flights of long duration. Patients with COPD are also at increased risk of VTE, particularly during exacerbations, possibly because of a hypercoagulable state secondary to hypoxia and/or heightened systemic inflammation. We investigated the effects of hypoxia on indices of coagulation and systemic inflammation in patients with COPD.

METHODS

Twenty clinically stable patients with mild COP<em>D</em> were recruited. Patients were randomized to receive either medical air or 100% nitrogen through a 40% venturi mask at a flow rate of 10 L/min for <em>2</em> h. Blood was sampled for thrombin-antithrombin complex (TAT), prothrombin activation fragments 1 + <em>2</em> (F(1 + <em>2</em>)), von Willebrand factor antigen (VWF:Ag), <em>D</em>-<em>dimer</em>, and interleukin-6 (IL-6) at baseline and after <em>2</em> h.

RESULTS

Patients in the hypoxia and control groups were similar in terms of age, sex, pack-years smoked, and severity of airflow obstruction. There was no difference in baseline TAT, F(1 + <em>2</em>), VWF:Ag, <em>D</em>-<em>dimer</em>, or IL-6 levels between groups. In the control group, there was no change in markers of coagulation or systemic inflammation over the <em>2</em>-h study. In patients who underwent hypoxic challenge, there was an increase in TAT (P < .001), F(1 + <em>2</em>) (P < .01), and IL-6 (P < .01), whereas <em>D</em>-<em>dimer</em> and VWF:Ag levels were unchanged.

CONCLUSIONS

This study demonstrates that a <em>2</em>-h hypoxic challenge in patients with COP<em>D</em> results in coagulation activation in conjunction with an increase in systemic inflammation.

The structural selectivity of the DNA-bin<em>d</em>ing antitumor <em>d</em>rug <em>d</em>itercalinium was investigate<em>d</em> by competition <em>d</em>ialysis with a series of nineteen <em>d</em>ifferent DNA substrates. The 7H-pyri<em>d</em>ocarbazole <em>dimer</em> was foun<em>d</em> to bin<em>d</em> to <em>d</em>ouble-stran<em>d</em>e<em>d</em> DNA with a preference for GC-rich species but can in a<em>d</em><em>d</em>ition form stable complexes with triplex an<em>d</em> qua<em>d</em>ruplex structures. The preferential interaction of the <em>d</em>rug with four-stran<em>d</em>e<em>d</em> DNA structures was in<em>d</em>epen<em>d</em>ently confirme<em>d</em> by electrospray mass spectrometry an<em>d</em> a <em>d</em>etaile<em>d</em> analysis of the bin<em>d</em>ing reaction was performe<em>d</em> by surface plasmon resonance (SPR) spectroscopy. The BIAcore SPR stu<em>d</em>y showe<em>d</em> that the kinetic parameters for the interaction of <em>d</em>itercalinium with the human telomeric qua<em>d</em>ruplex sequence are comparable to those measure<em>d</em> with a <em>d</em>uplex sequence. Slow association an<em>d</em> <em>d</em>issociation were observe<em>d</em> with both the qua<em>d</em>ruplex an<em>d</em> <em>d</em>uplex structures. The newly <em>d</em>iscovere<em>d</em> preferential bin<em>d</em>ing of <em>d</em>itercalinium to the antiparallel qua<em>d</em>ruplex sequence <em>d</em>(AG(3)[T(<em>2</em>)AG(3)](3)) provi<em>d</em>es new perspectives for the <em>d</em>esign of <em>d</em>rugs that can bin<em>d</em> to human telomeres.

HspA, a member of the GroES chaperonin family, is a small protein foun<em>d</em> in Helicobacter pylori with a unique histi<em>d</em>ine- an<em>d</em> cysteine-rich <em>d</em>omain at the C terminus. In this work, we overexpresse<em>d</em>, purifie<em>d</em>, an<em>d</em> characterize<em>d</em> this protein both in vitro an<em>d</em> in vivo. The apo form of the protein bin<em>d</em>s <em>2</em>.10 +/- 0.07 Ni(<em>2</em>+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a <em>d</em>issociation constant (K(<em>d</em>)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(<em>2</em>+) can reversibly bin<em>d</em> to the protein, as the boun<em>d</em> nickel can be release<em>d</em> either in the presence of a chelating ligan<em>d</em>, e.g. EDTA, or at an aci<em>d</em>ic pH (pH((1/<em>2</em>)) 3.8 +/- 0.<em>2</em>). In contrast, Bi(3+) bin<em>d</em>s almost irreversibly to the protein. Both gel filtration chromatography an<em>d</em> native electrophoresis <em>d</em>emonstrate<em>d</em> that apo-HspA exists as a heptamer in solution. Unexpecte<em>d</em>ly, bin<em>d</em>ing of Bi(3+) to the protein altere<em>d</em> its quaternary structure from a heptamer to a <em>dimer</em>, in<em>d</em>icating that bismuth may interfere with the biological functions of HspA. When culture<em>d</em> in Ni(<em>2</em>+)-supplemente<em>d</em> M9 minimal me<em>d</em>ium, Escherichia coli BL<em>2</em>1(DE3) cells expressing wil<em>d</em>-type HspA or the C-terminal <em>d</em>eletion mutant clearly in<em>d</em>icate<em>d</em> that the C terminus might protect cells from high concentrations of external Ni(<em>2</em>+). However, an opposite phenomenon was observe<em>d</em> when the same E. coli hosts were grown in Bi(3+)-supplemente<em>d</em> me<em>d</em>ium. HspA may therefore play a <em>d</em>ual role: to facilitate nickel acquisition by <em>d</em>onating Ni(<em>2</em>+) to appropriate proteins in a nickel-<em>d</em>eficient environment an<em>d</em> to carry out <em>d</em>etoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer <em>d</em>rug against H. pylori.

The endophytic fungus Stemphylium globuliferum was isolated from stem tissues of the Moroccan medicinal plant Mentha pulegium. Extracts of the fungus, which was grown on solid rice medium, exhibited considerable cytotoxicity when tested in vitro against L5178Y cells. Chemical investigation yielded five new secondary metabolites, alterporriol G (4) and its atropisomer alterporriol H (5), altersolanol K (11), altersolanol L (1<em>2</em>), stemphypyrone (13), and the known compounds 6-O-methylalaternin (1), macrosporin (<em>2</em>), altersolanol A (3), alterporriol E (6), alterporriol <em>D</em> (7), alterporriol A (8), alterporriol B (9), and altersolanol J (10). The structures were determined on the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry. Among the alterporriol-type anthranoid <em>dimers</em>, the mixture of alterporriols G and H (4/5) exhibited considerable cytotoxicity against L5178Y cells with an EC(50) value of <em>2</em>.7 microg/mL, whereas the other congeners showed only modest activity. The compounds were also tested for kinase inhibitory activity in an assay involving <em>2</em>4 different kinases. Compounds 1, <em>2</em>, 3, and the mixture of 4 and 5 were the most potent inhibitors, displaying EC(50) values between 0.64 and 1.4 microg/mL toward individual kinases.