The bifunctional CO <em>d</em>ehy<em>d</em>rogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Woo<em>d</em>-Ljung<em>d</em>ahl pathway of autotrophic CO(<em>2</em>) fixation. One structure of the Moorella thermoacetica enzyme reveale<em>d</em> that the active site of ACS (the A-cluster) consists of a [4Fe-4S] cluster bri<em>d</em>ge<em>d</em> to a binuclear CuNi center with Cu at the proximal metal site (M(p)) an<em>d</em> Ni at the <em>d</em>istal metal site (M(<em>d</em>)). In another structure of the same enzyme, Ni or Zn was present at M(p). On the basis of a positive correlation between ACS activity an<em>d</em> Cu content, we ha<em>d</em> propose<em>d</em> that the Cu-containing enzyme is active [Seravalli, J., et al. (<em>2</em>003) Proc. Natl. Aca<em>d</em>. Sci. U.S.A. 100, 3689-3694]. Here we have reexamine<em>d</em> this proposal. Enzyme preparations with a wi<em>d</em>er range of Ni (1.6-<em>2</em>.8) an<em>d</em> Cu (0.<em>2</em>-1.1) stoichiometries per <em>dimer</em> were stu<em>d</em>ie<em>d</em> to reexamine the correlation, if any, between the Ni an<em>d</em> Cu content an<em>d</em> ACS activity. In a<em>d</em><em>d</em>ition, the effects of o-phenanthroline (which removes Ni but not Cu) an<em>d</em> neocuproine (which removes Cu but not Ni) on ACS activity were <em>d</em>etermine<em>d</em>. EXAFS results in<em>d</em>icate that these chelators selectively remove M(p). Multifrequency EPR spectra (3-130 GHz) of the paramagnetic NiFeC state of the A-cluster were examine<em>d</em> to investigate the electronic state of this propose<em>d</em> interme<em>d</em>iate in the ACS reaction mechanism. The combine<em>d</em> results strongly in<em>d</em>icate that the CuNi enzyme is inactive, that the NiNi enzyme is active, an<em>d</em> that the NiNi enzyme is responsible for the NiFeC EPR signal. The results also support an electronic structure of the NiFeC-eliciting species as a [4Fe-4S](<em>2</em>+) (net S = 0) cluster bri<em>d</em>ge<em>d</em> to a Ni(1+) (S = (1)/(<em>2</em>)) at M(p) that is bri<em>d</em>ge<em>d</em> to planar four-coor<em>d</em>inate Ni(<em>2</em>+) (S = 0) at M(<em>d</em>), with the spin pre<em>d</em>ominantly on the Ni(1+). Furthermore, these stu<em>d</em>ies suggest that M(p) is inserte<em>d</em> <em>d</em>uring cell growth. The apparent vulnerability of the proximal metal site in the A-cluster to substitution with <em>d</em>ifferent metals appears to un<em>d</em>erlie the heterogeneity observe<em>d</em> in samples that has confoun<em>d</em>e<em>d</em> stu<em>d</em>ies of CODH/ACS for many years. On the basis of this principle, a protocol to generate nearly homogeneous preparations of the active NiNi form of ACS was achieve<em>d</em> with NiFeC signals of approximately 0.8 spin/mol.

We have produced a soluble form of a mouse alpha beta T-cell antigen receptor (TCR) by shuffling its variable (V) and constant (C) domains to the C region of an immunoglobulin kappa light chain. These chimeric molecules composed of V alpha C alpha C kappa and V beta C beta C kappa chains were efficiently secreted (up to 1 micrograms/ml) by transfected myeloma cells as noncovalent hetero<em>dimers</em> of about 95-kDa molecular mass. In the absence of direct binding measurement, we have refined the epitopic analysis of the soluble V alpha C alpha C kappa-V beta C beta C kappa <em>dimers</em> and shown that they react with an anti-clonotypic antibody and two antibodies directed to the C domain of the TCR alpha and beta chains. Conversely, we have raised three distinct monoclonal antibodies against the soluble TCR hetero<em>dimers</em> and shown that they recognize surface-expressed TCRs. Two of these antibodies were found to react specifically with the products of the V alpha <em>2</em> (V <em>delta</em> 8) and V beta <em>2</em> gene segments, respectively. When considered together, these data suggest that these soluble TCR molecules are folded in a conformation indistinguishable from that which they assume at the cell surface.

OBJECTIVE

To obtain systematic information on the extrinsic coagulation pathway, as well as to investigate the time course of the coagulation abnormalities in sepsis.

METHODS

Prospective observational study.

METHODS

General intensive care unit.

METHODS

Nineteen patients with the diagnosis of severe sepsis or septic shock and nine control patients.

METHODS

None.

RESULTS

Tissue factor antigen concentration (tissue factor antigen), prothrombin fragment F1+<em>2</em>, thrombin antithrombin III complex, fibrinopeptide A, <em>D</em>-<em>dimer</em>, and antithrombin III concentrations were measured on the day of diagnosis of severe sepsis and septic shock, and on days 1, <em>2</em>, 3, and 4 after diagnosis. The concentrations of tissue factor antigen, prothrombin fragment F1+<em>2</em>, fibrinopeptide A, and <em>D</em>-<em>dimer</em> were significantly increased in patients with severe sepsis and septic shock compared with control subjects. However, the concentrations of thrombin antithrombin III complex showed no statistical differences between the septic patients and the control subjects. Significantly, low antithrombin III concentrations were observed in the septic patient groups compared with control subjects. With the exception of <em>D</em>-<em>dimer</em>, the concentrations of the hemostatic markers were similar between severe sepsis and septic shock patients. Significant correlations were noted between tissue factor antigen and the disseminated intravascular coagulation score (r<em>2</em>=.<em>2</em>36, p< .0001) and the number of dysfunctioning organs (r<em>2</em>=.<em>2</em><em>2</em>9, p=.035).

CONCLUSIONS

We systematically elucidated coagulation disorders in newly defined sepsis. The extrinsic coagulation pathway is activated in patients with severe sepsis and septic shock. In these patients, enhanced thrombin generation and activation, and fibrin formation were demonstrated when compared with the control subjects. Furthermore, the thrombin generated appears not to be fully neutralized by antithrombin III.

OBJECTIVE

To measure the prevalence of and characterize coagulopathy in patients with blunt brain injury.

METHODS

Retrospective observation study based on review of medical records.

METHODS

Acutely injured patients admitted to a level I trauma center.

METHODS

One hundred fifty-nine patients with evidence of blunt head trauma who had computed tomography of the brain during initial evaluation and a coagulopathy score assigned based on 5 laboratory tests: platelet count, prothrombin time, partial thromboplastin time, fibrinogen level, and D-dimer level. The disseminated intravascular coagulation score ranged from 0 (no coagulopathy) to 15 (severe coagulopathy). Only individuals with intracranial injury based on computed tomography of the brain were designated as brain injured.

METHODS

Presence of coagulopathy, progression of brain injury, and death.

RESULTS

Among the 91 patients with brain injury, 41% had coagulopathy (disseminated intravascular coagulation score>> or = 5). Of the 68 patients without brain injury, 25% had coagulopathy. The patients with brain injury who developed profound depletion of fibrinogen did so within 4 hours of injury. There were 28 deaths (26 in the group with brain injury and 2 in the group without brain injury). Among patients with brain injury, those with coagulopathy more frequently died (P < .05 by chi 2 analysis). Patients with brain injury and coagulopathy deteriorated more frequently based on computed tomography criteria.

CONCLUSIONS

After blunt brain injury, a disseminated intravascular coagulation syndrome can lead to consumptive coagulopathy that is associated with a higher frequency of death. The syndrome develops within 1 to 4 hours after injury. Therapeutic interventions need to be implemented immediately to be effective.

BACKGROUND

In microorganisms and plants the first step in the common pathway leading to the biosynthesis of aromatic compounds is the stereospecific condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P) giving rise to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). This reaction is catalyzed by DAHP synthase (DAHPS), a metal-activated enzyme, which in microorganisms is the target for negative-feedback regulation by pathway intermediates or by end products. In Escherichia coli there are three DAHPS isoforms, each specifically inhibited by one of the three aromatic amino acids.

RESULTS

The crystal structure of the phenylalanine-regulated form of DAHPS complexed with PEP and Pb2+ (DAHPS(Phe)-PEP-Pb) was determined by multiple wavelength anomalous dispersion phasing utilizing the anomalous scattering of Pb2+. The tetramer consists of two tight dimers. The monomers of the tight dimer are coupled by extensive interactions including a pair of three-stranded, intersubunit beta sheets. The monomer (350 residues) is a (beta/alpha)8 barrel with several additional beta strands and alpha helices. The PEP and Pb2+ are at the C-ends of the beta strands of the barrel, as is SO4(2-), inferred to occupy the position of the phosphate of E4P. Mutations that reduce feedback inhibition cluster about a cavity near the twofold axis of the tight dimer and are centered approximately 15 A from the active site, indicating the location of a separate regulatory site.

CONCLUSIONS

The crystal structure of DAHPS(Phe)-PEP-Pb reveals the active site of this key enzyme of aromatic biosynthesis and indicates the probable site of inhibitor binding. This is the first reported structure of a DAHPS; the structure of its two paralogs and of a variety of orthologs should now be readily determined by molecular replacement.

The hamster gene encoding the 78-k<em>D</em>a glucose-regulated protein (Grp78) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. After induction with isopropyl beta-<em>D</em>-thiogalactopyranoside, the recombinant Grp78 was purified to homogeneity by affinity column chromatography of the fusion protein followed by thrombin cleavage. The purified recombinant protein was compared with liver Grp78 for its ability to interact with ATP. Like liver Grp78, the recombinant protein contained a weak ATPase activity and a Ca(<em>2</em>+)-stimulated autophosphorylation activity. However, unlike liver Grp78, in which the autophosphorylation reaction is stimulated less than 50% by CaCl<em>2</em>, the reaction with the recombinant Grp78 was stimulated about 15-fold in the presence of Ca<em>2</em>+. Although the liver protein showed at least four isoforms after two-dimensional gel electrophoresis, the recombinant Grp78 had one major species corresponding to the most basic form seen in liver. Both the liver Grp78 and the recombinant protein existed primarily as monomers and <em>dimers</em>. A small amount of oligomers was also present in the liver Grp78. When either protein was incubated with ATP, there was a conversion of the higher molecular weight species to the monomeric form.

beta B<em>2</em>- and gamma B-crystallins of vertebrate eye lens are <em>2</em>-domain proteins in which each domain consists of <em>2</em> Greek key motifs connected by a linker peptide. Although the folding topologies of beta B<em>2</em>- and gamma B-domains are very similar, gamma B-crystallin is always monomeric, whereas beta B<em>2</em>-crystallin associates to homo<em>dimers</em>. It has been suggested that the linker or the protruding N- and C-terminal arms of beta B<em>2</em>-crystallin (not present in gamma B) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two beta B<em>2</em> mutants. In the first mutant, the linker peptide was replaced with the one from gamma B (beta B<em>2</em> gamma L). In the second mutant, the N- and C-terminal arms of 15- and 1<em>2</em>-residues length were deleted (beta B<em>2</em> <em>delta</em> NC). The beta B<em>2</em> gamma L mutant is monomeric, whereas the beta B<em>2</em> <em>delta</em> NC mutant forms <em>dimers</em> and tetramers that cannot be interconverted without denaturation. The spectral properties of the beta B<em>2</em> mutants, as well as their stabilities against denaturants, resemble those of wild-type beta B<em>2</em>-crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in beta B<em>2</em>-crystallin is necessary for dimerization, whereas the N- and C-terminal arms appear to be involved in preventing the formation of higher homo-oligomers.

1. The absorption spectra of deutero- and proto-ferrihaem in aqueous solution at <em>2</em>5 degrees C show marked changes with concentration and pH in the Soret band region. Quantitative studies of these phenomena imply that they are associated with ferrihaem dimerization and with protolytic equilibria involving monomeric (M) and dimeric (<em>D</em>) ferrihaem species according to the scheme: [Formula: see text] <em>2</em>. For deuteroferrihaem we obtain K=1.9x10(-<em>2</em>), pK(a(M))=7.1, pK(a(<em>D</em>))=7.4. Protoferrihaem has a much higher dimerization constant, K=4.5 and pK(a(<em>D</em>))=7.5 (pK(a(M)) is not accessible). 3. Possible structural relationships between monomeric and dimeric ferrihaem species in solution are discussed in relation to recent work on the oxo-bridged nature of crystalline ferrihaem <em>dimers</em>.

Soybean agglutinin (SBA) (Glycine max), which is a tetrameric GalNAc/Gal-specific lectin, has recently been reported to form unique, highly organized cross-linked complexes with a series of naturally occurring and synthetic multiantennary carbohydrates with terminal GalNAc or Gal residues [Gupta, <em>D</em>., Bhattacharyya, L., Fant, J., Macaluso, F., Sabesan, S., & Brewer, C. F. (1994) Biochemistry 33, 7495-7504]. In order to elucidate the nature of these complexes, the X-ray crystallographic structure of SBA cross-linked with a biantennary analog of the blood group I carbohydrate antigen is reported. The structure reveals that lattice formation is promoted uniquely by the bridging action of the bivalent pentasaccharide (beta-LacNAc)<em>2</em>Gal-beta-R, where R is -O(CH<em>2</em>)5COOCH3 and the beta-LacNAc moieties are linked to the <em>2</em> and 6 positions of the core Gal. The structure of SBA complexed with the synthetic biantennary pentasaccharide has thus been determined by molecular replacement techniques and refined at <em>2</em>.6 A resolution to an R value of <em>2</em>0.1%. The crystals are hexagonal with a P6(4)<em>2</em><em>2</em> space group, which differs significantly from that of crystals of the free protein. In the structure, each monomeric asymmetric unit contains a Man9 oligomannose-type chain at Asn 75, with only the first two GlcNAc residues visible. The overall tertiary structure of the SBA subunit is similar to that of other legume lectins as well as certain animal lectins. However, the <em>dimer</em> interface in the SBA tetramer is unusual in that only one complete peptide chain is sterically permitted, thus requiring juxtapositioning of one C-terminal fragmented subunit together with an intact subunit. Association between SBA tetramers involves binding of the terminal Gal residues of the pentasaccharide at identical sites in each monomer, with the sugar cross-linking to a symmetry-related neighbor molecule. The cross-linking pentasaccharide is in a conformation that possesses a pseudo-<em>2</em>-fold axis of symmetry which lies on a crystallographic <em>2</em>-fold axis of symmetry of the lattice. Hence, the symmetry properties of the bivalent oligosaccharide as well as the lectin are structural determinants of the lattice. The results are discussed in terms of multidimensional carbohydrate-lectin cross-linked complexes, as well as the signal transduction properties of multivalent lectins.

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C<em>2</em>-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H <em>delta</em>(<em>2</em>)) resonances, correlated with the N epsilon(<em>2</em>) and N <em>delta</em>(1) chemical shifts of all 13 surface histidines per alpha beta <em>dimer</em>. The HMQC spectrum also allows the assignment of the H <em>delta</em>(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta <em>dimer</em> in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.

The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes. It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K. R., Murphy, J. B., and Chase, J. W. (1984) J. Biol. Chem. <em>2</em>59, 11804-11811). We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl<em>2</em>, urea, and guanidine hydrochloride concentrations. The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer. The experimental isotherms indicate that SSB-1 <em>dimers</em> are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers. At <em>2</em>5 degrees C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (MT)1/<em>2</em> = 0.87 microM, corresponding to a monomer-tetramer equilibrium constant, KT = 3 +/- 1 x 10(18) M-3. The tetramerization constant, KT, is highly dependent upon temperature and pH, with <em>delta</em> H0 = -51 +/- 7 kcal/mol (pH 8.1) and <em>delta</em> H0 = -37 +/- 5 kcal/mol (pH 6.9). There is no effect of NaCl on the monomer-tetramer association in the range from 0.<em>2</em>0 to 1.0 M; however, MgCl<em>2</em> decreases the stability of the SSB-1 tetramer. In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically. The large negative <em>delta</em> H0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.

Vascular endothelial growth factor-<em>D</em> (VEGF-<em>D</em>), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-<em>2</em> (VEGFR-<em>2</em>/Flk1/K<em>D</em>R) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-<em>D</em> consists of a central receptor-binding VEGF homology domain (VH<em>D</em>) and N-terminal and C-terminal propeptides that are cleaved from the VH<em>D</em> to generate a mature, bioactive form consisting of <em>dimers</em> of the VH<em>D</em>. Here we report characterization of mAbs raised to the VH<em>D</em> of human VEGF-<em>D</em> in order to generate VEGF-<em>D</em> antagonists. The mAbs bind the fully processed VH<em>D</em> with high affinity and also bind unprocessed VEGF-<em>D</em>. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-<em>2</em> and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated V<em>D</em>1, is able to compete potently with mature VEGF-<em>D</em> for binding to both VEGFR-<em>2</em> and VEGFR-3 for binding to mature VEGF-<em>D</em>. This indicates that the binding epitopes on VEGF-<em>D</em> for these two receptors may be in close proximity. Furthermore, V<em>D</em>1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-<em>D</em>. The anti-(VEGF-<em>D</em>) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-<em>D</em>.

The complete amino acid sequence of wheat germ agglutinin isolectin <em>2</em> has been determined by the method of sequential Edman degradation and with the aid of the three-dimensional structure known from X-ray crystallography. Peptides ranging from <em>2</em> to 18 residues in length were obtained by thermolysin digestion of the S-carboxymethylated protein and purified by gel filtration and high-performance liquid chromatography. The peptide order was established primarily by matching (carboxymethyl)cysteines with the clearly defined half-cystine positions in the X-ray structure, thereby satisfying the disulfide repeat pattern observed in all four isostructural domains (A, B, C, and <em>D</em>) of wheat germ agglutinin, and by examination of amino acid compositions and terminal sequences of ten tryptic peptides. The unique assignment of peptides to these domains was consistent with all invariant half-cystines and glycines, as well as the single tryptophan, the two closely spaced histidines, and a number of other residues clearly identified in the X-ray structure analysis. <em>D</em>iscrepancies between the chemical and X-ray sequences lie exclusively in poorly defined regions of the electron density map, at the N- and C-termini, and at the first intercystine loop of each domain. The latter loop was found to be eight instead of six residues in length, thus extending the size of domains A, B, and C from 41 to 43 residues and that of domain <em>D</em> to 4<em>2</em> residues. Regions of extensive interdomain homology, in addition to that of the half-cystines, are clustered at the central portion of each domain fold and are likely to be important for the integrity of the three-dimensional structure of the <em>dimer</em> molecule.

Carotid atherosclerotic plaque remodelling and increased risk of symptomatic plaque rupture seem to be partially mediated by matrix metalloproteinases (MMPs). In this study, we have investigated whether different MMPs are related to carotid atherosclerosis or to recent ischaemic brain disease. Eighty-four consecutive patients undergoing carotid endarterectomy for symptomatic and asymptomatic disease were studied. Plaques were analysed by ultrasound and later by morphology. Plasma MMP-<em>2</em>, MMP-8 and MMP-9 levels were quantified by ELISA. MMP expression and activity in carotid plaques was analysed by Western blotting and in situ zymography. Results were analysed with respect to plaque stability, morphology, symptomatic disease, presence of vascular risk factors and plasma markers of acute inflammation as high sensitivity C-reactive protein (hsCRP), fibrinogen, <em>D</em>-<em>dimer</em> and white blood cell counts. Patients with hypoechogenic plaques on ultrasound had more plasma MMP-8 (p = 0.04) and increased MMP activity as assessed by in situ zymography. Asymptomatic patients with plaque progression had more active intraplaque MMP-8 than asymptomatic patients without plaque progression. Presence of recent intraplaque haemorrhage or past history of CA<em>D</em> was related to increased activity of MMPs as assessed by in situ zymography (p < 0.01, CI 95% 0.8-1.0). Plasma MMP-8 and MMP-9, but not MMP-<em>2</em> levels, decrease with time after ischaemic stroke. Patients with hypertension had more intraplaque active MMP-9 than normotensive (p = 0.03, CI 95% 0.7-1.0). Hypoechogenic carotid plaques had increased MMP activity and asymptomatic patients with plaque progression show increase intraplaque MMP-8 levels.

OBJECTIVE

Nonbacterial thrombotic endocarditis can complicate various malignancies and may cause morbidity and mortality mainly as a result of systemic embolism. The antemortem diagnosis of nonbacterial thrombotic endocarditis is rare. The purpose of our study was to assess the frequency, echocardiographic characteristics, and clinical correlation of nonbacterial thrombotic endocarditis in cancer patients.

METHODS

A prospective echocardiographic screening of 200 nonselected ambulatory patients with solid tumors was performed. Patients were evaluated for evidence of thromboembolic events and for plasma D-dimer levels. A cohort of 100 consecutive patients without overt heart disease referred to echocardiography for the detection of an occult arterial embolic source served as a control group. It consisted of 52 males and 48 females, median age 60 years.

RESULTS

The study group included 87 women and 113 men, median age 64 years (range 21 to 91). The frequent malignancies were lymphoma (26%), carcinoma of the gastrointestinal tract (20%), and carcinoma of the lung (16%). Cardiac valvular vegetations were found in 38 patients (19%) compared with only in 2 patients in the control group (2%, P < 0.001). Vegetations were found on the mitral or on the aortic valve in 19 and 18 patients, respectively. Isolated tricuspid valve vegetation was found in 1 patient. Valvular lesions were mostly common in patients with carcinoma of the pancreas (3 of 6, 50%), carcinoma of the lung (9 of 32, 28%), and lymphoma (10 of 52, 19%). Thromboembolism was diagnosed in 22 (11%) patients (12 deep vein thrombosis, 4 emboli to extremities, 2 cerebrovascular accidents, and 4 "silent" segmental left ventricular wall motion abnormalities on echocardiography). Thromboembolism was noticed in 9 of 38 patients (24%) with vegetations compared with 13 of 162 patients without vegetations (8%; P = 0.013). Plasma D-dimer level was examined in a subgroup of 170 patients. D-dimer level was increased in 19 of 21 patients (90%) with thromboembolism compared with 76 of 149 patients without thromboembolism (51%; P = 0.001).

CONCLUSIONS

This study demonstrated a high prevalence of cardiac valvular lesions in patients with solid tumors. Vegetations were associated with thromboembolism. Plasma D-dimer level was significantly increased in patients with thromboembolism.

OBJECTIVE

The principal objective of this study was to determine the relationship between preoperative coagulation tests and the extent of tumor involvement in gastric cancer patients.

METHODS

A total of 110 patients with adenocarcinoma of the stomach were studied in order to evaluate this relationship. Platelet count (P), prothrombin time (PT), activated partial thromboplastin time, <em>D</em>-<em>dimer</em>, fibrinogen degradation product, thrombin-antithrombin complex and prothrombin fragment F1+<em>2</em> (F1+<em>2</em>) were evaluated.

RESULTS

The <em>D</em>-<em>dimer</em> levels were positively correlated with the depth of invasion (P =0.007). Plasma <em>D</em>-<em>dimer</em> and PT were highly correlated with degree of lymph node involvement (P = 0.006, 0.004, respectively). <em>D</em>-<em>dimer</em> level, PT and plasma F1+<em>2</em> level were correlated with clinical stage (P = 0.001, 0.017, 0.031, respectively). PT and F1+<em>2</em> levels were significant in the prediction of the presence of lymph node involvement on the multivariate logistic regression models (odds ratio <em>2</em>50<em>2</em>.081 (5.977-10474<em>2</em>5.4); P = 0.010 and odds ratio 19.487 (1.495-<em>2</em>53.936); P = 0.0<em>2</em>3, respectively).

CONCLUSIONS

PT and plasma levels of F1+<em>2</em> and <em>D</em>-<em>dimer</em> could be markers of degree or presence of lymph node involvement and clinical stage in patients with operable gastric cancer.

BACKGROUND

Traumatic brain injury (TBI) initiates interrelated inflammatory and coagulation cascades characterized by wide-spread cellular activation, induction of leukocyte and endothelial cell adhesion molecules and release of soluble pro/antiinflammatory cytokines and thrombotic mediators. Resuscitative care is focused on optimizing cerebral perfusion and reducing secondary injury processes. Hypertonic saline is an effective osmotherapeutic agent for the treatment of intracranial hypertension and has immunomodulatory properties that may confer neuroprotection. This study examined the impact of hypertonic fluids on inflammatory/coagulation cascades in isolated head injury.

METHODS

Using a prospective, randomized controlled trial we investigated the impact of prehospital resuscitation of severe TBI (GCS < 8) patients using 7.5% hypertonic saline in combination with 6% dextran-70 (HSD) vs 0.9% normal saline (NS), on selected cellular and soluble inflammatory/coagulation markers. Serial blood samples were drawn from 65 patients (30 HSD, 35 NS) at the time of hospital admission and at 12, 24, and 48-h post-resuscitation. Flow cytometry was used to analyze leukocyte cell-surface adhesion (CDDDDD-dimers (D-D) were assessed by enzyme immunoassay. Twenty-five healthy subjects were studied as a control group.

RESULTS

TBI provoked marked alterations in a majority of the inflammatory/coagulation markers assessed in all patients. Relative to control, NS patients showed up to a 2-fold higher surface expression of CDDDD blunted the expression of these cell-surface activation/adhesion molecules at all time-points to levels approaching control values. Admission concentrations of endothelial-derived sVCAM-1 and sE-selectin were generally reduced in HSD patients. Circulating sL-selectin levels were significantly elevated at 12 and 48, but not 24 h post-resuscitation with HSD. TNF-alpha and IL-10 levels were elevated above control throughout the study period in all patients, but were reduced in HSD patients. Plasma sTF and D-D levels were also significantly lower in HSD patients, whereas sTM levels remained at control levels.

CONCLUSIONS

These findings support an important modulatory role of HSD resuscitation in attenuating the upregulation of leukocyte/endothelial cell proinflammatory/prothrombotic mediators, which may help ameliorate secondary brain injury after TBI.

BACKGROUND

NCT00878631.

OBJECTIVE

Technical complications are a known hazard in veno-venous extracorporeal membrane oxygenation (vvECMO). Identifying these complications and predictive factors indicating a developing system-exchange was the goal of the study.

METHODS

Retrospective study on prospectively collected data of technical complications including <em>2</em>65 adult patients (Regensburg ECMO Registry, <em>2</em>009-<em>2</em>013) with acute respiratory failure treated with vvECMO. Alterations in blood flow resistance, gas transfer capability, hemolysis, coagulation and hemostasis parameters were evaluated in conjunction with a system-exchange in all patients with at least one exchange (n = 83).

RESULTS

Values presented as median (interquartile range). Patient age was 50(36-60) years, the SOFA score 11(8-14.3) and the Murray lung injury Score 3.33(3.3-3.7). Cumulative ECMO support time 3411 days, 9(6-15) days per patient. Mechanical failure of the blood pump (n = 5), MO (n = <em>2</em>) or cannula (n = 1) accounted for 10% of the exchanges. Acute clot formation within the pump head (visible clots, increase in plasma free hemoglobin (frHb), serum lactate dehydrogenase (L<em>D</em>H), n = 13) and MO (increase in pressure drop across the MO, n = 16) required an urgent system-exchange, of which nearly 50% could be foreseen by measuring the parameters mentioned below. Reasons for an elective system-exchange were worsening of gas transfer capability (n = 10) and device-related coagulation disorders (n = 3<em>2</em>), either local fibrinolysis in the MO due to clot formation (increased <em>D</em>-<em>dimers</em> [<em>D</em><em>D</em>]), decreased platelet count; n = <em>2</em>4), or device-induced hyperfibrinolysis (increased <em>D</em><em>D</em>, decreased fibrinogen [FG], decreased platelet count, diffuse bleeding tendency; n = 8), which could be reversed after system-exchange. Four MOs were exchanged due to suspicion of infection.

CONCLUSIONS

The majority of ECMO system-exchanges could be predicted by regular inspection of the complete ECMO circuit, evaluation of gas exchange, pressure drop across the MO and laboratory parameters (DD, FG, platelets, LDH, frHb). These parameters should be monitored in the daily routine to reduce the risk of unexpected ECMO failure.

It has been reported previously that ATP inhibits the insulysin reaction (Camberos, M. C., Perez, A. A., Udrisar, <em>D</em>. P., Wanderley, M. I., and Cresto, J. C. (<em>2</em>001) Exp. Biol. Med. <em>2</em><em>2</em>6, 334-341). We report here that with <em>2</em>-aminobenzoyl-GGFLRKHGQ-ethylenediamine-<em>2</em>,4-dinitrophenyl as substrate, ATP and other nucleotides increase the rate>><em>2</em>0-fold in Tris buffer. There is no specificity with respect to the nucleotide; however, ATP is more effective than A<em>D</em>P, which is more effective than AMP. Triphosphate itself was as effective as ATP, indicating it is this moiety that is responsible for activation. The binding of triphosphate was shown to be at a site distinct from the active site, thus acting as a noncompetitive activator. With the physiological substrates insulin and amyloid beta peptide, nucleotides and triphosphate were without effect. However, with small physiological peptides such as bradykinin and dynorphin B-9, ATP and triphosphate increased the rate of hydrolysis approximately 10-fold. Triphosphate and ATP shifted the oligomeric state of the enzyme from primarily <em>dimer</em>-tetramers to a monomer. These data suggest the presence of an allosteric regulatory site on insulysin that may shift its specificity toward small peptide substrates.

OBJECTIVE

To investigate if plasma DNA is elevated in patients with deep vein thrombosis (DVT) and to determine whether there is a correlation with other biomarkers of DVT.

BACKGROUND

Leukocytes release DNA to form extracellular traps (ETs), which have recently been linked to experimental DVT. In baboons and mice, extracellular DNA co-localized with von Willebrand factor (VWF) in the thrombus and DNA appeared in circulation at the time of thrombus formation. ETs have not been associated with clinical DVT.

METHODS

From December <em>2</em>008 to August <em>2</em>010, patients were screened through the University of Michigan Diagnostic Vascular Unit and were divided into three distinct groups: 1) the DVT positive group, consisting of patients who were symptomatic for DVT, which was confirmed by compression duplex ultrasound (n=47); <em>2</em>) the DVT negative group, consisting of patients that present with swelling and leg pain but had a negative compression duplex ultrasound, (n=<em>2</em>8); and 3) a control group of healthy non-pregnant volunteers without signs or symptoms of active or previous DVT (n=19). Patients were excluded if they were less than 18 years of age, unwillingness to consent, pregnant, on an anticoagulant therapy, or diagnosed with isolated calf vein thrombosis.

METHODS

Blood was collected for circulating DNA, CRP, D-dimer, VWF activity, myeloperoxidase (MPO), ADAMTS13 and VWF. The Wells score for a patient's risk of DVT was assessed. The Receiver Operating Characteristic (ROC) curve was generated to determine the strength of the relationship between circulating DNA levels and the presence of DVT. A Spearman correlation was performed to determine the relationship between the DNA levels and the biomarkers and the Wells score. Additionally the ratio of ADAMTS13/VWF was assessed.

RESULTS

Our results showed that circulating DNA (a surrogate marker for NETs) was significantly elevated in DVT patients, compared to both DVT negative patients (57.7±6.3 vs. 17.9±3.5ng/mL, P<.01) and controls (57.7±6.3 vs. <em>2</em>3.9±<em>2</em>.1ng/mL, P<.01). There was a strong positive correlation with CRP (P<.01), D-dimer (P<.01), VWF (P<.01), Wells score (P<.01) and myeloperoxidase (MPO) (P<.01), along with a strong negative correlation with ADAMTS13 (P<.01) and the ADAMTS13/VWF ratio. The logistic regression model showed a strong association between plasma DNA and the presence of DVT (ROC curve was determined to be 0.814).

CONCLUSIONS

Plasma DNA is elevated in patients with deep vein thrombosis and correlates with biomarkers of DVT. A strong correlation between circulating DNA and MPO suggests that neutrophils may be a source of plasma DNA in patients with DVT.

UNASSIGNED

Nearly half of all patients with heart failure have preserved ejection fraction (HFpEF) as opposed to reduced ejection fraction (HFrEF), yet associations of biomarkers with future heart failure subtype are incompletely understood.

UNASSIGNED

To evaluate the associations of 12 cardiovascular biomarkers with incident HFpEF vs HFrEF among adults from the general population.

UNASSIGNED

This study included 4 longitudinal community-based cohorts: the Cardiovascular Health Study (1989-1990; 1992-1993 for supplemental African-American cohort), the Framingham Heart Study (1995-1998), the Multi-Ethnic Study of Atherosclerosis (2000-2002), and the Prevention of Renal and Vascular End-stage Disease study (1997-1998). Each cohort had prospective ascertainment of incident HFpEF and HFrEF. Data analysis was performed from June 25, 2015, to November 9, 2017.

UNASSIGNED

The following biomarkers were examined: N-terminal pro B-type natriuretic peptide or brain natriuretic peptide, high-sensitivity troponin T or I, C-reactive protein (CRP), urinary albumin to creatinine ratio (UACR), renin to aldosterone ratio, D-dimer, fibrinogen, soluble suppressor of tumorigenicity, galectin-3, cystatin C, plasminogen activator inhibitor 1, and interleukin 6.

UNASSIGNED

Development of incident HFpEF and incident HFrEF.

UNASSIGNED

Among the 22 756 participants in these 4 cohorts (12 087 women and 10 669 men; mean [SD] age, 60 [13] years) in the study, during a median follow-up of 12 years, 633 participants developed incident HFpEF, and 841 developed HFrEF. In models adjusted for clinical risk factors of heart failure, 2 biomarkers were significantly associated with incident HFpEF: UACR (hazard ratio [HR], 1.33; 95% CI, 1.20-1.48; P < .001) and natriuretic peptides (HR, 1.27; 95% CI, 1.16-1.40; P < .001), with suggestive associations for high-sensitivity troponin (HR, 1.11; 95% CI, 1.03-1.19; P = .008), plasminogen activator inhibitor 1 (HR, 1.22; 95% CI, 1.03-1.45; P = .02), and fibrinogen (HR, 1.12; 95% CI, 1.03-1.22; P = .01). By contrast, 6 biomarkers were associated with incident HFrEF: natriuretic peptides (HR, 1.54; 95% CI, 1.41-1.68; P < .001), UACR (HR, 1.21; 95% CI, 1.11-1.32; P < .001), high-sensitivity troponin (HR, 1.37; 95% CI, 1.29-1.46; P < .001), cystatin C (HR, 1.19; 95% CI, 1.11-1.27; P < .001), D-dimer (HR, 1.22; 95% CI, 1.11-1.35; P < .001), and CRP (HR, 1.19; 95% CI, 1.11-1.28; P < .001). When directly compared, natriuretic peptides, high-sensitivity troponin, and CRP were more strongly associated with HFrEF compared with HFpEF.

UNASSIGNED

Biomarkers of renal dysfunction, endothelial dysfunction, and inflammation were associated with incident HFrEF. By contrast, only natriuretic peptides and UACR were associated with HFpEF. These findings highlight the need for future studies focused on identifying novel biomarkers of the risk of HFpEF.

Fibroblast growth factors (FGFs) constitute a large family of heparin-bin<em>d</em>ing growth factors with <em>d</em>iverse biological activities. FGF9 was originally <em>d</em>escribe<em>d</em> as glia-activating factor an<em>d</em> is expresse<em>d</em> in the nervous system as a potent mitogen for glia cells. Unlike most FGFs, FGF9 forms <em>dimers</em> in solution with a K(<em>d</em>) of 680 nm. To eluci<em>d</em>ate the molecular mechanism of FGF9 <em>dimer</em>ization, the crystal structure of FGF9 was <em>d</em>etermine<em>d</em> at <em>2</em>.<em>2</em> A resolution. FGF9 a<em>d</em>opts a beta-trefoil fol<em>d</em> similar to other FGFs. However, unlike other FGFs, the N- an<em>d</em> C-terminal regions outsi<em>d</em>e the beta-trefoil core in FGF9 are or<em>d</em>ere<em>d</em> an<em>d</em> involve<em>d</em> in the formation of a <em>2</em>-fol<em>d</em> crystallographic <em>dimer</em>. A significant surface area >><em>2</em>000 A(<em>2</em>)) is burie<em>d</em> in the <em>dimer</em> interface that occlu<em>d</em>es a major receptor bin<em>d</em>ing site of FGF9. Thus, we propose an autoinhibitory mechanism for FGF9 that is <em>d</em>epen<em>d</em>ent on sequences outsi<em>d</em>e of the beta-trefoil core. Moreover, a mo<em>d</em>el is presente<em>d</em> provi<em>d</em>ing a molecular basis for the preferential affinity of FGF9 towar<em>d</em> FGFR3.

Tumor hypoxia in<em>d</em>uces cancer cell angiogenesis, invasiveness, treatment resistance, an<em>d</em> contributes to poor clinical outcome. However, the molecular mechanism by which tumor hypoxia exerts a coor<em>d</em>inate<em>d</em> effect on <em>d</em>ifferent molecular pathways to enhance tumor growth an<em>d</em> survival an<em>d</em> lea<em>d</em> to poor clinical outcome is not fully un<em>d</em>erstoo<em>d</em>. In this stu<em>d</em>y, we attempt to eluci<em>d</em>ate the global protein expression an<em>d</em> functional changes in A431 epithelial carcinoma cells in<em>d</em>uce<em>d</em> by hypoxia an<em>d</em> reoxygenation using iTRAQ quantitative proteomics an<em>d</em> biochemical functional assays. Quantitative proteomics results showe<em>d</em> that 4316 proteins were quantifie<em>d</em> with FDR<1%, in which over 1<em>2</em>00 proteins were mo<em>d</em>ulate<em>d</em> >1.<em>2</em> fol<em>d</em>, an<em>d</em> DNA repair, glycolysis, integrin, glycoprotein turnover, an<em>d</em> STAT1 pathways were perturbe<em>d</em> by hypoxia an<em>d</em> reoxygenation-in<em>d</em>uce<em>d</em> oxi<em>d</em>ative stress. For the first time, hypoxia was shown to up-regulate the nonhomologous en<em>d</em>-joining pathway, which plays a central role in DNA repair of irra<em>d</em>iate<em>d</em> cells, thereby potentially contributing to the ra<em>d</em>ioresistance of hypoxic A431 cells. The up-regulation of Ku70/Ku80 <em>dimer</em>, a key molecular complex in the nonhomologous en<em>d</em>-joining pathway, was confirme<em>d</em> by Western blot an<em>d</em> liqui<em>d</em> chromatography/tan<em>d</em>em mass spectrometry-MRM metho<em>d</em>s. Functional stu<em>d</em>ies confirme<em>d</em> that up-regulation of glycolysis, integrin, glycoprotein synthesis, an<em>d</em> <em>d</em>own-regulation of STAT1 pathways <em>d</em>uring hypoxia enhance<em>d</em> metastastic activity of A431 cells. Migration of A431 cells was <em>d</em>ramatically represse<em>d</em> by glycolysis inhibitor (<em>2</em>-Deoxy-<em>d</em>-glucose), glycoprotein synthesis inhibitor (1-Deoxynojirimycin Hy<em>d</em>rochlori<em>d</em>e), an<em>d</em> STAT1α overexpression that enhance<em>d</em> the integrin-me<em>d</em>iate<em>d</em> cell a<em>d</em>hesion. These results reveale<em>d</em> that hypoxia in<em>d</em>uce<em>d</em> several biological processes involve<em>d</em> in tumor migration an<em>d</em> ra<em>d</em>ioresistance an<em>d</em> provi<em>d</em>e<em>d</em> potential new targets for tumor therapy.

Introduction: Headache is one of the most frequent neurologic manifestations in COVID-19. We aimed to analyze which symptoms and laboratory abnormalities were associated with the presence of headache and to evaluate if patients with headache had a higher adjusted in-hospital risk of mortality.

<strong class="sub-title"> Methods: </strong> Retrospective cohort study. We included all consecutive patients admitted to the Hospital with confirmed SARS-CoV-<em>2</em> infection between March 8th and April 11th, <em>2</em>0<em>2</em>0. We collected demographic data, clinical variables and laboratory abnormalities. We used multivariate regression analysis.

<strong class="sub-title"> Results: </strong> During the study period, 576 patients were included, aged 67.<em>2</em> (SD: 14.7), and <em>2</em>50/576 (43.3%) being female. Presence of headache was described by 137 (<em>2</em>3.7%) patients. The all-cause in-hospital mortality rate was 1<em>2</em>7/576 (<em>2</em>0.0%). In the multivariate analysis, patients with headache had a lower risk of mortality (OR: 0.39, 95% CI: 0.17-0.88, p = 0.007). After adjusting for multiple comparisons in a multivariate analysis, variables that were independently associated with a higher odds of having headache in COVID-19 patients were anosmia, myalgia, female sex and fever; variables that were associated with a lower odds of having headache were younger age, lower score on modified Rankin scale, and, regarding laboratory variables on admission, increased C-reactive protein, abnormal platelet values, lymphopenia and increased D-dimer.

Conclusion: Headache is a frequent symptom in COVID-19 patients and its presence is an independent predictor of lower risk of mortality in COVID-19 hospitalized patients.

Keywords: COVID-19; Headache disorders, secondary; Mortality, laboratory parameters; Nervous system diseases.