OBJECTIVE

We derive a decision rule to partition emergency department patients with suspected pulmonary embolism (PE) into a small, high-risk group (>40% pretest probability) that is unsafe for D -dimer testing and a larger group that is safe to have PE ruled out with either a whole-blood D -dimer plus alveolar deadspace measurement or a quantitative D -dimer assay.

METHODS

Nine hundred thirty-four patients with suspected PE were studied at 7 urban EDs in the United States. Patients were prospectively interviewed and examined for recognized symptoms, signs, and risk factors associated with PE. These data were collected before standard objective imaging for PE. Selected variables were analyzed by multivariate logistic analysis to determine factors associated with PE (P <.05). A decision rule was then constructed to categorize approximately 80% of ED patients as safe for D -dimer testing.

RESULTS

Pretest prevalence of PE was 19.4% (181/934; 95% confidence interval [CI] 16.3% to 21.7%). Six variables found to be significant on multivariate analysis were used to construct the decision rule. Unsafe patients had either a shock index (heart rate/systolic blood pressure) more than 1.0 or age older than 50 years, together with any one of the following conditions: unexplained hypoxemia (SaO (2) <95%; no prior lung disease), unilateral leg swelling, recent major surgery, or hemoptysis. These criteria were met by 197 (21.0%) of 934 patients, and 83 of 197 (42.1%; 95% CI 35.3% to 49.6%) patients had PE. Exclusion of these 197 unsafe patients significantly decreased the probability of PE in the remaining 737 (79.0%) safe patients to 13.3% (95% CI 10.9% to 15.9%).

CONCLUSIONS

Simple clinical criteria can permit safe D -dimer testing in the majority of ED patients with suspected PE. These criteria warrant prospective validation.

Overexposure to short- an<em>d</em> long-wave ultraviolet ra<em>d</em>iations (UVB, UVA) may contribute to melanoma <em>d</em>evelopment through combine<em>d</em> genotoxic an<em>d</em> mitogenic effects in melanocytes. This stu<em>d</em>y compares the impact of UVA-1 versus UVB, an<em>d</em> single versus fractionate<em>d</em> exposures on melanocyte proliferation in hairless SKH-<em>2</em> mice. A single erythemal <em>d</em>ose was compare<em>d</em> with an equal <em>d</em>ose fractionate<em>d</em> over 8 <em>d</em>, an<em>d</em> <em>d</em>ose-<em>d</em>epen<em>d</em>ency was stu<em>d</em>ie<em>d</em>. Proliferation (Ki-67 positive-sign) in melanocytes (melanoma antigen recognize<em>d</em> by T-cells-1 positive or micropthalmia transcription factor positive) was ascertaine<em>d</em> in <em>d</em>ouble-labele<em>d</em> skin sections. Single erythemal UVB exposures cause<em>d</em> a <em>d</em>elaye<em>d</em>, <em>d</em>ose-<em>d</em>epen<em>d</em>ent increase of melanocyte proliferation. The highest, 17-fol<em>d</em>, increase (from 0.05% to 0.8% of melanocytes) occurre<em>d</em> 4 <em>d</em> after UVB exposure, without any <em>d</em>etectable effect on overall melanocyte numbers. Correspon<em>d</em>ingly, DNA repair-<em>d</em>eficient xero<em>d</em>erma pigmentosum A (Xpa) mice prove<em>d</em> exquisitely sensitive to melanocyte proliferation in<em>d</em>uction by UVB exposure. No <em>d</em>iscernable effects were measure<em>d</em> from fractionate<em>d</em> suberythemal UVB exposures, or from any UVA-1 exposure regimen. Hence, melanocyte proliferation appears to be most efficiently in<em>d</em>uce<em>d</em> by a single UVB overexposure. Moreover, the ineffectiveness of UVA-1 ra<em>d</em>iation an<em>d</em> the enhance<em>d</em> sensitivity of Xpa mice point at pyrimi<em>d</em>ine <em>dimers</em> as causative DNA lesions. Consequently, murine nevi an<em>d</em> melanoma are expecte<em>d</em> to be most effectively in<em>d</em>uce<em>d</em> by intermittent UVB overexposures.

Magnetic switchable maleonitrile<em>d</em>ithiolate (mnt) complexes were stu<em>d</em>ie<em>d</em> by <em>d</em>ensity functional theory. The calculations were performe<em>d</em> for anion <em>dimers</em> of [RBzPyR'][Ni(mnt)(<em>2</em>)] (RBzPyR' = <em>d</em>erivatives of benzylpyri<em>d</em>inium) to eluci<em>d</em>ate magnetostructural correlations an<em>d</em> the nature of the weak intermolecular chemical bon<em>d</em>ing. The calculate<em>d</em> results showe<em>d</em> that the spin <em>d</em>elocalization, favore<em>d</em> by the eclipse<em>d</em> stacking an<em>d</em> the shorter interlayer <em>d</em>istance, was responsible for the <em>d</em>iamagnetic character of [1-benzyl-4-aminopyri<em>d</em>inium][Ni(mnt)(<em>2</em>)] at low temperature. The weak antiferromagnetic an<em>d</em> ferromagnetic interactions were also repro<em>d</em>uce<em>d</em> for [1-benzyl-4-aminopyri<em>d</em>inium][Ni(mnt)(<em>2</em>)] an<em>d</em> [1-(4'-fluorobenzyl)pyri<em>d</em>inium][Ni(mnt)(<em>2</em>)] at high temperature, respectively. The natural bon<em>d</em> orbital analysis suggeste<em>d</em> that the cooperative effect of the weak intermolecular bon<em>d</em>ings may be the intrinsic <em>d</em>riving force resulting in the switchable property, which is essentially similar to those in organic ra<em>d</em>icals exhibiting magnetic bistability. Further investigations with varying interlayer <em>d</em>istance <em>d</em>, the extent of slippage (slipping <em>d</em>istance r an<em>d</em> <em>d</em>eviation angle alpha), an<em>d</em> rotational angle theta suggeste<em>d</em> that the extent of slippage playe<em>d</em> an important role in magnetic interactions. Therefore, the abrupt mo<em>d</em>ulation of the extent of slippage in the [Ni(mnt)(<em>2</em>)](-) complexes by external perturbations provi<em>d</em>e<em>d</em> new possibilities for the <em>d</em>esign of molecular magnetic switching <em>d</em>evices.

Pregnancy is considered as a hypercoagulable state and an increased incidence of thromboembolic phenomena has been reported in pregnant women. Relevant changes in the hemostatic mechanism have been reported during physiological pregnancy: briefly, increased levels of coagulation factors, enhanced thrombin generation and suppression of fibrinolysis are commonly found in women with uncomplicated pregnancy. We recently described progressive increases in fibrinogen and <em>D</em>-<em>dimer</em> plasma levels during normal pregnancy. The increase in <em>D</em>-<em>dimer</em> levels makes difficult their interpretation for the exclusion of thromboembolic phenomena in pregnancy. The behavior of prothrombin fragment 1+<em>2</em> (F1+<em>2</em>) levels during physiological pregnancy is scarcely known. The aim of this preliminary study was to establish range values of F1+<em>2</em> plasma levels for different periods of normal pregnancy.

Two automated turbidimetric <em>D</em>-<em>dimer</em> assays (BC <em>D</em>-<em>dimer</em> Plus, <em>D</em>ade Behring, Marburg, Germany and Auto-<em>Dimer</em>, Biopool, Umeå, Sweden) were compared to two enzyme-linked immunosorbent assays (ELISAs) (Enzygnost <em>D</em>-<em>dimer</em> micro, <em>D</em>ade Behring and Asserachrome <em>D</em>-<em>dimer</em>, <em>D</em>iagnostica Stago, Asnières, France) and two rapid <em>D</em>-<em>dimer</em> assays (SimpliRed, Agen Biomedical, Brisbane, Australia and Minutex, Biopool) in out-patients with suspected deep vein thrombosis (<em>D</em>VT). In addition, the performance of prothrombin fragment 1+<em>2</em> (F1+<em>2</em>), thrombin-antithrombin complex (TAT), and soluble adhesion molecules VCAM-1 and P-selectin for <em>D</em>VT diagnosis was assessed. One hundred and thirty-five consecutive out-patients with suspected <em>D</em>VT of the lower limb were included, and in 5<em>2</em> (39%), <em>D</em>VT was confirmed by compression ultrasound. All <em>D</em>-<em>dimer</em> assays investigated reliably excluded <em>D</em>VT in those patients without <em>D</em>VT irrespective of their pre-test clinical probability of <em>D</em>VT. One <em>D</em>-<em>dimer</em> ELISA (<em>D</em>ade Behring) gave the highest area under the receiver operating characteristic (ROC) curve compared to other assays, and therefore, this was the most accurate assay in differentiating patients with from patients without <em>D</em>VT. The diagnostic performance of one automated turbidimetric assay (Auto <em>Dimer</em>, Biopool) was similar to ELISA and its convenience close to rapid latex agglutination assays. Most patients with a high pre-test clinical probability of <em>D</em>VT had positive <em>D</em>-<em>dimer</em> regardless of the presence or absence of <em>D</em>VT, which decreased the specificity of the tests and made <em>D</em>-<em>dimer</em> determination less useful for this group of patients. Because the diagnostic accuracy [sensitivity, specificity, negative (NPV) and positive predictive value (PPV)] of F1+<em>2</em>, TAT, VCAM-1 and P-selectin was inferior to <em>D</em>-<em>dimer</em> assay, these assays could not substitute or supplement <em>D</em>-<em>dimer</em> testing in diagnosis of <em>D</em>VT. Levels of VCAM-1 and P-selectin were increased in patients with <em>D</em>VT and should therefore be investigated further to clarify their role in <em>D</em>VT.

BACKGROUND

In inflammatory bowel disease (IBD), gut microvascular thrombosis as well as thromboembolic complications have repeatedly been observed. We examined the long-term course of markers of coagulation and fibrinolysis in relation to clinical disease activity.

METHODS

In a prospective study, prothrombin fragment 1 and <em>2</em> (F1.<em>2</em>), thrombin-antithrombin complex (TAT), antithrombin, <em>D</em>-<em>dimer</em>, plasmin-alpha(<em>2</em>)-antiplasmin complex (PAP) and plasminogen activator inhibitor-1 (PAI-1) were measured in <em>2</em>0 patients with Crohn's disease (C<em>D</em>), 18 with ulcerative colitis (UC), and 19 with giant cell arteritis during active and inactive disease, as well as in 51 controls without inflammation.

RESULTS

Levels of F1.<em>2</em>, TAT, <em>D</em>-<em>dimer</em>, PAP and PAI-1 were significantly higher in active versus inactive C<em>D</em> and UC. However, even after 1<em>2</em> months of follow-up, in C<em>D</em> and UC the mean levels of F1.<em>2</em>, <em>D</em>-<em>dimer</em> and PAP were significantly higher than the levels of the controls.

CONCLUSIONS

Levels of F1.<em>2</em>, <em>D</em>-<em>dimer</em> and PAP were markedly raised for a long time in clinically inactive IB<em>D</em>, underlining a chronic state of hypercoagulation and enhanced fibrinolysis.

The purpose of the present stu<em>d</em>y was to assess the therapeutic effect of hypothermic retrogra<em>d</em>e jugular vein flush (HRJVF) on heatstroke. HRJVF was accomplishe<em>d</em> by infusion of 4 <em>d</em>egrees C isotonic so<em>d</em>ium chlori<em>d</em>e solution via the external jugular vein (1.7 mL/100 g of bo<em>d</em>y weight over 5 min). Imme<em>d</em>iately after the onset of heatstroke, anesthetize<em>d</em> rats were <em>d</em>ivi<em>d</em>e<em>d</em> into <em>2</em> major groups an<em>d</em> given the following: 36 <em>d</em>egrees C or 4 <em>d</em>egrees C isotonic so<em>d</em>ium chlori<em>d</em>e solution, i.v. They were expose<em>d</em> to ambient temperature of 43 <em>d</em>egrees C to in<em>d</em>uce heatstroke. Another group of rats was expose<em>d</em> to room temperature (<em>2</em>4 <em>d</em>egrees C) an<em>d</em> use<em>d</em> as normothermic controls. When the 36 <em>d</em>egrees C saline-treate<em>d</em> rats un<em>d</em>erwent heat exposure, their survival time values were foun<em>d</em> to be <em>2</em>3 to <em>2</em>8 min. Imme<em>d</em>iately after the onset of heatstroke, resuscitation with an i.v. <em>d</em>ose of 4 <em>d</em>egrees C saline significantly improve<em>d</em> survival <em>d</em>uring heatstroke (<em>2</em>08-<em>2</em>5<em>2</em> min). All heat-stresse<em>d</em> animals <em>d</em>isplaye<em>d</em> systemic inflammation an<em>d</em> activate<em>d</em> coagulation, evi<em>d</em>ence<em>d</em> by increase<em>d</em> tumor necrosis factor alpha, prothrombin time, activate<em>d</em> partial thromboplastin time, an<em>d</em> <em>d</em>-<em>dimer</em>, an<em>d</em> <em>d</em>ecrease<em>d</em> platelet count an<em>d</em> protein C. Biochemical markers evi<em>d</em>ence<em>d</em> cellular ischemia an<em>d</em> injury/<em>d</em>ysfunction: plasma levels of bloo<em>d</em> urea nitrogen, creatinine, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, an<em>d</em> alkaline phosphatase; an<em>d</em> striatal levels of glycerol, glutamate, an<em>d</em> lactate/pyruvate; <em>d</em>ihy<em>d</em>roxy benzoic aci<em>d</em>, lipi<em>d</em> peroxi<em>d</em>ation, oxi<em>d</em>ize<em>d</em>-form glutathione re<em>d</em>uce<em>d</em>-form glutathione, <em>d</em>opamine, an<em>d</em> serotonin were all elevate<em>d</em> <em>d</em>uring heatstroke. Core an<em>d</em> brain temperatures an<em>d</em> intracranial pressure were also increase<em>d</em> <em>d</em>uring heatstroke. In contrast, the values of mean arterial pressure, cerebral perfusion pressure, an<em>d</em> striatal levels of local bloo<em>d</em> flow, partial pressure of oxygen, superoxi<em>d</em>e <em>d</em>ismutase, catalase, glutathione peroxi<em>d</em>ase, an<em>d</em> glutathions re<em>d</em>uctase activities were all significantly lower <em>d</em>uring heatstroke. The circulatory <em>d</em>ysfunction, systemic inflammation, hypercoagulable state, an<em>d</em> cerebral oxi<em>d</em>ative stress, ischemia, an<em>d</em> <em>d</em>amage <em>d</em>uring heatstroke were all significantly suppresse<em>d</em> by HRJVF. These fin<em>d</em>ings <em>d</em>emonstrate that brain cooling cause<em>d</em> by HRJVF therapy may resuscitate persons who ha<em>d</em> a stroke by attenuating cerebral oxi<em>d</em>ative stress, systemic inflammation, activate<em>d</em> coagulation, an<em>d</em> tissue ischemia/injury <em>d</em>uring heatstroke.

Factor VIIIa consists of subunits <em>d</em>esignate<em>d</em> A1, A<em>2</em>, an<em>d</em> A3-C1-C<em>2</em>. The limite<em>d</em> cofactor activity observe<em>d</em> with the isolate<em>d</em> A<em>2</em> subunit is marke<em>d</em>ly enhance<em>d</em> by the A1 subunit. A truncate<em>d</em> A1 (A1(336)) was previously shown to possess similar affinity for A<em>2</em> an<em>d</em> retain approximately 60% of its A<em>2</em> stimulatory activity. We now i<em>d</em>entify a secon<em>d</em> site in A1 at Lys(36) that is cleave<em>d</em> by factor Xa. A1 truncate<em>d</em> at both cleavage sites (A1(37-336)) showe<em>d</em> little if any affinity for A<em>2</em> (K(<em>d</em>>><em>2</em> microm), whereas factor VIIIa reconstitute<em>d</em> with A<em>2</em> plus A1(37-336)/A3-C1-C<em>2</em> <em>dimer</em> <em>d</em>emonstrate<em>d</em> significant cofactor activity ( approximately 30% that of factor VIIIa reconstitute<em>d</em> with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtaine<em>d</em> by fluorescence energy transfer using acrylo<em>d</em>an-labele<em>d</em> A<em>2</em> an<em>d</em> fluorescein-labele<em>d</em> A1. In contrast, factor VIIIa reconstitute<em>d</em> with A1(37-336) showe<em>d</em> little activity in a one-stage clotting assay. This resulte<em>d</em> in part from a 5-fol<em>d</em> increase in K(m) for factor X when A1 was cleave<em>d</em> at Arg(336). These fin<em>d</em>ings suggest that both A1 termini are necessary for functional interaction of A1 with A<em>2</em>. Furthermore, the C terminus of A1 contributes to the K(m) for factor X bin<em>d</em>ing to factor Xase, an<em>d</em> this parameter is critical for activity assesse<em>d</em> in plasma-base<em>d</em> assays.

BACKGROUND

Unstable angina pectoris is frequently associated with intracoronary thrombus formation. In a prospective study, we investigated in 35 patients with unstable angina pectoris markers of coagulation and the kallikrein-kinin and fibrinolytic systems in the acute and postacute phases.

RESULTS

We determined serially in the patients up to 10 days after admission factor XII and the beta-factor XIIa inhibition, kallikrein-like activity, prekallikrein, C1-esterase inhibitor, kallikrein inhibition, high molecular weight kininogen as indicators of the contact phase and bradykinin generation, thrombin-antithrombin III (TAT) complex as marker of the activated coagulation cascade, fibrinogen, plasminogen, plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (TPA), and <em>D</em>-<em>dimers</em> as indicators of the fibrinolytic system. <em>D</em>ata were compared with those from control subjects (n = <em>2</em>5) and from patients with stable angina pectoris (n = <em>2</em>5). In patients with unstable angina pectoris, initially the contact phase and the kallikrein-kinin system were markedly elevated (factor XII, 96 +/- 5% versus 117 +/- 5%; kallikrein-like activity, 35.7 +/- <em>2</em>.9 versus <em>2</em>7.4 +/- 1.3 U/L; high molecular weight kininogen, 5<em>2</em>.7 +/- 5.<em>2</em>% versus 87.7 +/- 3.9%; P < .01 versus control subjects). Contact-phase activation persisted for the following 10 days, whereas the initially enhanced bradykinin generation normalized after <em>2</em> days. Furthermore, we had evidence of a hypercoagulative state (TAT, 10.9 +/- 3.1 versus 4.5 +/- 0.7 micrograms/L, P < .05; <em>D</em>-<em>dimer</em>, 474 +/- 81 versus <em>2</em>7<em>2</em> +/- 71 ng/mL) persisting longer than the clinically symptomatic period in association with disturbed fibrinolysis (TPA, 15.9 +/- 1.9 versus 5.1 +/- 0.4 ng/mL; P < .01; PAI-1, 9.9 +/- <em>2</em>.6 versus 4.6 +/- 1.6 AU/mL; P = NS) in the presence of elevated fibrinogen levels.

CONCLUSIONS

Our data indicate that in patients with unstable angina pectoris, intracoronary thrombus formation is associated with a hypercoagulative state, including activation of the contact phase and of the kallikrein system and increased bradykinin generation. The persistence of this hypercoagulative state, together with a disturbed fibrinolysis, might indicate an increased risk for further coronary events.

The PK<em>D</em>1 and PK<em>D</em><em>2</em> genes are the genes that are mutated in patients suffering from autosomal dominant polycystic kidney disease. The human PK<em>D</em><em>2</em> gene codes for a 968-amino acid long membrane protein called polycystin-<em>2</em> that represents a cation channel whose activity can be regulated by Ca(<em>2</em>+) ions. By C<em>D</em>, fluorescence, and NMR spectroscopy, we have studied a 117-amino acid-long fragment of the cytoplasmic domain of polycystin-<em>2</em>, polycystin-<em>2</em>-(680-796) that was proposed to contain a Ca(<em>2</em>+)-binding site. NMR structure determination reveals the existence of two Ca(<em>2</em>+)-binding sites in polycystin-<em>2</em>-(680-796) arranged in a typical and an atypical EF-hand motif. In the absence of Ca(<em>2</em>+) the protein forms a <em>dimer</em> that is dissociated by Ca(<em>2</em>+) binding. This dissociation may be related to the Ca(<em>2</em>+) inactivation observed earlier. The calcium affinity of the protein was determined by fluorescence and NMR spectroscopy. At <em>2</em>93 K, the K(<em>D</em>) values for the high and low affinity sites are 55 mum and 179 mum, respectively.

Malignancy is a risk factor for thromboembolism and anti-cancer chemotherapy can increase this risk. Prophylaxis of thrombosis with very-low-dose warfarin given concurrently with chemotherapy has a significantly reduced rate of thromboembolism in a randomized trial in women with stage IV breast cancer. In a group of 3<em>2</em> patients randomized in one center (16 subjects on warfarin and 16 on placebo), we have prospectively studied the plasma levels of: 1. Markers of 'in vivo' clotting activation (thrombin-antithrombin complex [TAT], prothrombin fragment 1+<em>2</em> [F1+<em>2</em>] and <em>D</em>-<em>dimer</em>), <em>2</em>. Factor VII (FVII), and 3. Natural anticoagulants (protein C [PC] and antithrombin [AT]). The aims of this study were: 1. to examine whether laboratory tests predicted those patients who developed thrombosis, and <em>2</em>. to evaluate the effect of very-low-dose warfarin on hemostatic variables. The patients' hemostatic parameters were evaluated before entry into the study and after starting chemotherapy +/- prophylaxis, before each course for nine courses. Before-treatment results were compared to those of a sex and age-matched non-cancer control group. There was a significant elevation of plasma levels of TAT (p <0.001), F1+<em>2</em> (p <0.001), <em>D</em>-<em>dimer</em> (p <0.0001) and FVIIa (p <0.05), as well as an increase of FVII proteolysis (p <0.05), whereas plasma PC and AT concentrations were not different from controls. After starting chemotherapy, markers of clotting activation were progressively lower in the group receiving warfarin prophylaxis compared to the group on placebo. <em>D</em>ifferences between the groups became statistically significant (p <0.01) after the 4th course of chemotherapy. <em>D</em>eep vein thrombosis occurred in two patients in the placebo arm. The results of this study indicate that before therapy, an hypercoagulable state is present in stage IV breast cancer, and after starting chemotherapy, abnormalities of hypercoagulation markers persist, however they are reduced by very-low-dose-warfarin. None of the laboratory variables could predict thrombosis in the single patient.

OBJECTIVE

Plasma D-dimer levels are elevated in patients with a variety of solid tumors. Recently, it has been reported that the level before curative surgery is a prognostic factor for colorectal cancer (CRC). We investigated whether the plasma D-dimer level before systemic chemotherapies is a predictor for advanced or recurrent unresectable CRC.

METHODS

This study included 42 patients treated with systemic chemotherapies for advanced or recurrent unresectable CRC. Variables including clinicopathological factors, plasma D-dimer levels and the modified Glasgow Prognostic Factor Score (mGPS) were evaluated.

RESULTS

The plasma D-dimer level was closely related to the mGPS. Survival was shorter for patients with plasma D-dimer levels >5 µg/ml than for those with lower levels. Compared with an mGPS of 0 or 1, an mGPS of 2 was predictive of poor prognosis (p < 0.0001). Old age, advanced stage, plasma D-dimer level and mGPS were significantly associated with mortality, but plasma D-dimer level was the only independent risk factor in multivariate analysis, and was significant related to the clinical response to chemotherapy (p < 0.05).

CONCLUSIONS

Survival was significantly shorter in patients with elevated plasma D-dimer levels having advanced or recurrent CRC. The plasma D-dimer level before systemic chemotherapies was an independent mortality predictor.

N-methyl-<em>D</em>-aspartate (NM<em>D</em>A) receptors are glutamate-gated ion channels whose function is critical for normal excitatory synaptic transmission in the brain and whose dysfunction has been implicated in several neurologic conditions. NM<em>D</em>A receptor function is subject to extensive allosteric regulation both by endogenous compounds and by exogenous small molecules. Elucidating the structural determinants and mechanism of action by which allosteric regulators control gating will enhance our understanding of NM<em>D</em>A receptor activation and facilitate the development of novel therapeutics. Here, we investigated the structural determinants for (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-<em>2</em>(1H)-yl)methanone (CIQ), a GluN<em>2</em>C/<em>2</em><em>D</em>-selective positive allosteric modulator. We show that CIQ does not bind to the amino-terminal domain of the NM<em>D</em>A receptor and does not share structural determinants with modulators acting at the agonist-binding domain <em>dimer</em> interface or ion channel pore. Rather, we identified critical determinants of CIQ modulation in the region near the first transmembrane helix of GluN<em>2</em><em>D</em>, including in a putative pre-M1 cuff helix that may influence channel gating. We also show that mutations within the GluN<em>2</em><em>D</em> pre-M1 region alter open probability of the NM<em>D</em>A receptor. These results suggest a novel site of action for potentiation of NM<em>D</em>A receptors by small molecules and implicate the pre-M1 region in NM<em>D</em>A receptor gating.

Complementation of function after coexpression of pairs of nonfunctional G protein-coupled receptors that contain distinct inactivating mutations supports the hypothesis that such receptors exist as <em>dimers</em>. Chimeras between members of the metabotropic glutamate receptor-like family have been particularly useful because the N-terminal ligand binding and heptahelical transmembrane elements can be considered distinct domains. To examine the utility of a related approach for opioid receptors, fusion proteins were generated in which a pertussis toxin-resistant (Cys351Ile) variant of the G protein Gi1alpha was linked to the C-terminal tails of the <em>delta</em> opioid peptide (DOP), kappa opioid peptide, and mu opioid peptide receptors. Each was functional as measured by agonist stimulation of guanosine 5'-([gamma-35S]thio)triphosphate ([35S]GTPgammaS) binding in Gialpha immunoprecipitates from membranes of pertussis toxin-treated HEK<em>2</em>93 cells. Agonist function was eliminated either by fusion of the receptors to Gi1alphaGly<em>2</em>0<em>2</em>Ala,Cys351Ile or mutation of a pair of conserved Val residues in intracellular loop <em>2</em> of each receptor. Coexpression, but not simple mixing, of the two inactive fusion proteins reconstituted agonist-loading of [35S]GTPgammaS for each receptor. At equimolar amounts, reconstitution of DOP receptor function was more extensive than kappa or mu opioid receptor. Reconstitution of DOP function required two intact receptors and was not achieved by provision of extra Gi1alphaCys351Ile membrane anchored by linkage to DOP transmembrane domain 1. Inactive forms of all G protein alpha subunits can be produced by mutations equivalent to Gi1alphaGly<em>2</em>0<em>2</em>Ala. Because the amino acids modified in the opioid receptors are highly conserved in most rhodopsin-like receptors, this approach should be widely applicable to study the existence and molecular basis of receptor dimerization.

The repeating unit of the glycan chain in the S-layer of the bacterium Aneurinibacillus thermoaerophilus L4<em>2</em>0-91(T) is compose<em>d</em> of four alpha-<em>d</em>-rhamnose molecules an<em>d</em> two 3-acetami<em>d</em>o-3,6-<em>d</em>i<em>d</em>eoxy-alpha-<em>d</em>-galactose moieties (abbreviate<em>d</em> as Fucp3NAc). Formation of the glycan layer requires nucleoti<em>d</em>e-activate<em>d</em> sugars as the <em>d</em>onor molecules. Whereas the enzymes involve<em>d</em> in the synthesis of GDP-rhamnose have been well characterize<em>d</em>, less is known regar<em>d</em>ing the structures an<em>d</em> enzymatic mechanisms of the enzymes require<em>d</em> for the pro<em>d</em>uction of <em>d</em>TDP-Fucp3NAc. One of the enzymes involve<em>d</em> in the biosynthesis of <em>d</em>TDP-Fucp3NAc is a 3,4-ketoisomerase, hereafter referre<em>d</em> to as F<em>d</em>tA. Here we <em>d</em>escribe the first three-<em>d</em>imensional structure of this sugar isomerase complexe<em>d</em> with <em>d</em>TDP an<em>d</em> solve<em>d</em> to 1.5 A resolution. The F<em>d</em>tA <em>dimer</em> assumes an almost jellyfish-like appearance with the sole alpha-helices representing the tentacles. Formation of the F<em>d</em>tA <em>dimer</em> represents a classical example of <em>d</em>omain swapping whereby beta-stran<em>d</em>s <em>2</em> an<em>d</em> 3 from one subunit form part of a beta-sheet in the secon<em>d</em> subunit. The active site architecture of F<em>d</em>tA is characterize<em>d</em> by a cluster of three histi<em>d</em>ine resi<em>d</em>ues, two of which, His(49) an<em>d</em> His(51), appear to be strictly conserve<em>d</em> in the amino aci<em>d</em> sequences <em>d</em>eposite<em>d</em> to <em>d</em>ate. Site-<em>d</em>irecte<em>d</em> mutagenesis experiments, enzymatic assays, an<em>d</em> x-ray crystallographic analyses suggest that His(49) functions as an active site base.

BACKGROUND

Skin reactivity to the intra<em>d</em>ermal injection of autologous serum (autologous serum skin test - ASST) an<em>d</em>/or plasma (autologous plasma skin test - APST) is thought to i<em>d</em>entify chronic urticaria (CU) patients with an autoimmune/autoreactive <em>d</em>isease. Immune-me<em>d</em>iate<em>d</em> inflammation an<em>d</em> coagulation are strictly linke<em>d</em>, an<em>d</em> coagulation activation has been <em>d</em>escribe<em>d</em> in CU patients as shown by the elevation of plasma prothrombin fragment F1+<em>2</em> an<em>d</em>, in severe cases, of <em>d</em>-<em>dimer</em> as well.

OBJECTIVE

The aim of this study was to evaluate whether the coagulation cascade is activated in APST-negative CU patients as it has been described in CU patients with an autoreactive disease.

METHODS

A total of 43 a<em>d</em>ults with CU (M/F 15/<em>2</em>8; mean age 43.5 years; 16 APST-negative patients an<em>d</em> <em>2</em>7 APST-positive) an<em>d</em> 30 healthy subjects were stu<em>d</em>ie<em>d</em>. Prothrombin fragment F1+<em>2</em>, <em>d</em>-<em>dimer</em> an<em>d</em> C-reactive protein (CRP) plasma levels were measure<em>d</em> by ELISA.

RESULTS

Prothrombin fragment F1+<em>2</em> an<em>d</em> <em>d</em>-<em>dimer</em> were elevate<em>d</em> in seven of 16 APST-negative CU patients. The activation of the coagulation casca<em>d</em>e was associate<em>d</em> with <em>d</em>isease severity. Men were more prevalent in i<em>d</em>iopathic than in autoreactive CU patients (M/F: 10/6 vs. 5/<em>2</em><em>2</em>; P<0.001). In patients with APST-negative CU, mean F1 + <em>2</em> level [<em>2</em>4<em>2</em>.8 ± 33.7 pmol/L (ESM)] was higher than in normal controls (151.8 ± 9.09 pmol/L; P=0.00<em>2</em>) but lower than in autoreactive patients (5<em>2</em>6.<em>2</em> ± 97.8 pmol/L; P=0.05). Similarly, mean <em>d</em>-<em>dimer</em> level was higher than in normal controls (484.<em>2</em> ± 148.3 ng/mL vs. <em>2</em><em>2</em>9.5 ± 16.7 ng/mL; P=0.03) but lower than in autoreactive patients (114<em>2</em>.<em>2</em> ± 317.4 ng/mL; P=0.05). In contrast, mean CRP was lower than in autoreactive patients (1.06 ± 0.3<em>2</em> μg/mL vs. 3.09 ± 0.74 μg/mL; P=0.0<em>2</em>) but not <em>d</em>ifferent from normal subjects (0.78 ± 0.09 μg/mL; NS).

CONCLUSIONS

Autologous plasma skin test-negative CU prevails in men; in these patients the coagulation cascade is activated although with a lower intensity than in patients with autoreactive disease.

This study was performed to determine the accuracy of <em>D</em>-<em>Dimer</em> fibrin derivatives, thrombin-antithrombin III (TAT) complexes and prothrombin fragments 1 + <em>2</em> (F 1 + <em>2</em>) determinations for the diagnosis of deep vein thrombosis (<em>D</em>VT). One hundred and sixteen consecutive patients referred to the angiology unit of our hospital for a clinically suspected <em>D</em>VT were investigated. They were submitted to mercury strain gauge plethysmography and to ultrasonic duplex scanning examination; in cases of inconclusive results or of proximal <em>D</em>VT (n = 35), an ascending phlebography was performed. After these investigations were completed, the diagnosis of <em>D</em>VT was confirmed in 34 and excluded in 8<em>2</em>. One half of the patients were already under anticoagulant therapy at the time of investigation. The 3 biological markers were assayed using commercially available ELISA techniques and the <em>D</em>-<em>Dimer</em> was also assayed with a fast latex method. The normal distribution of these markers was established in 40 healthy blood donors. The most accurate assay for the diagnosis of <em>D</em>VT was the <em>D</em>-<em>Dimer</em> ELISA which had both a high sensitivity (94%) and a high negative predictive value (95%). The <em>D</em>-<em>Dimer</em> latex, TAT complexes and F 1 + <em>2</em> were far less sensitive and provided negative predictive values which ranged between 78 and 85%. In spite of positive and significant correlations between the levels of the 3 markers, their association did not improve their overall accuracy for detecting <em>D</em>VT. Therefore, with the exception of the <em>D</em>-<em>Dimer</em> ELISA, these markers were of little value for the diagnosis of <em>D</em>VT in this specific population.

The transcriptional activator RamA is involved in multidrug resistance (M<em>D</em>R) by increasing expression of the AcrAB-TolC RN<em>D</em>-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the <em>D</em>NA-binding activity of the RamR repressor with the ramA promoter (P(ramA)). As determined by high-resolution footprinting, the <em>2</em>8-bp-long RamR binding site covers essential features of P(ramA), including the -10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P(ramA) as a <em>dimer</em> of <em>dimers</em>, in a fashion that is structurally similar to the QacR-<em>D</em>NA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (K(<em>D</em>) [equilibrium dissociation constant] = 191 nM) for the <em>2</em>-bp-deleted P(ramA) of an M<em>D</em>R S. Typhimurium clinical isolate than for the wild-type P(ramA) (K(<em>D</em>) = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an M<em>D</em>R phenotype.

Ovarian cancer cells appear to be capable of both thrombin formation and induction of fibrin degradation which may be essential prerequisites for the development of deep vein thrombosis (<em>D</em>VT) as well as the spread of malignancy. To study further this coagulation-cancer interaction in 60 patients with untreated ovarian cancer of FIGO stage I-IV the incidence of <em>D</em>VT was recorded pre-operatively, post-operatively on day 1, 3, 5, 7, 10, before each of six cycles of Cisplatinum/ Epirubicin/Cyclophosphamide chemotherapy, during follow-up and in the post-operative period of second look surgery. In addition, blood coagulation tests results were determined prospectively. Two patients were excluded from these calculations due to previous <em>D</em>VT 5 to 6 weeks before the diagnosis of ovarian cancer but all patients were eligible for surgery and randomized to receive either daily low molecular weight heparin (LMWH) (n = <em>2</em>8) or unfractionated heparin (UFH) (n = 3<em>2</em>) for perioperative thrombosis prophylaxis until the 7th post-operative day. According to the FIGO stage, patients were equally distributed in the <em>2</em> heparin treatment groups. The predictive value of pre-operative coagulation test results, clinical parameters, and type of heparin used were tested in univariate and multivariate analysis for development of post-operative <em>D</em>VT and overall patients survival. Impedance plethysmography for <em>D</em>VT screening was used. The presence of <em>D</em>VT was then confirmed by phlebography. Only <em>D</em>-<em>dimer</em> and fibrinogen levels were correlated significantly with the FIGO stage while antithrombin, protein C, and plasminogen activator inhibitor activity were not. The incidence of <em>D</em>VT was 6.7% (4/60) up to the 7th and 8.3% (5/60) between the 8th and <em>2</em>9th post-operative day. <em>D</em>VT occurred in 10.6% (5/47) during chemotherapy. Pre-operative coagulation test results, the type of heparin used, and clinical parameters were not significant risk factors for post-operative <em>D</em>VT development in univariate analysis. The <em>D</em>-<em>dimer</em> and fibrinogen levels were significant risk factors for reduced overall survival in univariate analysis but only the FIGO stage was an independent predictor (in multivariate analysis). After a median follow up of <em>2</em>6.5 months (min. 8 months, max. 41 months), <em>2</em>1.4% of LMWH treated and 37.5% of UFH-treated patients died of cancer (p = 0.<em>2</em>6). Pre-operative test results were neither predictive for <em>D</em>VT nor the outcome of cancer but patients showed an improved though not statistically significant overall survival after LMWH treatment.

The env gene of Friend spleen focus-forming virus (SFFV) encodes a membrane glycoprotein (gp55) that is inefficiently (3 to 5%) processed from the rough endoplasmic reticulum to form a larger <em>dimer</em>ic plasma membrane derivative (gp55p). Moreover, the SFFV env glycoprotein associates with erythropoietin receptors (EpoR) to cause proliferation of infected erythroblasts [J.-P. Li, A. <em>D</em>. <em>D</em>'Andrea, H. F. Lodish, and <em>D</em>. Baltimore, Nature (London) 343:76<em>2</em>-764, 1990]. Interestingly, the mitogenic effect of SFFV is blocked in mice homozygous for the Fv-<em>2</em>r resistance gene, but mutant SFFVs can overcome this resistance. Recent evidence suggested that these mutants contain partial env deletions that truncate the membrane-proximal extracellular domain of the encoded glycoproteins (M. H. Majumdar, C.-L. Cho, M. T. Fox, K. L. Eckner, S. Kozak, <em>D</em>. Kabat, and R. W. Geib, J. Virol. 66:365<em>2</em>-3660, 199<em>2</em>). Mutant BB6, which encodes a gp4<em>2</em> glycoprotein that has a large deletion in this domain, causes erythroblastosis in <em>D</em>BA/<em>2</em> (Fv-<em>2</em>s) as well as in congenic <em>D</em><em>2</em>.R (Fv-<em>2</em>r) mice. Analogous to gp55, gp4<em>2</em> is processed inefficiently as a disulfide-bonded <em>dimer</em> to form cell surface gp4<em>2</em>p. Retroviral vectors with SFFV and BB6 env genes have no effect on interleukin 3-dependent BaF3 hematopoietic cells, but they cause growth factor independency of BaF3/EpoR cells, a derivative that contains recombinant EpoR. After binding 1<em>2</em>5I-Epo to surface EpoR on these factor-independent cells and adding the covalent cross-linking reagent disuccinimidyl suberate, complexes that had immunological properties and sizes demonstrating that they consisted of 1<em>2</em>5I-Epo-gp55p and 1<em>2</em>5I-Epo-gp4<em>2</em>p were isolated from cell lysates. Contrary to a previous report, SFFV or BB6 env glycoproteins did not promiscuously activate other members of the EpoR superfamily. Although the related env glycoproteins encoded by dualtropic murine leukemia viruses formed detectable complexes with EpoR, strong mitogenic signalling did not ensue. Our results indicate that the SFFV and BB6 env glycoproteins specifically activate EpoR; they help to define the glycoprotein properties important for its functions; and they strongly suggest that the Fv-<em>2</em> leukemia control gene encodes an EpoR-associated regulatory factor.

Because abnormalities in hemostatic factors may in part account for the risk of stroke and thromboembolism in atrial fibrillation, we measured plasma fibrinogen and fibrin <em>D</em>-<em>dimer</em> levels in 33 patients (18 men and 15 women, mean age 60.8 +/- 1.4 years [mean +/- SEM]) with paroxysmal atrial fibrillation (PAF) and 1<em>2</em> patients (3 men and 9 women, mean age 51.0 +/- 4.<em>2</em> years) with paroxysmal supraventricular tachycardia (PSVT). Levels of these markers were compared to levels in (1) patients with chronic atrial fibrillation; (<em>2</em>) hospital controls (age-matched [age +/- 5 years] and sex-matched patients in sinus rhythm with coronary artery disease and normal left ventricular function); and (3) healthy population controls in sinus rhythm. Patients with PAF had intermediate levels of median plasma fibrinogen and fibrin <em>D</em>-<em>dimer</em> when compared to patients with chronic atrial fibrillation and controls in sinus rhythm (both p < 0.001). There was no relation with atrial size or ventricular function on echocardiography. Patients with PSVT had plasma fibrinogen and fibrin <em>D</em>-<em>dimer</em> levels that were similar to the median levels of the population controls, suggesting that there was no excess in thrombogenesis. These findings are consistent with the hypothesis that atrial fibrillation is related to the increases in plasma fibrinogen and fibrin <em>D</em>-<em>dimer</em> levels. Patients with PAF have intermediate levels of these markers, a finding that is consistent with the intermediate risk of thromboembolism in such patients.

A sequence variation in the 3'-untranslated region of the prothrombin (PT) gene (<em>2</em>0<em>2</em>10 G->>A) was recently claimed to be associated with elevated plasma prothrombin levels and an increased risk for venous and arterial thrombosis. We examined the prevalence of the <em>2</em>0<em>2</em>10 A allele in the prothrombin gene in 400 healthy controls and in <em>2</em>63 patients with proven premature atherosclerotic disease. In addition, we measured prothrombin, prothrombin fragment 1 + <em>2</em>, thrombin-antithrombin (TAT) complex and <em>D</em>-<em>dimer</em> levels in plasma from carrier and non-carrier patients. The frequency of the variant allele was 1% in the control subjects and <em>2</em>.7% in the patient group, yielding a relative risk (RR) for the <em>2</em>0<em>2</em>10 A allele of <em>2</em>.7 (95% CI 0.8-9.4). All heterozygotes in the patient group were found to have had a myocardial infarction (MI), yielding a RR for MI of 4.<em>2</em> (95% CI 1.<em>2</em>-14.6). Plasma prothrombin levels in carriers (1<em>2</em>6+/-10) were higher than in non-carriers (103+/-1, P=0.0<em>2</em>). The levels of TAT complexes (16+/-9 v 6+/-1 microg/ml, P=0.0<em>2</em>) as well as of prothrombin fragment 1 + <em>2</em> (1.5+/-0.3 v 1.0+/-0.1 nmol/l, P=0.0<em>2</em>) were also elevated in carriers of the mutation. Our findings suggest that the <em>2</em>0<em>2</em>10 G->>A mutation in the prothrombin gene is a genetic risk factor for MI. In addition, our data provide evidence for an association of the mutation with excessive thrombin generation, which may contribute to the understanding of its role in venous and arterial disease.

We have clone<em>d</em> the M an<em>d</em> S genes of the restriction-mo<em>d</em>ification (R-M) system Ah<em>d</em>I an<em>d</em> have purifie<em>d</em> the resulting methyltransferase to homogeneity. M.Ah<em>d</em>I is foun<em>d</em> to form a 170 kDa tetrameric enzyme having a subunit stoichiometry M<em>2</em>S<em>2</em> (where the M an<em>d</em> S subunits are responsible for methylation an<em>d</em> DNA sequence specificity, respectively). Se<em>d</em>imentation equilibrium experiments show that the tetrameric enzyme <em>d</em>issociates to form a hetero<em>d</em>imer at low concentration, with K(<em>d</em>) approximately <em>2</em> microM. The intact (tetrameric) enzyme bin<em>d</em>s specifically to a 30 bp DNA <em>d</em>uplex containing the Ah<em>d</em>I recognition sequence GACN5GTC with high affinity (K(<em>d</em>) approximately 50 nM), but at low enzyme concentration the DNA bin<em>d</em>ing activity is governe<em>d</em> by the <em>d</em>issociation of the tetramer into <em>dimers</em>, lea<em>d</em>ing to a sigmoi<em>d</em>al DNA bin<em>d</em>ing curve. In contrast, only non-specific bin<em>d</em>ing is observe<em>d</em> if the <em>d</em>uplex lacks the recognition sequence. Methylation activity of the purifie<em>d</em> enzyme was assesse<em>d</em> by its ability to prevent restriction by the cognate en<em>d</em>onuclease. The subunit structure of the M.Ah<em>d</em>I methyltransferase resembles that of type I MTases, in contrast to the R.Ah<em>d</em>I en<em>d</em>onuclease which is typical of type II systems. Ah<em>d</em>I appears to be a novel R-M system with properties interme<em>d</em>iate between simple type II systems an<em>d</em> more complex type I systems, an<em>d</em> may represent an interme<em>d</em>iate in the evolution of R-M systems.

BACKGROUND

Growing evidence indicates that ambient air pollution is associated with exacerbation of chronic diseases like chronic pulmonary disease. A prospective panel study was conducted to investigate short-term changes of blood markers of inflammation and coagulation in response to daily changes in air pollution in Erfurt, Germany. 1<em>2</em> clinical visits were scheduled and blood parameters were measured in 38 male patients with chronic pulmonary disease during winter <em>2</em>001/<em>2</em>00<em>2</em>. Additive mixed models with random patient intercept were applied, adjusting for trend, weekday, and meteorological parameters. Hourly data on ultrafine particles (UFP, 0.01-0.1 mum), accumulation mode particles (ACP, 0.1-1.0 mum), PM10 (particulate matter <10 mum in diameter), elemental (EC) and organic carbon (OC), gaseous pollutants (nitrogen monoxide [NO], nitrogen dioxide [NO<em>2</em>], carbon monoxide [CO], and sulphur dioxide [SO<em>2</em>]) were collected at a central monitoring site and meteorological data were received from an official network. For each person and visit the individual <em>2</em>4-hour average of pollutants immediately preceding the blood withdrawal (lag 0) up to day 5 (lag1-4) and 5-day running means were calculated.

RESULTS

Increased levels of fibrinogen were observed for an increase in one interquartile range of UFP, PM10, EC, OC, CO, and NO revealing the strongest effect for lag 3. E-selectin increased in association with ACP and PM10 with a delay of one day. The ACP effect was also seen with the 5-day-mean. The pattern found for <em>D</em>-<em>dimer</em> was inconsistent. Prothrombin fragment 1+<em>2</em> decreased with lag 4 consistently for all particulate pollutants. Von Willebrand factor antigen (vWF) showed a consistent decrease in association with almost all air pollutants with all lags except for lag 0. No associations were found for C-reactive protein, soluble intercellular adhesion molecule 1, serum amyloid A and factor VII.

CONCLUSIONS

These results suggest that elevated concentrations of air pollution are associated with changes in some blood markers of inflammation and coagulation in patients with chronic pulmonary disease. The clinical implications of these findings need further investigation.