Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca(2+)-binding site in the protease domain.

The Fab fragment of a monoclonal antibody (mAb) reactive to the N-terminal half (residues 180-310) of the protease domain of human factor IX has been previously shown to inhibit the binding of factor IXa to its cofactor, factor VIIIa. These data suggested that this segment of factor IXa may participate in binding to factor VIIIa. We now report that the binding rate (kon) of the mAb is 3-fold higher in the presence of Ca2+ than in its absence for both factors IX and IXa; the half-maximal effect was observed at approximately 300 microM Ca2+. Furthermore, the off rate (koff) of the mAb is 10-fold higher for factor IXa than for factor IX with or without Ca2+. Moreover, like the kon for mAb binding, the incorporation of dansyl-Glu-Gly-Arg chloromethyl ketone (dEGR-CK) into factor IXa was approximately 3 times faster in the presence of Ca2+ than in its absence. Since steric factors govern the kon and the strength of noncovalent interactions governs the koff, the data indicate that the region of factor IX at residues 180-310 undergoes two separate conformational changes before expression of its biologic activity: one upon Ca2+ binding and the other upon zymogen activation. Furthermore, the dEGF-CK incorporation data suggest that both conformational changes also affect the active site residues. Analyses of the known three-dimensional structures of serine proteases indicate that in human factor IX a high-affinity Ca(2+)-binding site may be formed by the carboxyl groups of glutamates 235 and 245 and by the main chain carbonyl oxygens of residues 237 and 240. In support of this conclusion, a synthetic peptide including residues 231-265 was shown to bind Ca2+ with a Kd of approximately 500 microM. This peptide also bound to the mAb, although with approximately 500-fold reduced affinity. Moreover, like factor IX, the peptide bound to the mAb more strongly (approximately 3-fold) in the presence of Ca2+ than in its absence. Thus, it appears that a part of the epitope for the mAb described above is contained in the proposed Ca(2+)-binding site in the protease domain of human factor IX. This proposed site is analogous to the Ca(2+)-binding site in trypsin and elastase, and it may be involved in binding factor IXa to factor VIIIa.     

Association between neural reactivity and startle reactivity to uncertain threat in two independent samples

Prior studies indicate that anxiety disorders are associated with heightened sensitivity to uncertain threat (U threat). Individual differences in reactivity to U threat have been measured in the laboratory with two methodologies—startle eyeblink potentiation and fMRI. While startle and fMRI are purported to relate to each other, very little research exists on whether individual differences in one measure are associated with individual differences in another and, thus, whether startle and fMRI capture shared mechanisms. Therefore, the current study was designed to investigate if and where in the brain measures of startle potentiation and fMRI BOLD signal correlate during response to U threat across two independent samples. Participants in both studies completed two threat anticipation tasks—once during collection of startle potentiation and once during fMRI. In Study 1 (n = 43), the startle and fMRI tasks both used electric shock as the threat. As an extension, in Study 2 (n = 38), the startle task used electric shock but the fMRI task used aversive images. Despite these methodological differences, greater startle potentiation to U threat was associated with greater dorsal anterior cingulate, caudate, and orbitofrontal cortex reactivity to U threat in both samples. The findings suggest that startle and fMRI measures of responding to U threat overlap, and points toward an integrated brain‐behavior profile of aberrant U threat responding.     

Quantification of left and right ventricular function and myocardial mass: comparison of low-radiation dose 2nd generation dual-source CT and cardiac MRI

Objective

To prospectively evaluate the accuracy of left and right ventricular function and myocardial mass measurements based on a dual-step, low radiation dose protocol with prospectively ECG-triggered 2nd generation dual-source CT (DSCT), using cardiac MRI (cMRI) as the reference standard.

Materials and methods

Twenty patients underwent 1.5 T cMRI and prospectively ECG-triggered dual-step pulsing cardiac DSCT. This image acquisition mode performs low-radiation (20% tube current) imaging over the majority of the cardiac cycle and applies full radiation only during a single adjustable phase. Full-radiation-phase images were used to assess cardiac morphology, while low-radiation-phase images were used to measure left and right ventricular function and mass. Quantitative CT measurements based on contiguous multiphase short-axis reconstructions from the axial CT data were compared with short-axis SSFP cardiac cine MRI. Contours were manually traced around the ventricular borders for calculation of left and right ventricular end-diastolic volume, end-systolic volume, stroke volume, ejection fraction and myocardial mass for both modalities. Statistical methods included independent t-tests, the Mann–Whitney U test, Pearson correlation statistics, and Bland–Altman analysis.

Results

All CT measurements of left and right ventricular function and mass correlated well with those from cMRI: for left/right end-diastolic volume r = 0.885/0.801, left/right end-systolic volume r = 0.947/0.879, left/right stroke volume r = 0.620/0.697, left/right ejection fraction r = 0.869/0.751, and left/right myocardial mass r = 0.959/0.702. Mean radiation dose was 6.2 ± 1.8 mSv.

Conclusions

Prospectively ECG-triggered, dual-step pulsing cardiac DSCT accurately quantifies left and right ventricular function and myocardial mass in comparison with cMRI with substantially lower radiation exposure than reported for traditional retrospective ECG-gating.     

Quantification of Coronary Artery Calcium on the Basis of Dual-Energy Coronary CT Angiography

Purpose: To evaluate the feasibility of using virtual noncontrast material-enhanced (VNC) computed tomographic (CT) series derived from dual-energy CT imaging studies for coronary artery calcium quantification. Materials and Methods: This HIPAA-compliant study was institutional review board approved; all patients provided written informed consent. Thirty-six patients prospectively underwent noncontrast-enhanced CT calcium scoring followed by coronary CT angiography performed in dual-energy mode. By using different reconstruction algorithms, three VNC series were generated and evaluated for noise and efficiency of virtual iodine removal. Two readers independently quantified calcium on VNC images and true noncontrast-enhanced conventional calcium scoring series. A leave-one-out cross validation was used to assess the accuracy of calcium score prediction from VNC series by means of linear regression. Results: CT value histograms of the VNC series closely resembled the profile in the true noncontrast-enhanced series. There was excellent correlation between calcium volumes on the VNC series and true noncontrast-enhanced series on a per-patient (r = 0.94, P < .001, n = 36) and per-vessel (r = 0.94, 0.91, and 0.92 for the three coronary arteries, all P < .001, n = 36 each) level. The ability of a linear regression model to predict actual calcium scores from calcium volumes on VNC series was excellent (r = 0.82). Multiethnic Study of Atherosclerosis rankings that were derived from the predicted calcium scores also showed excellent agreement (intraclass correlation coefficient = 0.909). Conclusion: Coronary artery calcium identification and quantification based on dual-energy coronary CT angiographic studies may obviate the need for dedicated CT calcium scoring studies. ? RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112455/-/DC1.     

Relative to Quinine and Quinidine,Their 9-Epimers Exhibit Decreased Cytostatic Activity and Altered Heme Binding but Similar Cytocidal Activity versus Plasmodium falciparum

The 9-epimers of quinine (QN) and quinidine (QD) are known to exhibit poor cytostatic potency against P. falciparum (Karle JM, Karle IL, Gerena L, Milhous WK, Antimicrob. Agents Chemother. 36:1538–1544, 1992). We synthesized 9-epi-QN (eQN) and 9-epi-QD (eQD) via Mitsunobu esterification-saponification and evaluated both cytostatic and cytocidal antimalarial activities. Relative to the cytostatic activity of QN and QD, we observed a large decrease in cytostatic activity (higher 50% inhibitory concentration [IC₅₀s]) against QN-sensitive strain HB3, QN-resistant strain Dd2, and QN-hypersensitive strain K76I, consistent with previous work. However, we observed relatively small changes in cytocidal activity (the 50% lethal dose), similar to observations with chloroquine (CQ) analogues with a wide range of IC₅₀s (see the accompanying paper [A. P. Gorka, J. N. Alumasa, K. S. Sherlach, L. M. Jacobs, K. B. Nickley, J. P. Brower, A. C. de Dios, and P. D. Roepe, Antimicrob. Agents Chemother. 57:356–364, 2013]). Compared to QN and QD, the 9-epimers had significantly reduced hemozoin inhibition efficiency and did not affect pH-dependent aggregation of ferriprotoporphyrin IX (FPIX) heme. Magnetic susceptibility measurements showed that the 9-epimers perturb FPIX monomer-dimer equilibrium in favor of monomer, and UV-visible (VIS) titrations showed that eQN and eQD bind monomer with similar affinity relative to QN and QD. However, unique ring proton shifts in the presence of zinc(II) protoporphyrin IX (ZnPIX) indicate that binding of the 9-epimers to monomeric heme is via a distinct geometry. We isolated eQN- and eQD-FPIX complexes formed under aqueous conditions and analyzed them by mass, fluorescence, and UV-VIS spectroscopies. The 9-epimers produced low-fluorescent adducts with a 2:1 stoichiometry (drug to FPIX) which did not survive electrospray ionization, in contrast to QN and QD complexes. The data offer important insight into the relevance of heme interactions as a drug target for cytostatic versus cytocidal dosages of quinoline antimalarial drugs and further elucidate a surprising structural diversity of quinoline antimalarial drug-heme complexes.