Citations

AbstractWe have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea‐induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea–temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two‐state character. Global analysis of the urea–temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of and that define a complex temperature dependence of the m‐value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea‐denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions.

AbstractPlatelet attachment to von Willebrand factor (vWF) requires the interaction between the platelet GP1bα and exposed vWF‐A1 domains. Structural insights into the mechanism of the A1‐GP1bα interaction have been limited to an N‐terminally truncated A1 domain that lacks residues Q1238 − E1260 that make up the linker between the D3 and A1 domains of vWF. We have demonstrated that removal of these residues destabilizes quaternary interactions in the A1A2A3 tridomain and contributes to platelet activation under high shear (Auton et al., J Biol Chem 2012;287:14579–14585). In this study, we demonstrate that removal of these residues from the single A1 domain enhances platelet pause times on immobilized A1 under rheological shear. A rigorous comparison between the truncated A1‐1261 and full length A1‐1238 domains demonstrates a kinetic stabilization of the A1 domain induced by these N‐terminal residues as evident in the enthalpy of the unfolding transition. This stabilization occurs through site and sequence‐specific binding of the N‐terminal peptide to A1. Binding of free N‐terminal peptide to A1‐1261 has an affinity and this binding although free to dissociate is sufficient to suppress the platelet pause times to levels comparable to A1‐1238 under shear stress. Our results support a dual‐structure/function role for this linker region involving a conformational equilibria that maintains quaternary A domain associations in the inactive state of vWF at low shear and an intra‐A1‐domain conformation that regulates the strength of platelet GP1bα‐vWF A1 domain associations in the active state of vWF at high shear.

Abstract The selective estrogen receptor modulators (SERM) tamoxifen and raloxifene can reduce the occurrence of breast cancer in high-risk women by 50%, but this U.S. Food and Drug Administration-approved prevention therapy is not often used. We attempted to identify genetic factors that contribute to variation in SERM breast cancer prevention, using DNA from the NSABP P-1 and P-2 breast cancer prevention trials. An initial discovery genome-wide association study identified common single-nucleotide polymorphisms (SNP) in or near the ZNF423 and CTSO genes that were associated with breast cancer risk during SERM therapy. We then showed that both ZNF423 and CTSO participated in the estrogen-dependent induction of BRCA1 expression, in both cases with SNP-dependent variation in induction. ZNF423 appeared to be an estrogen-inducible BRCA1 transcription factor. The OR for differences in breast cancer risk during SERM therapy for subjects homozygous for both protective or both risk alleles for ZNF423 and CTSO was 5.71. Significance: This study identified novel, functionally polymorphic genes involved in the estrogen-dependent regulation of BRCA1 expression, as well as a novel mechanism for genetic variation in SERM therapeutic effect. These observations, and definition of their underlying mechanisms, represent steps toward pharmacogenomically individualized SERM breast cancer prevention. Cancer Discov; 3(7); 812–25. ©2013 AACR. See related commentary by Machiela and Chanock, p. 728 This article is highlighted in the In This Issue feature, p. 705

Genetic College Many genomic elements in humans are associated with behavior, including educational attainment. In a genome-wide association study including more than 100,000 samples, Rietveld et al. (p. 1467 , published online 30 May; see the Perspective by Flint and Munafò ) looked for genes related to educational attainment in Caucasians. Small genetic effects at three loci appeared to impact educational attainment.

ABSTRACTAnimal models of joint contracture may be used to elucidate the mechanisms of arthrofibrosis. Patients with joint contracture commonly undergo surgical capsular release. Previous animal models of joint contracture do not simulate this aspect of arthrofibrosis. We hypothesize that a surgical capsular release will decrease the severity of arthrofibrosis in this rabbit model. A capsular contracture was surgically created in 20 skeletally mature rabbits. Eight weeks later, ten rabbits underwent capsular release, which consisted of elevation of the posterior capsule through a lateral incision and manipulation under anesthesia. Ten rabbits had a sham incision, without release (control group). Immediately after release or sham surgery, extension loss (calculated by subtracting the knee extension angle (degrees) of the operative limb from the nonoperative, contralateral limb) was measured using fluoroscopy. All animals were sacrificed following 16 weeks of postoperative free cage activity. At sacrifice, joint contracture was measured using a custom, calibrated device. The histology of the posterior joint capsule was assessed at sacrifice. All animals survived both operations without complications. Immediately after surgical release or sham surgery, the average extension loss was 129.2 ± 10.7° in the control group versus 29.6 ± 8.2° in the capsular release group (p = 0.0002). Following 16 weeks of remobilization, the average extension loss of the control and capsular release animals were 49.0 ± 12.7° and 36.5 ± 14.2°, respectively (p = 0.035). There were no histological differences between the two groups. In this animal model, a surgical capsular release decreased the extension loss (flexion contracture) immediately after surgery, as well as following sixteen weeks of remobilization. There were no histological changes detected in the posterior joint capsule. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1529–1532, 2013