Oligomeric interactions of sarcolipin and the Ca-ATPase.

We have detected directly the interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a membrane protein that helps control contractility by regulating SERCA activity in fast-twitch and atrial muscle. Here we used FRET microscopy and spectroscopy with baculovirus expression in insect cells to provide direct evidence for: 1) oligomerization of SLN and 2) regulatory complex formation between SLN and the fast-twitch muscle Ca-ATPase (SERCA1a isoform). FRET experiments demonstrated that SLN monomers self-associate into dimers and higher order oligomers in the absence of SERCA, and that SLN monomers also bind to SERCA monomers in a 1:1 binary complex when the two proteins are coexpressed. FRET experiments further demonstrated that the binding affinity of SLN for itself is similar to that for SERCA. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased the binding affinity of SLN self-association and converted higher order oligomers into monomers and dimers. The I17A mutation also decreased SLN binding affinity for SERCA but maintained 1:1 stoichiometry in the regulatory complex. Thus, isoleucine-17 plays dual roles in determining the distribution of SLN homo-oligomers and stabilizing the formation of SERCA-SLN heterodimers. FRET results for SLN self-association were supported by the effects of SLN expression in bacterial cells. We propose that SLN exists as multiple molecular species in muscle, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (heterodimer), with transmembrane zipper residues of SLN serving to stabilize oligomeric interactions.

Radiographic landmarks for tunnel positioning in double-bundle ACL reconstructions.

PURPOSE: The purpose of this study was to establish quantitative and qualitative radiographic landmarks for identifying the femoral and tibial attachment sites of the AM and PL bundles of the native ACL and to assess the reproducibility of identification of these landmarks using intraclass correlation coefficients. It was hypothesized that the radiographic positions of the AM and PL bundles could be defined in relation to anatomic landmarks and radiographic reference lines. METHODS: The femoral and tibial attachment sites of the AM and PL bundles on twelve cadaveric knees were labeled with radio-opaque markers. The positions of the AM and PL bundle attachment sites were quantified on radiographs by three independent examiners. RESULTS: On the lateral femoral view, the AM bundle was located at 21.6 ± 5.6% of the sagittal diameter of the femur drawn along Blumensaat's line and 14.2 ± 7.7% distal to the notch roof along the maximum notch height. The PL bundle was located at 28.9 ± 4.6% of the sagittal diameter and 42.3 ± 6.0% of the notch height. The knee flexion angle at which the AM and PL bundle attachment sites were horizontally oriented was 115 ± 7.1°. On the tibial AP view, the AM and PL bundles were located at 44.2 ± 3.4 and 50.1 ± 2.1%, respectively, from the medial aspect of the tibia along its coronal diameter. On the lateral view, the distances from the AM and PL bundles to the anterior tibial margin measured along the tibial sagittal diameter were 36.3 ± 3.8 and 51.0 ± 4.0%, respectively. The center of the PL bundle attachment was located almost precisely at the center of the tibial plateau in both the coronal and sagittal planes. CONCLUSIONS: This study defines the radiographic locations of the femoral and tibial bundle attachment sites of the native ACL and a reliable and transferrable protocol for identifying these sites on radiographs in relation to surrounding landmarks and digitally projected reference lines. In addition, it was found that the femoral attachments of the AM and PL bundles were horizontally aligned at 115° of knee flexion and the PL bundle tibial attachment was located essentially at the center of the tibia.

Arthroscopically pertinent landmarks for tunnel positioning in single-bundle and double-bundle anterior cruciate ligament reconstructions.

BACKGROUND: Quantification of the overall anterior cruciate ligament (ACL) and anteromedial (AM) and posterolateral (PL) bundle centers in respect to arthroscopically pertinent bony and soft tissue landmarks has not been thoroughly assessed. HYPOTHESIS: A standardized anatomical measurement method can quantitate the locations of the ACL and AM and PL bundle centers in reference to each other and anatomical landmarks. STUDY DESIGN: Descriptive laboratory study. METHODS: Quantification of the ACL and its bundle attachments was performed on 11 cadaveric knees using a radio frequency-tracking device. RESULTS: The tibial ACL attachment center was 7.5 mm medial to the anterior horn of the lateral meniscus, 13.0 mm anterior to the retro-eminence ridge, and 10.5 mm posterior to the ACL ridge. The femoral ACL attachment center was 1.7 mm proximal to the bifurcate ridge and 6.1 mm posterior to the lateral intercondylar ridge. The tibial AM attachment center was 8.3 mm medial to the anteromedial aspect of the lateral meniscus anterior horn, 17.8 mm anterior to the retro-eminence ridge, and 5.6 mm posterior to the ACL ridge. The femoral AM attachment center was 4.8 mm proximal to the bifurcate ridge and 7.1 mm posterior to the lateral intercondylar ridge. The tibial PL bundle attachment center was 6.6 mm medial to the posteromedial aspect of the lateral meniscus anterior horn, 10.8 mm anteromedial to the root attachment of the lateral meniscus posterior horn, and 8.4 mm anterior to the retro-eminence ridge. The femoral PL bundle attachment center was 5.2 mm distal to the bifurcate ridge and 3.6 mm posterior to the lateral intercondylar ridge. CONCLUSION: The authors developed a comprehensive compilation of measurements of arthroscopically pertinent bony and soft tissue landmarks that quantitate the ACL and its individual bundle attachment centers on the tibia and femur. CLINICAL RELEVANCE: These clinically relevant arthroscopic landmarks may enhance single- and double-bundle ACL reconstructions through improved tunnel placement.

Kinematic impact of anteromedial and posterolateral bundle graft fixation angles on double-bundle anterior cruciate ligament reconstructions.

BACKGROUND: Currently in double-bundle anterior cruciate ligament (ACL) reconstructions, the range of knee flexion angles that surgeons use for anteromedial (AM) and posterolateral (PL) bundle graft fixation spans from 0 degrees to 90 degrees for both bundle grafts. Despite the recent popularity of this procedure, no consensus exists on an optimal set of AM and PL graft fixation angles. HYPOTHESIS: Graft fixation angles that simulate the native tensioning relationship of the AM and PL bundles will produce kinematic results similar to the intact knee, while graft fixation angles that do not simulate this relationship will under- or overconstrain the knee. STUDY DESIGN: Controlled laboratory study. METHODS: Twelve cadaveric knees were biomechanically tested in the intact state, ACL-sectioned state, and a randomized order of 7 double-bundle ACL reconstructed states at multiple graft fixation angle combinations. For each test state, data were collected for 88 N anterior tibial loads, 10 N.m valgus torques, 5 N.m internal rotation torques, and 2 simulated pivot shift loads consisting of a 5 N.m internal rotation torque coupled with either a 10 N.m valgus torque or an 88 N anterior tibial load at 0 degrees, 20 degrees, 30 degrees, 60 degrees, and 90 degrees of knee flexion. RESULTS: The AM and PL graft fixation angle combinations of 0 degrees /0 degrees (AM graft fixation angle/PL graft fixation angle), 60 degrees /0 degrees, 45 degrees /15 degrees, and 75 degrees /15 degrees restored normal laxity to the reconstructed knee in all of the biomechanical tests. The 30 degrees /30 degrees, 60 degrees /60 degrees, and 90 degrees /90 degrees graft fixation angle combinations significantly restricted knee laxity compared with the intact state in various biomechanical tests. CONCLUSION: We found that as long as the PL bundle graft was fixed between 0 degrees and 15 degrees , the AM graft could be fixed up to 75 degrees without restricting knee laxity. However, fixation of the PL graft at 30 degrees of knee flexion and above significantly overconstrained the knee. CLINICAL RELEVANCE: This study provides a range of angles that can be used in double-bundle ACL reconstructions to restore normal knee stability without causing overconstraint.

ARF6-mediated endosome recycling reverses lipid accumulation defects in Niemann-Pick Type C disease.

In human Niemann-Pick Type C (NPC) disease, endosomal trafficking defects lead to an accumulation of free cholesterol and other lipids in late endosome/lysosome (LE/LY) compartments, a subsequent block in cholesterol esterification and significantly reduced cholesterol efflux out of the cell. Here we report that nucleotide cycling or cellular knockdown of the small GTP-binding protein, ARF6, markedly impacts cholesterol homeostasis. Unregulated ARF6 activation attenuates the NPC phenotype at least in part by decreasing cholesterol accumulation and restoring normal sphingolipid trafficking. These effects depend on ARF6-stimulated cholesterol efflux out of the endosomal recycling compartment, a major cell repository for free cholesterol. We also show that fibroblasts derived from different NPC patients displayed varying levels of ARF6 that is GTP-bound, which correlate with their response to sustained ARF6 activation. These studies support emerging evidence that early endocytic defects impact NPC disease and suggest that such heterogeneity in NPC disease could result in diverse responses to therapeutic interventions aimed at modulating the trafficking of lipids.

Radiographic identification of the primary posterolateral knee structures.

BACKGROUND: It is often difficult to identify the attachment sites of the fibular collateral ligament, popliteus tendon, and popliteofibular ligament for chronic posterolateral knee injuries or during revision surgeries. Descriptions of radiographic landmarks for these attachment sites would assist in the intraoperative identification of their locations and also allow for postoperative assessment of the placement of reconstruction tunnels. HYPOTHESIS: Identification of qualitative and quantitative radiographic landmarks for the attachments of the main posterolateral knee structures are reproducible among observers of various experience levels and allow for improved intraoperative and postoperative identification of these attachment sites. STUDY DESIGN: Descriptive laboratory study. METHODS: Dissections were performed on 11 cadaveric knee specimens. The attachments and locations of the investigated structures were labeled with radiopaque markers. The positions of the attachments relative to other attachment sites, labeled bony landmarks, and superimposed reference lines were quantified on anteroposterior and lateral radiographs. Measurements were performed by 3 independent examiners. Intraobserver and interobserver reliability was determined using intraclass correlation coefficients. RESULTS: Overall intraclass correlation coefficients for intraobserver reproducibility and interobserver reliability were calculated to be 0.981 and 0.983, respectively. On the anteroposterior view, the perpendicular distances from a line intersecting the femoral condyles to the popliteus tendon, proximal fibular collateral ligament, and lateral gastrocnemius tendon were 14.5, 27.1, and 34.5 mm, respectively. On the lateral view, the femoral attachments of the fibular collateral ligament, popliteus tendon, and lateral gastrocnemius tendon were 4.3, 12.2, and 13.1 mm, respectively, from the lateral epicondyle. In addition, the fibular collateral ligament and popliteus tendon were located within 1 mm of a reference line projected along the posterior femoral cortex distally, and also were located within the posteroinferior quadrant bound by the posterior femoral cortex extension reference line and another reference line perpendicular to it at the posterior margin of Blumensaat's line. CONCLUSION: Comprehensive qualitative and quantitative guidelines for assessing posterolateral knee structures on both anteroposterior and lateral knee radiographs were described. CLINICAL SIGNIFICANCE: This radiographic information regarding the attachment sites of posterolateral structures can serve as a valuable reference for preoperative, intraoperative, and postoperative assessments of surgical reconstructions.