Chondrocytes Utilize a Cholesterol-Dependent Lipid Translocator to Externalize Phosphatidylserine

During endochondral ossification, growth plate chondrocytes release plasma membrane (PM) derived matrix vesicles (MV), which are the site of initial hydroxyapatite crystal formation. MV constituents which facilitate the mineralization process include the integral membrane ectoenzymes alkaline phosphatase (ALPase) and nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), along with a phosphatidylserine-(PS-) rich membrane surface that binds annexins and calcium, resulting in enhanced calcium entry into MV. In this study, we determined that chick growth plate MV were highly enriched in membrane raft microdomains containing high levels of cholesterol, glycophosphatidylinositol- (GPI-) anchored ALPase, and phosphatidylserine (PS) localized to the external leaflet of the bilayer. To determine how such membrane microdomains arise during chondrocyte maturation, we explored the role of PM cholesterol-dependent lipid assemblies in regulating the activities of lipid translocators involved in the externalization of PS. We first isolated and determined the composition of detergent-resistant membranes (DRMs) from chondrocyte PM. DRMs isolated from chondrocyte PM were enhanced in ganglioside 1 (GM1) and cholesterol as well as GPI-anchored ALPase. Furthermore, these membrane domains were enriched in PS (localized to the external leaflet of the bilayer) and had significantly higher ALPase activity than non-cholesterol- enriched domains. To understand the role of cholesterol-dependent lipid assemblies in the externalization of PS, we measured the activities of two lipid transporters involved in PS externalization, aminophospholipid translocase (APLT) and phospholipid scramblase (PLSCR1), during maturation of a murine chondrocytic cell line, N1511. In this report, we provide the first evidence that maturing chondrocytes express PLSCR1 and have scramblase activity. We propose that redistribution of PS is dependent on an increase in phospholipid scramblase activity and a decrease in APLT activity. Lastly, we show that translocator activity is most likely to be modulated by membrane cholesterol levels through a membrane raft microdomain. © 2006 American Chemical Society

Cholesterol-Rich Membrane Microdomains Mediate Cell Cycle Arrest Induced by Actinobacillus Actinomycetemcomitans Cytolethal-Distending Toxin

We have previously shown that Actinobacillus actinomycetemcomitans cytolethal-distending toxin (Cdt) is a potent immunosuppressive agent that induces G2/M arrest in human lymphocytes. In this study, we explored the possibility that Cdt-mediated immunotoxicity involves lipid membrane microdomains. We first determined that following treatment of Jurkat cells with Cdt holotoxin all three Cdt subunits localize to these microdomains. Laser confocal microscopy was employed to colocalize the subunits with GM1-enriched membrane regions which are characteristic of membrane rafts. Western blot analysis of isolated lipid rafts also demonstrated the presence of Cdt peptides. Cholesterol depletion, using methyl β-cyclodextrin, protected cells from the ability of the Cdt holotoxin to induce G2 arrest. Moreover, cholesterol depletion reduced the ability of the toxin to associate with Jurkat cells. Thus, lipid raft integrity is vital to the action of Cdt on host cells. The implications of our observations with respect to Cdt mode of action are discussed. © 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd

Cholesterol-Rich Membrane Microdomains Mediate Cell Cycle Arrest Induced by Actinobacillus Actinomycetemcomitans Cytolethal-Distending Toxin

We have previously shown that Actinobacillus actinomycetemcomitans cytolethal-distending toxin (Cdt) is a potent immunosuppressive agent that induces G2/M arrest in human lymphocytes. In this study, we explored the possibility that Cdt-mediated immunotoxicity involves lipid membrane microdomains. We first determined that following treatment of Jurkat cells with Cdt holotoxin all three Cdt subunits localize to these microdomains. Laser confocal microscopy was employed to colocalize the subunits with GM1-enriched membrane regions which are characteristic of membrane rafts. Western blot analysis of isolated lipid rafts also demonstrated the presence of Cdt peptides. Cholesterol depletion, using methyl β-cyclodextrin, protected cells from the ability of the Cdt holotoxin to induce G2 arrest. Moreover, cholesterol depletion reduced the ability of the toxin to associate with Jurkat cells. Thus, lipid raft integrity is vital to the action of Cdt on host cells. The implications of our observations with respect to Cdt mode of action are discussed

Cancellation of coherent artifacts in optical coherence tomography imaging

Coherent artifacts in optical coherence tomography ͑OCT͒ images can severely degrade image quality by introducing false targets if no targets are present at the artifact locations. Coherent artifacts can also add constructively or destructively to the targets that are present at the artifact locations. This constructive or destructive interference will result in cancellation of the true targets or in display of incorrect echo amplitudes of the targets. We introduce the use of a nonlinear deconvolution algorithm, CLEAN, to cancel coherent artifacts in OCT images of extracted human teeth. The results show that CLEAN can reduce the coherent artifacts to the noise background, sharpen the air-enamel and enamel-dentin interfaces, and improve the image contrast

Actinobacillus Actinomycetemcomitans Leukotoxin Requires Lipid Microdomains for Target Cell Cytotoxicity

Actinobacillus actinomycetemcomitans produces a leukotoxin (Ltx) that kills leukocyte function-associated antigen-1 (LFA-1)-bearing cells from man, the Great Apes and Old World monkeys. The unique specificity of Ltx for the β2 integrin, LFA-1, suggests it is capable of providing insight into the pathogenic mechanisms of Ltx and other RTX toxins. Using the Jurkat T cell line and an LFA-1-deficient Jurkat mutant (Jβ2.7) as models, we found the initial effect of Ltx is to elevate cytosolic Ca2+ [Ca2+]c, an event that is independent of the Ltx/LFA-1 interaction. [Ca2+]c increases initiate a series of events that involve the activation of calpain, talin cleavage, mobilization to, and subsequent clustering of, LFA-1 in cholesterol and sphingolipid-rich regions of the plasma membrane known as lipid rafts. The association of Ltx and LFA-1 within lipid rafts is essential for cell lysis. Jβ2.7 cells fail to accumulate Ltx in their raft fractions and are not killed, while cholesterol depletion experiments demonstrate the necessity of raft integrity for Ltx function. We propose that toxin-induced Ca2+ fluxes mobilize LFA-1 to lipid rafts where it associates with Ltx. These findings suggest that Ltx utilizes the raft to stimulate an integrin signalling pathway that leads to apoptosis of target cells

Actinobacillus Actinomycetemcomitans Leukotoxin Requires Lipid Microdomains for Target Cell Cytotoxicity

Actinobacillus actinomycetemcomitans produces a leukotoxin (Ltx) that kills leukocyte function-associated antigen-1 (LFA-1)-bearing cells from man, the Great Apes and Old World monkeys. The unique specificity of Ltx for the β2 integrin, LFA-1, suggests it is capable of providing insight into the pathogenic mechanisms of Ltx and other RTX toxins. Using the Jurkat T cell line and an LFA-1-deficient Jurkat mutant (Jβ2.7) as models, we found the initial effect of Ltx is to elevate cytosolic Ca2+ [Ca2+]c, an event that is independent of the Ltx/LFA-1 interaction. [Ca2+ ]c increases initiate a series of events that involve the activation of calpain, talin cleavage, mobilization to, and subsequent clustering of, LFA-1 in cholesterol and sphingolipid-rich regions of the plasma membrane known as lipid rafts. The association of Ltx and LFA-1 within lipid rafts is essential for cell lysis. Jβ2.7 cells fail to accumulate Ltx in their raft fractions and are not killed, while cholesterol depletion experiments demonstrate the necessity of raft integrity for Ltx function. We propose that toxin-induced Ca2+ fluxes mobilize LFA-1 to lipid rafts where it associates with Ltx. These findings suggest that Ltx utilizes the raft to stimulate an integrin signalling pathway that leads to apoptosis of target cells. © 2006 The Authors; Journal compilation © 2006 Blackwell Publishing Ltd

Genetic mapping in mice identifies DMBT1 as a candidate modifier of mammary tumors and breast cancer risk

Low-penetrance breast cancer susceptibility alleles seem to play a significant role in breast cancer risk but are difficult to identify in human cohorts. A genetic screen of 176 N2 backcross progeny of two Trp53+/- strains, BALB/c and C57BL/6, which differ in their susceptibility to mammary tumors, identified a modifier of mammary tumor susceptibility in an ∼25-Mb interval on mouse chromosome 7 (designated SuprMam1). Relative to heterozygotes, homozygosity for BALB/c alleles of SuprMam1 significantly decreased mammary tumor latency from 70.7 to 61.1 weeks and increased risk twofold (P = 0.002). Dmbt1 (deleted in malignant brain tumors 1) was identified as a candidate modifier gene within the SuprMam1 interval because it was differentially expressed in mammary tissues from BALB/c-Trp53+/- and C57BL/6-Trp53+/- mice. Dmbt1 mRNA and protein was reduced in mammary glands of the susceptible BALB/c mice. Immunohistochemical staining demonstrated that DMBT1 protein expression was also significandy reduced in normal breast tissue from women with breast cancer (staining score, 1.8; n = 46) compared with cancer-free controls (staining score, 3.9; n = 53; P < 0.0001). These experiments demonstrate the use of Trp53+/- mice as a sensitized background to screen for low-penetrance modifiers of cancer. The results identify a novel mammary tumor susceptibility locus in mice and support a role for DMBT1 in suppression of mammary tumors in both mice and women

Combined Forward-Backward Asymmetry Measurements in Top-Antitop Quark Production at the Tevatron

The CDF and D0 experiments at the Fermilab Tevatron have measured the asymmetry between yields of forward- and backward-produced top and antitop quarks based on their rapidity difference and the asymmetry between their decay leptons. These measurements use the full data sets collected in proton-antiproton collisions at a center-of-mass energy of $\sqrt s =1.96$ TeV. We report the results of combinations of the inclusive asymmetries and their differential dependencies on relevant kinematic quantities. The combined inclusive asymmetry is $A_{\mathrm{FB}}^{t\bar{t}} = 0.128 \pm 0.025$. The combined inclusive and differential asymmetries are consistent with recent standard model predictions

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo