Localization of nitration and chlorination sites on apolipoprotein A-I catalyzed by myeloperoxidase in human atheroma and associated oxidative impairment in ABCA1-dependent cholesterol efflux from macrophages

We recently reported that apolipoprotein A-I (apoA-I), the major protein component of high density lipoprotein, is a selective target for myeloperoxidase (MPO)-catalyzed nitration and chlorination in both and serum of subjects with cardiovascular disease. We further showed that the extent of both apoA-I nitration and chlorination correlated with functional impairment in reverse cholesterol transport activity of the isolated lipoprotein. Herein we used tandem mass spectrometry to map the sites of MPO-mediated apoA-I nitration and chlorination in vitro and in vivo and to relate the degree of site-specific modifications to loss of apoA-I lipid binding and cholesterol efflux functions. Of the seven tyrosine residues in apoA-I, Tyr-192, Tyr-166, Tyr-236, and Tyr-29 were nitrated and chlorinated in MPO-mediated reactions. Site-specific liquid chromatography-mass spectrometry quantitative analyses demonstrated that the favored modification site following exposure to MPO-generated oxidants is Tyr-192. MPO-dependent nitration and chlorination both proceed with Tyr-166 as a secondary site and with Tyr-236 and Tyr-29 modified only minimally. Parallel functional studies demonstrated dose-dependent losses of ABCA1-dependent cholesterol acceptor and lipid binding activities with apoA-I modification by MPO. Finally tandem mass spectrometry analyses showed that apoA-I in human atherosclerotic tissue is nitrated at the MPO-preferred sites, Tyr-192 and Tyr-166. The present studies suggest that site-specific modifications of apoA-I by MPO are associated with impaired lipid binding and ABCA1-dependent cholesterol acceptor functions, providing a molecular mechanism that likely contributes to the clinical link between MPO levels and cardiovascular disease risk.     

A comparison of scholastic and collegiate longsnapping techniques

The purpose of this study was to identify the differences that exist between collegiate (CS) and scholastic (SS) longsnappers. Six CS (21.4 +/- 1.37 years) and 7 SS (16.7 +/- 1.11 years) longsnappers were filmed performing 10 longsnaps each. The CS and SS longsnappers had 7.0 +/- 0.89 and 2.7 +/- 0.95 years experience longsnapping, respectively. Each of the 10 longsnaps for all subjects were analyzed for takeoff velocity and accuracy. The snap that most closely approximated the individual snapper's median values for takeoff velocity and accuracy was digitized using a 20-point model. CS were both faster (0.85 +/- 0.10 seconds vs. 1.25 +/- 0.19 seconds) and more accurate in terms of mean radial error (29.36 +/- 8.4 cm vs. 47.2 +/- 8.26 cm) than their SS counterparts. These differences were related to body positioning both before and during the longsnap. CS exhibited more shoulder flexion (135 +/- 6.33 degrees ; vs. 98 +/- 9.01 degrees ) and greater elbow extension (133 +/- 8.1 degrees vs. 95 +/- 6.77 degrees ) in the set position phase, along with greater center of mass movement (0.27 +/- 0.02 m vs. 0.14 +/- 0.04 m) in the anterior-posterior direction and less hip flexion (72 +/- 1.85 degrees vs. 49 +/- 9.42 degrees ) during the preflight phase. Longsnapping experience and accuracy were significantly related (r = -0.82, p < 0.05). These results suggest that body positioning both before and during the longsnap motion significantly influence the velocity and accuracy of the longsnap.     

The two tempos of nuclear pore complex evolution: highly adapting proteins in an ancient frozen structure

Background  

The origin of the nuclear compartment has been extensively debated, leading to several alternative views on the evolution of the eukaryotic nucleus. Until recently, too little phylogenetic information was available to address this issue by using multiple characters for many lineages.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label: the effect of cholesterol

X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, Smol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.     

Molecular cytogenetic definition of the chicken genome: the first complete avian karyotype

Chicken genome mapping is important for a range of scientific disciplines. The ability to distinguish chromosomes of the chicken and other birds is thus a priority. Here we describe the molecular cytogenetic characterization of each chicken chromosome using chromosome painting and mapping of individual clones by FISH. Where possible, we have assigned the chromosomes to known linkage groups. We propose, on the basis of size, that the NOR chromosome is approximately the size of chromosome 22; however, we suggest that its original assignment of 16 should be retained. We also suggest a definitive chromosome classification system and propose that the probes developed here will find wide utility in the fields of developmental biology, DT40 studies, agriculture, vertebrate genome organization, and comparative mapping of avian species.     

Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 10⁶ M⁻¹ s⁻¹ and 6.7 ± 2.5 × 10⁻² s⁻¹, respectively, which corresponded to an average equilibrium dissociation constant (K_D) of 2.6 ± 1.4 × 10⁻⁸ M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.     

Kinetic attributes of Na+, K+ ATPase and lipid/phospholipid profiles of rat and human erythrocyte membrane

Kinetic properties of Na+, K+ ATPase of membranes from rat and human erythrocytes were examined. The enzyme stability decreased with incubation time. The Vmax of the human enzyme was about 4 times lower than the values of the rat enzyme. However the energies of activation were higher. Phase transition temperature for the rat and the human enzyme was 24 degrees C and 17 degrees C, respectively. The human erythrocyte membranes were characterized by lower total phospholipid and cholesterol contents and were relatively more fluid. The human membranes contained lower proportions of acidic phospholipids which correlated well with the lower Vmax of the enzyme; the proportion of lysophosphoglyceride and sphingomyelin was higher in the human membrane.     

Comparison of normalization methods for CodeLink Bioarray data

Background  

The quality of microarray data can seriously affect the accuracy of downstream analyses. In order to reduce variability and enhance signal reproducibility in these data, many normalization methods have been proposed and evaluated, most of which are for data obtained from cDNA microarrays and Affymetrix GeneChips. CodeLink Bioarrays are a newly emerged, single-color oligonucleotide microarray platform. To date, there are no reported studies that evaluate normalization methods for CodeLink Bioarrays.