Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.     

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.     

Maintained contractile reserve in a transgenic mouse model of myocardial stunning

Cardiac excitation-contraction (E-C) coupling is impaired at the myofilament level in the reversible postischemic dysfunction known as "stunned" myocardium. We characterized tension development and calcium cycling in intact isolated trabeculae from transgenic (TG) mice expressing the major proteolytic degradation fragment of troponin I (TnI) found in stunned myocardium (TnI(1-193)) and determined the ATPase activity of myofibrils extracted from TG and non-TG mouse hearts. The phenotype of these mice at baseline recapitulates that of stunning. Here, we address the question of whether contractile reserve is preserved in these mice, as it is in genuine stunned myocardium. During twitch contractions, calcium cycling was normal, whereas tension was greatly reduced, compared with non-TG controls. A decrease in maximum Ca2+-activated tension and Ca2+ desensitization of the myofilaments accounted for this contractile dysfunction. The decrease in maximum tension was paralleled by an equivalent decrease in maximum Ca2+-activated myofibrillar ATPase activity. Exposure to high calcium or isoproterenol recruited a sizable contractile reserve in TG muscles, which was proportionately similar to that in control muscles but scaled downward in amplitude. These results suggest that calcium regulatory pathways and beta-adrenergic signal transduction remain intact in isolated trabeculae from stunned TG mice, further recapitulating key features of genuine stunned myocardium.     

Hip and knee joint kinematics during a diagonal jump landing in anterior cruciate ligament reconstructed females

Anterior cruciate ligament (ACL) injury is a common injury encountered by sport medicine clinicians. Surgical reconstruction is the recommended treatment of choice for those athletes wishing to return to full-contact sports participation and for sports requiring multi-directional movement patterns. The aim of ACL reconstruction is to restore knee joint mechanical stability such that the athlete can return to sporting participation. However, knowledge regarding the extent to which lower limb kinematic profiles are restored following ACL reconstruction is limited. In the present study the hip and knee joint kinematic profiles of 13 ACL reconstructed (ACL-R) and 16 non-injured control subjects were investigated during the performance of a diagonal jump landing task. The ACL-R group exhibited significantly less peak knee joint flexion (P=0.01). Significant between group differences were noted for time averaged hip joint sagittal plane (P<0.05) and transverse plane (P<0.05) kinematic profiles, as well as knee joint frontal plane (P<0.05) and sagittal plane (P<0.05) kinematic profiles. These results suggest that aberrant hip and knee joint kinematic profiles are present following ACL reconstruction, which could influence future injury risk.     

Synthesis and SAR of selective small molecule neuropeptide Y Y2 receptor antagonists

Highly potent and selective small molecule neuropeptide Y Y2 receptor antagonists are reported. The systematic SAR exploration of a hit molecule N-(4-ethoxyphenyl)-4-[hydroxy(diphenyl)methyl]piperidine-1-carbothioamide, identified from HTS, led to the discovery of highly potent NPY Y2 antagonists 16 (CYM 9484) and 54 (CYM 9552) with IC(50) values of 19 nM and 12 nM respectively.     

Insertion mutagenesis of Escherichiacoli GroEL

To gain insights into the in vivo folding and assembly of bacterial chaperonins, groEL was subjected to insertion mutagenesis using transposon ISlacZ/in. Four GroEL-LacZ fusions and the corresponding insertion mutants were obtained after residues 34, 90, 291, and 367. Apical domain insertion mutants GroEL291 and GroEL367 were degraded into monomeric 30- and 40-kDa fragments, respectively. Only the latter was fully soluble, suggesting that proper isomerization of an essentially complete apical domain is required for efficient protomer folding. Truncated variants were inactive as minichaperones as they failed to restore the growth of groEL140 cells at 43 degrees C whether or not GroES was co-expressed. A 31-residue insertion in equatorial helix D led to complete degradation of GroEL90. By contrast, extraneous amino acids were tolerated at equatorial position 34, indicating that this region is highly flexible. Nevertheless, GroEL34 did not fold as efficiently as authentic GroEL and reached only a heptameric conformation.     

Sequence-function analysis of the K+-selective family of ion channels using a comprehensive alignment and the KcsA channel structure

Sequence-function analysis of K(+)-selective channels was carried out in the context of the 3.2 A crystal structure of a K(+) channel (KcsA) from Streptomyces lividans (Doyle et al., 1998). The first step was the construction of an alignment of a comprehensive set of K(+)-selective channel sequences forming the putative permeation path. This pathway consists of two transmembrane segments plus an extracellular linker. Included in the alignment are channels from the eight major classes of K(+)-selective channels from a wide variety of species, displaying varied rectification, gating, and activation properties. Segments of the alignment were assigned to structural motifs based on the KcsA structure. The alignment's accuracy was verified by two observations on these motifs: 1), the most variability is shown in the turret region, which functionally is strongly implicated in susceptibility to toxin binding; and 2), the selectivity filter and pore helix are the most highly conserved regions. This alignment combined with the KcsA structure was used to assess whether clusters of contiguous residues linked by hydrophobic or electrostatic interactions in KcsA are conserved in the K(+)-selective channel family. Analysis of sequence conservation patterns in the alignment suggests that a cluster of conserved residues is critical for determining the degree of K(+) selectivity. The alignment also supports the near-universality of the "glycine hinge" mechanism at the center of the inner helix for opening K channels. This mechanism has been suggested by the recent crystallization of a K channel in the open state. Further, the alignment reveals a second highly conserved glycine near the extracellular end of the inner helix, which may be important in minimizing deformation of the extracellular vestibule as the channel opens. These and other sequence-function relationships found in this analysis suggest that much of the permeation path architecture in KcsA is present in most K(+)-selective channels. Because of this finding, the alignment provides a robust starting point for homology modeling of the permeation paths of other K(+)-selective channel classes and elucidation of sequence-function relationships therein. To assay these applications, a homology model of the Shaker A channel permeation path was constructed using the alignment and KcsA as the template, and its structure evaluated in light of established structural criteria.