A high‐speed data‐collection system for large‐unit‐cell crystals is presented, using the Fuji Imaging Plate as an X‐ray detector and a rotating‐anode generator as the X‐ray source. It is an automatic data‐acquisition system that requires almost no manual intervention. The quality of data collected on the system is discussed. Merging R values ranged from 0.04 to 0.05. Compared with a four‐circle diffractometer, data reproducibility was better, isomorphous/anomalous Patterson maps were almost identical in quality and data from a small‐molecule crystal, cytidine, were of almost the same quality. Protein structures were refinable using the data measured on the system, the final crystallographic R value of the 2.2 Å 3‐isopropylmalatedehydrogenase structure being 0.185 and that of the 1.88 ÅFlammulina veltipes agglutinin structure being 0.199.

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Aim:The ATP binding cassette transporters A1 and G1 (ABCA1/G1) and scavenger receptor class B type (SR-B ) are key molecules in cholesterol efflux and atherogenesis. These genes are regulated by peroxisome proliferator-activated receptor (PPAR ) and liver X receptor (LXR). Telmisartan is an angiotensin type 1 receptor blocker which has been reported to act as a ligand for PPAR . We investigated whether PPAR -activating ARBs affect the expression of these genes and cholesterol efflux from macrophages. Methods and Results: Telmisartan increased ABCA1, ABCG1 and SR-B mRNA levels in THP-1 macrophages in a dose-and time-dependent fashion. It also increased their protein levels and enhanced apoA--and HDL-mediated cholesterol efflux from macrophages. The knockdown of PPAR by siRNA abolished the telmisartan-induced expression of these genes. The knockdown of LXR resulted in the complete and partial abolishment of telmisartan-induced ABCA1 and ABCG1 expression, respectively. We also demonstrated that telmisartan-induced SR-B expression was dependent on the PPAR pathway but not on the LXR pathway. A luciferase assay using an ABCA1 promoter construct showed that telmisartan activated ABCA1 transcription, which was abolished if the LXR binding element was mutated, indicating that increased ABCA1 transcription by telmisartan is LXRdependent. Conclusion: Our results showed that telmisartan enhanced both apoA--and HDL-mediated cholesterol efflux from macrophages by increasing ABCA1, ABCG1 and SR-B expression via PPAR -dependent and LXR-dependent/independent pathways.

ATP-binding cassette transporter (ABC) A7 is an ABC family protein that is a so-called full-size ABC transporter, highly homologous to ABCA1, which mediates the biogenesis of high-density lipoprotein (HDL) with cellular lipid and helical apolipoproteins. ABCA7 mediates the formation of HDL when exogenously transfected and expressed; however, endogenous ABCA7 was shown to have no significant impact on the generation of HDL and was found to be associated with phagocytosis regulated by sterol regulatory element binding protein 2. Since phagocytosis is one of the fundamental functions of animal cells as an important responsive reaction to infection, injury and apoptosis, ABCA7 seems to be one of the key molecules linking sterol homeostasis and the host defense system. In this context, HDL apolipoproteins were shown to enhance phagocytosis by stabilizing ABCA7 against calpain-mediated degradation and increasing its activity, shedding light on a new aspect of the regulation of the host-defense system. Roles of ATP-Binding Cassette Transporter (ABC) A7 in cholesterol homeostasisHigh-density lipoprotein (HDL) apolipoproteins, such as apolipoprotein (apo) A-and apoA-, are helical amphiphilic proteins bound to the HDL lipid surface in equilibrium with an aqueous phase. These helical apolipoproteins interact with the cell surface in their free form and generate HDL particles by removing cellular phospholipid and cholesterol [1][2][3] . Fibroblasts from patients with Tangier disease, a genetic HDL deficiency, lack the interaction with apolipoprotein 4, 5) due to mutations in ABCA1 [6][7][8] , indicating that this reaction is the main source of plasma HDL. The reaction is one of the major pathways of cellular cholesterol release along with diffusion-mediated nonspecific efflux 9) . Thus, ABCA1 is a key membrane protein for cholesterol homeostasis, for the generation of HDL and the release of cell cholesterol for its catabolism 10) . ABCA1 is one of the ABC family proteins consisting of two sets of multiple membranespanning domains plus the Walker motifs for ATP interaction 11, 12) , and is thereby classified as a so-called full-size ABC transporter.Human ABCA7 is another full-size ABC transporter showing the highest homology among those known to human ABCA1 (54%) and human ABCA4 (49%) 13) . On the other hand, its interspecies identity of the protein sequences is 79% between humans and mice, less than that of ABCA1 (95%) and ABCA4 (88%) 14)

Objective-The ATP-binding cassette transporter-A1 (ABCA1) regulates cholesterol efflux from cells and is involved in high-density lipoprotein metabolism and atherogenesis. The objective of this study was to investigate the effect of dexamethasone (Dex) and other glucocorticoid receptor (GR) ligands on apolipoprotein AI-mediated cholesterol efflux from macrophages and ABCA1 expression in them. Methods and Results-Dex, a GR agonist, decreased ABCA1 mRNA levels in a dose-and time-dependent fashion, and RU486, a GR antagonist, reversed the inhibitory effect of Dex. The effects of Dex and RU486 on ABCA1 protein levels and apolipoprotein AI-mediated cholesterol efflux from the macrophages were consistent with these changes in mRNA levels. Transfected RAW264.7, together with a human ABCA1 promoter-luciferase construct, inhibited transcriptional activity by Dex and overexpression of human GR. Transrepression by GR was not mediated by liver X receptor (LXR), because there were no differences in the effects of the GR ligands on promoter activity between a reporter construct with mutations at the LXR binding site and one without the mutations, and no changes were brought about in ABCG1 and ABCG4 expression by GR ligands. Conclusions-Our results showed that GR ligands affected ABCA1 expression and cholesterol efflux from macrophages, which are regulated by GR through a LXR-independent mechanism. (Arterioscler Thromb Vasc Biol. 2006;26:163-168.)

Thermodynamic parameters for complexation of polyvalent cyclodextrin (CD) cation and anion with oppositely charged guests have been determined in D2O containing 0.02 M NaCl by means of 1H-NMR spectroscopy. Protonated heptakis(6-amino-6-deoxy)-beta-CD (per-NH3+-beta-CD) forms stable inclusion complexes with monovalent guest anions. The enthalpy (deltaH) and entropy changes (deltaS) for complexation of per-NH3+-beta-CD with p-methylbenzoate anion (p-CH3-Ph-CO2-) are 3.8 +/- 0.7 kJ mol(-1) and 88.6 +/- 2.2 J mol(-1) K(-1), respectively. The deltaH and deltaS values for the native beta-CD-p-CH3-Ph-CO2- system are -8.6 +/- 0.1 kJ mol(-1) and 15.3 +/- 0.7 J mol(-1) K(-1), respectively. The thermodynamic parameters clearly indicate that dehydration from both the host and guest ions accounts for the entropic gain in inclusion process of p-CH3-Ph-CO2- into the per-NH3+-beta-CD cavity. The fact that the neutral guests such as 2,6-dihydroxynaphthalene and p-methylbenzyl alcohol hardly form the complexes with per-NH3+-beta-CD exhibits that van der Waals and/or hydrophobic interactions do not cause the complexation of the polyvalent CD cation with the monovalent anion. The acetate anion is not included into the per-NH3+-beta-CD cavity, while the butanoate and hexanoate anions form the inclusion complexes. The complexation of the alkanoate anions is entropically dominated. Judging from these results, it may be concluded that Coulomb interactions cooperated with inclusion are required for realizing the large entropic gain due to extended dehydration. Entropically favorable complexation was also observed for the anionic CD-cationic guest system. The present study might present a general mechanism for ion pairing in water.

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Objective-To investigate the interaction of ATP-binding cassette transporter A1 (ABCA1) with calmodulin in relation to its calpain-mediated degradation because many calpain substrates bind calmodulin to regulate cellular functions. Methods and Results-The activity of ABCA1 is regulated through proteolysis by calpain. An immunoprecipitation and glutathione S-transferase pull-down assay revealed that ABCA1 directly binds calmodulin in a Ca 2ϩ -dependent manner. The cytoplasmic loop of ABCA1 contains a typical calmodulin binding sequence of 1-5-8-14 motifs (1245 to 1257 amino acids). The peptide of this region showed binding to calmodulin, and deletion of the 1-5-8-14 motif abolished this interaction. This motif is located near the ABCA1 Pro-Glu-Ser-Thr sequence, and the presence of calmodulin/Ca 2ϩ protected the peptides from proteolysis by calpain. The knockdown of calmodulin by a specific small and interfering RNA increased the degradation of ABCA1 and decreased ABCA1 protein and apolipoprotein A-I-mediated lipid release. Surprisingly, calmodulin inhibitor W7 increased calmodulin binding to ABCA1 and protected it from calpain-mediated degradation, consistent with our previous finding that this compound increased apolipoprotein A-I-mediated cell cholesterol release. Conclusion-Calmodulin directly binds and stabilizes ABCA1 in the presence of Ca 2ϩ and increases the generation of high-density lipoprotein.