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Reverse cholesterol transport (RCT) is pivotal in the return of excess cholesterol from peripheral tissues to the liver for excretion in bile and eventually feces. RCT from macrophages is a critical anti-atherogenicity mechanism of HDL. As the cholesterol absorption inhibitor ezetimibe promoted RCT in mice, which lack cholesterol ester transfer protein (CETP), we investigated its effects in hamsters, which have CETP. A high-cholesterol diet (HC) increased cholesterol levels throughout lipoprotein fractions and ezetimibe markedly reduced VLDL/LDL cholesterol levels under both normal chow (NC) and HC. However, ezetimibe did not affect and reduced HDL-cholesterol levels under NC and HC, respectively. Intraperitoneal injection of (3)H-cholesterol pre-labeled macrophages in an in vivo RCT assay increased tracer accumulation in the liver but reduced it in bile under HC, and these changes were completely cancelled by ezetimibe. Under both NC and HC, ezetimibe reduced tracer levels in the liver but increased them in feces, indicating promotion of RCT in vivo. We performed a RCT assay using hamsters subjected to bile duct ligation (BDL) to clarify whether a transintestinal cholesterol efflux (TICE) pathway contributes to ezetimibe's enhancement of RCT. BDL markedly inhibited macrophage-derived (3)H-cholesterol excretion to feces and cancelled ezetimibe's stimulatory effect on RCT, suggesting that biliary cholesterol excretion is a major contributor in RCT promotion by ezetimibe but the contribution of the TICE pathway is minimal. In conclusions, ezetimibe exerts an additive anti-atherogenic property by enhancing RCT in hamsters. Our findings suggest that this property is independent of the TICE pathway., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Stearoyl-coenzyme A desaturase 1 (SCD1) is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. However, the impact of SCD1 on atherosclerosis remains unclear. The aim of this study was to determine whether SCD1 affects macrophage reverse cholesterol transport (RCT) in mice. Compared to the control, adenoviral-mediated SCD1 overexpression in RAW264.7 macrophages increased cholesterol efflux to HDL, but not to apoA-I, without clear changes in ABCA1, ABCG1 and SR-BI expressions. While knockdown of ABCG1 and SR-BI did not affect the SCD1-induced cholesterol efflux to HDL, SCD1-overexpressing macrophages promoted the formation of both normal- and large-sized HDL in media, accompanying increased apolipoprotein A-I levels in HDL fractions. Transformation to larger particles of HDL was independently confirmed by nuclear magnetic resonance-based lipoprotein analysis. Interestingly, media transfer assays revealed that HDL generated by SCD1 had enhanced cholesterol efflux potential, indicating that SCD1 transformed HDL to a more anti-atherogenic phenotype. To study macrophage RCT in vivo, (3)H-cholesterol-labeled RAW264.7 cells overexpressing SCD1 or the control were intraperitoneally injected into mice. Supporting the in vitro data, injection of SCD1-macrophages resulted in significant increases in (3)H-tracer in plasma, liver, and feces compared to the control. Moreover, there was a shift towards larger particles in the (3)H-tracer distribution of HDL fractions obtained from the mice. In conclusion, macrophage-specific SCD1 overexpression promotes overall RCT through increased cholesterol efflux to HDL, suggesting that macrophage SCD1 achieves an anti-atherogenic effect by enhancing RCT., (Copyright © 2013 Elsevier B.V. All rights reserved.)

ABC transporter G1 (ABCG1) plays a pivotal role in HDL-mediated cholesterol efflux and atherogenesis. We investigated whether, and how, retinoic acid receptors (RARs) regulate ABCG1 expression in macrophages. All-trans retinoic acid (ATRA), an RAR ligand, increased ABCG1 protein levels and apoA-I/HDL-mediated cholesterol efflux from the macrophages. Both ATRA and other RAR agonists, TTNPB and Am580, increased major transcripts driven by promoter B upstream of exon 5, though minor transcripts driven by promoter A upstream of exon 1 were only increased by ATRA. The stimulatory effects of ATRA on ABCG1 expression were completely abolished in the presence of RAR/RXR antagonists but were only partially canceled in the presence of an LXR antagonist. Adenovirus with overexpressed oxysterol sulfotransferase abolished the LXR pathway, as previously reported, and ATRA-responsiveness in ABCA1/ABCG1 expressions were respectively attenuated by 38 and 22% compared to the control virus. Promoter assays revealed that ABCG1 levels were regulated more by promoter B than promoter A, and ATRA activated promoter B in a liver X receptor-responsive element (LXRE)-dependent manner. Further, LXRE-B in intron 7, but not LXRE-A in intron 5, enhanced ATRA responsiveness under overexpression of all RAR isoforms-RARα/β/γ. In contrast, the activation of promoter B by TTNPB depended on LXRE-B and RARα, but not on RARβ/γ. Finally, chromatin immunoprecipitation and gel-shift assays revealed a specific and direct repeat 4-dependent binding of RARα to LXRE-B. In conclusion, RAR ligands increase ABCA1/G1 expression and apoA-I/HDL-mediated cholesterol efflux from macrophages, and modulate ABCG1 promoter activity via LXRE-dependent mechanisms., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Apoptotic cell death was induced in human lung cancer DMS114 cells by treatment with beta-hydroxyisovalerylshikonin (beta-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases. Changes in phosphoprotein profiles were analyzed by two-dimensional-polyacrylamide gel electrophoresis (2D-PAGE) after the cells were treated with beta-HIVS. One spot on the 2D gel showed a marked decrease in intensity and the corresponding protein was identified by mass spectrometry as dUTP nucleotidohydrolase (dUTPase). The beta-HIVS-induced decrease of dUTPase in the phosphoprotein fraction of DMS114 cells was confirmed using immunoblotting. Treatment of the cells with beta-HIVS-induced rapid reduction of dUTPase activity. An antioxidant N-acetyl-cysteine inhibited both the reduction of phosphorylated dUTPase and the induction of apoptosis by beta-HIVS treatment of DMS114 cells. Introduction of siRNA directed against dUTPase mRNA into DMS114 cells enhanced the susceptibility of beta-HIVS-induced apoptosis. Treatment of DMS114 cells with beta-HIVS and 5-fluorouracil, a specific inhibitor of thymidylate synthase used as a chemotherapeutic drug, revealed the synergistic effects of these drugs on the inhibition of cell growth. These results suggest that dUTPase activity is one of the crucial factors involved in apoptotic cell death in lung cancer cells.

We have developed an enzyme immunoassay method for curculin, a new type of taste-modifying protein. This method can accurately quantify 0.05-20 ng of curculin, a sensitivity about 3000-times that of the psychometric method. The content of curculin in the fruit of Curculigo latifolia increased gradually until 3 weeks after artificial pollination and dramatically at 4 weeks, to finally reach 1.3 mg per fruit. Immunoblot analysis indicated that antiserum to curculin was faintly reactive with miraculin, but not with thaumatin or monellin.

The uptake of 1-methyl-4-phenylpyridinium (MPP+) by intact mitochondria was measured by an electrode sensitive to MPP+. The electrode was constructed with a polyvinyl chloride membrane that contained tetraphenylboron (TPB) as an ion-exchange. MPP+ was taken up by mitochondria in an energy-dependent process. TPB rapidly enhanced MPP+ uptake by mitochondria, and then induced release of MPP+ from mitochondria in medium containing glutamate and malate. No release of MPP+ from mitochondria after addition of TPB could be observed in medium containing succinate, the oxidation of which is not inhibited by MPP+. The release of MPP+ was caused by respiratory inhibition by MPP+ taken up in mitochondria. Since the release of MPP+ did not increased O2 uptake in mitochondria, the major part of MPP+ released from the matrix, where no respiratory enzyme inhibited by MPP+ exists. We concluded the following effect of TPB on MPP+ uptake from the results: (1) The increase of MPP+ concentration in matrix by addition of TPB increased the amount of bound to the inner membranes of mitochondria. (2) The increase of the amount of MPP+ in the inner membranes enhanced the respiratory inhibition. (3) The respiratory inhibition induced to release MPP+ from the matrix. The relation between MPP+ distribution in the membrane of mitochondria and the respiratory inhibition by MPP+ are discussed.

The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.

Fluorescence changes of rhodamine 6G in synaptosomal suspension, which are correlated to changes in membrane potential in synaptosomes, were measured in the presence of various monosaccharides and organic acids. Addition of D-glucose, D-mannose, pyruvate and L-lactate hyperpolarized the membrane potential, whereas D-fructose, L-glucose, D-galactose, citrate, succinate and L-glutamate were without effect on the membrane potential. Hyperpolarization induced by D-glucose was inhibited by cytochalasin B, phloretin, iodoacetate, F- and 2-deoxy-D-glucose, but not inhibited by oligomycin or phlorizin. On the other hand, hyperpolarization induced by pyruvate was inhibited by alpha-cyanocinnamate or phloretin, but not inhibited by cytochalasin B or F-. Elimination of Na+ in physiological saline depressed hyperpolarization of membrane potential induced by addition of D-glucose, L-lactate or pyruvate. These results suggest that the activity of (Na+ + K+)-ATPase in plasma membranes of synaptosomes is increased by ATP formed by glycolysis, and that the accumulated K+ in synaptosomes hyperpolarizes the membrane potential.

Tryptic activity was competitively inhibited by cationic detergents which contain a cetyl group or longer hydrocarbon chains. Since the cetyl group is much longer than the side chains of lysine or arginine residues of substrates for trypsin, the nature of inhibition by cetyldimethylbenzylammonium chloride was examined using Na-benzoyl-L-arginine-p-nitroanilide as substrate and compared to that by butylamine. The inhibition by cetyldimethylbenzylammonium chloride occurred instantaneously and was completely reversible. The inhibitory effect of cetyldimethylbenzylammonium chloride was strongly dependent on both pH and salt concentration, contrasting with inhibition by butylamine which was relatively indifferent to these changes. The Ki value of cetyldimethylbenzylammonium chloride at pH 7.5 and 25 degrees C was calculated to be 2.0 +/- 0.3 mM, which is equal to that of butylamine within experimental errors. The standard entropy change of binding of cetyldimethylbenzylammonium chloride (44 +/- 2 cal/mol/degree) was much larger than for butylamine, indicating the formation of an efficient hydrophobic bond between the cetyl group and the enzyme.

The intensity of rhodamine 6G fluorescence was found to be a useful scale for measuring the membrane potential in synaptosomes. The fluorescence of rhodamine 6G in synaptosomal suspensions increases with depolarization in the synaptosomes induced by the replacement of cations in the medium or by the addition of agents known to depolarize the membrane potential. Considering the character of the dye, we have derived an equation which gives the relation between the fluorescence intensity of the dye and the membrane potential. The change in membrane potential (diffusion potential) of synaptosomes was calculated using the equation. The calculated membrane potential was proportional to the logarithm of the K+ concentration above 20 mM, and the slope of membrane potential against log [K+] was about 52 mV per decade of concentration. The permeability ratio (Px/Pk; the ratio of the permeability constants of a given cation, X+, and K+) was estimated from the calculated membrane potential.

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.

Effects of three probes for measuring membrane potential, tetraphenylphosphonium (TPP+), rhodamine 6G and 3,3'-dipropylthiocarbocyanine (diS-C3-(5)) on energy metabolism in synaptosomes were investigated. None of the three probes had any effect on lactate production in synaptosomes. TPP+ and rhodamine 6G did not inhibit the respiration of synaptosomes with pyruvate and succinate as exogenous substrate and were only weakly inhibitory with endogenous substrates. In contrast, diS-C3-(5) markedly inhibited the respiration of synaptosomes with glucose, pyruvate and endogenous substrates. All three probes reduced ATP content in synaptosomes and depolarized the membrane potential in synaptosomes with increasing concentrations of the probes. It is, therefore, preferable to estimate membrane potential with TPP+ or rhodamine 6G at their low concentrations where their effect on metabolism is negligible.

The detergents which contain a hydrocarbon side chain longer than 16 cabron atoms were used as a perturbant for the study of protein structure. ta low concentration of cetyldimethylbenzylammonium chloride (CDBA) caused difference spectra for Ac-Trp-OEt and AC-Tyr-OEt. The delta e values at their difference maxima became constant above 30 mM of cetyldimethylbenzylammonium chloride, 1430 at 294 nm for Ac-Trp-OEt and 450 at 288 nm for Ac-Tyr-OEt. These delta e values are higher than any other delta e values resulting from solvent effects by such a remarkably low concentration of organic reagents described in the literature so far. The absence of denaturation blue shift in the difference spectra and the fact that the optical rotatory dispersion of the proteins examined in the present study was not changed significantly by cetyldimethylbenzylammonium chloride indicate that the secondary and tertiary structures of the proteins were not destroyed by cetyldimethylbenzylammonium chloride. These characteristics, together with small overlapping of their difference spectra at 288 and 294 nm were advantageous in the determination of tryptophan and tyrosine residues exposed in glucagon, insulin and alcohol dehydrogenase from yeast. No tyrosine residues in ribonuclease A was accessible to cetyldimethylbenzylammonium chloride. Unusual difference spectrum with a peak at 298 nm was observed for lysozyme which is known to contain tryptophan residues in special environments. Ovalbumin gave a novel unusual difference spectrum with a peak at 290 nm and a shoulder at 298 nm, showing the existence of unusual tryptophan and probably tyrosine residues in the molecule.

A major glycoprotein of the plasma membranes of AH-66 hepatoma ascites cells was isolated in essentially pure form and in milligram amounts. The plasma membranes were solubilized with a solution containing both 0.3 M lithium diiodosalycylate and 0.2% cetylpyridinium chloride, and further extracted with 50% phenol, followed by gel filtration on Sepharose 6B in the presence of 0.1% Ammonyx-LO at pH 8.0. The apparent molecular weight of the purified glycoprotein was estimated to be 165 000 in 5.6% polyacrylamide gels, of which 54% was carbohydrate and 46% was protein. The chemical composition of the glycoprotein resembles glycophorin A from human erythrocyte membranes in that it has a high content of N-acetylgalactosamine, N-acetylglucosamine, galactose and sialic acid and a particularly large proportion of serine, threonine, aspartic acid and glutamic acid.