Fushiki (2014) Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36, Bioscience, Biotechnology, and Biochemistry, 78:2, 238-244,

CD36 is an integral membrane protein that mediates the cellular uptake of oxidized low-density lipoprotein (oxLDL) through recognition of the oxidized glycerophospholipids (oxPLs) formed during LDL oxidation. We aimed to devise an assay system to detect binding between CD36 and oxLDL/oxPL without using recombinant proteins. A peptide corresponding to amino-acid residues 149-168 of mouse CD36 with biotin at its N-terminus (named biotin-CD36(149-168)) and variants of it were synthesized and immobilized onto streptavidin-coated plates. oxLDL labeled with Alexa-Fluor-488 bound specifically and saturably to immobilized biotin-CD36(149-168), but poorly or not at all to the variants, such as that with a scrambled amino-acid sequence. The binding of fluorescence-labeled oxLDL to biotin-CD36(149-168) was inhibited efficiently by an oxPL species, but not by a nonoxidized glycerophospholipid. This assay system using biotin-CD36(149-168) provides a convenient means not only of characterizing binding profiles between CD36 and oxLDL/oxPL but also of finding competitors for the binding.

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(2014) Further validation of unsaturated long-chain fatty acids as inhibitors for oxidized low-density lipoprotein binding to CD36 via assays with synthetic CD36 peptide-cross-linked plates, Bioscience, Biotechnology, and Biochemistry, 78:5,[839][840][841][842]

SummaryThe present study was conducted to identify reliable gene biomarkers for the adverse effects of excessive leucine (Leu) in Sprague-Dawley rats by DNA microarray. It has long been known that the adverse effects of excessive amino acid intake depend on dietary protein levels. Male rats were divided into 12 groups (n56) and fed for 1 wk a diet containing low (6%), moderate (12%) or high (40%) protein. Different levels of Leu (0, 2, 4, and 8%) were added to the diets. Consumption of diets containing more than 4% Leu in 6% protein resulted in growth retardation and reduced liver weight, whereas the administration of the same dose of Leu with 12% or 40% protein did not affect them. By a process of systematic data extraction, 6 candidate gene markers were identified. The liver gene expression data obtained from another experiment with 0, 2, 3, 4, and 8% Leu in a low-protein diet was used to examine the validity of these biomarker candidates with receiver operating characteristic (ROC) curve analysis. All of AUC values of the biomarker candidates were more than 0.700, suggesting the effectiveness of the marker candidates as the indices of Leu excess. The cut-off value for the ROC curve of the gene-marker panel, which was obtained by multiple regression analysis of gene markers, indicated that Leu levels higher than 3% have adverse effects. In conclusion, the gene-marker panel suggested that for male rats dietary Leu supplementation of 2% is the NOAEL dose in low-protein (6%) diets. Key Words leucine, transcriptome, biomarker, DNA microarray, dietary protein * To whom correspondence should be addressed. E-mail: akatoq@mail.ecc.u-tokyo.ac.jp Abbreviations: Actb, actin, beta; Ccnb2, cyclin B2; Ccnd1, cyclin D1; Cdkn1a, cyclin-dependent kinase inhibitor 1A; Fgf21, fibroblast growth factor 21; Igf1, insulin-like growth factor 1; Igfals, insulin-like growth factor binding protein, acid labile subunit; Igfbp1, insulin-like growth factor binding protein 1; Inhbe, inhibin beta E; Leu, leucine; NOAEL, no observable adverse effect level; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROC curve, receiver operating characteristic curve; SD rat, Sprague-Dawley rat; Stat5a, signal transducer and activator of transcription 5A; Stat5b, signal transducer and activator of transcription 5B; UL, tolerable upper intake level.