Analysis of the acrolein-modified sites of apolipoprotein B-100 in LDL

We have reported that acrolein-conjugated low-density lipoprotein (Acro-LDL) uptake by scavenger receptor class A type 1 (SR-A1) can mediate macrophage foam cell formation. The purpose of this study was to determine which amino acid residues of apoB protein in LDL are conjugated with acrolein. Acro-apoB was prepared by incubation of LDL with acrolein (10 to 60 μM) at 37 °C for 7 days. Identification of acrolein-conjugated amino acid residues in apoB was performed using LC-MS/MS. The levels of acrolein-conjugated amino acid residues of apoB as well as crosslinking apoB increased in proportion to acrolein concentration. The level of LDL uptake by macrophages was parallel with the acrolein-conjugated monomer apoB. Acrolein-conjugated amino acid residues in apoB were C212, K327, K742, K949, K1087, H1923, K2634, K3237 and K3846. The NH₂-teriminal four amino acid residues (C212, K327, K742 and K949) were located at the scavenger receptor SR-A1 recognition site, suggesting that these four acrolein-conjugated amino acids are involved in the rapid uptake of Acro-LDL by macrophages. It is proposed that the rapid uptake of LDL by macrophages is dependent on acrolein conjugation of four amino acids residues at the scavenger receptor recognition site of apoB in LDL.     

Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

Characterization of acrolein-induced protein cross-links

Lipid peroxidation products contribute to protein aggregation that occurs during oxidative stress in a number of degenerative disorders. Acrolein (ACR), a highly toxic lipid peroxidation aldehyde, is a strong cross-linking agent of cellular components such as proteins. To understand the mechanisms of oxidative stress-induced protein aggregation, this study characterized the ACR modification of chain B from bovine insulin by mass spectrometry. To identify the cross-linking sites, the ACR-treated peptide was digested with a protease and the resulting peptides were analysed by liquid chromatography-tandem mass spectrometry. Inter- and intra-molecular cross-linking adducts were identified between amino groups and the side chain of histidine in the peptide. These results indicated that the ACR-induced cross-links were accompanied by two reactions, namely Michael addition and Schiff base formation. In conclusion, the use of mass spectrometric techniques provided chemical evidence for protein cross-linking with ACR.     

A Novel Method for the Synthesis of ddA and F-ddA Via Regioselective 2′-O-Deacetylation of 9-(2,5-DI-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine

Abstract

Regioselective 2′-O-deacetylation of 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine (1) is achieved by treatment of 1 with β-cyclodextrin (β-CyD) / aq. NaHCO₃ or N₂H₄·H₂O / EtOH. The 9-(5-O-Acetyl-3-bromo-3-deoxy-β-D-xylo-furanosyl)adenine (2) obtained is a common intermediate for the synthesis of 2′,3′-dideoxy-adenosine (ddA) (7) and 9-(2-fluoro-2,3-dideoxy-β-D-threo-pentofuranosyl)-adenine (F-ddA) (9).     

DNA-Binding Properties of the Antibody Specific for the Dewar Photoproduct of Thymidylyl-(3′′-5′′)-Thymidine

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3′-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.     

Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets

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Highlights? Triacylglyceride (TG) synthesis is coupled with lipid droplet (LD) growth ? Two LD populations exist: growing LDs, containing TG enzymes, and small LDs ? Specific TG synthesis enzymes move from the ER to LDs through membrane bridges ? LD localization of TG enzymes mediates expansion of a subset of LDs     

Increase of Circulating CD11b+Gr1+ cells and Recruitment into the Synovium in Osteoarthritic Mice with Hyperlipidemia

Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F₂ animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b⁺Gr1⁺ cells in spleens and peripheral blood was increased, and CD11b⁺Gr1⁺ cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b⁺Gr1⁺ cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b⁺Gr1⁺ cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F₂ mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b⁺Gr1⁺ cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b⁺Gr1⁺ cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.