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  • In Ovo injection of betaine affects hepatic cholesterol metabolism through epigenetic gene regulation in newly hatched chicks. 25860502

    Betaine is reported to regulate hepatic cholesterol metabolism in mammals. Chicken eggs contain considerable amount of betaine, yet it remains unknown whether and how betaine in the egg affects hepatic cholesterol metabolism in chicks. In this study, eggs were injected with betaine at 2.5 mg/egg and the hepatic cholesterol metabolism was investigated in newly hatched chicks. Betaine did not affect body weight or liver weight, but significantly increased the serum concentration (P less than 0.05) and the hepatic content (P less than 0.01) of cholesterol. Accordingly, the cholesterol biosynthetic enzyme HMGCR was up-regulated (P less than 0.05 for both mRNA and protein), while CYP7A1 which converts cholesterol to bile acids was down-regulated (P less than 0.05 for mRNA and P = 0.07 for protein). Moreover, hepatic protein content of the sterol-regulatory element binding protein 1 which regulates cholesterol and lipid biosynthesis, and the mRNA abundance of ATP binding cassette sub-family A member 1 (ABCA1) which mediates cholesterol counter transport were significantly (P less than 0.05) increased in betaine-treated chicks. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and adenosylhomocysteinase-like 1 were increased (P less than 0.05), which was associated with global genomic DNA hypermethylation (P less than 0.05) and diminished gene repression mark histone H3 lysine 27 trimethylation (P less than 0.05). Furthermore, CpG methylation level on gene promoters was found to be increased (P less than 0.05) for CYP7A1 yet decreased (P less than 0.05) for ABCA1. These results indicate that in ovo betaine injection regulates hepatic cholesterol metabolism in chicks through epigenetic mechanisms including DNA and histone methylations.
    Document Type:
    Reference
    Product Catalog Number:
    17-622
    Product Catalog Name:
    ChIPAb+ Trimethyl-Histone H3 (Lys27) - ChIP Validated Antibody and Primer Set

  • Highly efficient gene transfer into hepatocyte-like HepaRG cells: new means for drug metabolism and toxicity studies. 20213646

    HepaRG progenitor cells are capable of differentiating into hepatocyte-like cells that express a large set of liver-specific functions. These cells, however, only express small amounts of an important cytochrome P450, the CYP2E1, which limits their use for toxicological studies of drugs metabolized by this pathway. Our aim was to establish an efficient transfection protocol to increase CYP2E1 expression in HepaRG cells. Transfection protocols of the green fluorescent protein (GFP) reporter gene were evaluated using electroporation and cationic lipids belonging to the lipophosphonates, lipophosphoramidates and lipids derived from glycine betaine. Following optimization of the charge ratios, plasmid DNA and formulations with neutral co-lipids, the lipophosphoramidate compounds KLN47 and BSV10, allowed expression of the GFP in approximately 50% of adherent progenitor HepaRG cells, while electroporation targeted GFP expression in approximately 85% of both progenitor and differentiated cells in suspension. Transient enforced expression of active CYP2E1 was also achieved in progenitors and/or differentiated HepaRG cells using the electroporation and the lipophosphoramidate compound BSV10. Importantly, in electroporated cells, CYP2E1 expression level was correlated with a significant increase in CYP2E1-specific enzymatic activity, which opens new perspectives for this CYP-dependent drug metabolism and toxicity studies using HepaRG cells.
    Document Type:
    Reference
    Product Catalog Number:
    AB1254
    Product Catalog Name:
    Anti-Cytochrome P450 Enzyme CYP3A4 Antibody

  • A critical analysis of Atoh7 (Math5) mRNA splicing in the developing mouse retina. 20808762

    The Math5 (Atoh7) gene is transiently expressed during retinogenesis by progenitors exiting mitosis, and is essential for ganglion cell (RGC) development. Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa polypeptide. Mouse Math5 mutants have essentially no RGCs or optic nerves. Given the importance of this gene in retinal development, we thoroughly investigated the possibility of Math5 mRNA splicing by Northern blot, 3'RACE, RNase protection assays, and RT-PCR, using RNAs extracted from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA clones. Because Math5 mRNA contains an elevated G+C content, we used graded concentrations of betaine, an isostabilizing agent that disrupts secondary structure. Although approximately 10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (less than 1%). Rare deleted cDNAs do arise via RT-mediated RNA template switching in vitro, and are selectively amplified during PCR. These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs. Our findings clarify the architecture of the Math5 gene and its mechanism of action. These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments.
    Document Type:
    Reference
    Product Catalog Number:
    AB5694

  • The axonal gamma-aminobutyric acid transporter GAT-1 is sorted to the apical membranes of polarized epithelial cells. 8308038

    Recent studies suggest that epithelial cells and neurons employ similar mechanisms to target proteins to the distinct subdomains of their polarized cell surface membranes. We have examined the sorting behavior of the neuronal gamma-aminobutyric acid (GABA) transporter GAT-1 expressed by transfection in the polarized epithelial Madin-Darby canine kidney (MDCK) cell line. We find that the GABA transporters endogenously expressed by polarized hippocampal neurons in culture are restricted to axonal plasma membranes. In transfected MDCK cells, the GABA transporter is found to be localized primarily to the apical cell surface when examined by immunocytochemistry, cell surface biotinylation, and transport assay. MDCK cells exposed to hyperosmotic stress express a close relative of GAT-1, the betaine transporter (BGT-1). We find that BGT-1 expressed by transfection in MDCK cells accumulates predominantly at the basolateral cell surface. These observations suggest that the sorting information required for axonal targeting may be similar to that which mediates apical localization in epithelia. Furthermore, it would appear that despite their high degree of homology, the BGT-1 and GAT-1 transporters manifest sorting signals which specify their targeting to distinct cell surface domains.
    Document Type:
    Reference
    Product Catalog Number:
    05-369
    Product Catalog Name:
    Anti-Na+/K+ ATPase α-1 Antibody, clone C464.6

  • Soy protein modification of rat polycystic kidney disease. 9530270

    We undertook a study to determine whether soy protein feeding would ameliorate renal injury in the Han:SPRD-cy rat model of polycystic kidney disease (PKD). Male offspring of Han:SPRD-cy heterozygotes received isocaloric diets based on 20% casein or 20% heat-treated soy protein at weaning ad libitum for 8 wk. Soy-fed animals demonstrated lower serum creatinine (66 vs. 125 mumol/l; P = 0.002), lower urinary ammonium excretion (0.080 vs. 0.173 mmol/kg; P = 0.01), reduced renal cysts (0.98 vs. 4.92 ml/kg body wt, P 0.0001), renal fibrosis (0.79 vs. 1.4 ml/kg; P = 0.016), macrophage infiltration, renal tubular cell proliferation, and apoptosis. Proton nuclear magnetic resonance (1H-NMR) studies of urine demonstrated that soy diet was associated with increased losses of citric acid cycle organic anions. 1H-NMR of perchloric acid-extracted tissue found that levels of succinate were not depleted in soy-fed animals, despite increased urinary losses. Soy-fed animals had marked elevation of tissue betaine (P 0.001), with reduced taurine and cholines, compared with casein-fed animals (P 0.001). Soy feeding dramatically reduces both tubular and interstitial pathology in the Han:SPRD-cy rat model of PKD, through mechanisms that remain to be determined.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1435
    Product Catalog Name:
    Anti-Macrophages/Monocytes Antibody, clone ED-1
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