It has been proposed that plasma low-density lipoprotein (LDL) undergoes oxidative modification before it can give rise to foam cells in atherosclerosis. Oxidation of LDL generates a variety of reactive aldehyde products including 4-hydroxy-2-nonenal (HNE), which may covalently attach to the LDL apolipoproteins. We here present direct evidence that HNE derivatization of LDL forms Michael addition-type adducts of HNE with histidine and lysine residues of apolipoprotein B-100 (apoB) and also demonstrate the utility of an antibody specific to the HNE adducts generated in the LDL treated with HNE or oxidatively modified by Cu2+ or cultured endothelial cells. HNE adducts present in the LDL that had been treated with HNE were attested to be Michael addition-type adducts on the basis of the fact that incubation of LDL with 1 mM HNE (2 h, 37 degrees C) resulted primarily in the formation of Michael addition-type HNE-histidine (39.9 mol/mol of LDL) and HNE-lysine (19.3 mol/mol of LDL) adducts. An enzyme-linked immunosorbent assay (ELISA) and an SDS-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot analysis of HNE-modified LDL demonstrated that these HNE adducts were detectable with the HNE-specific antibody affinity-purified with the Michael adduct (HNE-histidine) as a ligand. The following lines of evidence indicated the presence of Michael addition-type HNE adducts in the oxidatively modified LDL in vitro: (i) Amino acid analysis of LDL that had been treated with Cu2+ (24 h, 37 degrees C) demonstrated the presence of a Michael addition-type HNE-histidine adduct (7-9 mol/mol of LDL).(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocyte nuclear factor-6 (HNF-6) is a liver-enriched transcription factor that contains a single cut domain and a novel type of homeodomain. Here we have studied the developmental expression pattern of HNF-6 in the mouse. In situ hybridization experiments showed that HNF-6 mRNA is detected in the liver at embryonic day (E) 9, at the onset of liver differentiation. HNF-6 mRNA disappeared transiently from the liver between E12.5 and E15. In transfection experiments HNF-6 stimulated the expression of HNF-4 and of HNF-3 beta, two transcription factors known to be involved in liver development and differentiation. HNF-6 was detected in the pancreas from E10.5 onward, where it was restricted to the exocrine cells. HNF-6 was also detected in the developing nervous system. Both the brain and the spinal cord started to express HNF-6 at E9-9.5 in postmitotic neuroblasts. Later on, HNF-6 was restricted to brain nuclei, to the retina, to the ventral horn of the spinal cord, and to dorsal root ganglia. Our observations that HNF-6 contributes to the control of the expression of transcription factors and is expressed at early stages of liver, pancreas, and neuronal differentiation suggest that HNF-6 regulates several developmental programs.

The effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (HC) diet (10g/kg) to rats was examined. When various amounts of taurine (0.25, 0.5, 1, 2.5, 5, 10, 20, 30, 40 or 50 g/kg diet) were supplemented to HC for 2 wk, serum total cholesterol gradually and significantly decreased in a dose-dependent manner and normalized at the dose of 10 g taurine/kg, compared with the control (cholesterol free) diet group. By contrast, serum HDL-cholesterol was elevated by taurine supplementation. The HC diet caused a significant decrease in the concentration of taurine in serum, liver and heart compared to that in the control group, and the effective dose of supplemental taurine to improve its reduction was 2.5 g/kg diet. In the hypercholesterolemic rats fed the HC diet, the excretion of fecal bile acids and hepatic cholesterol 7 alpha-hydroxylase (CYP7A1) activity and its mRNA level increased significantly, and the supplementation of taurine further enhanced these indexes, indicating an increase in cholesterol degradation. The abundance of mRNA for Apo A-I, one of the main components of HDL, was reduced by HC and recovered by taurine supplementation. Agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the HC diet, the serum level of the heavier VLDL increased significantly, but taurine repressed this increase and normalized this pattern. Significant correlations were observed between the time- and dose-dependent increases of CYP7A1 gene expression and the decrease of blood cholesterol concentration in rats fed the HC diet supplemented with taurine (time, r = -0.538, P < 0.01, n = 32; dose, r = -0.738, P < 0.001, n = 20). These results suggest that the hypocholesterolemic effects of taurine observed in the hypocholesterolemic rats fed the HC diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid.

Resetting the peripheral clock and understanding the integration between the circadian rhythm and metabolic pathways are fundamental questions. To test whether insulin acts as a synchronizer for the hepatic clock by cell-autonomous mechanisms, the phase-resetting capabilities of insulin were investigated in cultured hepatic cells. We provide evidence that three-dimensional (3D) cell culture conditions that preserve the differentiated state of primary hepatocytes sustained the robustness of the molecular clock, while this robustness rapidly dampened under classical monolayer cell culture conditions. Herein, we established a 3D cell culture system coupled with a real-time luciferase reporter, and demonstrated that insulin directly regulates the phase entrainment of hepatocyte circadian oscillators. We found that insulin-deficient diabetic rats had a pronounced phase advance in their hepatic clock. Subsequently, a single administration of insulin induced phase-dependent bi-directional phase shifts in diabetic rat livers. Our results clearly demonstrate that insulin is a liver clock synchronizer.

Functional genes can be introduced into mammalian cells in vitro by a variety of physical methods, including direct microinjection, electroporation, and co-precipitation with calcium phosphate. Most of these techniques, however, are impractical for delivering genes into tissues of intact animals. In contrast, receptor-mediated gene transfer has been shown to successfully introduce DNA into suitable recipient cells, both in vitro and in vivo (1-12). This procedure involves the formation of a complex between DNA and a polycation (such as poly-L-lysine), which bears a covalently linked ligand moiety specific for a given receptor on the surface of cells in the target tissue. The gene is internalized by the tissue, transported to the nucleus, and expressed in the cell for varying lengths of time (1,3,6,11). The overall level of expression of the transgene in the target tissue is dependent on several factors, such as the stability of the DNA/ligand⅐poly-L-lysine complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-DNA/ligand interaction, endocytosis of the DNA complex and the efficiency of gene transcription in the nucleus of the target cells.DNA in the nucleus of a higher eukaryote is intimately associated with basic nuclear proteins rich in lysine (i.e. histones) or arginine (i.e. protamines). The interaction of DNA with these basic proteins is responsible for the control of the condensation process that occurs upon chromosome formation during metaphase and is thought to play a role in the regulation of gene expression. DNA condensation, which occurs naturally in viruses, bacteria, and eukaryote nuclei, has been extremely difficult to reproduce in the laboratory (13,14). Due to the high negative charge of the DNA phosphate backbone, an increase in the degree of charge neutralization of the DNA theoretically results in extensive condensation and the separation of the DNA phase in the form of insoluble compact structures (15, 16). We have found, however, that the structure and stoichiometry of DNA⅐polycation complexes in solution can be manipulated by means of the process by which DNA⅐cationic polypeptide complexes are formed.Specific complexes of DNA (⌿-DNA) are formed with cationic homo-polypeptides (poly-L-lysine, poly-L-arginine, or poly-L-ornithine) after "annealing" both components in a step-down dialysis from NaCl concentrations of 3 to 0.010 M (11, 16). In contrast, direct addition of basic polypeptides to DNA at physiological salt concentrations results in reversible molecular aggregation and the formation of precipitates (7,17,18). Shapiro et al. (16) elucidated changes in DNA secondary structure in DNA⅐poly-L-lysine complexes prepared by directly mixing poly-L-lysine and DNA. The physical properties of the resulting soluble complexes were investigated by circular dichroism (CD) and optical rotatory dispersion. A change in the magnitude of the molar residue rotation was found, with a characteristic red shift and a strong negative rotatory transition center...