Epidemiological, pathological and genetic studies show a strong positive correlation between elevated plasma concentrations of low-density lipoprotein (LDL) cholesterol and the risk of premature coronary heart disease. Apolipoprotein (apo) B-100 is the sole protein component of LDL and is the ligand responsible for the receptor-mediated uptake and clearance of LDL from the circulation. Apo B-100 is made by the liver and is essential for the assembly of triglyceride-rich very low-density lipoproteins (VLDL) in the cisternae of the endoplasmic reticulum and for their secretion into the plasma. VLDL transports triglyceride to peripheral muscle and adipose tissue, where the triglyceride is hydrolysed by lipoprotein lipase. The resultant particle, relatively enriched in cholesteryl ester, constitutes LDL. LDL delivers cholesterol to peripheral tissues where it is used for membrane and steroid hormone biosynthesis and to the liver, the only organ which can catabolize and excrete cholesterol. Plasma LDL levels are therefore determined by the balance between their rate of production from VLDL and clearance by the hepatic LDL (apo B/E) receptor pathway. Here we report the complete 4,563-amino-acid sequence of apo B-100 precursor (relative molecular mass (Mr) 514,000 (514K] determined from complementary DNA clones. Numerous lipid-binding structures are distributed throughout the extraordinary length of apo B-100 and must underlie its special functions as a nucleus for lipoprotein assembly and maintenance of plasma lipoprotein integrity. A domain enriched in basic amino-acid residues has been identified as important for the cellular uptake of cholesterol by the LDL receptor pathway.

We previously generated transgenic mice expressing human apolipoprotein (apo-) B and demonstrated that the plasma of chow-fed transgenic animals contained markedly increased amounts of LDL (Linton, M. F., R. V. Farese, Jr., G. Chiesa, D. S. Grass, P. Chin, R. E. Hammer, H. H. Hobbs, and S. G. Young 1992. J. Clin. . In this study, we fed groups of transgenic and nontransgenic mice either a chow diet or a diet high in fat (16%) and cholesterol (1.25%). Lipid and lipoprotein levels were assessed, and after 18 wk of diet, the extent of aortic atherosclerotic lesions in each group of animals was quantified. Compared with the female transgenic mice on the chow diet, female transgenic mice on the high-fat diet had higher plasma levels of cholesterol (312±17 vs 144±7 mg/dl; P < 0.0001) and human apo-B (120±8 vs 84±3 mg/dl; P < 0.0001). The higher human apo-B levels were due to increased plasma levels of human apo-B48; the human apo-B100 levels did not differ in animals on the two diets. In mice on the high-fat diet, most of the human apo-B48 and apo-B100 was found in LDL-sized particles. Compared with nontransgenic mice on the high-fat diet, the transgenic animals on the high-fat diet had significantly increased levels of total cholesterol (312±17 vs 230±19 mg/dl; P < 0.0001) and non-HDL cholesterol (283±17 vs 193±+19 mg/dl; P < 0.0001). The extent of atherosclerotic lesion development within the ascending aorta was quantified by measuring total lesion area in 60 progressive sections, using computerassisted image analysis. Neither the chow-fed transgenic mice nor the chow-fed nontransgenic mice had significant atherosclerotic lesions. Nontransgenic animals on the highfat diet had relatively small atherosclerotic lesions ( < 15,000 imm2/section), almost all of which were confined to the proximal 400 ,im of the aorta near the aortic valve. In contrast, transgenic animals on the high-fat diet had extensive athero- Thus, human apo-B expression, in the setting of a diet rich in fats, causes severe atherosclerosis in mice. (J. Clin. Invest. 1995Invest. . 95:2246Invest. -2257

The fluorogenic reaction of o-phthalaldehyde (OPTA) and /3-mercaptoethanol (MERC) with primary amino acids gives a 1-alkylthio-2-alkylisoindole as the product. The structure of this group of previously unknown isoindoles was determined (1) from an in situ analysia of the adducts formed in solution from OPTA, MERC or ethanethiol (ET), and n-propylamine, (2) from the characterization of solid derivatives of these MERC and ET adducts, and (3) from the studies of two isolable isoindoles (an OPTAltert-butylthiol/n-propylamine adduct and a dimeric adduct formed from OPTA, ethanedithiol, and ri-propylamine). The reaction is found to be quite general as OPTA and numerous thiols rapidly react with n-propylamine or leucine to give isoindoles in excellent yield. Most adducts were not isolated, but their physical properties in solution were qualitatively identical with those of the tert-butyl and dimeric ethanedithiol adducts. Analyses of the chemical shifts of the isoindole alkyl substituents in the NMR spectra support the previous conclusions that these heterocycles have relatively low levels of aromatic character. The addition sequence of the reaction is important; the best results are obtained by mixing OPTA and thiol before adding the amine. With some thiols, this procedure results in the initial formation of a l-alkylthio-3-hydroxy-1,3-dihydroisobenzofuran. However, these 1:l adducts do not appear to be obligatory intermediates in the subsequent reaction with primary amines to form 1-thio-sulistituted isoindoles.The fluorogenic reaction of o -phthalaldehyde (OPTA) and 6-mercaptoethanol (MERC) with amineslJ and amino acids and protein^^-^ has recently attracted much attention due to the high sensitivity of the assay which can be conducted in aqueous solutions. Thus, picomole quantities of amino acids can be readily detected. In recent preliminary communications we deduced that these intensely fluorescent OPTA reaction products are 1-alkylthio-2-alkyl-substituted isoindoles 1.2.10 d-R' N-R' 1 R ' = alkyl or aryl; RZ = alkyl Isoindoles in general are quite reactive and eluded isolation until 1951.11812 In spite of their 10-a-electron apparently aromatic structure, N-substitution and especially halogen s u b~t i t u t i o n l~-'~ are required to increase the stability of isoindoles not conjugated with other unsaturated functional groups. Very few 0xygen-~3%1~ or nitrogen-substituted17 isoindoles have been reported, and the effect of such subsdtution on isoindole stability is not yet clear. For this reason, thiosubstituted isoindoles l are an important new class of hetero-substituted isoindoles which should be useful in further defining the physical and chemical properties of these interesting 10-a-electron bicyclic heterocycles. In this paper we present the details of our preliminary communicati o n~,~J~ describe the preparation and physical properties of several new 1-alkyl(and aryl)thio-2-alkylisoindoles, and assess the scope and mechanism of the OPTA reaction. A discussion of the fluorescence properties of these compoun...

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