The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal with familial hypercholesterolemia, produces a mutant receptor for plasma low-density lipoprotein (LDL) that is not transported to the cell surface at a normal rate. Cloning and sequencing of complementary DNA's from normal and WHHL rabbits, shows that this defect arises from an in-frame deletion of 12 nucleotides that eliminates four amino acids from the cysteine-rich ligand binding domain of the LDL receptor. A similar mutation, detected by S1 nuclease mapping of LDL receptor messenger RNA, occurred in a patient with familial hypercholesterolemia whose receptor also fails to be transported to the cell surface. These findings suggest that animal cells may have failsafe mechanisms that prevent the surface expression of improperly folded proteins with unpaired or improperly bonded cysteine residues.Receptors and other cell surface proteins follow a complex path from their sites of synthesis in the rough endoplasmic reticulum to their sites of function in the plasma membrane (1). The orderly progression of such proteins through the endoplasmic reticulum and the Golgi complex was elucidated through study of model proteins, such as those of lipid-enveloped viruses. The signals that direct this movement and the fail-safe mechanisms that prevent denatured proteins from reaching the cell surface are still largely unknown. One way to solve the problem is by studying mutant proteins that do not reach the cell surface. Such mutations have been created artificially through in vitro mutagenesis of genes encoding viral envelope proteins (2). A second approach is through study of naturally occurring mutations in which transport is blocked. In human and animal cells, the most informative group of these mutations occurs in the gene for the low density lipoprotein (LDL) receptor and gives rise to a genetic disease called familial hypercholesterolemia (FH) (3, 4).FH is an autosomal dominant disorder characterized by an elevation of cholesterol in plasma and severe atherosclerosis (3, 4). The disease is caused by defects in the cell surface receptor for LDL, which is a cholesterol transport protein (5). When the LDL receptor is defective, LDL cannot enter cells by receptor-mediated endocytosis and the lipoprotein accumulates in plasma, eventually producing atherosclerosis (3). Some mutations alter the receptor in such a way that it cannot move from the rough endoplasmic reticulum to the Golgi complex (6, 7). Such transport-deficient mutations have been observed frequently in humans with FH (5- WHHL rabbits are homozygous for a mutant allele that produces an LDL receptor that is of normal apparent molecular size but is transported to the cell surface at only one-tenth the normal rate (7). The newly synthesized receptor receives its normal complement of high mannose N-linked sugars and the core N-acetylgalactosamine of the O-linked sugars (5). However, these carbohydrate chains are not processed to their mature form, apparently because the mutant receptor does no...

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme.

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The procedures currently used by the U.S. Army Center for Health Promotion and Preventive Medicine (USACHPPM) for the analysis of energetics and related compounds in water and soils are presented. These procedures are based on the use of isoamyl acetate to extract the analytes of interest from their environmental matrices with subsequent analysis using gas chromatography with electron capture detection. The suite of compounds included are those that have been of environmental significance for years (such as 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and dinitrotoluenes) and are the subject of several U.S. Environmental Protection Agency SW-846 methods. The procedures presented in this study are the product of years of development and refinement of methods used for the analysis of many real-world samples by the USACHPPM explosives analysis laboratory. The development, performance, advantages, and details of these procedures are described. The extension of these methods to the analysis of other media is also briefly discussed.