G protein-coupled receptors can exist as dimers and higher-order oligomers in biological membranes. The specific oligomeric assembly of these receptors is believed to play a major role in their function, and the disruption of native oligomers has been implicated in specific human pathologies. Computational predictions and biochemical analyses suggest that two molecules of rhodopsin (Rho) associate through the interactions involving its fifth transmembrane helix (TM5). Interestingly, there are several pathogenic loss-of-function mutations within TM5 that face the lipid bilayer in a manner that could potentially influence the dimerization of Rho. Though several of these mutations are known to induce misfolding, the pathogenic defects associated with V209M and F220C Rho remain unclear. In this work, we utilized a variety of biochemical and biophysical approaches to elucidate the effects of these mutations on the dimerization, folding, trafficking, and function of Rho in relation to other pathogenic TM5 variants. Chemical cross-linking, bioluminescence energy transfer, and pulsed-interleaved excitation fluorescence cross-correlation spectroscopy experiments revealed that each of these mutants exhibits a wild type-like propensity to self-associate within the plasma membrane. However, V209M and F220C each exhibit subtle defects in cellular trafficking. Together, our results suggest that the RP pathology associated with the expression of the V209M and F220C mutants could arise from defects in folding and cellular trafficking rather than the disruption of dimerization, as has been previously proposed.

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Soybean is believed to be a rich source of sphingolipids, a class of polar lipids with desirable biological activities. Analytical methods for sphingolipids vary, and quantitative data for sphingolipids in foods, including soybeans, are scarce. The objectives of this study were to establish a method for quantification of sphingolipids in soybeans and to determine whether genotype, stage of maturity, and growing location affect sphingolipid content in soybeans. Separation of neutral lipids and interfering polar lipids from sphingolipids by saponification, transesterification, and solvent partition was studied. Solvent partition accompanied by TLC purification was determined to be the most accurate sample preparation method for HPLC quantification of cerebroside. There were significant differences in cerebroside concentration among genotypes, with a range of 142 to 492 nmol/g seed (dry wt basis). The differences in cerebroside concentration between immature and mature seeds of one genotype and between two seed production locations of one genotype were considerable but not statistically significant.Sphingolipids are found primarily in the plasma membrane of all eukaryotes, some prokaryotes, and in all foods, with soybeans considered a rich source (1). Sphingolipids include free sphingoid bases, ceramides, sphingophospholipids, and glycosphingolipids. Sphingoid bases, usually 18-carbon amino alcohols, are N-acetylated to a long-chain FA to form ceramides. Polar head groups, such as sugar residues and phosphorylcholine, attach to the 1-ol position of ceramide to form more complex sphingolipids.Soybean contains two classes of sphingolipids, ceramide (Cer) and cerebroside. Cerebroside is the predominating class in soybeans (2), and it is the simplest glycosphingolipid because it contains only one sugar residue. The only type of cerebroside found in soybean is glucosylceramides (GlcCer), which contain a glucose molecule (2).Until recently, sphingolipids were recognized only as structural lipids. It has been discovered that their metabolites (i.e., ceramides and sphingosine) are involved in intracellular signaling, cell growth, differentiation, and apoptosis (3). Dietary sphingolipids have been shown to protect mice from skin and colon cancer (3) and decrease plasma cholesterol by 30% in rats (4).Dietary sphingolipids have important positive health implications, but information on their total content in foodstuffs, including soybeans, is sparse and may not be accurate. Certain data have been obtained from incomplete, single studies in which the main sphingolipid class may not have been measured and/or the effects of processing/preparation or other aspects influencing sphingolipid content were not considered (2). Almost all quantification studies have used chemical hydrolysis or derivatization, both of which require many steps and may produce artifacts, causing over-or underestimation of sphingolipid concentration. The possible degradation or hydrolysis during these quantification studies often has not been reported. Becau...

Soybeans are believed to be a rich source of sphingolipids, a class of polar lipids that has received attention for their possible cancer-inhibiting activities. The effect of processing on the sphingolipid content of various soybean products has not been determined. Glucosylceramide (GlcCer), the major sphingolipid type in soybeans, was measured in several processed soybean products to illustrate which product(s) GlcCer is partitioned into during processing and where it is lost. Whole soybeans were processed into full-fat flakes, from which crude oil was extracted. Crude oil was refined by conventional methods, and defatted soy flakes were further processed into alcohol-washed and acid-washed soy protein concentrates (SPC) and soy protein isolates (SPI) by laboratory-scale methods that simulated industrial practices. GlcCer was isolated from the samples by solvent extraction, solvent partition, and TLC and was quantified by HPLC. GlcCer remained mostly within the defatted soy flakes (91%) rather than in the oil (9%) after oil extraction. Only 52, 42, and 26% of GlcCer from defatted soy flakes was recovered in the acid-washed SPC, alcohol-washed SPC, and SPI products, respectively. All protein products had a similar GlcCer concentration of about 281 nmol/g (dry wt basis). The minor quantity of GlcCer in the crude oil was almost completely removed by water degumming. FIG. 2.Overall soybean processing scheme and mass balance of GlcCer. Product masses (dry wt basis) are based on the average of duplicate processing.

CHAPTER I.GENERAL INTRODUCTION Literature Review Sphingolipid structure Sphingolipids were discovered by J.L. W. Thudichum, a German practitioner, in 1884 (1). Thudichum first isolated and characterized sphingolipid molecules from bovine brain extracts (2) and named their backbones "sphingosin" (1). Today, the generic term for a nonspecific sphingolipid base is sphingoid. Sphingolipids are considered a very complex lipid group and include the following classes: free sphingoid bases, ceramides, sphingophospholipids, and glycosphingolipids (2). The sphingophospholipid and glycosphingolipid classes also contain subclasses based on headgroup compositions. All sphingolipids contain a sphingoid base which share a common core structure, 2-amino, 1, 3dihydroxy-octadecane or named sphinganine (abbreviated d18:0, d=dihydroxy base) (fig. 1) (3). Bases may deviate from the core structure through the following variations: 1) alkyl chain length, although C 18 is most common, 2) double bonds at C4 and/or C8 (for example, trans-4-sphingenine or commonly named sphingosine), 3) branching methyl groups, and 4) the presence of an additional hydroxyl group, usually at C4 (for example, 4-hydroxysphinganine or t18:0, t= trihydroxy base) (3). Over 70 sphingoid species have been identified, but the most common mammalian sphingolipid backbone is sphingosine (d 18: 1 M). Other less common backbones are sphinganine (d18:0) and 4-hydroxysphinganine (t18:0) (4). In plants, the major backbone is 4,8-sphingadienine (d18:2 trans 4 ' cis or trans 8), and other less common backbones include sphinganine (dl 8:0), 8-sphingenine (d 18: 1 trans orcis 8), 4-2 Corresponding Author