Thesis (M.S.)--University of Wisconsin--Madison, 1936. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [48-51]).

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Characterization of DAP1/YPL170W: the Saccharomyces cerevisiae Membrane Associated Progesterone Receptor (MAPR) Homologue Christopher D. Banna 135 pages Directed by Dr. Jung H. Choi MAPRs (Membrane Associated Progesterone Receptors) from several sources have been isolated, studied and minimally characterized in mammalian systems, yet the specific role of this protein family has not been fully determined. Early worked characterized this protein family as a type of steroid binding protein, unrelated to the classical nuclear receptors, and linked this family to non-genomic cellular responses. The MAPR homologues as a group have been suggested to play widely varying roles from axon guidance and neuronal formation, to steroid hydroxylation, to influencing reproductive behavior. Their specific role has not yet been clearly demonstrated in any organism. There is some debate as to whether MAPRs do indeed bind steroid compounds, but there is clear evidence this family of proteins is involved in steroid perception. Recent work has begun to link a specific member of the MAPR family, IZAg from rat, to steroid metabolism/production, specifically, in the hydroxylation step of glucocorticoid production from progesterone. In the yeast Saccharomyces cerevisiae, the MAPR homologue is DAP1. Preliminary work on haploid strains demonstrated several phenotypes associated with the DAP1 deletion mutant, most notably an altered sterol profile. Previous characterization of diploid homozygous mutant strain has shown a differential sensitivity to alcohol, an altered sterol profile, and a strong yeast two-hybrid interaction with Ypr118wp; methylthioribose-1-phosphate isomerase. Work in this study link the localization of Dap1p to lipid particles and on the ER, both sites of sterol synthesis. The sterol profiles of the control strain and the dap1Ġdeletion mutant strain were examined in detail. The most notable difference was the presence of an additional sterol compound associated with the deletion mutant strain. The structure of this compound does not correspond to normal sterols in the ergosterol biosynthetic pathway, but does correspond to structure of sterols in so-called alternate aberrant sterol pathways. The data presented in this study demonstrates that Dap1p was involved in sterol processing, although its specific role is unknown. Two possible scenarios are proposed; one where Dap1p is involved in regulating the flux of sterols from one internal membrane to another, and another where Dap1p is involved in aberrant sterol pathways.

The major issue that hinder the widespread use of biodiesel is its poor cold weather stability and operability. This is attributed minor components identified as monoglycerides (MG), diglycerides (DG) and sterol glycosides (SG). There is currently no standard method to determine SG levels in biodiesel. A method to isolate and measure SG concentration in biodiesel and FAME was first developed. This was accomplished by decompatibilizing SG from the biodiesel matrix using n-dodecane and purifying the solids using a Folch liquid-liquid extraction. The extracted SG was analyzed by GC-FID; the tricaprin internal standard was detected at 21.5 min and SG from 26-26 min. Recovery using this method was 100% ± 2.5% when 3 commercial canola biodiesel samples were spiked with 38 ppm SG and extracted. This method was used to measure SG concentration of filtered FAME produced using 0.3wt%, 0.5wt% and 0.7wt% at a MeOH:Oil (mol/mol) ratio of 4:1, 5:1, 6:1, 7:1 and 9:1. The biodiesel produced was characterized according to ASTM D6584; MG, DG and TG decreased with increasing catalyst concentration and MeOH:Oil ratios. The SG solubility in reactive FAME was found to be lowest at high glycerol catalyst concentration. High levels of TG were found to solubilize SG in the reactive FAME. Finally, the solubility of SG in reactive FAME increased when high ratios of methanol were used.

When grown in the presence of the sterol biosynthesis inhibitor, paclobutrazol, suspension cultures of Apium graveolens (celery) accumulate substantial amounts of I 4a-methylsterols, at the expense of 4-demethylsterols. These changes have been correlated with reduced synthesis of phosphatidylcholine (PC) via the CDP-base pathway (Roiph & Goad, 1991). It was subsequently proposed that changes in the membrane sterol composition of plant cells may regulate the activity of CTP: cholinephosphate cytidylyltransferase (CT), the rate-determining enzyme of this pathway (Kinney & Moore, 1989). In preliminary studies, the membrane-associated form of CT in A.graveolens, was found to exhibit optimal activity at pH 7.7, in the presence of 8.0 mM CTP and 3.5 mM Mg2t Microsomal membrane fractions, in which a large proportion of CT activity was found to reside, were analysed in terms of lipid composition. The predominant phospholipid in such membranes, PC, constituted approximately 70% of the total phospholipid content. Other, more minor constituents, included phosphatidylethanolamine (PE), phosphatidylglycerol (PU), phosphatidylinositol (P1), phosphatidylserine (PS), and phosphatidic acid (PA). All phospholipids present in A.graveolens were found to be rich in linoleate (18:2) and palmitate (16:0). Lesser amounts of stearate (18:0), oleate (18:1), and a-linolenate (a- 18:3), were also present. The major phytosterols in microsomes were identified as campesterol, stigmasterol, sitosterol, and isoflicosterol, with trace amounts of cholesterol and 24-methylene cholesterol. The sterol biosynthesis inhibitors, miconazole, terbinafine, fenpropimorph, and tomatidine, proved to be useflul tools in the manipulation of membrane sterol composition in suspension cultures of A.graveolens. Treatment with these inhibitors caused significant alterations in lipid composition with corresponding changes in the activity of membrane-associated CT. Terbinaflne and fenpropimorph caused a large increase in the stigmasterol/sitosterol ratio of cells with a concomitant stimulation of CT activity. The latter compound also resulted in the accumulation of various 9$, 19- cyclopropyl sterols. Similarly, the azasterol inhibitor, tomatidine, resulted in an enhancement of CT activity, but with very lift le change in the stigmasterol/sitosterol ratio of cells. Conversely, miconazole resulted in a decline in the stigmasterol/sitosterol ratio, corresponding to lower membrane-associated CT activity. The latter inhibitor also caused an accumulation of oleoyl residues in the PC fraction of cells, suggesting an inhibition of A 1 2-desaturase activity. Radiolabelling studies with [3IflS-adenosyl-L-methionine revealed a degree of coordinate regulation between the CDP-base and methyltransferase pathways of PC biosynthesis. Consequently, despite changes in CT activity, levels of phospholipid in most inhibitor-treated cultures remained relatively constant. Supplementation of miconazole-treated cultures with free fatty acids partially overcame the cytostatic nature of the azole inhibitor, with a concomitant reactivation of CT. Mono- and diunsaturated fatty acids were found to be the most effective compounds in this respect. The addition of stigmasterol or sitosterol to miconazoletreated cultures also resulted in partial growth restoration and reactivation of membrane-bound CT.

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Chemical investigation of the soft coral Cladiella hirsuta, collected off the coast of Penghu Islands, has led to the isolation of natural products including ten new compounds hirsutalins A-D (1, 2, 3, 5) and hirsutosterols A-F (6, 7, 8, 9, 10, 11), and one known compound 3-butanoyloxycladiell-11(17)-en-6,7-diol (4). All of the chemical structures were established by detailed spectral data analysis (NMR, IR, MS) and by comparison of the spectral data with those of the related known compounds. The absolute structure of compound 1 was determined applying a modified Mosher¡¦s method. On the part of the cytotoxicity assay with six human cancer cell lines, HepG2 (liver cancer cell), Hep3B (liver cancer cell), Ca9-22 (mouth cavity cancer cell), A549 (lung cancer cell), MCF7 (breast cancer cell) and MDA-MB-231 (breast cancer cell), compound 5 showed the significant cytotoxicity against the cancer cell line HepG2; compound 7 showed the moderate cytotoxicity against all of the above cancer cell lines; compounds 6, 8 and 10 also showed the moderate cytotoxicity against two of them, Hep3B and Ca9-22. The products 6a and 7a , obtained by hydorlysis of 6 and 7, respectively, did not show obvious difference in cytotoxity by comparison of the IC50 values of compounds 6 and 7. On the part of anti-inflammation assay, compound 2 showed the effectively inhibiting activity against the induced iNOS and COX-2 proteins. Compound 3 showed nearly the same activity against the induced iNOS protein as that of 2.

Oxysterol binding protein (OSBP) binds 25-hydroxycholesterol (25OH) and cholesterol, which regulates PH and FFAT domain interaction with the Golgi apparatus and endoplasmic reticulum, respectively. Adjacent to these domains is a phosphorylated serine-rich motif (SRM, T379, S381, S384, S387, S388, S391) that we hypothesize controls sterol transport by OSBP. To test this, OSBP dephospho-mimics or phospho-mimics were expressed in CHO cells. Western blot analysis showed that the S381 is phosphorylated by PKA and is required for phosphorylation of down-stream serine residues. When expressed in OSBP-null CHO cells, there was no difference in the localization of the OSBP mutants, and all mutants restored SM synthesis in response to 25OH. Recombinant OSBP 5S?5E had increased cholesterol binding and extraction, and decreased cholesterol transfer to liposomes compared to OSBP. OSBP 5S?5E also bound VAP more efficiently. A model is proposed wherein SRM phosphorylation facilitates VAP association with the ER and increases cholesterol extraction.

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Structured phospholipids with functional ingredients like conjugated linoleic acid (CLA) and plant sterols to deliver their physiological effects in different food formulations were synthesized. The lipase and phospholipase A2 catalyzed enzymatic acidolysis reaction between phospholipids (PLs) and CLA was used for fatty acid modification, while the phospholipase D catalyzed transphosphatidylation reaction between PLs and sterol was used for head group modification. Enzymatic processes were an effective way to produce structured phospholipids. Screening of four lipases and immobilized phospholipase A2 and combination of lipase and phospholipase showed that only Lipozyme RM IM and Lipozyme TL IM were effective in incorporation of CLA into PLs. The maximum incorporation achieved by the latter enzyme was 16% with soy PLs in 72 h. The class of phospholipids had a significant effect on the rate of incorporation of CLA compare to source of PLs. A method capable of predicting the rate of incorporation of CLA into phospholipids was developed using response surface methodology. A three-level four-factor Central Composite Rotatable Design (CCRD) was used. The four factors selected were lipase dosage (Ed, wt.% of substrate), substrate ratio (Sr,mol%), reaction time (ti, h) and reaction temperature (Te,oC). The enzyme load and substrate ratio had a greater effect on the rate of incorporation than did reaction time and temperature. A polynomial regression equation was developed to predict the reaction rate. The new phosphatidyl derivative, phosphatidyl-sitosterol, was found to be synthesized by the transfer reaction of phosphatidyl residue from phosphatidylcholine to β-sitosterol by phospholipase D from Streptomyces sp. in biphasic medium. The novel phosphatidyl .sitosterol derivative was identified by MALDI-TOF mass spectrometry. Plant sterols were modified to a more polar lipid class by synthesizing phospholipid derivatives of them. When these structured phospholipids were added to a whey protein based oil-in-water emulsion, the CLA incorporated structured phospholipids (CLA-PL) had higher heat stability and oxidative stability compared to the controls.