Thesis (M.S.)--University of Wisconsin--Madison, 1966. / Typescript eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 59-62)

Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. eContent provider-neutral record in process. Description based on print version record.

Two kinds of affinity chromatography, CDP-choline- and CTP-Sepharose 4B, were investigated for purification of the cytosolic CTP:phosphocholine cytidylyltransferase from rat liver. The enzyme did not show strong affinity for the CDP-choline Sepharose resin, but bound to the CTP-Sepharose column in the presence of 14 mM magnesium acetate. The combination of CTP affinity chromatography with ion-exchange techniques provided about 70-fold purification of the cytosolic enzyme with a specific activity of about 90 units per milligram protein. The influence of diphenylsulfone compounds on the synthesis of phosphatidylcholine by the CDP-choline pathway was examined in isolated rat hepatocytes and HeLa cells. The administration of the sulfones (100 ug/ml), except dapsone, to HeLa cells inhibited the total [methyl-³H]choline incorporation into the cells, but did not change the rate of conversion of choline to phosphatidylcholine. The addition of the sulfones (100 ug/ml) to rat hepatocytes did not inhibit the biosynthesis of phosphatidylcholine and choline metabolism. The effect of vasopressin on the distribution of cytidylyl-transferase between cytosol and microsomes in rat hepatocytes was also investigated. The digitonin-mediated release of cytosolic cytidylyltransferase was reduced from the cells treated with vasopressin (5-20 nM) , while the enhanced rate of incorporation of [methyl-³H]choline into phosphatidylcholine was not observed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Class of 2009 Abstract / OBJECTIVES: The primary objective of this experiment was to characterize the pH stability range of 20% Pluronic lecithin organogel (PLO). We intended to determine the viscosity at varying pHs. We prepared six samples of 20% PLO. METHODS: An initial rheological reading of each sample was recorded by a dynamic stress rheometer. Each sample was titrated drop-wise with citric acid or KOH in 0.5 pH increments. When the desired pH was obtained, a 0.5 mL sample was analyzed with a dynamic stress rheometer, RS-200, using Rheos software. RESULTS: Since PLO is a non-Newtonian substance, viscosity changed relative to shear stress and we were not able to examine a correlation of pH with viscosity. Instead we inputted the data into Microsoft Excel® and plotted a shear stress versus viscosity curve for each sample to identify trends. CONCLUSIONS: We were unable to achieve our primary objective of determining the viscosity characterization of 20% PLO at varying pHs due to the non-Newtonian nature of the material. Subjectively, we determined the viscosity of 20% PLO is not substantially affected by pH. Other factors such as temperature, excess liquid, and surfactant ability may influence viscosity and need to be examined in the future.

Phospholipids are the major components of cell membranes and have a wide variety of structures, shapes and properties. Different ratios of phospholipid species confer different properties to membranes and contribute to the normal function of organelles. We have previously shown that acute phosphatidylcholine (PC) biosynthesis inhibition leads to a severe form of lipid imbalance that disrupts ER morphology and structure. Furthermore, our previous studies also revealed a mechanism for ER proteostasis under conditions of lipid-imbalance-induced ER stress in yeast, whereby unfolded ER proteins are removed by lipid droplets (LDs) and targeted to the vacuole for degradation by microlipophagy. Here, we find that LDs also contribute to ER proteostasis during chemically induced ER stress. Furthermore, we find that ER stress results in an increase in ubiquitinated proteins in LDs as well as recruitment of cytosolic and ER heat shock proteins, as well as ER proteins to LDs. ER stress-induced microlipophagy does not require core ATG genes and can occur in the absence of lipid ordered microdomains (Lo) in the vacuolar membrane. Instead, we find that the ESCRT machinery is up-regulated and localizes to the vacuolar membrane in response to ER stress induced microlipophagy and that ESCRT I, II and III complexes are required for microlipophagy in response to each of these stressors. Similar to yeast, we find that lipid imbalance in skeletal muscle from CHKB CMD, new autosomal recessive CMD (Congenital Muscular Dystrophy) caused by a mutation of choline kinase beta (CHKB), results in abnormal SR/ER morphology. CHKB is the first enzyme in the de novo PC biosynthesis pathway and causes phospholipid imbalance in cell membranes similar to that observed in yeast. Besides the disruption of SR morphology, we also detect a dysfunction of the Ryanodine Receptor (RyR), the Ca2+ channels responsible for initiating muscle contraction. Specifically, we observe abnormal RyR morphology and increased association of RyRs with lipid droplets (LD) in muscle fibers from a CHKB CMD patient. Finally, we detect ER stress and pronounced UPR activation in rmd mice, a mouse model of CHKB CMD. Given these results, we propose that inhibition of PC biosynthesis leads to phospholipid imbalance in the SR, which in turn, causes RyR leakage and ER stress which lead to mitochondrial dysfunction and dystrophy in CHKB CMD.

Graduation date: 1998

In cellular studies of patients with lipid related disorders such as mammary cancers, leukemia, and artheroscierosis, separation of molecular species of oxidized phosphatidylcholine (PC) can be an important assistance in research or diagnosis. Goals of this project were to optimize separation of oxidized and unoxidized PC molecular species in a single HPLC chromatogram followed by in line identification of hydroperoxides. Oxidized egg PC's were produced using UV light exposure in air. Separations were performed on an Ultrasphere ODS column and an Asahipak ODPVA column using a Waters 2695 system with photodiode array. The ODPVA column routinely gave 10 times larger plate numbers. Various mobile phase mixtures (methanol, acetonitrile, water) and gradients were tested. The optimum gradient on our system is (1) 5 minutes, 47 % methanol/40 % acetonitrile/13 water in a linear gradient to (2) 17 minutes, 49 % methanol/40 % acetonitrile/11 % water to (3) 18 minutes, 29 % methanol/60 % acetonitrile/11 % water linearly to (4) 50 minutes, 31 % methanol/60 % acetonitrile/9 % water continued isocratically to 110 minutes. Oxidized hydroperoxides were detected by fluorescence using a post column reaction with diphenyl-1 pyrenylphosphine (DPPP). Both iron (III) and pyridine were tested as catalysts for this reaction. / Department of Chemistry

Thesis (Ph. D.)--University of Michigan, 1935. / "By A.L. Ferguson, L.O. Case and G. Harlowe Evans." From Journal of chemical physics, v. 3, May, 1935.

The last step in sphingomyelin's de novo biosyn-thetic pathway was investigated in Baby Hamster Kidney (BHK-21F) cells in tissue culture. Three types of pulse-chase experiments were done to try to identify the precursor for sphingomyelin's phosphocholine moiety. First, [Me- ³H]-choline was used to monitor the movement of the choline moiety in all the possible phosphocholine donors: ie. phosphocholine, CDP-choline, phosphatidylcholine, lysophospha-tidylcholine, and glycerophosphocholine. Radioactivity was observed in phosphocholine before appearing in phosphatidylcholine, glycerophosphocholine, and sphingomyelin. Specific radioactivities of phosphatidylcholine and sphingomyelin revealed a peculiar pattern, if representative of a precursor-product relationship between these two phospholipids. Their specific radioactivities became equal at 22 hours of chase and remained quite similar for the next 24 hours. The other two types of pulse-chase experiments both utilized prelabeled BHK phosphatidyl[Me- ³H]choline in phospholipid vesicles as their 'pulse' source. Phospholipid Exchange Protein(PLEP)-mediated exchange and polyethylene glycol/phytohemagglutinin (PEG/PHA)-mediated fusion between phospholipid vesicles and BHK cells were used to introduce the labeled phosphatidylcholine. In addition, in the 'fusion' experiments, other labeled compounds (glycerophosphocholine and sphingomyelin) were substituted for labeled phosphatidylcholine. PLEP- mediated exchange of labeled phosphatidylcholine did not result in enough transfer of radioactivity into the cell to adequately monitor individual cell phospholipids or any transfer of label - ie. from phosphatidylcholine to sphingomyelin. However, PEG/PHA-mediated fusion of vesicles and cells did result in enough radioactivity showing up in the cells. When labeled phosphatidylcholine was used, the radioactivity ratio between it and sphingomyelin averaged around 16, depending on the length of the chase (2-52 hours)m The use of either labeled glycerophosphocholine or sphingomyelin resulted in a ratio of about 1.8. One 'cold-trap1 experiment was done by including a large amount of unlabeled glycerophosphocholine with labeled phosphatidylcholine in the vesicle preparation. The resultant radioactivity in sphingomyelin was 50% less than previously, but phosphatidylcholine's had remained the same. The evidence seems to indicate a reversible precursor-product relationship between phosphatidylcholine and sphingomyelin, but does not clearly show whether or not any other intermediate (such as glycerophosphocholine) is also involved. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Drug-induced tardive dyskinesia, an irreversible involuntary movement disorder caused by neuroleptic drugs, may reflect cholinergic hypofunction in the corpus striatum. Therapeutic results have been reported in trials of choline and lecithin, nutritional substrates which may enhance cholinergic neurotransmission. Lecithin's effects on dyskinetic symptoms were examined in 50 male patients in a double-blind, placebo-controlled trial. Patients were randomly assigned to treatment or control groups; 31 patients were retained in the analytic cohort. Experimental patients were treated with 60 gm/day lecithin (55% phosphatidyl choline) for 11 days. Symptom frequency was rated from videotapes made at baseline, 3 and 11 days of treatment, and 1 week follow-up.