The aim of this study was to investigate molecular interactions between Quil A and phosphatidylcholine in the solid state using diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS). Analysis of the interactions was characterized on the different regions of phosphatidylcholine: hydrophobic chain, interfacial and headgroup regions. The spectra of the hydrocarbon region of phosphatidylcholine alone compared to that for the binary mixture of Quil A and phosphatidylcholine were similar. These findings suggest that Quil A did not cause conformational disorder of the fatty acyl chains of the phospholipid. In contrast, a shift in the wavenumber of the choline group and a broad band in this moiety indicate a modification of the phospholipid in the headgroup region due to interaction between Quil A and phosphatidylcholine. These results suggest possibly ionic interactions between the negatively charged glucuronic acid moiety of the Quil A molecule with the positively charged choline group. The findings could also be the result of conformational changes in the choline group because of the intercalation of sugar moieties in Quil A between the choline and phosphate groups due to hydrogen bonding. Shift of wavenumbers to lower values on the carbonyl group was observed suggesting hydrogen bonding between Quil A and phosphatidylcholine. The difference in degrees of wavenumber shift (choline > phosphate > carbonyl group) and observed broad bands indicated that Quil A preferentially interacted with phosphatidylcholine on the hydrophilic headgroup. Cholesterol influenced such interactions at relatively high concentration (60%, w/w).

Phosphatidylcholine (PC) is an essential component of biological membranes and is synthesized by the CDP-choline pathway under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase-alpha (CCT?). Ras transformed cells have increased lipid synthesis; the aim of this study was to determine if upregulation of CCT? was part of this transformed phenotype. Rat intestinal epithelial cell lines (IEC) and three oncogenic H-ras expressing IEC (IEC-Ras) were used to investigate the role of CCT? and phosphatidylcholine (PC) synthesis in resistance to detachment dependant apoptosis, termed anoikis. IEC-Ras have increased CCT? expression within the nucleus. Reduction of CCT? expression with lentiviral short hairpin RNA sensitized IEC-Ras to anoikis and decreased PC degradation, but did not change PC synthesis. Thus, in addition to CCT? being involved in anoikis-resistance in IEC-Ras these data indicate the possibility that it may also have nuclear-specific functions.

No description available.

Phosphatidylcholine (PC) is made in all nucleated mammalian cells via the CDP-choline pathway. Another major pathway for PC biosynthesis in liver is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We have identified 3T3-L1 adipocytes as a cell culture model that expresses PEMT endogenously. Analysis of the proximal PEMT promoter in 3T3-L1 adipocytes revealed an important regulatory region. Sp1 binds to a GC-rich site within this section of the promoter and inhibits PEMT transcriptional activity. Tamoxifen is an anti-estrogen drug widely used for the treatment of hormone-responsive breast cancer but has a frequent side-effect of increasing accumulation of lipid in the liver (hepatic steatosis). Tamoxifen represses PEMT gene expression by promoting Sp1 binding to the promoter. However, decreased catalytic activity of PEMT was not a major initial contributor to tamoxifen-mediated hepatic steatosis. We found that increased de novo fatty acid synthesis is the primary event which leads to tamoxifen-induced steatosis in mouse liver. Tamoxifen did not significantly alter hepatic fatty acid uptake, triacylglycerol secretion or fatty acid oxidation. Finally, we provide evidence that deletion of the PEMT gene in a well-established mouse model of atherosclerosis (apolipoprotein E deficient) reduces the formation of aortic lesions and prevents the associated development of dilated cardiomyopathy. This beneficial effect is likely due a reduction of atherogenic lipoproteins. These results indicate that treatment strategies aimed at the inhibition of PEMT could prevent the development of atherosclerosis that predisposes individuals to heart failure.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Previous studies in yeast in this laboratory have found carnitine to be both protective against oxidative stress induced by hydrogen peroxide and to increase the detrimental effect of dithiothreitol. These phenotypes were found to be independent of the role of carnitine within the carnitine shuttle. A screen for suppressor mutations for these carnitine-dependent phenotypes identified, among others, Δcho2 and Δopi3. Cho2p and Opi3p catalyse the sequential methylation reactions in the formation of phosphatidylcholine from phosphatidylethanolamine. Therefore, this study aimed to investigate the relationship between choline, phosphatidylcholine and the carnitine phenotypes. Liquid growth assays of Δcho2 and Δopi3 cultures revealed that addition of choline can restore the protective effects of carnitine against hydrogen peroxide. The connection between the cellular phospholipid composition and the carnitine-dependent shuttleindependent phenotypes was also investigated. Analysis of the lipid composition of cells by LCMS showed that Δcho2 and Δopi3 had a largely different lipid composition compared with the wild type, most notably, a reduction in phosphatidylcholine and an increase in triacylglycerol content were observed for both mutants. These changes were reversed by supplementation with choline. However, no effects on the lipid composition of cells in response to carnitine treatment were observed, either when supplemented alone or in combination with DTT and hydrogen peroxide. Carnitine has also been investigated in mammalian systems for its potential to protect cells from oxidative stress, an effect which would be of benefit in various neurodegenerative disorders. Several studies have documented the positive effects of carnitine against oxidative stress in mammalian cells however the mechanism behind this action remains unknown. It is therefore thought that, provided similar effects for carnitine can be shown in mammalian cells as was observed in yeast, it would be beneficial to use yeast as a model system for the study of the molecular changes induced by carnitine. In view of this, the effects of carnitine on toxicity induced by oxidative stress in mammalian neural cells were compared to that which has been observed in yeast. For this purpose the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, a measure of reductive capacity of cells, was used. However, no effects for carnitine were observed in the MTT assay in combination with either dithiothreitol or paraquat. / AFRIKAANSE OPSOMMING: Vorige studies op gis in hierdie laboratorium het bevind dat karnitien beskermend is teenoor oksidatiewe stres wat deur waterstofperoksied geïnduseer word en ook die nadelige effek van ditiotreitol verhoog. Hierdie fenotipes is gevind om onafhanklik te wees van die rol van karnitien binne die karnitien-pendel. Die sifting vir onderdrukker-mutasies van hierdie karnitienafhanklike fenotipes het onder andere Δcho2 en Δopi3 geïdentifiseer. Cho2p en Opi3p kataliseer die opvolgende metileringsreaksies tydens die vorming van fosfatidielcholien vanaf fosfatidieletanolamien. Hierdie studie het dus gepoog om die verhouding tussen cholien, fosfatidielcholien en die karnitienfenotipes te ondersoek. Vloeistofanalises van Δcho2- en Δopi3-kulture het aangedui dat die byvoeging van cholien die beskermende effekte van karnitien teenoor waterstofperoksied kan herstel. Die verband tussen die sellulêre fosfolipiedsamestelling en die karnitienafhanklike pendel-onafhanklike fenotipes is ook ondersoek. Die analise van die lipiedsamestelling van selle deur middel van LCMS het getoon dat Δcho2 en Δopi3 ‘n grootliks verskillende samestelling het in vergelyking met die wilde tipe, en daar is veral ‘n afname in fosfatidielcholien en ‘n verhoging in triasielgliserol-inhoud vir beide mutante waargeneem. Hierdie veranderinge is omgekeer deur aanvulling met cholien. Geen effekte op die lipiedsamestelling van die selle is egter in reaksie op die karnitienbehandelings waargeneem nie, hetsy toe dit alleen aangevul is of in kombinasie met ditiotreitol en waterstofperoksied. Karnitien is ook in soogdierstelsels ondersoek vir sy potensiaal om selle teen oksidatiewe stres te beskerm, ‘n effek wat groot voordeel sal inhou vir verskeie neurodegeneratiewe steurings. Verskeie studies het reeds die positiewe effekte van karnitien teen oksidatiewe stres in soogdierselle opgeteken, hoewel die meganisme agter hierdie werking nog onbekend is. Daar word dus vermoed dat, gegewe dat soortgelyke effekte vir karnitien in soogdierselle getoon kan word as wat in gis waargeneem is, dit voordelig sou wees om gis as ‘n modelsisteem vir die studie van die molekulêre veranderinge wat deur karnitien geïnduseer word, te gebruik. In die lig hiervan is die effekte van karnitien op giftigheid wat deur oksidatiewe stres in soogdiersenuselle geïnduseer is, vergelyk met dít wat in gis waargeneem is. Om hierdie rede is die 3-[4,5-dimetieltiasool-2-iel]-2,5-difeniel tetrasoliumbromied (MTT) essaiëring, ‘n meting van die verminderende kapasiteit van selle, gebruik. Geen effekte vir karnitien is egter met die MTT essaiëring in kombinasie met óf ditiotreitol óf parakwat waargeneem nie.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Deficiency of the surfactant leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. The generation of DPPtdCho in cells occurs via two integral routes: the de novo and remodeling pathways. The interplay between these pathways has not been investigated. Overexpression of the remodeling enzyme, acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT1), in epithelia decreases de novo PtdCho synthesis without significantly altering cellular phospholipid mass; this occurs through increased degradation of cholinephosphotransferase (CPT1), the terminal enzyme of the de novo pathway. CPT1 is degraded by multi-ubuiquitination and trafficking via the lysosomal pathway. When expressed in lung epithelia, CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysine residues exhibited proteolytic resistance to effects of LPCAT1 overexpression. Cellular expression of these CPT1 mutants also restores de novo PtdCho synthesis to levels normally observed in lung epithelia. Further studies demonstrate that the SCF (Skip-Cullen-F-box) ubiquitin E3 ligase component, β-TrCP, was sufficient to degrade CPT1. Similar to CPT1, LPCAT1 levels are also regulated at the level of protein stability. However, LPCAT1 is a polyubiquitinated enzyme processed within the proteasome. Similar to CPT1, β-TrCP is the putative E3 ubiquitin ligase subunit responsible for LPCAT1 ubiquitination. β-TrCP appears to dock and ubiquitinate LPCAT1 within its amino-terminus. Collectively, these observations indicate the presence of cross-talk between the phospholipid remodeling and de novo pathways; this involves tight regulation by site-specific ubiquitination of indispensable regulatory enzymes catalyzed by SCF ubiquitin E3 ligase members that mechanistically provide homeostatic control of cellular phospholipid content.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong pro-inflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTalpha). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTalpha from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTalpha does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha monoubiquitination masks its NLS resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTalpha for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTalpha and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsis-induced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

Stuttgart, Univ., Diss., 2000.

No description available.

Orientador: Celio Kenji Miyasaka / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-12T23:28:45Z (GMT). No. of bitstreams: 1 Fonseca_CarenFernandaNavarroda_M.pdf: 642682 bytes, checksum: 5ba563cf4536604afdecde132b47d3fe (MD5) Previous issue date: 2009 / Resumo: A Fosfatidilcolina (PC) exerce vários efeitos no organismo humano. Este fosfolipídeo é encontrado em abundância nas membranas celulares e, juntamente com a fosfatidiletanolamina, somam mais de 75% do total de fosfolipídeos presentes nestas. A PC vem sendo utilizada em clínicas dermatológicas, de estética e emagrecimento, pois, parece ser eficaz para eliminar gordura localizada, em especial no abdome, quadril, joelhos, pescoço e pálpebras inferiores. Este trabalho teve por objetivo, estudar os efeitos da PC solubilizada em diferentes compostos (deoxicolato de sódio, etanol, detergente Tween® 80 e albumina) sobre os adipócitos epididimais isolados de ratos machos da linhagem Wistar. Atualmente, não há estudos científicos conclusivos liberando o seu uso farmacológico e, portanto, há a necessidade de mais pesquisas que definam seus mecanismos de ação. Para atingirmos o objetivo proposto, utilizou-se o método de isolamento e contagem dos adipócitos e também a determinação do glicerol no meio de incubação. Sendo assim, o tratamento que obteve maior quantidade de glicerol produzido foi o Tween® 80 e Tween® 80 + PC no tempo de incubação de 15 minutos e o DS + PC no tempo de incubação de 30 minutos / Abstract: The phosphatidylcholine (PC) has severa effects on the human body. This phospholipid is found in abundance in cell membranes and, together with phophatidylethanolamine, up over 75% of total phospholipids present in these. The PC has been used in clinical dermatology, weight loss of aesthetics and therefore appears to be effective for removing localized fat, especially in the abdomen, hips, knees, neck and lower eyelids. This work was aimed at, to study the effects of PC solubilized in different compounds (sodium deoxycholate, ethanol, Tween ® 80 detergent and albumin) on adipocytes isolated epididymis of male rats of Wistar strain. Currently, there is no conclusive scientific studies drug releasing its use and therefore there is a need for more research to define its mechanisms of action. To achieve the proposed objective, we used the method of isolation and counting of adipocytes and the determination of glycerol in the middle of incubation. Thus, the treatment they received higher amount of glycerol produced was the Tween ® 80 and Tween ® 80 + PC in the incubation period of 15 minutes and DS + PC in the incubation time of 30 minutes / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição