Thesis (Ph.D)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Physiology. Title from title-page of electronic thesis. Includes bibliographical references.

No description available.

Sphingomyelin (SM), a sphingolipid that is concentrated in the extracellular leaflet of the plasma membrane, can interact with cholesterol to form more ordered raft domains. The hydrolysis of SM by sphingomyelinase (SMase) generates ceramide and may redistribute cholesterol molecules to other less ordered domains. I employed carbon fibre amperometry to examine whether SM hydrolysis affected the kinetics of release of catecholamines from individual granules of rat chromaffin cells when exocytosis was triggered by elevated extracellular [K+]. Similar to cholesterol overload, SMase treatment selectively increased the proportion of stand-alone foot signals and the duration of the pre-spike foot signals; both effects could be reduced by extraction of cellular cholesterol. In contrast, the application of an exogenous ceramide did not mimic the effects of SMase. My results suggest that SMase treatment liberated cholesterol from lipid rafts to increase the persistence of the semi-stable fusion pore before the onset of rapid dilation.

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

Sphingolipid metabolites play a role in the initiation and perpetuation of inflammatory responses. Since intestinal inflammation is a driving force in the development of colon cancer, in the present study, we investigated the suppression of dextran sodium sulfate (DSS)-induced colitis by dietary sphingomyelin in mice that lack functional peroxisome proliferator-activated receptor γ (PPAR-γ) in intestinal epithelial and immune cells. Dietary spingomyelin decreased colonic inflammation in mice of both genotypes but more efficiently in mice expressing PPAR-γ. Using a real-time polymerase chain reaction array, we detected an up-regulation in genes involved in Th1 (interferon γ) and Th17 (interleukin [IL]-17 and IL-23) responses despite the reduced inflammation. However, the genes involved in Th2 (IL-4, IL-13 and IL-13ra2) and Treg (IL-10rb) anti-inflammatory responses were up-regulated in a PPAR-γ-dependent manner. In order to direct mechanistic studies of how PPAR-γ expression is involved in SM-induced suppression of DSS colitis, we investigated the effect of dietary SM in DSS-treated mice that lack PPAR-γ in the CD4+ T-cells. While the pathogenesis of colitis was independent of PPAR-γ expression in CD4+ T-cells, dietary SM decreased disease activity and colonic inflammation in mice of both genotypes but more efficiently in mice expressing PPAR-γ, indicating both PPAR-γ dependent and independent signaling pathways. In conclusion, in contrast to endogenous sphingolipid metabolites, dietary SM modulated both pro- and anti-inflammatory responses at the early stages of the disease in a partially PPAR-γ dependent manner resulting in a suppression of inflammation that may be critical for the suppression of inflammation-driven colon cancer. / Master of Science

Die saure Sphingomyelinase (Asm), ein Enzym des Sphingolipidmetabolismus, spaltet Sphingomyelin zu Ceramid und Phosopocholin. Aktiviert wird die Asm unter anderem durch Stimulation des CD28 Rezeptors. CD28 Signale werden auch für die Aktivierung von konventionellen T-Zellen (Tconv) und für die Kostimulation benötigt und sind essentiell für die Differenzierung von regulatorischen T-Zellen (Treg) im Thymus und deren Erhalt in der Peripherie. Wir konnten zeigen, dass sich Tconv und Treg Zellen hinsichtlich der Asm unterscheiden. Treg haben eine höhere "basale" Asm Aktivität, widergespiegelt im höheren Ceramidgehalt und haben eine niedrigere Lipidordnung als Tconv Zellen. Die Abwesenheit der Asm in defizienten Mäusen bewirkt einen relativen Anstieg der Treg-Frequenz innerhalb der CD4+ T-Zellen. Außerdem führt die Asm-Defizienz in Treg Zellen zu einer erhöhten Umsatzrate des immunsupprimierenden Moleküls CTLA-4 und zu einer verstärkten Suppressivität von Treg Zellen aus Asm-/- Mäusen gegenüber Wildtyp Zellen. Ein Anstieg in der Treg-Frequenz, äquivalent zur genetischen Defizienz, kann auch durch Inhibition der Asm, d. h. durch Wirkstoffe wie Amitriptylin und Desipramin erreicht werden. Es konnte gezeigt werden, dass die Inhibitorbehandlung die absolute Anzahl der Tconv Zellen selektiv verringert, da Treg Zellen gegenüber dem Asm Inhibitor-induzierten Zelltod resistenter sind. Mechanistisch erklärbar sind die Unterschiede gegenüber den proapoptotischen Inhibitoreffekten zwischen Tconv und Treg Zellen dadurch, dass Treg Zellen durch die Anwesenheit von IL-2 geschützt sind. In Abwesenheit von IL-2 sterben die Treg Zellen ebenfalls. Die gezielte Veränderung des Verhältnisses von Treg zu Tconv durch den Einsatz von Asm-inhibitorischen Medikamenten kann hilfreich bei der therapeutischen Behandlung von inflammatorischen- und Autoimmunerkrankungen sein. Inwiefern die Asm für die Funktion von T-Zellen in der anti-viralen Immunantwort entscheidend ist, wurde im Masernvirus-Infektionsmodell näher untersucht. In Asm-/- Mäusen und Amitriptylin-behandelten Mäusen konnte gezeigt werden, dass in Abwesenheit der Asm die Kontrolle der Masernvirusinfektion verschlechtert ist. Treg sind auch hier von entscheidender Bedeutung, da die Asm-abhängige, verstärkte Masernvirusinfektion bei Fehlen der Asm nur in Gegenwart von Treg auftritt. In der akuten Phase gibt es in Asm-/- Mäusen weniger masernvirusspezifische T-Zellen und dadurch eine verringerte Beseitigung der Viruslast. In der chronischen Phase ist die Anzahl masernvirusspezifischer T-Zellen zwischen WT und Asm-/- Mäusen vergleichbar. In Letzteren ist allerdings die Anzahl und Frequenz von T-Zellen im Gehirn infizierter Mäuse noch deutlich erhöht, was die verstärkte Maserninfektion widerspiegelt. Zusammenfassend zeigt sich, dass die Asm die Funktion von Treg moduliert und einen Einfluss auf das Verhältnis von Tconv und Treg zueinander hat. Im Masernvirus-Infektionsmodell kann die Veränderung des Tconv zu Treg Verhältnisses in Abwesenheit der Asm ursächlich für die verringerte Viruskontrolle sein. Die Asm Inhibitor-induzierte Treg-Aktivierung und die Beeinflussung des Treg zu Tconv Verhältnisses können wiederum für therapeutische Zwecke genutzt werden, wie beispielsweise bei Multipler Sklerose und Rheumatoider Arthritis. / The acid sphingomyelinase (Asm), an enzyme of the sphingolipid metabolism, hydrolyses sphingomyelin into ceramide and phosphocholine. Besides other stimuli the Asm is activated by ligation of the costimulatory molecule CD28. CD28 signaling is necessary to activate conventional T-cells (Tconv) and is crucial for the differentiation and maintenance of thymus-derived regulatory T-cells (Treg). We could demonstrate that Tconv and Treg cells differ with respect to Asm activity. Treg cells have an increased "basal" Asm activity resulting in an elevated level of ceramide and show a decreased lipid order compared to Tconv cells. The absence of Asm leads to a relative increase in Treg frequency among CD4+ T-cells in Asmdeficient mice. Furthermore, the Asm deficiency results in an increased turnover rate of the immunosuppressive molecule CTLA-4 and strengthens the suppressive capacity of Treg cells from Asm-/- mice compared to wild type Treg cells. An increase of the Treg cell frequency, equivalent to that seen with genetic deficiency, can be achieved by drugs like amitriptyline and desipramine too. The inhibitor treatment selectively decreases the absolute numbers of Tconv cells as Treg cells are more resistant towards Asm inhibitor induced cell death. The mechanistic explanation for the difference concerning the proapotptotic effects of Asm inhibitors between Treg and Tconv cells is that Treg cells are protected by IL-2. In the absence of IL-2 Treg cells die too. Therapeutically shifting the balance of Treg and Tconv cells by Asminhibiting drugs can be beneficial in inflammatory and autoimmune diseases. Whether the Asm is necessary for the function of T-cells during anti-viral immune responses was investigated in a measles virus infection model. In Asm-/- mice and amitriptyline treated mice control of the measles virus was impaired. Treg cells are of critical relevance as the Asm dependent boost in measles infection was only visible in the presence of Treg cells. During the acute phase of infection less measles virus specific T-cells were present leading to a decreased clearance of virus from the brains of Asm-/- mice. In the chronic phase the number of measles virus specific Tcell was comparable between wt and Asm-/- mice. But in the latter the number and frequency of T-cells in brains of infected mice was increased, which mirrors the enhanced measles virus infection. In conclusion, the Asm modulates the function of Treg cells and influences the Treg- Tconv ratio. The changed Treg-Tconv ratio in the absence of Asm expression might be responsible for the reduced virus control in the measles virus infection model. Additionally, the Asm inhibitor induced Treg cell activation and its effects on the Treg- Tconv ratio can be used for therapeutical approaches in diseases like multiple sclerosis or rheumatoid arthritis.

Depression is one of the most commonly seen psychiatric diseases in the population in recent years. Treatment of depression is mainly carried out by psychiatric drugs. In the past few years, agomelatine which is released to the market with a trade name, Valdoxane, has been thought to have far less side effects due to its non-addictive nature, not having trouble when the drug is quitted, and also due to its property of binding only to the specific receptor that the drug interacts with. The action mechanism of agomelatine on the membrane structure has not been clarified yet, for instance, no study has been found in the literature about the interaction of agomelatin with the lipids of biological membranes. In this current study, the interaction of agomelatine with the model membranes of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylgylcerol (DPPG) and sphingomyelin (SM) is examined by Fourier transform infrared spectroscopy (FTIR) and Differential scanning calorimetry (DSC). DSC and FTIR studies show that, agomelatine shifts the phase transition temperature of DPPC and DPPG multilamellar membrane to the lower degrees, however, it shifts the phase transition temperature of SM membrane to the higher degrees. Agomelatine addition increases the lipid order of the DPPC and SM liposome, whereas, it decreases the lipid order of DPPG liposome. Moreover this drug enhances the membrane fluidity among all types of liposome studied. The increase of v lipid order and increase of fluidity at DPPC and SM liposome indicates domain formation upon drug addition (Vest et al., 2004). This was also confirmed by DSC studies. Agomelatine enhances H bonding capacity of all types of liposomes have been studied. However it has different effects on glycerol backbones of the DPPC and DPPG liposomes. At low agomelatine concentrations the increase in the frequency values indicates a decrease in the hydrogen bonding capacity of the glycerol skeleton of DPPC. In contrast, at high concentrations of agomelatine, a decrease in the frequency values was observed as an indicator of the enhancement of the hydrogen bonding capacity. So it enhances H-bonding capacity at gel phase but lowers it at liquid chrystalline phases. A progressive decreases in Tm was observed at DPPG and DPPC liposomes where it increased the Tm at SM. The pretransition peak is abolished and the Tm peak becomes broad, indicating a larger perturbation to the membrane. These observations indicate the possible interaction of agomelatine with the head group as well. The shoulder seen at the thermograms of DPPC and DPPC liposomes at high doses may indicate the lateral phase separation in to drug-rich and drug-poor domains (D&rsquo / Souza et al., 2009). These results may indicate that agomelatine is partially buried in the hydrocarbon core of the bilayer, interacting primarily with the C2-C8 methylene region of the hydrocarbon chains. All these results highlight the fact that agomelatine interacts around the head group in such a manner that it destabilizes the membrane architecture to a large extent.

Naja naja atra Cardiotoxins (CTXs), basic polypeptides of 60 amino acid residues adopt a three-fingered loop-folding topology and show cytotoxicity for human tissues in targeting cell membrane. Despite having highly similar sequence, the six CTX isoforms also display different cytotoxic potencies and hemolytic activities. The goal of these studies is to explore the mechanical processes that involved in membrane-damaging activities of CTXs on vesicles composed of different cell membrane components, and to delineate the events that lead to different biological activities of CTXs. The studies were performed by estimating the color transformation of phospholipid/polydiacetylene vesicles and the fluorescence enhancement of fluorescein-labeled phospholipid/protein or fluorescein released from vesicles. It was found that vesicles consisted of unsaturated phospholipids improve membrane-damaging activity of CTXs and adopt a vital membrane-bound conformation of CTXs. In contract, the characteristic of vesicles consisted of saturated phospholipids was against CTXs adopting an essential membrane-damaging structure. It was also found that not only electrostatic force but also hydrophobic force were involved in the interaction between CTXs and membrane. Comparing with phosphatidylcholine-only vesicles, CTXs displayed higher membrane-damaging activity for the sphingomyelin-containing vesicles, and the loop2 region of CTXs play a crucial role for the membrane-damaging activity of sphingomyelin-containing vesicles. Besides, the CTX3 and CTX5 would interact with the H-antigen of blood group O red blood cells, but only the binding of CTX3 with H-antigen reduce its membrane-damaging activity for red blood cells membrane. Moreover, the fusogenicity of CTXs is responsible for the membrane-damaging activity of CTXs toward bacterial membrane-mimicking vesicles. The cardiolipin have the potency to improve the fusogenicity of CTX3, which induced the bactericidal activity toward the cardiolipin-containing bacterium.

No description available.

Non Melanoma Skin Cancer (NMSC) affects 3.3 million Americans each year and results from Ultra Violet Radiation (UVR) damage to DNA in the form of pyrimidine dimers and photoproducts [1]–[5]. Cells directly detect the damage and initiate apoptosis, cell cycle arrest, or DNA repair by modulating p53 and p21 levels [6]–[9]. Current methods of photoprotection include sunscreen, but controversy over safety of some active ingredients necessitates research into more natural alternatives [10]–[12]. In particular, 24 hour incubation with bovine milk sphingomyelin (BSM) has demonstrated photoprotective potential by reducing p21 and p53 levels in keratinocytes (KRTs) after UV radiation [13], [14]. This thesis aims to expand on past BSM research by exploring the mechanism for photoprotection. Normally, sphingomyelin (SM) is metabolically degraded to ceramide which then leads to cell apoptosis [6]. The goals of this thesis were to characterize a fluorescent SM (FSM) to assess changes in intracellular fluorescence distribution after various incubation and post-UV exposure times. FSM was deemed functionally equivalent to BSM by reducing levels of p21 after UV. Furthermore, quantification demonstrated that FSM trafficking and intracellular fluorescence were independent of continuous incubation time, warranting further investigation into shorter timepoints like 1 hour. Across several post-UV timepoints, the 1 hour incubation had a consistently higher average cytoplasmic mean gray value compared to 24 hour incubation. In addition, the no UV control was significantly lower compared to the 24 hour and 12 hour post-UV timepoints. No post-UV differences were observed for the 24 hour incubation, suggesting future work is necessary for the 1 hour incubation, which potentially streamlines future experiments. Two immunofluorescence stains for endogenous SM (lysenin) and ceramide were also optimized for preliminary fluorescence distribution studies and colocalization with FSM. Finally, a 3T3 fibroblast spheroid model was utilized as proof-of-concept for future 3D KRT cultures and depth of dye penetration quantification methods. These findings suggest FSM is an appropriate model for BSM trafficking, a shorter FSM incubation time could potentially be adopted in future studies, dual immunofluorescence staining for SM and ceramide is viable, and spheroids provide a promising model for future 3D KRT studies.