Two swine kidney proteins (PI 4.8 and 5.8) both possessing 9-prostaglandin ketoreductase (9-PGKR) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) activities were purified to homogeneity. Purification increased specific activities in parallel. Molecular weight, subunit size, amino acid composition, coenzyme and substrate specificity and antigenicity of both proteins were similar. Gel filtration and SDS-polyacrylamide gel electrophoresis molecular weights of 29,500 and 29,000, respectively, suggested a single subunit. Although a variety of prostaglandins served as substrates, the best for 15-PGDH was PGB, while PGA_1-GSH showed the lowest Km for 9-PGKR. Rabbit antibody against the PI 5.8 protein crossreacted with both purified renal enzymes and with extracts from rat spleen, lung, heart, aorta, and liver.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 122-146.

Thesis (PhD.(Physiology)--Faculty of Health Sciences)-University of Pretoria, 2005. / Summary in English and Afrikaans. Also available on the Internet via the World Wide Web.

No / Lipidomics aim to generate qualitative and quantitative information on different classes of lipids and their species, and when applied in conjunction with proteomic and genomic assays, facilitate the comprehensive study of lipid metabolism in cellular, organ or body systems. Advances in mass spectrometry have underpinned the expansion of lipidomic methodologies. Prostanoids are potent autacoids present in a plethora of cellular systems, known best for their intimate role in inflammation. Electrospray ionisation (ESI) allows the efficient ionisation of prostanoids in aqueous systems. ESI can be readily coupled to liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS)-based detection, thus allowing the development of a potent and selective LC/ESI-MS/MS quantitative assays. The protocol we describe in this chapter outlines the steps we follow to a) extract prostanoids from solid or liquid samples, b) semi-purify the metabolites using solid phase extraction c) set-up the HPLC separation using reverse phase chromatography and d) set up the MS/MS assay using a triple quadrupole mass spectrometer. The experimental details and notes presented here are based on the detailed protocols followed in our group

Call number: LD2668 .T4 1985 L825 / Master of Science

Modulation of PGs by inhibition or stimulation is a promising approach for the management of pain and inflammation in patients with rheumatic disease. Based on recent results from our laboratories as well as on the literature, we hypothesise that Prostaglandin D[subscript 2] (PGD[subscript 2]) is an important anabolic agent for osteoblasts. Our results show that the PGD[subscript 2] decreases the osteoblasts proliferation acting probably through the CRTH2 receptor. Surprisingly, when DK-PGD[subscript 2] was used alone or with Naproxen, although the proliferation decreased with the dose, it seemed to be restored to the control level at higher concentrations of DK-PGD[subscript 2] . Thus, we hypothesise the existence of other compensatory mechanisms. The PGD[subscript 2] had no relevant effect alone or when used with Naproxen, but seemed to decrease the osteoblast differentiation when used with Diclofenac at a higher concentration only. When vitamin D was added to all conditions, PGD[subscript 2] had an inhibitory effect on the differentiation (dose-response), but this could not be replicated when Naproxen was used. In a test with Diclofenac, we can assume a decreasing trend-line for differentiation when augmenting the PGD[subscript 2] dose, but the effect is not statistically relevant. In a competition test with PGD[subscript 2] and DP/CRTH2 antagonists, blocking DP receptor yielded no effect on differentiation, and blocking the CRTH2 receptor showed a relevant decrease at high concentration of PGD[subscript 2]. The effect was similar in a test with PGD[subscript 2] and PPAR[gamma] antagonist suggesting that it might have a compensatory, positive effect that reversed DP activation. The PGD[subscript 2] has a slight positive effect on the osteoblast matrix mineralisation (with Naproxen), but not through its receptors since use of DP/CRTH2 antagonists did not abrogate this. In a competition test with PGD[subscript 2] and DP/CRTH2 antagonists we had no response. When we used the PGD[subscript 2] in the presence of PPAR[gamma] antagonist, the calcification decreased significantly, indicating that the positive effect of PGD[subscript 2] on calcification works rather through this receptor.

The progesterone (P4) produced by the corpus luteum (CL) is essential for maintenance of pregnancy. On the other hand, the interferon tau (IFNT) produced by the embryo during elongation process, besides being the primary signal for recognition, also is responsible for maintenance of the CL during early pregnancy. The presence of oxytocin receptors (OXTR) in endometrium during expected time of luteolysis is determinant for trigger the uterine release of prostaglandin F2∝ (PGF), which is in charge of CL regression. The IFNT avoid the luteolysis by suppressing the OXTR appearance. However, during second month, accessory CLs are able to regress, indicating that the PGF release occurs with the advancing of the pregnancy and the mechanisms that initiated luteolysis are recovered. Therefore, failures in maintenance of the CL can cause luteolysis and pregnancy loss during this period of 30 to 60 days, which is one of the most important problems in reproductive efficiency in cattle, specially when in vitro produced (IVP) embryos are transferred. Two experiments were designed to study this factors, focused on point when uterus recover its PGF release during pregnancy and to identify possible differences between those mechanisms on pregnancies from IVP or artificial insemination (AI) embryos. The first study evaluated circulating PGF metabolite (PGFM) after an oxytocin challenge throughout first two months of pregnancy in lactating Holstein cows. Treatment with oxytocin did not affected PGFM concentration in d11 pregnant (P) and non-pregnant (NP), on d18 had a little increase in P cows, while increased 2-fold in NP cows. Oxytocin-induced PGFM in P cows on day 25 was greater than d18 P, however was lower than P cows on d53 and d60. Days 32, 39 and 46 of pregnancy had intermediate response. The second study evaluated the oxytocin-induced PGFM in Nelore cows pregnant from AI or IVP embryos on days 17 and 31, and its association with factors that can impact in success of the pregnancy, such as P4 levels, conceptus length on d18 and size of the embryo on d32. Also, OXTR and interferon-stimulated gene 15 (ISG15) gene expression were evaluated and located in uterine endometrium. Embryo size on d32 and P4 on d31, were higher in AI than IVP. Cows from IVP on d17 presented lower oxytocin-induced PGFM than AI in the same day, however, d31 for both groups had higher PGF release after oxytocin. On d31 there was similar PGFM increase in synchronized non-inseminated group (NI). The OXTR are highly suppressed on pregnant cows on d18, especially in IVP group, but were high expressed in NI cows and on d32 for both groups, AI being higher than IVP at this day. The ISG15 had irrelevant expression on NI and d32 groups, while had extremely high expression in d18 pregnant cows for both groups. Concluding, the CL in early pregnancy is maintained by PGF release suppression, while during second month there is oxytocin-induced PGF release, suggesting that other mechanisms are responsible for maintaining CL after d25. In addition, these results demonstrate there are signaling differences between IVP and AI pregnancies, impacting the molecular and endocrine environment that influences PGF release during these time points. / A progesterona (P4) produzida pelo corpo lúteo (CL) é essencial para a manutenção da gestação. Por sua vez, o interferon tau (IFNT) produzido pelo embrião durante o processo de alongamento, além de ser o sinal primário para reconhecimento e manutenção da gestação também é responsável pela manutenção do CL durante a gestação inicial. A presença de receptores de ocitocina (OXTR) no endométrio no momento esperado da luteólise é determinante para liberação uterina de prostaglandina F2∝ (PGF), a qual é responsável pela regressão do CL. O IFNT evita a ocorrência da luteólise por meio da supressão da expressão de OXTR no endométrio. Entretanto, durante o segundo mês de gestação, CLs acessórios, principalmente contralaterais são capazes de regredir, indicando que ocorre liberação de PGF pelo útero conforme a gestação avança, e os mecanismos que iniciam a luteólise são restabelecidos. Portanto, falhas na manutenção do CL podem causar luteólise e perdas gestacionais de 30 para 60 dias, um dos importantes problemas de eficiência reprodutiva em bovinos, principalmente quando embriões produzidos in vitro (PIV) são transferidos. Dois estudos foram delineados para estudar estes fatores, com foco em determinar o momento em que o útero gravídico retoma a liberação de PGF, e identificar prováveis diferenças entre estes mecanismos em gestações de embriões PIV ou de inseminação artificial (IA). O primeiro estudo avaliou a concentração circulante do metabólito de PGF (PGFM) após desafio com ocitocina durante os primeiros dois meses de gestação em vacas Holandesas lactantes. O tratamento com ocitocina não afetou a concentração de PGFM em vacas de d11 prenhes (P) e não-prenhes (NP), no d18 apresentou um ligeiro aumento em vacas P, enquanto aumentou cerca de duas vezes em relação ao nível basal em vacas NP. O aumento de PGFM induzido por ocitocina em vacas P no dia 25 foi maior que P em d18, entretanto foi menor que vacas P nos dias 53 e 60. Os dias 32, 39 e 46 da gestação tiveram resposta intermediária. O segundo estudo avaliou a PGFM circulante em resposta a ocitocina em vacas Nelore prenhes de embriões PIV ou IA, nos dias 17 e 31 de gestação, e sua associação com fatores que podem impactar no sucesso da prenhes, como P4 circulante, tamanho de concepto no d18, e o tamanho de embrião no d32. Além disso, foi avaliada e localizada a expressão de OXTR e do gene estimulado por interferon 15 (ISG15) no endométrio uterino. O tamanho de embrião no dia 32 e a P4 circulante no dia 31 foram maiores no grupo IA. Vacas do grupo PIV d17 apresentaram menor resposta a ocitocina na concentração de PGFM do que as de IA no mesmo dia, contudo no dia 31 ambos os grupos tiveram maior resposta do que PIV d17. As vacas do d31 dos dois grupos tiveram aumento na PGFM similar às vacas não-inseminadas (NI). Os OXTR foram altamente suprimidosnas vacas prenhes do d18, especialmente no grupo PIV, mas com alta expressão em vacas NI e no dia 32 para os dois grupos, sendo a IA com maior expressão que a PIV neste dia. O gene ISG15 apresentou expressão irrelevante em NI e d32 para IA e PIV, mas apresentou expressão extremamente alta no d18 nos dois grupos prenhes. Conclui- se que o CL na gestação inicial é mantido pela supressão da liberação de PGF, enquanto que no segundo mês, ocitocina induz liberação de PGF, sugerindo que outros mecanismos regem a manutenção do CL a partir do dia 25. Além disso, nossos resultados demonstram que há diferenças entre a sinalização de gestações provenientes de embriões PIV e IA, que impactam no ambiente molecular e endócrino, influenciando a liberação de PGF nestes momentos.

O processo de morte por apoptose pode ser dividido em duas vias: intrínseca e extrínseca. A sinalização via FAS (extrínseca) pode ocorrer independente (células Tipo I) ou dependente da mitocôndria (células Tipo II). É importante considerar que: 1) Resultados prévios mostraram que doses subletais de CHX foram capazes de sensibilizar células Tipo I e Tipo II à apoptose e de converter células Tipo II em Tipo I; 2) Um dos mecanismos envolvidos pode ser o recrutamento de FAS para as \"balsas lipídicas\"; 3) A PGE2 ativa PKA pelo aumento de cAMP via EP2 e EP4, que fosforila ezrina, envolvida nesse processo; 4) A PGE2 pode induzir apoptose em linhagens celulares e sensibilizá-las a esse processo. Assim, formulamos a hipótese de que a PGE2 poderia, assim como a CHX, sensibilizar certas células à apoptose e converter células Tipo II em Tipo I. Esse efeito não foi observado em células DO11.10 nas quais a apoptose foi induzida por CD95L solúvel e em células Tipo I e Tipo II, nas quais a apoptose foi induzida pelo anticorpo agonista anti-FAS. / The death process by apoptosis can be divided into two pathways: intrinsic and extrinsic. The signaling by FAS (extrinsic) may occur in a mitochondrial independent (Type I cells) or dependent (Type II cells) manner. It is important to consider that: 1) Previous results demonstrated that sub-lethal doses of CHX were able to sensitize type I and type II cells to apoptosis and to convert type II cells into type I; 2) One of the mechanisms involved can be FAS recruitment to \"lipid rafts\"; 3) PGE2 activates PKA by increasing cAMP via EP2 and EP4, which phosphorylates of Ezrin, involved in this process; 4) PGE2 can induces apoptosis in cell lines and can to sensitize them to this process. So, we hypothesized that PGE2 could, similarly to CHX, sensitize certain cells to apoptosis and convert type II cells into type I. This effect was not observed in DO11.10 cells in which apoptosis was induced by soluble CD95L and in type I and type II cells, in which apoptosis was induced by agonist anti-FAS antibody.

The parallel actin filament bundling protein Fascin is a critical protein in both disease and development. Overexpression of Fascin is linked to increased aggressiveness in a number of cancer types, including breast and colon carcinomas. Importantly, Fascin is not normally expressed in adult epithelial cells from which many of these cancers arise. Therefore, Fascin is increasingly cited as both a potential biomarker and therapeutic target in many types of cancer. Fascin is most commonly associated with the formation of filopodia and invadapodia (parallel actin filament bundle structures) to drive migration and invasion. However, Fascin activity and regulation remain poorly understood. In order for Fascin to be an effective target for cancer therapeutics, a better understanding of the mechanisms regulating Fascin activities in the cell is necessary. Prostaglandins (PGs) are short-lived lipid signaling molecules that mediate a wide range of biological activities. PGs act through G protein-coupled receptors to initiate signaling cascades that affect downstream targets, including actin cytoskeletal remodeling. Importantly, the key enzymes in the synthesis of PGs, cyclooxygenase (COX) 1 and 2, are the targets of non-steroidal anti-inflammatory drugs like aspirin. Interestingly, like Fascin, PGs have been independently implicated in cancer development and metastasis and aspirin may reduce the risk of aggressive cancer. However, the exact mechanisms by which PGs mediate cancer development are unknown. The work presented in this thesis focused on novel PG-dependent regulation and activity of Fascin. The research presented here utilized Drosophila oogenesis as a model system to analyze PG-dependent Fascin activity. Drosophila oogenesis is an ideal model in which to study the activity and regulation of actin binding proteins like Fascin. Oogenesis consists of 14 morphologically defined stages, which are observable many times over within a single isolated pair of ovaries. A developing follicle consists of 16 germline cells – 15 nurse, or support cells, and a single oocyte. The nurse cells are of particular interest because they are the sites of dynamic actin remodeling during mid-late oogenesis. During stage 10B, an array of radially-aligned actin filament bundles form at the nurse cell membranes and extend inwards towards the nucleus. A network of cortical actin is also strengthened during this stage. These actin structures are essential for the completion of oogenesis, and ultimately female fertility. Importantly, PGs and Fascin are required for this actin remodeling; genetic loss of Fascin or the Drosophila COX-like enzyme Pxt (Peroxinectin-like) leads to disruption of cytoplasmic actin remodeling, and ultimately, female sterility. Using this model system, work presented here describes the discovery of Fascin as a downstream target of PGs to promote actin bundle formation, described in Chapter 2. Additionally, Fascin is required for strengthening of the cortical actin network downstream of PGs. This observation is one of the first to describe a role for Fascin in a branched actin network. Additionally, Fascin is regulated by a specific PG – PGF2α – during S10B to promote follicle development. Finally, Chapter 2 shows that PGs target specific actin binding proteins to promote cytoskeletal remodeling; Villin, another actin bundling protein, does not interact with PGs. Chapter 3 describes the novel observation that Fascin localizes to the nucleus and the nuclear periphery in Drosophila nurse cells. This finding is significant, as it is the first to describe Fascin in a context other than cytoplasmic. Fascin localization in and around the nucleus is specific and dynamic, and changes throughout late stage oogenesis, suggesting regulated functions at these sites. Fascin localization is regulated by PGs, and loss of Pxt leads to reduced nuclear Fascin localization and failure to localize to the nuclear periphery. Additionally, Fascin has novel potential functions in the nucleus and at the nuclear periphery. Loss of Fascin leads to disruption of nucleolar morphology in the nurse cell nuclei. Additionally, loss of PGs, which cause reduced nuclear Fascin levels, also causes abnormal nucleolar morphology. These data suggest that PGs regulate Fascin to control nucleolar organization. At the nuclear periphery, Fascin localization requires components of the protein complex that links the nucleoplasm to the cytoplasm, termed the LINC complex. Loss of an essential LINC complex protein, Koi, leads to a loss of nuclear periphery Fascin localization. These data suggest that Fascin may be a novel component of the LINC complex. Finally, Chapter 4 describes regulation of Fascin by phosphorylation at conserved serine residues. PGs affect Fascin phosphorylation, and loss of PGs leads to more heavily phosphorylated Fascin. Additionally, phosphorylation of Fascin alters localization to the nucleus and to the nuclear periphery. These data suggest that one mechanism by which PGs regulate Fascin is to control its phosphorylation status to affect subcellular distribution. In summary, the work presented in this thesis has demonstrated novel regulation and function of the actin bundling protein Fascin using Drosophila oogenesis as a model. Importantly, these functions and regulation of Fascin are likely conserved in mammals, and may have implications in human health and disease. Continued study of the activity and regulation of actin binding proteins like Fascin in Drosophila will likely have great effect on our understanding of many human diseases.

Prostaglandins (PGs) are small, lipid signaling molecules produced downstream of cyclooxygenase (COX) enzymes. PG signaling regulates many processes including pain, inflammation, fertility, cardiovascular function and disease, and cancer. One mechanism by which PG signaling exerts its function is by regulating the dynamics of the actin cytoskeleton; however, the exact mechanisms remain largely undefined. Drosophila oogenesis provides an ideal system to determine how PG signaling regulates the actin cytoskeleton. Drosophila follicles, or eggs, pass through 14 well- characterized, morphologically defined stages of development. Each developing follicle is comprised of 16 interconnected germline-derived cells (15 nurse cells and 1 oocyte) that are surrounded by a layer of somatically derived epithelial cells. During Stage 10B (S10B), the nurse cells form a cage-like network of parallel actin filament bundles that extend from the nurse cell membranes inward, toward the nurse cell nuclei. During Stage 11 (S11), the nurse cells rapidly transfer their cytoplasmic contents into the oocyte in an actomysoin-dependent contraction termed nurse cell dumping. Previous work uncovered that the Drosophila COX-like enzyme, Peroxinectin-like (Pxt), and thus PG signaling, is required to promote both actin filament bundle formation during S10B and subsequent nurse cell dumping. This finding established Drosophila oogenesis as a genetically tractable model in which to elucidate the conserved mechanisms underlying PG- dependent actin remodeling. The research presented in this dissertation focused on identifying actin-binding proteins that are regulated by PG signaling during Drosophila oogenesis. To identify these downstream effectors, we performed a dominant modifier screen to uncover factors that could suppress or enhance the ability of COX inhibitors to block nurse cell dumping in vitro. This screen revealed a number of actin-binding proteins that enhance the dumping defects caused by COX-inhibition, including the actin bundling protein, Fascin (Drosophila Singed, Sn); the actin filament elongation factor, Enabled (Ena); and the actin filament capper, Capping protein (Drosophila Capping protein alpha, Cpa, and beta, Cpb). Through a collaborative effort between Christopher Groen and myself, Fascin was shown to mediate PG-dependent cortical actin integrity and actin bundle formation during Drosophila ooogenesis. Ena and Capping protein regulate actin filament elongation through opposing actions: Ena promotes their elongation, while Capping protein binds to, or caps, the growing end of actin filaments to prevent their further elongation. However, genetic reduction of either Ena or Capping protein enhance the nurse cell dumping defects caused by COX inhibition. These findings suggest that Ena activity must be balanced to promote proper actin remodeling during S10B. Ena localization to the growing ends of actin filament bundles is reduced in pxt mutants during S10B, suggesting that PG signaling is required to promote Ena localization at this stage. Together, these data support a model in which PG signaling promotes actin remodeling during S10B, at least in part, by modulating Ena-dependent actin remodeling. While PG signaling promotes parallel actin filament bundle formation during S10B, PGs also restrict actin remodeling during Stage 9 (S9). Loss of Pxt results in early actin remodeling, including the formation of extensive actin filaments and actin aggregate structures within the posterior nurse cells of S9 follicles. Wild-type follicles exhibit similar structures at a low frequency. Ena preferentially localizes to the early actin structures observed in pxt mutants and reduced Ena levels strongly suppress early actin remodeling in pxt mutants. These data indicate that PG signaling temporally restricts actin remodeling during Drosophila oogenesis, at least in part, through negative regulation of Ena localization or activity during S9. The data presented here support a model in which PG signaling coordinates the concerted activity of a number of actin-binding proteins to regulate actin remodeling during Drosophila oogenesis. Specifically, PG signaling temporally restricts actin remodeling during S9 of Drosophila oogenesis, but promotes parallel actin filament bundle formation during S10B. PG signaling achieves this temporal regulation, at least in part, through differential regulation of Ena-dependent actin remodeling. Based on prior pharmacologic studies, we hypothesize that PGE2 is required to restrict Ena-dependent actin remodeling during S9, while PGF2Α; is required to promote Ena-dependent actin remodeling during S10B. Determining how these signaling cascades achieve differential regulation of Ena throughout Drosophila oogenesis is an important area for future investigation. As both the actin cytoskeletal machinery and PG signaling are conserved across species, the data presented here provide new and significant insights into the likely conserved mechanisms by which PG signaling regulates actin remodeling across species.