Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a debilitating disease characterized by an increase in cerebrospinal fluid (CSF) in the brain, leading to increases in pressure that can ultimately result in death. Current treatments for hydrocephalus include only invasive brain surgery. Therefore, the need for a pharmaceutical therapy is great. In order to develop a suitable treatment, we first must be able to study the disease and the mechanisms by which it develops. By characterizing appropriate in vivo and in vitro models, we are better able to study this disease. In this thesis, the Wpk rat model and the PCP-R cell line are described as such appropriate models. In addition to suitable models, we also require a target for drug treatment. Transient Receptor Potential Vanilloid 4 (TRPV4) is a non-selective cation ion channel present in the main CSF-producing organ in the brain, the choroid plexus (CP). Preliminary data suggest this channel plays a role in the development of hydrocephalus. In the following work, some of the mechanisms by which TRPV4 functions in the brain are also described, including through calcium-sensitive potassium channels and inflammation. From this research, we are able to achieve a better understanding of the function of TRPV4 and how it can affect the development and progression of hydrocephalus.

Although synthetic analogs of alkylglycerol (AG), such as dodecylglycerol, possess potent biological activities, their mechanism of action has not been determined. We recently detected substantial amounts of AG in unstimulated MDCK cells (Warne, T.R. and Robinson, M. (1991) Anal. Biochem. 198, 302-307) raising the possibility mediator. In this study, we examined the effects of synthetic AG on the release of arachidonic acid and arachidonate metabolites (AA) from Madin Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to characterize its effects on this signalling pathway. Treatment of MDCK with AG potently inhibited the release of AA during subsequent stimulation with TPA. Dodecylglycerol, the most effective of a series of alkylgycerols tested, was active at concentrations as low as 3 μM. The sn-1 and sn-3 forms of AG were found to be equally potent inhibitors. The effects of AG on AA release were not the result of arachidonic acid redistribution among cellular lipids and were independent of the phospholipid source of the released AA. AG did not inhibit the release of AA from MDCK cells when bradykinin was used as a stimulus, indicating selectivity for the effects produced by phorbol esters. These results show that AG can function as a potent and specific inhibitor of TPA-mediated AA release. The ability of AG to regulate this signalling pathway in intact MDCK cells, together with its natural occurrence, suggests a potential bioregulatory role for the endogenous compound as an inhibitor of protein kinase C.

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The main objective of this thesis was to gain new insights in three of Florida’s high value food and stock enhancement finfish nutrition (Common snook, Florida pompano and red drum) to improve larviculture protocols. The main bottleneck in snook production is the extremely low larval survival rate, which hinders subsequent research. This work first focused on the source of the larvae by looking at potential nutritional deficiencies in captive broodstock. The lipid composition of wild and captive common snook broodstock were compared to identify disparities and gain the information necessary for the formulation of a suitable diet for captive stocks. Results showed that captive snook lipid content was significantly higher than that of wild fish. However, cholesterol and arachidonic acid (ARA) levels were significantly lower compared to wild broodstock, with potential impact on steroid and prostaglandin production, reproductive behavior and gametogenesis. Eggs from captive broodstock incorporated high docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) levels and low ARA levels. Consequently, ARA/EPA ratio in captive eggs was more than half of that in wild eggs (2.3 ± 0.6 and 0.9 ± 0.1 respectively), with a probable perturbation in eicosanoid production and adverse effects on embryo and larval development. The large differences observed between wild and captive broodstock most likely contributed to the reproductive dysfunctions observed in captive snook broodstock (e.g. incomplete oocyte maturation, low milt production and poor egg quality). In addition, the presence of hydrocarbons was detected in the liver of most of the wild snook sampled. This requires further investigation to identify the source of the contamination, monitor a potential impact on reproductive performances and protect the species habitat. Another major bottleneck in marine fish rearing occurs during the transition from endogenous feeding to exogenous feeding, with mass mortality events linked to inadequate first feeding diets. To gain insight on the early fatty acid requirements and mobilization of pompano and snook larvae, the pattern of conservation and loss of fatty acids from the yolk sac during the endogenous feeding period and subsequent starvation was studied. In both species, fatty acids were utilized as an energy source after hatching. Mono-unsaturated fatty acids were catabolized, while saturated and poly-unsaturated fatty acids were conserved. High levels of arachidonic acid (ARA) in pompano and snook eggs (respectively 2.44 ± 0.1 and 5.43 ± 0.3 % of total fatty acids), as well as selective retention in the unfed larvae, suggested a high dietary requirement for this fatty acid during the early stages of larval development. The effect of an ARA supplementation was therefore investigated in snook larvae at the rotifer feeding stage. Larvae receiving the supplementation did incorporate higher levels of ARA, and DHA/EPA and ARA/EPA ratios were successfully modified to match those observed in wild eggs. No significant improvements in growth or survival were observed, however the success in fatty acid profile modification suggest a possible impact of the supplementation on a longer period of time and a possible effect on stress resistance. Probiotics have been shown to enhance larval performances of several species and this strategy was therefore investigated to evaluate a potential impact on Florida pompano, red drum and common snook larvae. The effect of a commercial mix of Bacillus sp. was studied on larval survival, growth and digestive enzyme activities. Larvae were fed either live feed enriched with Algamac 3050 (Control), Algamac 3050 and probiotics (PB), or the previous diet combined with a daily addition of probiotics to the tank water (PB+). Microbiological analyses were performed at the end of the pompano trial. Numbers of presumptive Vibrio sp. were low and not statistically different between treatments, therefore no additional microbiological analyses were performed on the system. At the end of the pompano and snook trial, standard lengths of larvae from the PB and PB+ treatments were significantly greater than for the control larvae. For both pompano and snook, trypsin specific activity was higher in PB and PB+ larvae compared to the control larvae. Similarly, alkaline phosphatase activity was higher for the pompano larvae fed the PB and PB+ treatments and for the snook larvae fed the PB+ treatment compared to the control larvae. No enhancement of growth or digestive enzymes activities was observed in red drum larvae. Yet, no negative effects were noticed and a longer trial period and the study of additional parameters could reveal different effects. In all three species, survival was not affected by the supplementation; however, stress exposure should be further investigated as the supplementation may strengthen the larvae, especially pompano and snook larvae where the Bacillus sp. supplementation appears to promote growth through an early maturation of the digestive system. Another key challenge in marine fish larval rearing resides in weaning the larvae onto dry micro-diets. This step is commonly concurrent with larvae metamorphosis into juveniles, with extensive morphological and physiological changes that are likely to influence nutritional requirements. In the present project, three microdiets were tested on weaning of Florida pompano larvae: Otohime, Gemma and a reference diet LR803. The experimental system was stocked with 11-day-old larvae, which were co-fed micro-diets and live food from 11 dph to 17 dph then micro-diets only until 28 dph. Survival from 11 dph to 28 dph was similar for all treatments, with an average of 33 %. At the end of the trial, the Gemma larvae were significantly longer and heavier than larvae fed the other diets. Fatty acid composition of the diets and larvae varied significantly between treatments. The Gemma larvae incorporated the lowest amount of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA). However, they presented the highest DHA/EPA and ARA/EPA ratios, supporting the concept that the proportions of polyunsaturated fatty acids are of greater importance than their absolute amount. Results from the enzyme analysis showed that fishmeal is a suitable main source of protein for Florida pompano larvae and demonstrated the full functionality of the pancreas at 16 days post hatch. These results provide the basis of a suitable weaning diet for pompano larvae and indicate the possibility of a weaning time prior to 16 days post hatch, which is of high interest in commercial production. Overall, this research provides new data on common snook, pompano and red drum nutritional requirements with results that can be directly applied to help overcome major bottlenecks in the hatchery phase and improve rearing protocols.

Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl– system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils.

Ceramide-1-phosphate (C1P) is a bioactive lipid that has been implicated in many biological processes. Our laboratory has conclusively demonstrated its role in inflammation via activation of cPLA2α. The only known enzyme to date responsible for direct synthesis of C1P is ceramide kinase. Very little was known about this enzyme in terms of its enzyme kinetics and substrate specificity. As CERK is an enzyme that acts on membrane lipids, its kinetics cannot be studied using standard bulk dilutions methods. Thus we developed a surface dilution approach using Triton X 100 mixed micelles for studying the kinetics of CERK. We discovered that ceramide kinase has an affinity for naturally occurring long chain ceramides while ceramides containing shorter than 8 carbons are very poor substrates for the enzyme. Also of note is the discovery that there is no discrimination between the naturally occurring long chain ceramides leading to the conclusion that the preponderance of D-e-C16 C1P in cells are due to an availability effect. We also investigated the chain length specificity of interaction between C1P and cPLA2α. Our data indicate that cPLA2α is activated by C1P’s containing acyl chains longer than two carbons. The study showed C2 C1P as being unable to activate cPLA2α thus establishing a tool for the investigation of cPLA2α dependent and independent effects of C1P. In the course of the study we investigated the ethanol/dodecane delivery system as a means of safely delivering lipids to cells. Our data conclusively demonstrate that this delivery system successfully delivers lipids to the internal membranes where their biological action takes place and that at low lipid concentration (<1µM), is non toxic to cells. A significant technical hurdle in the study of C1P was the lack of accurate and reproducible method of quantitatively and qualitatively analyzing the lipid. Using a mass spectrometric approach we developed an accurate technique that now allows us to quantify the lipids in cells. Using this and radiolabeling studies we discovered evidence for production of C1P from S1P via an acyl transferase pathway. Further studies are currently being carried out to identify the enzyme/s responsible for this pathway.

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