The marine sponge Geodia barretti produces a range of secondary metabolites. Two of these compounds were isolated and elucidated guided by their ability to inhibit settlement of cypris larvae of the barnacle Balanus improvisus. The compounds barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]) as E/Z mixture and 8,9-dihydrobarettin (cyclo-[6-bromo-tryptophan)-arginine]) were determined by using mass spectrometry, nuclear magnetic resonance and quantitative amino acid analysis.The bioactivity of these brominated dipeptides is in the range of antifouling substances used today: EC50 values of 0.9 µM (barettin) and 7.9 µM (8,9-dihydrobarettin). The compounds were successfully synthesised and then tested in a field experiment to evaluate their antifouling properties. The compounds were incorporated in four different commerical, non-toxic marine coatings. The concentrations of the compounds were 0.1 and 0.01% (w/w) and coated panels were exposed to field conditions for eight weeks. The experiment evaluated the effect of barettin and 8,9-dihydrobarettin on recruitment of the barnacle B. improvisus and the blue mussel Mytilus edulis (major Swedish foulers). The most efficient paint was a SPC polymer, for which the reduction of recruitment of B. improvisus was 89% with barettin (0.1%) and 61% with 8,9-dihydrobarettin (0.1%). For M. edulis the reduction of recruitment was 81% with barettin (0.1%) and 72% with 8,9-dihydrobarettin (0.1%) with the same SPC paint. Furthermore, 14 analogs of barettin and dipodazine were synthesised and tested for their ability to inhibit larval settlement. Two of the analogs have a barettin scaffold and twelve have a dipodazine scaffold. Six of the analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine (EC50 value 0.034 µM). The effect of benzo[g]dipodazine was also shown to be reversible. Finally, an investigation of the mode of action was performed on 5-HT receptors. Barettin demonstrated a specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted only with 5-HT2C of the receptor subtypes tested (5-HT1-5-HT7).

This thesis is based on three inter-related studies: the first part deals with the microbial consortium, the identification of microbes and their volatiles, the second part deals with the study of bio-chemical control methods of two conifer pests; the pine weevil Hylobius abietis (L.) and the root rot fungi Heterobasidion spp., and the third part describes the production of styrene by a fungus using forest waste.The large pine weevil (Hylobius abietis L.) is an economically important pest insect of conifers in reforestation areas of Europe and Asia. The female weevils protect their eggs from feeding conspecifics by adding frass (mixture of weevil feces and chewed bark) along with the eggs. In order to understand the mechanism behind frass deposition at the egg laying site and to find repellents/antifeedants for pine weevils, microbes were isolated from the aseptically collected pine weevil frass. Microbial produced volatile organic compounds (VOCs) were collected by solid phase micro extraction and analyzed by GC-MS after cultivating them on weevil frass broth. The major VOCs were tested against pine weevils using a multi-choice olfactometer. Ewingella sp., Mucor racemosus, Penicillium solitum, P. expansum, Ophiostoma piceae, O. pluriannulatum, Debaryomyces hansenii and Candida sequanensis were identified as abundant microbes. Styrene, 6-protoilludene, 1-octene-3-ol, 3-methylanisole, methyl salicylate, 2-methoxyphenol and 2-methoxy-4-vinylphenol were the VOCs of persistently isolated microbes. In behavioral bioassay, methyl salicylate, 3-methylanisole and styrene significantly reduced the attraction of pine weevils to their host plant volatiles. Heterobasidion spp. are severe pathogenic fungi of conifers that cause root and butt rot in plants. Bacterial isolates were tested for the antagonistic activity against fungi on potato dextrose agar. Bacillus subtilis strains significantly inhibited the growth of H. annosum and H. parviporum. Styrene is an industrial chemical used for making polymeric products, currently produced from fossil fuel. A strain of Penicillium expansum isolated from pine weevil frass was investigated for the production of styrene using forest waste. Grated pine stem bark and mature oak bark supplemented with yeast extract produced greater amounts of styrene compared to potato dextrose broth. / <p>QC 20130507</p>

Nevirapine (NVP) can cause serious skin rashes and hepatotoxicity. It also causes an immune-mediated skin rash in rats but not hepatotoxicity. There is strong evidence that the rash is due to 12-hydroxynevirapine (12-OH-NVP), which is further metabolized to a reactive benzylic sulfate in the skin. This could both act as a hapten and induce a danger signal. In contrast, most of the covalent binding in the liver appears to involve oxidation of the methyl group leading to a reactive quinone methide. In this study we examined the effects of NVP and 12-OH-NVP on gene expression in the liver and skin. Both NVP and 12-OH-NVP induced changes in the liver, but the list of genes was different, presumably reflecting different bioactivation pathways. In contrast, many more genes were up-regulated in the skin by 12-OH-NVP than by NVP, which is consistent with the hypothesis that the 12-hydroxylation pathway is involved in causing the rash. Some genes up-regulated by 12-OH-NVP were Trim63, S100a7a, and IL22ra2, etc. Up-regulation of genes such as S100a7a, which is considered a danger signal, supports the danger hypothesis. Up-regulation of genes such as the ubiquitin ligase and Trim63 are consistent with protein-adduct formation. Up-regulation of IL-22ra2 gene suggests an immune response. These results provide important clues to how NVP causes induction of an immune response, in some cases leading to an idiosyncratic drug reaction.

Nevirapine (NVP) can cause serious skin rashes and hepatotoxicity. It also causes an immune-mediated skin rash in rats but not hepatotoxicity. There is strong evidence that the rash is due to 12-hydroxynevirapine (12-OH-NVP), which is further metabolized to a reactive benzylic sulfate in the skin. This could both act as a hapten and induce a danger signal. In contrast, most of the covalent binding in the liver appears to involve oxidation of the methyl group leading to a reactive quinone methide. In this study we examined the effects of NVP and 12-OH-NVP on gene expression in the liver and skin. Both NVP and 12-OH-NVP induced changes in the liver, but the list of genes was different, presumably reflecting different bioactivation pathways. In contrast, many more genes were up-regulated in the skin by 12-OH-NVP than by NVP, which is consistent with the hypothesis that the 12-hydroxylation pathway is involved in causing the rash. Some genes up-regulated by 12-OH-NVP were Trim63, S100a7a, and IL22ra2, etc. Up-regulation of genes such as S100a7a, which is considered a danger signal, supports the danger hypothesis. Up-regulation of genes such as the ubiquitin ligase and Trim63 are consistent with protein-adduct formation. Up-regulation of IL-22ra2 gene suggests an immune response. These results provide important clues to how NVP causes induction of an immune response, in some cases leading to an idiosyncratic drug reaction.

Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the meta­bolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification. The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small mole­cules purification. Specifically, this investigation aims to see if it is achievable to make a bio­tinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis. Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermi­dine, histamine and nicotinamide have been selected. The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control non­biotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.

<p>Over the past 200 years, the Earth's atmospheric carbon dioxide (CO₂) concentration has increased by more than 35%, and climate experts predict that CO₂ levels may double by the end of this century. Understanding the mechanisms of resource management in plants is fundamental for predicting how plants will respond to the increase in atmospheric CO₂. Plant productivity sustains life on Earth and is a principal component of the planet's system that regulates atmospheric CO₂ concentration. As such, one of the central goals of plant science is to understand the regulatory mechanisms of plant growth in a changing environment. Short-lived positron-emitting radiotracer techniques provide time-dependent data that are critical for developing models of metabolite transport and resource distribution in plants and their microenvironments. To better understand the effects of environmental changes on resource transport and allocation in plants, we have developed a system for real-time measurements of metabolite transport in plants using short-lived positron-emitting radiotracers. This thesis project includes the design, construction, and demonstration of the capabilities of this system for performing real-time measurements of metabolite transport in plants.</p><p>The short-lived radiotracer system described in this dissertation takes advantage of the combined capabilities and close proximity of two research facilities at Duke University: the Triangle Universities Nuclear Laboratory (TUNL) and the Duke University Phytotron, which are separated by approximately 100 meters. The short-lived positron-emitting radioisotopes are generated using the 10-MV tandem Van de Graaff accelerator located in the main TUNL building, which provides the capability of producing short-lived positron-emitting isotopes such as carbon-11 (¹¹C; 20 minute half-life), nitrogen-13 (¹³N; 10 minute half-life), fluorine-18 (¹⁸F; 110 minute half-life), and oxygen-15 (¹⁵O; 2 minute half-life). The radioisotopes may be introduced to plants as biologically active molecules such as ¹¹CO₂, ¹³NO₃^-, ¹⁸F^--[H₂O], and H₂<sup>15<\sup>O. Plants for these studies are grown in controlled-environment chambers at the Phytotron. The chambers offer an array of control for temperature, humidity, atmospheric CO₂ concentration, and light intensity. Additionally, the Phytotron houses one large reach-in growth chamber that is dedicated to this project for radioisotope labeling measurements.</p><p>There are several important properties of short-lived positron-emitting radiotracers that make them well suited for use in investigating metabolite transport in plants. First, because the molecular mass of a radioisotope-tagged compound is only minutely different from the corresponding stable compound, radiotracer substances should be metabolized and transported in plants the same as their non-radioactive counterparts. Second, because the relatively high energy gamma rays emitted from electron-positron annihilation are attenuated very little by plant tissue, the real-time distribution of a radiotracer can be measured <em>in vivo</em> in plants. Finally, the short radioactive half-lives of these isotopes allow for repeat measurements on the same plant in a short period of time. For example, in studies of short-term environmental changes on plant metabolite dynamics, a single plant can be labeled multiple times to measure its responses to different environmental conditions. Also, different short-lived radiotracers can be applied to the same plant over a short period of time to investigate the transport and allocation of various metabolites.</p><p>This newly developed system provides the capabilities for production of ¹¹CO₂ at TUNL, transfer of the ¹¹CO₂ gas from the target area at TUNL to a radiation-shielded cryogenic trap at the Phytotron, labeling of photoassimilates with ¹¹C, and <em>in vivo</em> gamma-ray detection for real-time measurements of the radiotracer distribution in small plants. The experimental techniques and instrumentation that enabled the quantitative biological studies reported in this thesis were developed through a series of experiments made at TUNL and the Phytotron. Collimated single detectors and coincidence counting techniques were used to monitor the radiotracer distribution on a coarse spatial scale. Additionally, a prototype Versatile Imager for Positron Emitting Radiotracers (VIPER) was built to provide the capability of measuring radiotracer distributions in plants with high spatial resolution (~2.5 mm). This device enables detailed quantification of real-time metabolite dynamics on fine spatial scales.</p><p>The full capabilities of this radiotracer system were utilized in an investigation of the effects of elevated atmospheric CO₂ concentration and root nutrient availability on the transport and allocation of recently fixed carbon, including that released from the roots via exudation or respiration, in two grass species. The ¹¹CO₂ gas was introduced to a leaf on the plants grown at either ambient or elevated atmospheric CO₂. Two sequential measurements were performed per day on each plant: a control nutrient solution labeling immediately followed by labeling with a 10-fold increase or decrease in nutrient concentration. The real-time distribution of ¹¹C-labeled photoassimilate was measured <em>in vivo</em> throughout the plant and root environment. This measurement resulted in the <em>first</em> observation of a rapid plant response to short-term changes in nutrient availability via correlated changes in the photoassimilate allocation to root exudates. Our data indicated that root exudation was consistently enhanced at lower nutrient concentrations. Also, we found that elevated atmospheric CO₂ increased the velocity of photoassimilate transport throughout the plant, enhanced root exudation in an annual crop grass, and reduced root exudation in a perennial native grass.</p> / Dissertation

Soft corals of the genus Briareum (Briareidae) have been well known as a rich source for marine natural products with novel structural features. Briarane-related natural products attracted the attentions of researchers because of the structural complexity and interesting biological activity associated with numerous compounds of this type. Previous studies on the secondary metabolites of wild-type and cultured Formosan octocoral Briareum excavatum were collected around the sea area of Kenting. In the thesis of our studies on secondary metabolites from marine organisms, the acetone-soluble of the Formosan octocoral B. excavatum collected at Orchid Island has led to the isolation of eleven briarane-type diterpenoids (1−11), compounds 3, 4, and 6−10 are new compounds. The structures of these compounds were determined on the basis of their spectroscopic analysis (1H NMR, 13C NMR, 1H−1H COSY, HSQC, HMBC, NOESY, IR and mass spectra) and physical data by comparison of the physical and spectral data with those of the related literatures. The antiviral activity against HCMV (human cytomegalovirus) cells of these secondary metabolites was evaluated. Metabolite 8 exhibited significant activity against HCMV cells and compound 11 showed anti-inflammatory activity.−

Lysophosphatidic acid (LPA) is the simplest of the glycerol lipids and regulates a number of cellular processes such as morphological changes, migration, proliferation, and inhibition of apoptosis. LPA exerts these effects through activation of the G-protein coupled receptors (GPCRs) LPA1-6 and the intracellular fatty acid receptor peroxisome proliferator-activated receptor-gamma (PPARγ). The overall goal of this thesis was to determine the mechanisms by which LPA enhances cell survival by inhibiting apoptosis. The project was divided into three studies: 1) to determine the mechanism of LPA-mediated inhibition of p53 in A549 lung carcinoma cells, 2) to investigate the regulation of growth plate chondrocytes by LPA, and 3) to determine the mechanisms of LPA-mediated effects in the growth plate. In the first study, evidence is provided that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells. The LPA effect depends upon increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. The result of LPA-mediated inhibition of p53 in A549 cells is enhanced resistance to chemotherapeutic-induced apoptosis. In the second study, the role of LPA in resting zone chondrocytes (RC cells) was investigated. RC cells are regulated by 24,25-dihydroxyvitamin D3 [24,25(OH)[subscript2]D [subscript 3]] via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)[subscript 2]D[subscript 3] stimulates rat costochondral RC cells to release LPA. Additionally, we demonstrated that RC cells respond to LPA with increased proliferation, maturation, and inhibition of apoptosis. In the final study, the mechanism of LPA and 24R,25(OH)[subscript 2]D[subscript 3]-mediated inhibition of chondrocyte apoptosis was further investigated. Our data show that 24R,25(OH)[subscript 2]D[subscript 3] inhibits apoptosis through Ca⁺⁺, PLD, and PLC signaling and through LPA/Gαi/PI[subscript 3]K/mdm2-mediated degradation of p53, resulting in decreased caspase-3 activity. Collectively, our data establish LPA, vitamin D, and p53 as an anti-apoptotic signaling axis.

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 134-151).

D. Tech. Chemistry. / Discusses the extent of mango cultivar susceptibility to gall fly infestation can be predicted by the composition of secondary metabolites produced by the tree.