STUDENT RESEARCH DAY 2005

MBES AWARD WINNERS

Karenia brevis is a dinoflagellate whose expressed genome is of significant interest because of its role in producing harmful algal blooms. The longevity of a K. brevis bloom is dependant on the cell's ability to adapt to the coastal environment. Transcription of general stress response genes is known to be a key regulatory step in the development of a broad stress resistance. To gain a better understanding of gene regulation in K. brevis, we developed a DNA microarray containing 10,265 unigenes. To identify if a general stress response program exists in K. brevis, the microarray was used to measure transcriptional changes in response to acute heat, peroxide, lead, paraquat, or sodium nitrite. Consistent with a general stress response that includes a transient shut-off of general mRNA transcription, genes involved in ATP driven processes were downregulated following each of the treatments. However, transcription of stereotypical heat shock proteins and other stress related genes, known to be induced at the protein level in K. brevis, were not seen, implicating post-transcriptional regulation of these mRNAs. The lack of transcriptional regulation found following acute environmental stress along with the distinctive nuclear organization in dinoflagellates suggests that K. brevis may have evolved alternative regulatory mechanisms for regulating gene expression. There are several similarities between the gene organization of dinoflagellates and their sister group, the trypanosomes, including polycistronic transcription and the lack of identifiable transcriptional regulators suggesting that dinoflagellate RNA transcription may be similar to trypanosomes. In trypanosomes, long stretches of chromosome are constitutively transcribed into polycistronic units with concurrent splice leader trans-splicing and polyadenylation of the nascent chains. The presence of this regulatory mechanism in a closely related genus prompted us to investigate trypanosome-like trans-splicing in K. brevis. Following sequence analysis of our K. brevis unigene set, we identified several mRNA sequences that contain a consensus 22-bp leader sequence. Furthermore, we isolated a gene from genomic DNA encoding a potential SL RNA. This gene shares several key features to the previously characterized SL RNAs. This is the first evidence for the occurrence of SL trans-splicing in a dinoflagellate and lends tremendous insight into regulation of gene expression in these early branching eukaryotes.

Acropora spp., once dominant corals on Caribbean reefs, have suffered region-wide declines over the past two decades.  A. palmata and A. cervicornis are the first corals named as threatened species under the U.S. Endangered Species Act.  These corals harbor diverse microbiota suspected of participating in both health and disease processes.  Several investigators have previously attempted to ascertain the structure of microbial communities associated with these corals by varying methods, often with conflicting results.  This study used a larger-scale genomic approach to analyze the microbiota.  Replicate biopsies from apparently healthy and diseased acroporid colonies were collected at six sites in the Florida Keys and Dry Tortugas.  Three different processing methods were used in parallel during collection of these samples.  Individual 16S rRNA gene libraries were generated from each sample with over 10,000 clones sequenced to date.  Results indicate that the methodology employed to process a given sample introduces significant variability into the microbial community composition detected.  Analysis of coral mucus samples reveal consistent microbial composition between similar samples, with increased levels of variation introduced by differing health status, geographic location, and species.  Several groups of bacteria differentially detected in healthy versus diseased samples are currently under investigation.

Presentations

A new Lophotoxin derivative has been isolated from the deep water octacoral Plumarella pourtalesii. The structure of this new derivative has been solved through the analysis of 2-D NMR experiments at 600MHz and through spectral comparison with known Lophotoxin. Cytotoxicity studies have revealed that this compound is biologically active against the pancreatic cancer cell line Panc-1, the colon cancer cell line DLD-1, and the NCI drug resistant cancer cell line NCI/ADR. Presented here is the biological activity, purification and structure elucidation of this new derivative.

The red tide dinoflagellate, Karenia brevis, is responsible for harmful algal blooms (HABs) that occur annually in the Gulf of Mexico. The mechanisms controlling cell proliferation in this dinoflagellate are of interest due to their central role in bloom development. In dinoflagellates the cell division process is characterized by a number of novel adaptations including the presence of permanently condensed chromatin throughout the cell cycle, a lack of nucleosomes to aid in chromatin packaging, a permanently intact nuclear envelope, and an extranuclear mitotic spindle that interacts with chromosomes via cytoplasmic channels. Our laboratory has identified a number of conserved cell cycle genes in K. brevis by high throughput screening of a cDNA library. Unlike typical eukaryotes, microarray and Real-Time PCR studies in our lab suggest that cell cycle genes are not regulated at the transcriptional level in K. brevis. Therefore, it is probable that these cell cycle gene products are regulated at the translational or post-translational level. In order to further study the regulation of cell cycle genes in K. brevis, we are using the proteomic approach of two dimensional gel electrophoresis (2DE). We have optimized extraction procedures for 2DE of dinoflagellate proteins and assessed inter-gel variability. We found that extraction of proteins using phenol/chloroform reagents resulted in gels containing a greater number of protein spots compared to gels run with traditional urea/detergent based homogenization buffers. This method is compatible with newer quantitative methods of 2DE protein quantification such as difference gel electrophoresis (DIGE). In the current study, we are using DIGE to study protein abundances over the diel cycle. In this way, we will be able to compare diel changes in the global transcript profile with global changes in protein abundances. This study will enable a more thorough understanding of the regulation of key proteins involved in proliferation of HABs in the Gulf Coast region.

Blake C. Ellis

The Atlantic bottlenose dolphin (BND) has recently attracted attention as a potential sentinel for human health. Greater knowledge of how BND responds to environmental stress and how that response compares to humans is needed, but such studies are limited by its status as a protected species. We previously established cell cultures and cell lines from the skin of BND as an in vitro tool for measuring the impact of the environment at the molecular level on this protected marine mammal. These cell models are being used to investigate the role of the vitamin D3 pathway in the dolphin's response to ultraviolet (UV) radiation. Vitamin D is of importance, not only because of its cutaneous, UV-induced synthesis, but for its various immunomodulatory, antimicrobial, and chemo-preventive effects. In the skin of humans and other terrestrial mammals, UVB radiation stimulates the conversion of 7-dehydrocholesterol into the active, hormonal form of vitamin D: 1,25-dihydroxy-D3 (1,25[OH]2D3). This hormone can interact with the nuclear vitamin D receptor (VDR), a potent ligand-activated regulator of gene transcription that is expressed in skin along with many other organs. Whether aquatic mammals also possess this pathway and gain the same immune and cancer-fighting benefits from vitamin D3 as terrestrial mammals is unknown. Consequently, we are interested in measuring the VDR-mediated transcriptomic changes observed when dolphin skin is exposed to exogenous 1,25(OH)2D3 and to UV light. We have detected in the BND cell cultures expression of VDR and the 1alpha-hydroxylase (CYP27B1) enzyme responsible for 1,25(OH)2D3 generation, suggesting that dolphins also possess this pathway in their skin. Furthermore, we found VDR levels to be up-regulated with exogenous 1,25(OH)2D3 administration in a time- and dosage-dependent manner. This project takes advantage of a novel dolphin cDNA microarray, biased towards immune- and stress-related genes, as a tool for evaluating global transcriptomic changes induced by this up-regulation of VDR. Such in vitro methods provide a means for studying vitamin D pathway components and vitamin D-mediated gene regulation induced by UV exposure, specifically for the dolphin. Differences identified between BND and humans will help us better understand the role of vitamin D on innate immunity in skin, in general, as well as the function of the dolphin's skin as a crucial interface between the organism and its ocean environment.

Michelle H. Hsia

The chain-forming dinoflagellate Alexandrium monilatum, formerly known as Gonyaulaux, was first observed in 1936 on the Texas coast in Offats Bayou where it caused annual formation of red colored water, as well as severe fish mortality. According to Connell and Cross, local residents of that area observed red water and dead fish virtually every year 15-20 years prior to their 1949 report. It has also been reported to be associated with widespread discolored water and increased fish mortality in the Mississippi Sound, off the eastern and western coasts of Florida, and in the Gulf of Nicoya, Costa Rica. Early studies have found that A. monilatum produces a harmful substance(s) that is predominantly contained in the cell mass and increases toxicity when the organism cytolyses. Our studies corroborate with other research demonstrating that the toxin has low water solubility, casting doubt on the presence of saxitoxin-like toxins that are water soluble. Using sophisticated chemical, chromatographic, and analytical techniques, we have successfully purified and identified the molecular structure of the toxin produced by A. monilatum. To solve the molecular structure of the toxin, we utilized a 500MHz NMR for the following experiments: 1H, 13C, COSY, HSQC, HMBC. In addition, mass analysis utilized ESI-MS, MALDI-TOF MS, and Q-TOF MS. The toxin represents a polyether macrolide with an empirical formula of C43H60O12. This toxic compound is identified as Goniodomin A, identical to the one produced by the rock pool-blooming dinoflagellate, Alexandrium pseudogoniaulax. This compound had not been previously determined for A. monilatum. We have successfully solved the structure of a toxin that has caused fish mortalities in the Gulf of Mexico for over 60 years.

Wesley Jackson

Rhodopsin was the first described G-coupled receptor protein (GCRP) and later additional classes of this protein have been found to be essential sensory proteins in bacteria, plant and animal systems for various different sensory stimuli. These GCRP's are able to detect changes in pH, smell, taste, light in a non-visual sense. Rhodopsin (found in rod cells) and cone opsins (found in cone cells) are the main light sensing proteins of the retina and work together with the nervous system to create vision. The intracellular C-terminal part of rhodopsin is similar in all known vertebrate visual systems and known to have several modification sites. These modifications, phosphorylation and acylation, are thought to be essential for G-coupled protein interaction and activation with all GCRPs. Bovine rhodopsin contains 348 amino acids, ten of which are cysteines. Two of these cysteines are thought to be constitutively acylated residues at position 322 and 323 close to the c-terminus. Acylation modifications are common features amongst G-coupled receptor proteins. In general, acylation modifications consist of sixteen-carbon palmitate groups bound to cysteine through thioester bonds. Reports from other laboratories have alluded to the liability of the thioester bond linkage on rhodopsin but it has never been quantified. We report here that roughly 15-25% of the native bovine rhodopsin proteins are singly palmitylated with less than 2% non-palmitylated. These results were obtained through ESI/MS analysis of CNBr digested rod outer segments. The singly- and non-palmitoylated native state of rhodopsin also occurs in other species including mouse, rat and pig. Through the analysis of the singly palmitylated species, we found that the primary site of this palmitate loss is at the second cysteine 323, as palmitylation on 322 was two times greater than on 323. Primary site data were collected using ESI/MS/MS after CNBr and AspN cleavage. Additional findings in this presentation document palmitate loss by the use of hydroxylamine. Hydroxylamine is a commonly used agent in regeneration experiments measuring the active rhodopsin proteins from retina outer segment preparations. Other laboratories have shown that palmitate loss occurs with use of hydroxylamine. Using mass spectrometry techniques we have been able to measure palmitate loss with increasing concentrations of hydroxylamine. Native bovine rhodopsin has a molar ratio of 1.77 moles of palmitate per rhodopsin molecule. All palmitate groups are removed when exposed to approximately one molar hydroxylamine for thirty minutes. This knowledge is important for eventually comparing rhodopsins to cone opsins that normally have a molar ratio of less than one palmitate per cone opsin. The clinical relevance to these studies focuses on poorly understood genetic disorders of the retina such as retina degeneration, this disease begins with rod photoreceptor loss causing night blindness that eventually moves to the cones causing complete loss of vision. Supported by NIH grants EY04939, EY08235, EY 14793, a NIH minority supplement training grant to EY04939, and an unrestricted grant from RPB.

Mara L. Lennard

The enhancer (E“3') of the immunoglobulin heavy chain locus (IgH) of the channel catfish (Ictalurus punctatus) has been well characterized. The functional core region consists of two variant Oct transcription factor binding octamer motifs and one E-protein binding “E5 site. The core region of the mammalian IgH enhancer E“ contains two ETS factor binding sites and a “E3 site. This indicates an evolutionary shift in the transcription factors driving transcription of the vertebrate IgH locus. Homologues to two important Oct transcription factors (Oct1, Oct2) and one co-activator (BOB.1) have been cloned from the catfish. The overall structure and expression of these transcription factors have been evolutionarily conserved. Catfish Oct2 has previously been shown to be active in driving transcription but both catfish Oct1 and BOB.1 appear to be inactive, as measured using reporter constructs containing a variety of octamer-dependent Pol II promoters. Electrophoretic mobility shift assays (EMSA) demonstrate that catfish Oct1 is capable of binding to both consensus and variant octamer motifs. Gal4 fusion experiments indicate that catfish Oct1, unlike catfish Oct2, lacks functional activation domains. Catfish Oct1 is dominant negative over Oct2 and appears to be acting as a negative regulator of transcription. Additional studies to investigate the lack of activity of the co-activator BOB.1 have been conducted. EMSAs have shown that both catfish Oct2 and catfish Oct1 bind to both the human BOB.1 and the catfish BOB.1. Chimeric BOB.1 proteins were then constructed to test the theory that catfish BOB.1 lacks activation domains. These results suggest that the reason catfish BOB.1 does not enhance transcriptional activation is also due to a lack of functional activationThe enhancer (E“3') of the immunoglobulin heavy chain locus (IgH) of the channel catfish (Ictalurus punctatus) has been well characterized. The functional core region consists of two variant Oct transcription factor binding octamer motifs and one E-protein binding “E5 site. The core region of the mammalian IgH enhancer E“ contains two ETS factor binding sites and a “E3 site. This indicates an evolutionary shift in the transcription factors driving transcription of the vertebrate IgH locus. Homologues to two important Oct transcription factors (Oct1, Oct2) and one co-activator (BOB.1) have been cloned from the catfish. The overall structure and expression of these transcription factors have been evolutionarily conserved. Catfish Oct2 has previously been shown to be active in driving transcription but both catfish Oct1 and BOB.1 appear to be inactive, as measured using reporter constructs containing a variety of octamer-dependent Pol II promoters. Electrophoretic mobility shift assays (EMSA) demonstrate that catfish Oct1 is capable of binding to both consensus and variant octamer motifs. Gal4 fusion experiments indicate that catfish Oct1, unlike catfish Oct2, lacks functional activation domains. Catfish Oct1 is dominant negative over Oct2 and appears to be acting as a negative regulator of transcription. Additional studies to investigate the lack of activity of the co-activator BOB.1 have been conducted. EMSAs have shown that both catfish Oct2 and catfish Oct1 bind to both the human BOB.1 and the catfish BOB.1. Chimeric BOB.1 proteins were then constructed to test the theory that catfish BOB.1 lacks activation domains. These results suggest that the reason catfish BOB.1 does not enhance transcriptional activation is also due to a lack of functional activation domains. These results suggest that the redundancy in transcriptional regulation of the IgH locus involving Oct1, Oct2 and BOB.1 in mammals is not conserved at the phylogenetic level of the teleost fish. domains. These results suggest that the redundancy in transcriptional regulation of the IgH locus involving Oct1, Oct2 and BOB.1 in mammals is not conserved at the phylogenetic level of the teleost fish.

Kelli L. Margot

Blooms of the red tide causing organism Karenia brevis occur annually along the Gulf coast of Florida. K. brevis produces brevetoxins, which are lipid soluble biotoxins responsible for adverse health and economic effects. Brevetoxins are known to act as both neurotoxins and respiratory toxins, resulting in neurotoxic shellfish poisoning and profound altered lung function. Brevetoxins have proven harmful to marine mammals, seabirds, and humans following inhalation, and multiple marine mammal mortality events have been attributed to brevetoxin exposure. Analysis of manatee lungs following one mortality event identified brevetoxin accumulation in lung macrophages and lymphocytes. Therefore, the goal of this report is to identify the effects of brevetoxins on alveolar macrophages. Two alveolar macrophage cell lines, NR8383 and MH-S, were treated with 1-2 “g/ml brevetoxin-2, brevetoxin-3, or brevetoxin-2 + brevetoxin-3. Cell viability decreased following exposure to brevetoxins but varied depending upon brevetoxin congener, with brevetoxin-2 resulting in the greatest negative effect. Macrophage phagocytosis of latex beads appeared to increase, along with labeling of the lipid raft marker GM1. However, analysis of cytokine levels with a cytokine antibody array indicated few changes following brevetoxin exposure. The results indicate that brevetoxins are exhibiting effects on alveolar macrophages, and that these effects appear to be on cytotoxic and/or functional (phagocytic) levels rather than that of an acute inflammatory response.

Emily Monroe

Karenia brevis is a toxic dinoflagellate endemic to the Gulf of Mexico where it forms near annual blooms and causes harmful environmental and human health effects through the production of a suite of neurotoxins, the brevetoxins. Brevetoxins are synthesized in a similar manner to other polyektide compounds through polyketide synthase enzymes (PKS). PKS genes have been identified in fungi, bacteria, and several species of dinoflagellates. We have identified 10 PKS genes in a K. brevis EST database; however, whether these genes are expressed by K. brevis or co-occurring bacteria remains controversial. The current study analyzes the origin of the PKS genes identified in our EST database through sequence analysis and PKS gene expression in the absence of the normal bacterial flora. To obtain full sequences of the PKS genes, rapid amplification of cDNA ends (RACE) was performed and sequences were analyzed for the presence of poly(A)+ tails and 3' untranslated regions (UTRs), characteristics typically seen in eukaryotic genes. All of the PKS genes are polyadenylated with 3'- UTRs suggesting these genes are encoded by K. brevis. To further investigate the origin of these genes, cultures of K. brevis were treated with antibiotics to eliminate associated bacteria, and PKS gene expression was analyzed using microarrays. All of the PKS genes are present in the bacteria-free cultures at expression levels similar to control cultures. These analyses suggest that the PKS genes we have identified in K. brevis are encoded by K. brevis and not associated bacteria. Further characterization of these genes and their expression will provide insight into molecular mechanisms involved in brevetoxin biosynthesis.

Benjamin A. Neely

Tracey B. Schock

Trichodesmium thiebautii is a toxin producing non-heterocystous cyanobacterium ubiquitous in tropical, subtropical, and temperate seas. It is known for its ability to fix nitrogen and for its massive blooms, making it the major component of oceanic primary production and nitrogen cycling.  The toxin(s) produced by this cyanophyte has been observed as a potential cause of death of fish, crabs and bivalves.  It has demonstrated neurotoxic effects in laboratory studies, as well as caused respiratory distress and contact dermatitis of humans at collection sites.  To date, a T. thiebautii toxin has not been isolated or structurally characterized.  Here, we report the extraction of a toxin(s) from T. thiebautii cell mass. We have established a purification method with chromatographic methods, demonstrated cytotoxic activity of T. thiebautii extracts using GH4C1 rat pituitary cells and N2A mouse neuroblastoma cells, and we are in the process of completing a chemical structure using nuclear magnetic resonance (NMR), mass spectroscopy (MS) and fourier transformed-infrared spectroscopy (FT-IR). With a structure in hand, we will conduct studies as to the biological activity it may possess, be it, antibacterial, anticancer or another function that the structure may define. 

 Jessica E. Shockey

As invertebrates, penaeid shrimp rely solely upon an innate, non-adaptive immune system to survive in marine environments under a variety of stresses. These animals are subject to bacterial, fungal, and viral pathogens, both in aquaculture and their natural habitats. Little is known regarding shrimp immunity, especially at the molecular level. However, it is known that the invertebrate immune response includes both cellular responses, such as phagocytosis, and humoral responses, such as the secretion of antimicrobial peptides. Antimicrobial peptides are important in defense against pathogens in most organisms. Preliminary work on a putative antimicrobial, the crustins, indicates activity against Gram-positive bacteria. Sequence homology also indicates a possible C-terminal protease inhibitor domain that could be acting in conjunction with or independent from its bactericidal activity. In order to study the activity of crustins in vivo, a series of RNA knockdown experiments were completed. First, the efficiency of knockdown was determined by injecting long, dsRNAs homologous to a portion of the crustin sequence into the animals; successful knockdown was observed via reverse transcription (RT)-PCR and western blotting at the RNA and protein levels, respectively. After determining that crustin message and protein could be knocked down in the shrimp, experiments began to test the activity of crustin by challenging knockdown animals with different immune effectors, to mimic microbial and fungal infections.

Petra A Tsuji

The use of dietary flavonoids as potential chemopreventive agents is a concept of increasing interest. Recent findings indicate that methylated flavones have the advantage of increased metabolic stability. One such compound, the naturally-occurring 5,7-dimethoxyflavone (5,7-DMF), has been shown to be a potential chemopreventive agent in human cancer originating from the liver, mouth, esophagus and lung. As bioavailability is a key issue for potential in vivo effects, the tissue accumulation and biliary elimination of 5,7-DMF and its non-methylated analog chrysin were examined in a small fish model (Fundulus heteroclitus). The fish were exposed to 5,7-DMF, chrysin or vehicle control (DMSO < 0.01%) in seawater for 8 h. Toxicity was not observed at the 5 microM exposure level. Tissues and bile were harvested and analyzed by HPLC and LC/MS for quantitation and identification of parent compound and metabolites. 5,7-DMF accumulated 20- to 100-fold in all tissues examined, with the highest accumulation in liver and brain, whereas chrysin was barely detectable in any tissues except the liver. The bile of chrysin-exposed fish contained very low concentrations of unchanged chrysin but high concentrations of two glucuronic acid conjugates. In the bile of 5,7-DMF-exposed fish, the parent compound was detectable in significant amounts along with glucuronic acid conjugates of O-demethylated 5,7-DMF. In conclusion, our study demonstrated high tissue accumulation and limited metabolism of 5,7-DMF compared to chrysin in vivo, making this flavone a promising chemopreventive molecule

CORAL-ASSOCIATED MICROBIAL NATURAL PRODUCTS AND THEIR INFLUENCE ON PSEUDOPTEROGORGIA AMERICANA CORAL HEALTH, Maria Vizcaino, Jennifer Delaney, Katherine Williams, Peter D.R. Moeller, Pamela J. Morris, Marine Biomedicine and Environmental Sciences Center, MUSC, Hollings Marine Laboratory, Ft Johnson, Charleston, SC, Grice Marine Laboratory, College of Charleston, Ft. Johnson, Charleston, SC, Toxin Chemistry, NOAA National Ocean Service, Ft. Johnson, Charleston, SC, Department of Cell Biology and Anatomy, MUSC, Center for Coastal Environmental Health and Biomolecular Research, NOAA National Ocean Service, Ft. Johnson, Charleston, SC.

Brominated flame retardants, such as polybrominated diphenyl ethers (PBDEs), are chemical additives to consumer products which reduce their flammability and the risk of fire. Piscivorous marine mammals, including bottlenose dolphins, feed at top trophic levels within aquatic ecosystems and are therefore vulnerable to accumulating heavy burdens of many different organic contaminants, including PBDEs. Assessing the potential health effects of contaminant exposure in wild marine mammals has proven difficult, partly as a result of their chronic exposure to extremely complex contaminant mixtures. To assess the toxicity of these biologically relevant contaminant mixtures using lab based tests, a measure of exposure in wild marine mammal populations is needed and the variability of contaminant mixtures between individuals of a wild population must be understood to determine whether all individuals are exposed to comparable mixtures or whether subsets of the population are exposed differentially. To assess exposure to brominated flame retardant mixtures in a wild marine mammal population, 14 PBDE congeners were measured in 106 blubber biopsies, 40 plasma samples and 18 milk samples collected from free-ranging bottlenose dolphins during capture and release health assessments in Sarasota Bay, FL between 2000 and 2005. Total PBDE levels ranged from 20 to 1508 ng/g wet mass in blubber. Concentrations were detectable, but lower in plasma and milk. No relationship between age and total PBDE blubber concentrations was evident for male bottlenose dolphins. However, upon reaching sexual maturity at 10 years of age, PBDE levels in females appear to drop and remain low for life, suggesting substantial offloading of PBDEs during parturition and lactation. PBDE congener mixtures also appeared to vary with age and sex. Shifts in PBDE mixtures in male bottlenose dolphins may be a result of age related metabolism or ontogenetic changes in prey preference or location. In females, shifts in PBDE mixtures may result from a differential offloading of PBDE congeners during lactation as a comparison of PBDE profiles in milk and reproductive female blubber suggest that larger, higher brominated PBDEs are retained in blubber while smaller, less lipophillic PBDE congeners are offloaded through the milk.