This accomplishment-based renewal of the Pacific Research Center for Marine Biomedicine (PRCMB) will be a collaboration between the University of Hawaii, Stanford, and Jackson State University. Research activities will be focused on two areas: (1) pathogens in tropical marine waters and (2) marine toxins and pharmaceutical lead discovery. This new research follows logically from accomplishments and discoveries made during the first phase of PRCMB funding.

The pathogen and pathogen indicator work will focus on two streams that discharge to the ocean, one tropical (Kaelepulu on the island of Oahu) and the other temperate (San Pedro Creek in California). Monthly sampling at the two streams will be used to characterize seasonal variability in pathogen indicators and pathogen (bacterial and viral) concentrations using both culture-dependent and culture-independent (PCR-based) methods. These studies will be complemented by high-frequency sampling (ten-minute and hourly) during storm and dry-weather conditions to provide insights concerning spatial and temporal variability. Health-risk models (Quantitative Microbial Risk Assessment) will be developed using pathogen and pathogen indicator concentrations as input.

The marine toxin work will concern ciguatoxin and beta-N-methylamino-L-alanine (BMAA), both of which were the focus of recent PRCMB studies. Taking advantage of (1) the refinement of an assay (n2a) that is now capable of detecting ciguatoxin in fish tissue at concentrations ten times below the threshold associated with ciguatera symptomology and (2) a collaboration with Hawaiian recreational fishermen, PRCMB scientists will extract and concentrate sufficient ciguatoxin from fish tissue to obtain a molecular structure and will then, in collaboration with scientists at the University of Washington, work toward the development of an antibody-based assay for ciguatoxin. Ciguatoxin research will also involve (1) exploratory refinement of the n2a assay based on generation of nitric oxide by the n2a cell line and (2) an investigation of environmental conditions that trigger ciguatoxin production by taxonomically defined strains of the dinoflagellate Gambierdiscus. The BMAA work will build on initial studies to determine the prevalence of BMAA in marine cyanobacteria and the transfer of this toxin to higher trophic levels. The pharmaceutical studies will take advantage of the more than 2,500 microbial isolates in the PRCMB culture collection that have not yet been screened for bioactivity. Extracts of these cultures will be used in cell-based and molecular assays to determine if they affect the growth of microbial pathogens (Candida albicans, Escherichia coli, Staphlyococcus aureus, multi-drug resistant S. aureus, and vancomycin resistant Enterococcus faecium) or if they exert effects on human adenocarcinma cells, on protein kinase C, or on mitogen-activated protein kinase. Identification of the compounds responsible for reproducible bioactivity in these extracts will be accomplished through bioassay-guided fractionation and spectroscopic analysis (e.g., mass spectrometry, high-field nuclear magnetic resonance spectrometry)

The broader impacts of the proposed work fall into several categories. One is certainly the public health endpoints: (i) credible methods that can be used to determine whether coastal waters are safe for recreational use, (ii) a simple, inexpensive assay for ciguatoxin in fish, (iii) a better appreciation of the threat to human health associated with the movement of BMAA through marine food chains, and (iv) the discovery of novel compounds with application to the treatment of human health problems. Second, this is a collaborative proposal with an historically black university (Jackson State University) and a female principal investigator from Stanford. Finally, the PRCMB will continue to train students and to collaborate with other institutions and Ocean and Human Health centers.

Project Report

(PRCMB) was a collaborative research effort involving the University of Hawaii at Manoa (PI: Edward Laws) and Stanford University (PI: Alexandria Boehm). PRCMB scientists at the University of Hawaii included faculty from the School of Ocean and Earth Science and Technology, John A. Burns School of Medicine, College of Natural Sciences, Water Resources Research Center, and the Hawaii Institute of Marine Biology. PRCMB research projects included studies of (1) pathogens in natural waters, (2) ciguatera fish poisoning, (3) the prevalence and concentration of the neurotoxin b-N-methylamino-L-alanine (BMAA) in marine organisms, and (4) the presence in marine organisms of natural products with potential use in the treatment of human disease. Important results of the research have included the following discoveries: Through collaborations with local spearfishermen and the PRCMB Fish4Science web site, PRCMB scientists were able to obtain more than 2500 specimens of the carnivorous peacock grouper (Cephalopholis argus). The fish were tested for the presence of ciguatoxin. No portion of any scoastline was free of ciguatoxic C. argus. For all islands, the frequency of positively ciguatoxic C. argus specimens from the leeward areas was approximately twice that from the windward areas (38% and 18%, respectively). Tissues from fish that tested positive for ciguatoxin were processed so as to isolate the toxin and determine its molecular structure. There was no correlation between the concentration of ciguatoxin (the neurotoxin responsible for ciguatera fish poisoning (CFP)) in the carnivorous peacock grouper (Cephalopholis argus), an invasive fish that is widely distributed on coral reefs in the Hawaiian Islands, and the body weight of the grouper. There was, however, a significant positive correlation between the frequency of occurrence of ciguatoxin in peacock groupers and the body weight of the fish. Larger fish were more likely to contain ciguatoxin than smaller fish. Roughly 40% of fish weighing more than 630 grams (n.b., log10(630) = 2.8) contained ciguatoxin, whereas only 15–20% of smaller peacock groupers contained ciguatoxin. beta-methylamino-L-alanine? (BMAA) is a potent neurotoxin, chronic exposure to which has been linked to neurogenerative illnesses such as Alzheimers Disease, amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease), and Parkinson's disease. BMAA is a molecule that resembles a number of other compounds with which it can be easily confused analytically, including in particular diaminopimelic acid, which is ubiquitous in cyanobacteria (blue-green algae). PRCMB scientists developed a BMAA-specific analytical protocol based on liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) that can quantify BMAA with a precision of 2.4% over roughly a 400-fold range of BMAA concentrations. PRCMB scientists used this newly developed analytical technique to measure BMAA concentrations in oysters and blue crabs from the Gulf of Mexico. PRCMB scientists completed the structure elucidation of a pair of highly brominated antibiotics from a marine bacterium. One compound, a benzofuro(2,3-b)pyrrole, is unprecedented, whereas another compound is related to known marine antibacterials, such as bromophene, and differs from these known compounds in terms of extent of bromination.Both compounds are effective against multi-drug resistant Staphylococus aureus at low micromolar concentrations. The new compound is likely biogenetically related to pentabromopseudeline through intramolecular ring closure between a phenolic oxygen and one bromine-bearing carbon atom of the tribromopyrrole moiety of pentabromopseudeline. This ring closure results in a loss of biological potency by an order of magnitude. PRCMB scientists demonstrated the possibility of using human enteric viruses as an alternative to traditional bacterial fecal indicator organism to monitor for human sewage contamination in recreational waters with a rapid nucleic acid extraction technique followed by detection of specific viruses by using highly sensitive polymerase chain reaction (PCR) protocols. PRCMB scientists used this newly established PCR array methodology to perform a surveillance of 20 different sites around O‘ahu for sewage contamination. With the exception of Bellows Air Field Beach Park and West Loch Community Shoreline Park, results indicated that water at all of the sites was contaminated with human enteric viruses. With appropriate modifications, the PCR array methodology will be applicable to many other tasks and scenarios including (1) testing river and stream waters and recreational beaches for the presence of enteric viruses in water, shellfish, and mollusks, (2) testing for arboviruses in birds and mosquitoes, and Leptospira in rodents, and (3) assessing the threat to humans and ecobiological system from related diseases. PRCMB research activities resulted in the training of four doctoral students, three M.S. students, fifteen undergraduate students, and four high school students. Sixty percent of the students were women, and 40% were minorities. Three of the undergraduate students were supported with funding from the NSF Research Experience for Undergraduates program. The activities of the undergraduate students included training in general laboratory safety protocols, documentation and archiving of samples, a variety of processing and chemical extraction procedures, and data compilation and reporting.

National Science Foundation (NSF)
Division of Ocean Sciences (OCE)
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Donald L. Rice
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University of Hawaii
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