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This paper provides an outline of a collaborative research project between researchers at the University of California, San Diego, University of Rhode Island, and the Bristol-Myers Squibb Pharmaceutical Research Institute, with participating members from the Developmental Therapeutics Branch of the National Cancer Institute. The program, formally funded by the National Cancer Institute under the National Cooperative Drug Discovery Groups (NCDDG) program, seeks to discover new anticancer drugs from marine organisms, in particular invertebrates such as sponges and ascidians, and marine microalgae, marine bacteria and fungi. In this report, the program and results obtained since its beginning in 2000 will be summarized.

The isolation of a class of bioactive aromatic alkaloid compounds known as pyridoacridines from members of four phyla (Porifera, Chordata–Subphylum Tunicata, Mollusca and Cnidaria) caused some to speculate that they were produced by associated symbionts. We tested this hypothesis by localizing specific metabolites in cells using a combination of visualization methods, including laser-scanning confocal, epifluorescence, and transmission electron microscopy, as well as cell-separation techniques, and chemical analysis. This study demonstrates that large quantities of the pyridoacridine alkaloid dercitamide (=Kuanoniamine C) are localized exclusively in bacteria-free sponge cells in the marine sponge Oceanapia sagittaria (Sollas), and are probably not produced by intracellular symbiotic organisms. We hypothesize that it is unlikely that the pyridoacridines are produced by extracellular bacteria and then transferred to specific sponge cells. The localization of dercitamide in significant concentrations in specific cells throughout the sponge suggests important biological and ecological functions, such as chemical defense against predators and possibly microbial pathogens. If pyridoacridines are produced by the host organism in other phyla, this may be a case of convergent evolution of an efficient and useful biosynthetic pathway.

The Palauan sponge Theonella swinhoei (class Demospongiae, order Lithistida, family Theonellidae) harbors filamentous bacterial symbionts that contain theopalauamide, an antifungal, bicyclic glycopeptide. In this study, the filamentous symbionts were shown to be novel bacteria belonging to the δ-subdivision of proteobacteria. The 16S rRNA gene sequence was determined using a combination of denaturing gradient-gel electrophoresis (DGGE) and specific polymerase chain-reaction (PCR) primers, and its source was confirmed by in situ hybridization. In a series of culture experiments, the filamentous bacteria were propagated in a mixed culture on agar plates. Related 16S rRNA gene sequences were isolated from related sponges with slightly different chemistry. The taxonomic status “Candidatus Entotheonella palauensis” is proposed for the theopalauamide-containing filamentous bacteria from T. swinhoei.

This review covers the marine natural products literature for the year 2000 and is organized phylogenetically, with sections on marine microorganisms and phytoplankton, green algae, brown algae, red algae, sponges, coelenterates, bryozoans, molluscs, tunicates. echinoderms and miscellaneous marine organisms. There is an emphasis on new structures, stressing their biological activities, source organisms and countries of origin, and also syntheses that confirm the structures of known compounds. The review contains 869 structures and 592 references, of which 434 appeared between January and December 2000.

HIV-1 integrase is an attractive target for anti-retroviral chemotherapy, but to date no clinically useful inhibitors have been developed. We have screened diverse marine natural products for compounds active against integrase in vitro and found a series of ascidian alkaloids, the lamellarins, that show selective inhibition. A new member of the family named lamellarin alpha 20-sulfate (1), the structure of which was determined from spectroscopic data, displayed the most favorable therapeutic index. The site of action of lamellarin alpha 20-sulfate on the integrase protein was mapped by testing activity against deletion mutants of integrase. Inhibition of isolated catalytic domain was detectable though weaker than inhibition of full length integrase; possibly lamellarin alpha 20-sulfate binds a site composed of multiple integrase domains. Lamellarin alpha 20-sulfate also inhibited integration in vitro by authentic HIV-1 replication intermediates isolated from infected cells. Lamellarin alpha 20-sulfate was tested against wild type HIV using the MAGI indicator cell assay and found to inhibit early steps of HIV replication. To clarify the inhibitor target, we tested inhibition against an HIV-based retroviral vector bearing a different viral envelope. Inhibition was observed, indicating that the HIV envelope cannot be the sole target of lamellarin alpha 20-sulfate in cell culture. In addition, these single round tests rule out action against viral assembly or budding. These findings provide a new class of compounds for potential development of clinically useful integrase inhibitors.

The AC current transfer ratios for BJT current mirrors operating with nanoampere DC bias currents have been measured and analysed. The new measurement technique uses current-feedback topology instrumentation amplifiers to sense indirectly the input and output currents of a current mirror under test. Sample results are reported for the popular two-transistor and Wilson current mirrors. The measured frequency response data show that bipolar integrated circuit current mirrors, biased at nanoampere DC currents, have current transfer ratios in the range 0.7 to 1 with sufficient bandwidth for use in micropower circuit design. There is close agreement between measured and theoretical data. However, the results of SPICE simulation deviate at very low currents, confirming previous reports of inadequacies in the BJT model under these conditions.

The marine sponge Theonella swinhoei (lithistid Family Theonellidae, Order Astrophorida) has yielded many important, bioactive natural products, most of which share structural features with bacterial natural products. The presence of microbial symbionts in T. swinhoei has been reported, and it was originally suggested that the cytotoxic macrolide swinholide A and many of the bioactive cyclic peptides from T. swinhoei were all produced by simbiotic cyanobacteria. By transmission electron microscopy, we found four distinct cell populations to be consistently present in T. swinhoei: eukaryotic sponge cells, unicellular heterotrophic bacteria, unicellular cyanobacteria and filamentous heterotrophic bacteria. Purification and chemical analyses of each cell type showed the macrolide swinholide A to be limited to the mixed population of unicellular heterotrophic bacteria, and an anti-fungal cyclic peptide occurred only in the filamentous heterotrophic bacteria. Contrary to prior speculation, no major metabolites were located in the cyanobacteria or sponge cells.

The dictyoceratid marine sponge Dysidea herbacea (Keller, 1889) is common in shallow waters of the tropical Pacific Ocean. Polybrominated biphenyl ethers such as 2-(2′,4′-dibromophenyl)-4,6-dibromophenol (1) are characteristic secondary metabolites of some specimens of this sponge and may represent as much as 12% of the dry weight. We have found 1 to be deposited as conspicuous crystals throughout the sponge tissue. The dominant prokaryotic endosymbiont in the mesohyl of the sponge is a filamentous cyanobacterium (Oscillatoria spongeliae), although a vacuole-containing, heterotrophic bacterium is also present. The cyanobacteria were separated from the sponge cells and heterotrophic bacteria by flow cytometry. Coupled gas chromatography—mass spectrometry and proton nuclear magnetic-resonance spectroscopy revealed that the major brominated Compound 1 isolated from the intact symbiotic association is found in the cyanobacteria and not in the sponge cells or heterotrophic bacteria. This suggests that the production of the compound is due to the cyanobacterium, and not to the sponge or symbiotic heterotrophic bacteria, as had been suggested earlier.

The tropical marine spongeDysidea herbacea contains large numbers of a symbiotic filamentous cyanobacterium identified on the basis of a detailed ultrastructural study asOscillatoria spongeliae. We report the flow-cytometric separation of the symbiont from the sponge cells, and demonstrate by chemical analyses that a unique group of polychlorinated compounds isolated from the whole sponge tissue is limited to the cyanobacterial filaments, whereas the accompanying sesquiterpenoids are found only in the sponge cells. This is the first demonstration that secondary metabolites ascribed to a sponge are localized in prokaryotic symbiont cells.

This review of phospholipase A2 (PLA2) inhibitors from marine organisms presents a compilation of research in this field over the past decade. As an introduction to the research on marine natural products, there is an overview of the role of PLA2 in inflammation that provides a rationale for seeking inhibitors of PLA2 as anti-inflammatory agents. A radiometric assay to measure inhibition of bee venom PLA2 is described in detail. Examples of marine natural products that inhibit PLA2 are manoalide and its derivatives, scalaradial and related compounds, the pseudopterosins, the vidalols, and a group of terpenoids that contain masked 1,4-dicarbonyl moieties.

Four novel alkaloids derived from tryptophan and tyrosine subunits have been isolated from the marine sponge Chelonaplysilla sp. collected from a marine lake in Palau. The structures of chelonin A (3), chelonin B (4), bromochelonin B (5), and chelonin C (6) were determined by interpretation of spectral data and chemical conversions. Chelonin A (3) and C (6) are the first natural products incorporating a 2,6-disubstituted morpholine ring. Chelonin A (3), chelonin B (4), and bromochelonin B (5) exhibited antimicrobial activity against Bacillus subtilis, while chelonin A (3) showoed in vivo antiinflammatory activity

Lipid-soluble toxins were isolated from a Caribbean strain of the epiphytic dinoflagellate Prorocentrum concavum Fukuyo. The major lipid-soluble toxin (LD50 = 210 +/- 15 micrograms/kg i.p. in mice) was purified by normal and reversed-phase column chromatography and characterized by 1H NMR and mass spectrometry. The toxin was identified as okadaic acid by interpretation of the spectral data. Okadaic acid was previously identified as a toxic component of the related species P. lima (Ehrenberg) Dodge. The finding of okadaic acid production in P. concavum and P. lima, abundant primary producers in the ciguatera-endemic Caribbean, suggests that the role of this toxin in the etiology of ciguatera may be more significant than previously thought.

Advances in digital computer technology since the middle decades of the twentieth century have transformed many subject areas in astrophysics. Topics which had previously been dealt with by analytic approximations (usually to a very limited number of special cases) suddenly became amenable to detailed numerical modelling for all cases. Frequently, insights derived from this modelling ran ahead of other techniques in predicting physical phenomena before they were either observed or discerned in purely analytic treatments. It has sometimes been said that the second half of this century has seen the advent of a totally new modus operandi in scientific research, which ranks alongside the two traditional approaches–experimentation and theory. The most powerful computers now available have greatly accelerated these developments. They employ simultaneous computational techniques (either vector processing or parallel processing, or both), and their throughput is so large that, for most problems, the only way in which the human mind can fully appreciate the scientific content of the numerical results being calculated is by transforming those results into pictorial representations. This paper draws on my experience as Academic Director of the ANU Supercomputer Facility during the first eighteen months of its operation, to describe the place which I believe super-computers will occupy in the development of astrophysics during the 1990s and into the next century.

Evolutionary calculations have been made for a star of initial mass 17.5 M⊙ in order to emulate the evolution of the precursor of SN1987A. The results give good agreement (i) with the observed properties of Sanduleak -69°202 (star 1) when the model reaches the supernova epoch, and (ii) with the observed distribution of red and blue supergiants in the LMC. Mass loss throughout the life of the star at rates reasonably consistent with observed values is required to produce these results. At the time of explosion, the supernova has a mass of only ∼ 5.4 M⊙ and it is surrounded by about 8 M⊙ of wind material in a shell of ∼0.4 pc radius.

Adociasulfates 1−6 (1−6) were isolated from an extract of the Palauan sponge Haliclona (aka Adocia) sp. that inhibited the transport of stabilized microtubules by the motor protein kinesin, which was immobilized on a microscope slide. The structures of adociasulfates 1−6, the relative stereochemistry of adociasulfates 1, 2, 5, and 6, and the relative stereochemistry of subunits of adociasulfates 3 and 4 were determined by interpretation of spectroscopic data. In a quantitative assay that measures ATP hydrolysis by kinesin, adociasulfates 2 and 6 were the most active.

Marine invertebrates are sources of a diverse array of bioactive metabolites with great potential for development as drugs and research tools. In many cases, microorganisms are known or suspected to be the biosynthetic source of marine invertebrate natural products. The application of molecular microbiology to the study of these relationships will contribute to basic biological knowledge and facilitate biotechnological development of these valuable resources. The bryostatin-producing bryozoan B. neritina and its specific symbiont "Candidatus Endobugula sertula" constitute one promising model system. Another fertile subject for investigation is the listhistid sponges that contain numerous bioactive metabolites, some of which originate from bacterial symbionts.

Marine organisms have provided a large proportion of the bioactive natural products reported over the last 20 years, but none of these compounds have reached the pharmaceutical marketplace. This review describes current progress in the development of a selection of new antiinflammatory and anticancer agents, discusses some difficulties encountered during the development process and suggests how these difficulties may be overcome in the near future through applications of recent advances in biotechnology.