Program

The order Zoantharia contains at least 350 species, which can be found from shallow warm-water coral reefs to cold seeps in the deep sea. While our understanding of the diversity of these benthic cnidarians is far from complete, studies over the past three decades combining molecular phylogenetic studies with an increased focus on in situ observation have greatly increased the robustness of their systematics. Still, zoantharians are often overlooked in marine biodiversity assessments due to difficulty in their identification and a lack of experts.
Important in many marine ecosystems, some zoantharians are well-known for their marine natural products. Examples include Palythoa spp. with palytoxin and related products and Zoanthus spp. containing norzoanthamine. However, Palythoa and Zoanthus are the most commonly encountered zoantharians and are easily sampled, while the majority of zoantharians remain to be properly examined for natural products. In this presentation, several less well-known zoantharian groups will be introduced, and based on recent findings of a novel trisguanidine alkaloid from Epizoanthus species in Palau, the promise of undiscovered marine natural products in this understudied order is high. A systematic study of natural products combining field work and in situ monitoring with genetic and genomic methods should help to unravel patterns of chemical diversity within the order. While we can expect continued discovery of exciting natural products, discovering the ultimate source and reason for their production remains the elusive final goal of such studies from a biological point of view, and more work is needed in this area.

Marine dinoflagellates of the genus Amphidinium are well-known as a producer of unique bioactive metabolites. Cytotoxic macrolides designated as amphidinolides or caribenolide have been isolated from symbiotic or free-swimming dinoflagellates Amphidinium species. some of these macrolides exhibit potent cytotoxicity against tumor cell lines in vitro and antitumor activity in vivo. In our investigation for new metabolites from the Amphidinium dinoflagellates, we have performed the screening of macrolide-producing Amphidinium strains using genomics and metabonomics analyses, and then the selected strains were cultivated using deep-seawater in a large-scale tank system. Detailed investigation of algal extracts led to the isolation of several unique secondary metabolites with cell-proliferation regulating activity. Amphirionins-1 ~ 3 are linear polyketides with cell-growth inhibitory activity, while linear polyketides, amphirionin-4 and 5 show potent proliferation-promoting activity. This presentation will report on structures and biological activities of these unique secondary metabolites.

Important commercial edible red seaweeds Bangia fusco-purpurea are abundantly cultivated in Fujian province of China and proposed to be superior to Porphyra sp. in nutritional value and taste. In this study, an acidic heteropolysaccharide (BFP) isolated from B. fusco-purpure was prepared and characterized, and its apparent molecular mass was estimated to be 12 kDa. The composition analysis showed that BFP mainly consisted of galactose (77.14 ± 0.11%) and arabinose (15.16 ± 0.09%), together with a small amount of uronic acid (5.76 ± 0.16%), glucose (0.69 ± 0.03%) and sulfate groups (0.07 ± 0.02%). Galactose and arabinose accounted for about 93.47 % of the total monosaccharide residues in BFP suggesting that BFP is mainly composed of arabinogalactans. Meanwhile, the analytic results of fourier transform infrared spectra indicated that the presence of galactopyranose structure and alpha-type glycosidic linkages with the possible presence of O-acetyl groups in BFP. Furthermore, the enzyme activity analysis demonstrated that BFP significantly inhibited the activities of alpha-amylase and alpha-glycosidase, the key enzymes for starch/sugar digestion and absorption, in a concentration-dependent manner through the reversible inhibition, and the IC₅₀ values of BFP against alpha-amylase and alpha-glucosidase were estimated to be 1.26 ± 0.11 mg/mL and 1.34 ± 0.07 mg/mL, respectively. Interestingly, our results further revealed that the inhibition types of BFP against alpha-amylase and alpha-glucosidase were obviously discrepant, in which BFP exerted competitive inhibition against alpha-amylase, whereas the inhibition of BFP against alpha-glucosidase followed non-competitive (or mixed) inhibition. Our results suggest that BFP may possess the benefit potential to be developed as a diet food or food additive allowed to long-term consumption for the postprandial hypoglycemic control, and this study also provides a useful foundation for extending the application of BFP and seaweed B. fusco-purpure in the fields of food industry.

Sex pheromone is one of major subject in natural product chemistry. I identifications of sex pheromonal molecules and artificial production of pheromones of crabs have potential to develop new technology for fisheries by controlling courtship and mating behavior of the target species. However the identification process may be slow due to a number of issues. First, the short mating season of some crabs can limit the number of assays that can be run. The pheromones may be mixtures of molecules that lose activity in the process of purification because each component molecule alone may have no or very limited pheromonal activity. Testing the activity of various combinations of purified molecules requires running many bioassays, which is time-consuming. The pheromonal molecules are high polar water soluble molecues that are difficult purify by reversed phase chromatography. To cope with these problems, we have utilized metabolomics approaches and electrophysiological methods on antennules in two species of crabs, helmet crab Telmessus cheiragonus and blue crab Callinectes sapidus. NMR based metabolomics discovered a premolt biomarker metabolite together with unidentified molecules in urine of blue crab and helmet crab as a candidate sex pheromone molecule. Multiunit electrophysiological recording from olfactory receptors on outer flagellum of the antennule showed that the male’s antennule can detect differences between male and female urine. Calcium imaging of olfactory receptor cells in antennule of blue crab showed that the antennule can detect a candidate sex pheromone molecule. The combination of metabolomics, electrophysiological assays, and behavioral assays with separation of candidate molecules from urine may elucidate the sex pheromone molecules of the pheromones.

The effect of partitioned trypsin from albacore tuna (Thunnus alalunga) spleen using an aqueous two-phase system on the recovery and properties of carotenoprotein from Pacific white shrimp (Litopenaeus vannamei) shells was investigated. The partitioned trypsin from albacore tuna spleen showed an efficient hydrolysis and recovery of carotenoprotein from Pacific white shrimp shells. The recovery of carotenoprotein was maximized by the hydrolysis of shrimp shells using 0.8 trypsin units/g shrimp shells for 45 min at 25oC and shrimp shells/buffer ratio of 1:2 (w/v). Carotenoprotein consisted of 72.37% protein, 18.79% lipid, 7.14% ash, 1.61% chitin, and 73.25 µg total astaxanthin/g sample. It was rich in essential amino acid and Asp/Asn and Glu/Gln were found as dominant amino acids. When hydrolytic activities of albacore tuna and bovine trypsins toward carotenoprotein in Pacific white shrimp shells were compared, the recovery efficacy of protein and pigment by albacore tuna trypsin was similar to that achieved by bovine trypsin. Therefore, carotenoprotein from Pacific white shrimp shells could be successfully extracted using partitioned trypsin from albacore tuna spleen and could serve as the value-added nutritive food ingredients.

The ladder-shaped polyethers (LSPs) are secondary metabolites produced by marine phytoplanktons and their structural features are trans/syn fused ladder-shaped ether rings as core structures constructed by 5-9 membered ether rings. They have attracted many researchers for their unique structures and strong bioactivity. Their structural determination and total synthesis have been progressed remarkably. On the other hand, biosynthesis of LSPs are rarely reported. Because the amount of LSPs produced by dinoflagellates are extremely small, and the genome analysis of dinoflagellates is not proceeded adequately due to their large genome size. A plausible biosynthetic mechanism was proposed by Prof Nakanishi in 1985. A trans-olefin precursor is epoxidized and followed by fused ether ring formation occurs in a stepwise or cascading series of an endo-tet epoxide-opening process. A disfavored endo-tet epoxide-opening reaction is deduced to be catalyzed by an enzyme. However, biosynthetic enzymes are still to have not been disclosed experimentally. To explore an endo-tet epoxide-opening enzyme, we designed truncated epoxide precursors of yessotoxin (YTX) as substrates of an epoxide-opening enzyme, which had the AB ring portion of yessotoxin, produced by a dinoflagellate Protoceratium reticulatum. The truncated epoxide precursors were synthesized from D-glucose. The synthesized model epoxides were incubated with extracts from dinoflagellate cells. Products catalysed by enzymes were detected by LC-MS detection methods.

Sponges are one of the most ancient metazoans, unique and very important organisms in marine benthic ecosystems. Sponge tissues are extremely rich in bacteria that can comprise up to 30% of their biomass. Symbiotic bacteria of sponges are considered to produce useful bioactive compounds, some of which are developing huge market. Thus sponges draw attention from both basic and applied sciences, especially from the pharmaceutical area.
In our recent study, Japanese sponge species are revealed to include 636 species plus 16 subspecies, represented by all four classes of the phylum Porifera. However, the number of species in total is still underestimated, considering the diversity of marine habitat around Japan, e.g. seamounts or submarine caves around southern part of Japan. By adding these sponge fauna and unstudied soft-bodied or thinly encrusting species living in both shallow and deep-water habitat, the number of the species will be more diverse, at least double.
In this talk, I introduce preliminary report of diversity of sponges in southern part of Japan especially in submarine caves based on recent faunistic survey. And I suggest to use these undiscovered sponges for future collaborative studies of natural products chemistry.

Marine-derived actinomycetes are significant biological resources to provide structurally diverse secondary metabolites. In recent years, a large number of secondary metabolites with a variety of biological activities have been isolated from marine-derived actinomycetes, and some of them are considered to be promising drug leads.
In the course of our continuing research to discover biologically active natural products from Marine-derived actinomycetes, we isolated several compounds and determined their structures. In this presentation, isolation, structure elucidation, and biological activities of novel compounds will be discussed.

[Objective] Exposure of UV light can cause skin rashes and sunburn, thus stimulating the formation of melanin. Long term UV exposure also can cause skin cancer. Sunscreen is known to reduce the negative impact of UV radiation. The application of sunscreen can reduce the intensity of UV radiation that affect the skin. The aims of this study were to obtain the best ratio of seaweed P. australis and E. cottonii slurry to produce sunscreen cream and as well as obtaining sunscreen formulation with good stability.
[Material and Methods] Creams were formulated consist of cream with different ratio of seaweed P. autralis and E. cottonii slurry, i.e. A (control), B (1:1), C (2:1) and D (1:2). The commercial creams product also used as a comparator (KI and KII).
[Result and Discussion] The best ratio of P. australis and E. cottonii slurry was showed on cream B (1:1) with IC50 value 103,46 ppm, pH 7,21, and SPF value 5,229±0,02. Microbes were not detected either in seaweed slurry or creams. Sunscreen cream had good stability with no occurance of phase separation, discoloration and odor. Sunscreen creams have at least 1 year shelf life as there was no phase separation after centrifugal force at 3.800 rpm for 5 hours.

Keywords: antioxidant activity, Euchema cottonii, Padina australis, SPF, stability

The effects of formulation parameters on the particle size (Z-Ave), zeta potential, polydispersity index, and peptide loading efficiency (LE) were investigated for biomaterial -loaded chitosan (CS)/sodium alginate (SA) particles (CS-SA-Ps) formed via the external gelation technique. The CS viscosity played a significant role in particle formation, with a lower viscosity yielding particles with lower Z-Ave and higher LE. The FT-IR spectra and XRD indicated lysozyme/CS-SA-NPs had similar chemical characteristics as that of CS-SA-NPs except peak shifts. An assessment of thermodynamic parameters proved that the binding of lysozyme to SA involved self-assembly via exothermic interactions in which the binding stoichiometry showed a slight decrease in the presence of calcium chloride. Moreover, a study of the release behaviour revealed that 100% of lysozyme was released in PBS over 1800 minutes for lysozyme/CS-SA-Ps prepared by 0.5 mM calcium chloride, while just no more than 60% lysozyme released from those particles produced with 1.0 or 3.0 mM calcium chloride, where the release rate could be slowed by an increase in the calcium cation concentration or quickened by an increase in SA concentration. The bioactivities of the particle as well as its application in inhibiting micro-bacterial growth will be analyzed, which may have great potential in the preservation of aquatic products.

Saxitoxin (STX), one of the paralytic shellfish toxins (PST), is a guanidine toxin that specifically and strongly blocks the voltage-gated Na+ channels. Shimizu et al. first proposed biosynthetic pathway of STX that begins with a Claisen condensation of arginine and acetyl-CoA. After that, putative biosynthetic genes of STX were reported in several toxin-producing freshwater cyanobacteria and marine dinoflagellates, and its biosynthetic pathway was predicted based on the predicted enzymes coded by these genes, while enough chemical evidences had not been collected. Recently, we chemically synthesized genetically predicted and also unpredicted biosynthetic intermediates, and identified them in the toxic cyanobacterium Anabaena circinalis (TA04) and the toxic dinoflagellate Alexandrium tamarense (Axat-2).1-4 These results combined with the incorporation experiments of stable isotope labeled intermediates enabled us to propose a novel route to STX and its shunt pathway, in addition to confirm the previously proposed steps.
References:
1)Tsuchiya, S.; Cho, Y.; Konoki, K.; Nagasawa, K.; Oshima, Y.; Yotsu-Yamashita, M. Org. Biomol. Chem. 2014, 12, 3016-3020.
2)Tsuchiya, S.; Cho, Y.; Konoki, K.; Nagasawa, K.; Oshima, Y.; Yotsu-Yamashita, M. Chem. Eur. J. 2015, 21, 7835-7840.
3)Tsuchiya, S.; Cho, Y.; Konoki, K.; Nagasawa, K.; Oshima, Y.; Yotsu-Yamashita, M. Sci. Rep. 2016, 6, 20340.
4) Tsuchiya, S.; Cho, Y.; Yoshioka, R.; Konoki, K.; Nagasawa, K.; Oshima, Y.; Yotsu-Yamashita, M. Angew. Chem. Int. Ed., 2017, 56, 5327-5331.

Mycalolides are cytotoxic macrolides isolated from the marine sponge genus Mycale collected from all over the world, and having characteristic tris-oxazole substructure in the macrocyclic core. Molecular target of the mycalolides is actin filament, and sold as actin depolymerizing reagents.
Recently, several marine sponge derived compounds have been revealed to be produced by symbiotic bacteria. However, real producers of the bioactive molecules from Mycale sponges have not been determined yet, in spite of their potent secondary metabolites production ability. In order to clarify the producer of mycalolides, we attempted identification of the biosynthetic gene cluster of mycalolides by direct metagenome sequencing approach.
Metagenomic DNA was prepared from a symbiotic bacteria fraction of the Mycale sp. collected at Ariake Sea, and subjected to the direct NGS sequencing to obtain total 2 Gb data. It was assembled to form about 5,000 contigs whose total length is about 40 Mb. Result of the antiSMASH analysis, a secondary metabolites biosynthetic gene detection tool, nine PKS and NRPS containing contigs were found, and predicted structures of the five of them highly matched with the mycalolide core structure. These five contigs were connected by PCR gap closing to form putative mycalolide biosynthetic genes designated as myc cluster whose length is about 120 kb. By sequence binning method and homology search analysis of the all generated contigs, it was showed that a novel bacterium most likely belongs to the phylum Verrucomicrobia has myc cluster in its genome.
Detailed analysis of the myc cluster suggested that mycalolides are initially produced as a pre-form and modified later to be known mycalolides, but predicted initial product has not been reported from the nature. In order to detect the predicted product, we attempted metabolomics analysis of the extract of the fresh sponge specimen, and resulted in finding a novel mycalolide analogue.

Most reef-building corals acquire symbiotic dinoflagellates, Symbiodinium spp., from surrounding environments and establish symbiosis with Symbiodinium. Since Symbiodinium occur in low densities in the water column, corals would attract symbionts using chemotactic compounds to acquire them. We found a coral Acropora tenuis crude water extract attracted specific Symbiodinium strains, including strains NBRC102920 (clade A) and CCMP2556 (clade D), but not strains GTP-A6-Sy (clade A), CCMP421 (clade E) and CS-156 (clade F). The chemotactic activity in crude A. tenuis water extract remained after dialysis, but lost completely after heat treatment, indicating that the chemotactic compounds are water-soluble, heat-labile macromolecules. As juvenile A. tenuis tend to acquire the strains which attracted by the crude water extract and an factor involved in the acquisition was a carbohydrate-binding protein, lectin, chemoattraction seems to be inhibited by carbohydrates. As expected, N-acetyl-glucosamine (GlcNAc) inhibited the chemotactic activity. The GlcNAc-bining lectin purified from crude water extract, ActL, demonstrated 70% of the chemotactic activity in the crude water extract, and this chemoattraction was inhibited by addition of anti-ActL antibody, suggesting that ActL is a major factor for attraction. ActL and crude water extract attracted same strains which were acquired by juvenile A. tenuis. Moreover, rearing medium of 10 juvenile A. tenuis contained 34 ng/ml of ActL. It estimated that a polyp releases ActL at 3.4 ng/ml/juvenile polyp/day, and ActL at 3.4 ng/ml showed the chemotactic activity in vitro. These results suggest that ActL was released from juvenile A. tenuis and selectively attract some specific Symbiodinium.