The Spirocyclic Imine from a Marine Benthic Dinoflagellate, Portimine, Is a Potent Anti-Human Immunodeficiency Virus Type 1 Therapeutic Lead Compound.

In this study, we aimed to find chemicals from lower sea animals with defensive effects against human immunodeficiency virus type 1 (HIV-1). A library of marine natural products consisting of 80 compounds was screened for activity against HIV-1 infection using a luciferase-encoding HIV-1 vector. We identified five compounds that decreased luciferase activity in the vector-inoculated cells. In particular, portimine, isolated from the benthic dinoflagellate Vulcanodinium rugosum, exhibited significant anti-HIV-1 activity. Portimine inhibited viral infection with an 50% inhibitory concentration (IC50) value of 4.1 nM and had no cytotoxic effect on the host cells at concentrations less than 200 nM. Portimine also inhibited vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped HIV-1 vector infection. This result suggested that portimine mainly targeted HIV-1 Gag or Pol protein. To analyse which replication steps portimine affects, luciferase sequences were amplified by semi-quantitative PCR in total DNA. This analysis revealed that portimine inhibits HIV-1 vector infection before or at the reverse transcription step. Portimine has also been shown to have a direct effect on reverse transcriptase using an in vitro reverse transcriptase assay. Portimine efficiently inhibited HIV-1 replication and is a potent lead compound for developing novel therapeutic drugs against HIV-1-induced diseases.

Brevenal, a Marine Natural Product, is Anti-Inflammatory and an Immunomodulator of Macrophage and Lung Epithelial Cells.

Chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, are some of the leading causes of illness and fatalities worldwide. The search for novel treatments led to the exploration of marine natural products as drug candidates to combat the debilitating effects of mucus accumulation and chronic inflammation. Previous research showed that an alga-derived compound, brevenal, could attenuate the effects of inflammatory agents, but the mechanisms by which it exerted its effects remained unclear. We investigated the effects of brevenal on lipopolysaccharide (LPS) induced cytokine/chemokine production from murine macrophages and human lung epithelial cells. It was found that brevenal reduces proinflammatory mediator secretion while preserving anti-inflammatory secretion from these cells. Furthermore, we found that brevenal does not alter cell surface Toll-like receptor 4 (TLR4) expression, thereby maintaining the cells' ability to respond to bacterial infection. However, brevenal does alter macrophage activation states, as demonstrated by reduced expression of both M1 and M2 phenotype markers, indicating this putative anti-inflammatory drug shifts innate immune cells to a less active state. Such a mechanism of action would be ideal for reducing inflammation in the lung, especially with patients suffering from chronic respiratory diseases, where inflammation can be lethal.

Structures of the Largest Amphidinol Homologues from the Dinoflagellate Amphidinium carterae and Structure-Activity Relationships.

Amphidinols are polyketide metabolites produced by marine dinoflagellates and are chiefly composed of a long linear chain with polyol groups and polyolefins. Two new homologues, amphidinols 20 (AM20, 1) and 21 (AM21, 2), were isolated from Amphidinium carterae collected in Korea. Their structures were elucidated by detailed NMR analyses as amphidinol 6-type compounds with remarkably long polyol chains. Amphidinol 21 (2) has the longest linear structure among the amphidinol homologues reported so far. The congeners, particularly amphidinol 21 (2), showed weaker activity in hemolysis and antifungal assays compared to known amphidinols.

Screening and transcriptomic analysis of Crypthecodinium cohnii mutants with high growth and lipid content using the acetyl-CoA carboxylase inhibitor sethoxydim.

The heterotrophic microalga Crypthecodinium cohnii is well known for its lipid accumulation, with a high proportion of docosahexaenoic acid (DHA). In this study, we report a novel screening approach to obtain mutants of C. cohnii with high growth and lipid content using the acetyl-CoA carboxylase (ACCase) inhibitor sethoxydim. C. cohnii mutants were generated using atmospheric and room-temperature plasma (ARTP) and then screened for two rounds in media supplemented with sethoxydim. These efforts led to the identification of mutant M-1-2, which had 24.32% higher growth and 7.05% higher lipid content than the wild type, demonstrating the effectiveness of the sethoxydim-based screening. Consistently, the M-1-2 mutant displayed a 16.15% increase in ACCase enzymatic activity and 1.53-fold upregulation of its ACCase-encoding gene based on comparative ACCase activity analysis and transcriptomic analysis, respectively. In addition, transcriptomic analysis showed that transcripts involved in fatty acid biosynthesis, energy, central carbohydrate, and amino acid metabolism were upregulated in the mutant compared to the wild type.

Iriomoteolides-9a and 11a: two new odd-numbered macrolides from the marine dinoflagellate Amphidinium species.

Iriomoteolides-9a (1) and 11a (2), new 15- and 19-membered macrolides, respectively, have been isolated from the marine dinoflagellate Amphidinium species (strain KCA09052). Compounds 1 and 2 were obtained from the extracts of the algal cells inoculated in the PES and TKF seawater medium, respectively. The structures of 1 and 2 were assigned on the basis of detailed NMR analyses. Compounds 1 and 2 exhibited cytotoxic activity against human cervix adenocarcinoma HeLa cells.

Effects of N and P availability on carbon allocation in the toxic dinoflagellate Ostreopsis cf. ovata.

Blooms of the toxic dinoflagellate Ostreopsis cf. ovata are usually associated with shallow and calm coastal waters, characterized by low nutrient concentrations. The algal cells typically cover the benthic substrates, such as the macroalgal and invertebrate communities and rocks, forming a mucilaginous film. Data reported on O. cf. ovata toxin production observed under both field and culture conditions show high variability in terms of toxic profile and cellular content; little is known about the environmental and physiological aspects which regulate the toxin dynamics. In this study, O. cf. ovata physiology was investigated using batch cultures supplied with nutrient concentrations similar to those found in the Adriatic Sea during the recurrent blooms and the observed cellular dynamics were compared with those found in a culture grown under optimal conditions, used as a reference. Data on the cellular C, N and P content during the growth highlighted a possible important role of the cellular nutritional status in regulating the toxin production that resulted to be promoted under specific intervals of the C:N and C:P ratios. The variable toxicity found for O. cf. ovata in various geographic areas could be related to the different in situ prevalent environmental conditions (e.g., nutrient concentrations) which affect the cellular elemental composition and carbon allocation. The obtained results strongly suggest that in the environment toxin production is steadily sustained by a low and constant nutrient supply, able to maintain appropriate cellular C:N (>12) or C:P (>170) ratios for a long period. These results explain to some extent the variability in toxicity and growth dynamics observed in blooms occurring in the different coastal areas.

Laser desorption/ionization mediated by bionanostructures from microalgae.

RATIONALE: Organic matrices are the state-of-the-art ionization mediators in Laser Desorption/Ionization Mass Spectrometry (LDI-MS). Despite improvements in understanding matrix chemistry, interfering matrix-related signals complicate the analysis. Surface-assisted LDI techniques like desorption/ionization on silicon (DIOS) or nanostructure initiator mass spectrometry (NIMS) provide promising alternatives but rely often on elaborate materials. METHODS: We introduce nanopatterned biomineralized cell walls of microalgae as easily accessible biological surfaces that support the ionization of embedded molecules in LDI-MS. Microalgae cell walls were cleaned through oxidation and washing before pipetting on a stainless-steel matrix-assisted laser desorption/ionization (MALDI) target. Added molecules were efficiently ionized in positive and negative ionization mode in common MALDI sources. The method was rigorously validated by comparison with established MALDI experiments. RESULTS: Ionization of PEG600, D-sphingosine and raffinose was successfully mediated by nanostructured cell wall preparations from two different microalgae. Without any change in protocol, steric acid could be detected in the negative ionization mode. Ionization is also supported by commercially available celite, a material containing mineralized diatom cell walls. Characteristic ingredients of fresh coffee were detected in LDI-MS after pipetting it on celite without further sample preparation. Caffeine and saccharose were detected in positive and characteristic fatty acids in negative ionization mode. Detection limits were comparable to established MALDI experiments. CONCLUSIONS: Bionanostructure-enhanced ionization allows the analysis of a diverse selection of analytes including polymers, sugars, amino alcohols, and organic acids without interfering matrix signals. We also show that celite, a commercially available porous material containing mineralized algal bionanostructures, supports LDI-MS.

FTIR spectra of algal species can be used as physiological fingerprints to assess their actual growth potential.

Fourier transform infrared (FTIR) spectra were measured from cells of Microcystis aeruginosa and Protoceratium reticulatum, whose growth rates were manipulated by the availability of nutrients or light. As expected, the macromolecular composition changed in response to the treatments. These changes were species-specific and depended on the type of perturbation applied to the growth regime. Microcystis aeruginosa showed an increase in the carbohydrate-to-protein ratio with decreased growth rates, under nutrient limitation, whereas light limitation induced a decrease of the carbohydrate-to-protein ratio with decreasing proliferation rates. The macromolecular pools of P. reticulatum showed a higher degree of compositional homeostasis. Only when the lowest light irradiance and nutrient availability were supplied, an increase of the carbohydrate-to-protein FTIR absorbance ratio was observed. A species-specific partial least squares (PLS) model was developed using the whole FTIR spectra. This model afforded a very high correlation between the predicted and the measured growth rates, regardless of the growth conditions. On the contrary, the prediction based on absorption band ratios generally used in FTIR studies would strongly depend on growth conditions. This new computational method could constitute a substantial improvement in the early warning systems of algal blooms and, in general, for the study of algal growth, e.g. in biotechnology. Furthermore, these results confirm the suitability of FTIR spectroscopy as a tool to map complex biological processes like growth under different environmental conditions.

A review on the effects of environmental conditions on growth and toxin production of Ostreopsis ovata.

Since the end of the 1990s the occurrence of blooms of the benthic dinoflagellates Ostreopsis spp. is spreading in many tropical and temperate regions worldwide, sometimes causing benthonic biocenosis suffering and occasional human distress. Ostreopsis ovata has been found to produce palytoxin-like compounds, a class of highly potent toxins. As general, the highest abundances of Ostreopsis spp. are recorded during warmer periods characterized by high temperature, salinity, and water column stability. Moreover, as these cells are easily resuspended in the water column, the role of hydrodynamism in the blooms development and decline has been highlighted. The environmental conditions appear, therefore, to be one of the main factors determining the proliferation of these species as testified by several field surveys. Laboratory studies on the effect of environmental parameters on growth and toxicity of O. ovata are rather scarce. With regard to the effects of temperature, culture results indicate that different strains blooming along Italian coasts displayed different optima, in accordance to blooming periods, and that higher toxin levels correlated with best growth conditions. Additionally, in relation to an Adriatic strain, cell growth positively correlated with the increase in salinity, while toxicity was lowest at the highest salinity value (i.e. 40). For the same strain, both nitrogen and phosphorus limitation determined a decrease in cell toxicity showing different behaviour with respect to many other toxic dinoflagellates.

Symbiopolyol, a VCAM-1 inhibitor from a symbiotic dinoflagellate of the jellyfish Mastigias papua.

A super-carbon-chain compound, symbiopolyol (1a), was isolated from a symbiotic dinoflagellate of the jellyfish Mastigias papua. Although a direct comparison between symbiopolyol (1a) and lingshuiol B has not been completed, symbiopolyol (1a) is suggested to be the enantiomer of lingshuiol B. The structure of 1a, including its partial relative configuration, was elucidated on the basis of interpretation of spectroscopic data and chemical transformations. This compound exhibited significant inhibitory activity against the expression of VCAM-1 in human umbilical vein endothelial cells (HUVEC).

Evaluation of the efficiency of metabolism of dinoflagellate phosphorus and carbon by a planktonic ciliate.

The trophic transfer of nutrients through the microbial food web is a key top-down control in aquatic ecosystems which is notoriously difficult to evaluate, particularly for planktonic protists. In this study, a sensitive dual-radioactive tracer technique was developed to simultaneously assess the ingestion rate, and carbon- and phosphorus-specific assimilation efficiencies, of the marine planktonic ciliate Strobilidium neptuni feeding on the autotrophic dinoflagellate Heterocapsa triquetra. Dinoflagellate prey were simultaneously 16h pulse labelled with NaH(14)CO(3) and H(3)(33)PO(4) before being fed to the ciliate, and radioactive labels were traced into ciliate biomass and the experimental medium, as well as being monitored in the prey cells. Rates measured in short-term (10min) incubations, as commonly used to estimate protist uptake of fluorescently labelled prey, were approximately 6 times higher and 3-6 times more variable than rates measured in longer 3-5h incubations. The efficiency of accumulation of prey carbon (54+/-9%) by ciliates was lower than that of prey phosphorus (68+/-3%) suggesting that the phosphorus to carbon ratio in the ciliates was 1.3 times higher than in the labelled dinoflagellate biomass. Rates of phosphorus accumulation and release were combined to reveal that ciliates consumed 3.2+/-0.6 dinoflagellates cell(-1)h(-1). The assessment of carbon tracer release by ciliates was less reliable due to (14)CO(2) exchange between the experimental media and air. The study concludes that the dual phosphorus-carbon radioactive tracer labelling of algal prey allowed the quantification of protist herbivory and nutrient remineralisation in laboratory experiments, thereby providing a potential technique for studying planktonic microbial trophic interactions in situ.

Antioxidant properties of aqueous extracts from red tide plankton cultures.

The antioxidant properties of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined. An electron spin resonance (ESR)-spin trapping method coupled with steady state kinetic analysis showed that all of the extracts directly scavenge superoxide, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. As for hydroxyl radical scavenging, the Fenton reaction and the method of ultraviolet radiation to hydrogen peroxide were used as hydroxyl radical generation systems. All of extracts reduced the level of hydroxyl radicals in both of the systems, indicating that the extracts also directly scavenge hydroxyl radicals. Since the levels of phenolic compounds did not correlate with the antioxidant activities of the extracts, substances other than phenolic compounds also appeared to be attributable to the activities. It is of our interest that the scavenging activities of extract from G. impudicum against superoxide and hydroxyl radicals were increased by heat exposure at 100 degrees C and 200 degrees C respectively. Although the reason for the increased activities of the aqueous extract from G. impudicum is not clear, the heat-resistance of the extract from G. impudicum might make it a desirable antioxidant.

Nitrogen:Phosphorus supply ratio may control the protein and total toxin of dinoflagellate Alexandrium tamarense.

The protein and total toxin of dinoflagellate Alexandrium tamarense at the exponential growth phase were studied at four N:P supply ratios=8, 16, 24 and 48 by maintaining the N concentration at 880 microM with variable P concentrations without nutrient limitation. Because A. tamarense grew well at all N:P supply ratios, they might synthesize RNA which contains high phosphorus and consequently low N:P atomic ratio of cells might be retained during exponential growth phase. Cellular protein:C ratio and toxin:C ratio depended on N:P supply ratio, suggesting that intracellular biochemical composition of A. tamarense might vary due to N:P supply conditions. These biochemical changes could not be detected by only investigations of cellular N:C atomic ratio which was independent on N:P supply ratio. Total cellular toxin contents of A. tamarense increased with increasing N:P supply ratio, indicated that total cellular toxin contents of A. tamarense might be stimulated by relative P-deficiency. In situ P concentration of the Seto Inland Sea of Japan has been reduced since 1980s the environmental regulation issued by Japanese Government, and therefore N:P supply ratio of input water from adjacent rivers has became higher than the Redfield ratio. The present study may suggest that the reduction of P supply into ambient sea water might cause A. tamarense to accumulate more toxin within the cell, in result, noxious A. tamarense would be more influential to marine organisms in coastal ecosystem.

Nitrate and phosphate supplementation to increase toxin production by the marine dinoflagellate Alexandrium tamarense.

Alexandrium tamarense toxins have great value in biotechnology research as well as important in connection with shellfish poisoning. The influence of nitrate or nitrate and phosphate supplementation on cell biomass and toxin content were investigated in batch cultures. When cultures at low nitrate (88.2 microM NaNO(3)) were supplemented with 793.8 microM NaNO(3) at day 10 the cell density and cellular toxin contents were increased by 6-29% and 20-76%, respectively, compared with controls, and maximal values were 43,600 cells/ml (day 38) and 0.91 pg/cell (day 31). Supplementation with nitrate at day 14 or with nitrate and phosphate at day 10/14 to the cultures did not increase the cell density compared with the non-supplemented middle nitrate or high phosphate (108 microM NaH(2)PO(4)) cultures, respectively, but increased the cellular toxin contents by an average of 52%. The results showed that supplementation with nitrate or with nitrate and phosphate at different growth phases of the cultures increased toxin yield by an average of 46%. Supplementation with nitrate at selected times to maintain continuous low level of nitrate might contribute to the effective increase of toxin yield of A. tamarense.

Relevance of the dinoflagellate Gonyaulax fragilis in mucilage formations of the Adriatic Sea.

Oceanographic cruises were carried out monthly from June 1999 to July 2002 to follow the mucilage formation process in the Northern Adriatic Sea. Results show that in correspondence with these events the dinoflagellate Gonyaulax fragilis (Schütt) Kofoid was observed both in the water column and within mucilage aggregates. In the water column, increasing abundances were observed from May until July, with values never exceeding 8500 cells l(-1). Much higher densities were observed within superficial gelatinous aggregates (22800-3400000 cells l(-1)). In mucilage samples, a large number of decomposing cells were present, together with abundant alive cells, enveloped in exudates. G. fragilis isolated from mucilage samples was cultured in three different culture media; it was characterized by a low growth rate but it produced a high amount of polysaccharides. The highest yield both in terms of cell number and carbohydrate production was observed in the medium having the highest nitrogen and phosphorus content and the lowest N/P ratio. The monomeric composition of G. fragilis carbohydrates, compared with that of mucilage samples, showed that in both natural and cultured samples galactose was the most abundant sugar; in addition, an overall good correlation, especially between the monomeric carbohydrate composition of G. fragilis grown in f/2 medium and that of a mucilage sample in which this species was present in high density, was observed.

Chemical characterization of different typologies of mucilaginous aggregates in the Northern Adriatic Sea.

The chemical composition of mucilage aggregates found during summer 2000, 2001 and 2002 in the North Adriatic Sea depends on the nature of the organic matter during aggregation, on the environmental conditions of the site of formation and on the transformations during ageing. The mucilages were composed of organic matter, together with a significant inorganic fraction. Elemental analysis revealed 12.5-32.2% of organic carbon, 0-7.3% of inorganic carbon and 1.0-3.7% of nitrogen. The C(org)/N ratios of most aggregates were between 7.5 and 12.6, values close to those found in the suspended matter; higher ratios were found in large-size (>5 m) aggregates which are probably older. The content of carbohydrates and proteins determined in the aggregates, respectively, 15.4+/-8.9% and 7.9+/-4.8%, w/w, showed a prevalence of carbohydrates over proteins. Neutral carbohydrate analysis of purified polysaccharides from mucilage samples showed very similar signatures with high relative abundance of galactose and glucose. Humic, fulvic and humin substances extracted from the mucilages constitute an important fraction of the organic matter in the aggregates. The humin (a fraction insoluble in acidic and basic media) was present in all mucilage samples, indicating the refractory nature of a part of the organic matter in the mucilage. The iron and calcium could play a role during the aggregation process to form a complex with polysaccharides and humic fractions. The C(org)/N ratio 10+/-2 found in the humic acids extracted from the Adriatic aggregates disclosed a marine origin. The low phosphorus content and the high C(org)/P ratio found in the aggregates might depend from high bacteria activity or from the aggregation of organic fractions depleted of phosphorus. The principal inorganic species contained aluminium and silicon, part of which was of biogenic origin and was more significant in the offshore mucilage aggregates than in the coastal ones. The Si(biog)/C(org) ratio showed that diatoms were always present in the aggregates, although it cannot be established whether these are the producers or these develop within the aggregates.

Studies of the biosynthesis of DTX-5a and DTX-5b by the dinoflagellate Prorocentrum maculosum: regiospecificity of the putative Baeyer-Villigerase and insertion of a single amino acid in a polyketide chain.

The biosynthetic origins of the diarrhetic shellfish poisoning toxins DTX-5a and DTX-5b have been elucidated by supplementing cultures of the producing organism Prorocentrum maculosum with stable isotope labeled precursors and determining the incorporation patterns by 13C NMR spectroscopy. The amino acid residue in the sulfated side chain is found to originate from glycine, and oxygen insertion in the chain is shown to occur after polyketide formation.

Dynamics of C2 toxin and chlorophyll-a formation in the dinoflagellate Alexandrium tamarense during large scale cultivation.

The production of paralytic shellfish toxins (PSTs) by the dinoflagellate Alexandrium tamarense ATCI01, a toxigenic strain isolated from South China coastal waters, was studied in batch cultures in relatively large volumes (20l). Under nutrient-replete conditions, this strain produced C2 toxin (C2T) as a predominant PST. In a 15-day production culture, phosphate was depleted by day 4, the stationary phase began at day 6, and the toxin productivity peaked at day 10, in which the cell content of C2T reached 76 fmol per cell. Much of the toxin was produced after the depletion of phosphate in the medium suggesting that C2T is a secondary metabolite. Aeration with small bubbles was useful in increasing cell mass and toxin yield. Chlorophyll-a (Chl-a) was formed in algal cells until the culture entered the stationary phase, after which Chl-a began to disappear rapidly from the culture while the C2T content continued to rise. These results suggest a metabolic relationship between Chl-a and C2T.