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1.
Mar Pollut Bull ; 145: 105-117, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31590766

RESUMO

The Beagle Channel is a remote subantarctic environment where mussel aquaculture initiatives have existed since the early 1990s. Here we analyze phytoplankton biomass and composition, and the occurrence of harmful microalgae species and their toxins at three sites during the period 2015-2016. The occurrence of potentially harmful algae was observed throughout the study period, including toxigenic dinoflagellates such as Alexandrium catenella (Group I of the A. tamarense complex), A. ostenfeldii, Dinophysis acuminata, Gonyaulax spinifera, Azadinium sp., and the diatoms Pseudo-nitzschia australis and P. fraudulenta. Toxic dinoflagellates were detected in low densities whereas a Pseudo-nitzschia bloom was observed in late February. Isolates of A. catenella and P. delicatissima sensu stricto were phylogenetically characterized. The toxin profile of A. catenella was dominated by GTX4, while P. delicatissima sensu stricto showed no production of the neurotoxin domoic acid in culture conditions. The results provide base-line information for the management of harmful algal blooms in this little explored subantarctic area.


Assuntos
Aquicultura , Diatomáceas/química , Dinoflagelados/química , Toxinas Marinhas/análise , Fitoplâncton/química , Animais , Biomassa , Diatomáceas/classificação , Diatomáceas/crescimento & desenvolvimento , Dinoflagelados/classificação , Dinoflagelados/crescimento & desenvolvimento , Proliferação Nociva de Algas , Microalgas/química , Microalgas/classificação , Microalgas/crescimento & desenvolvimento , Fitoplâncton/classificação , Fitoplâncton/crescimento & desenvolvimento , América do Sul
2.
J Agric Food Chem ; 67(34): 9667-9682, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31415166

RESUMO

This study assessed the feasibility of an NMR metabolomics approach coupled to multivariate data analysis to monitor the naturally present or stresses-elicited metabolites from a long-term (>170 days) culture of the dinoflagellate marine microalgae Amphidinium carterae grown in a fiberglass paddlewheel-driven raceway photobioreactor. Metabolic contents, in particular, in two members of the amphidinol family, amphidinol A and its 7-sulfate derivative amphidinol B (referred as APDs), and other compounds of interest (fatty acids, carotenoids, oxylipins, etc.) were evaluated by altering concentration levels of the f/2 medium nutrients and daily mean irradiance. Operating with a 24 h sinusoidal light cycle allowed a 3-fold increase in APD production, which was also detected by an increase in hemolytic activity of the methanolic extract of A. carterae biomass. The presence of APDs was consistent with the antitumoral activity measured in the methanolic extracts of the biomass. Increased daily irradiance was accompanied by a general decrease in pigments and an increase in SFAs (saturated fatty acids), MUFAs (monounsaturated fatty acids), and DHA (docosahexaenoic acid), while increased nutrient availability lead to an increase in sugar, amino acid, and PUFA ω-3 contents and pigments and a decrease in SFAs and MUFAs. NMR-based metabolomics is shown to be a fast and suitable method to accompany the production of APD and bioactive compounds without the need of tedious isolation methods and bioassays. The two APD compounds were chemically identified by spectroscopic NMR and spectrometric ESI-IT MS (electrospray ionization ion trap mass spectrometry) and ESI-TOF MS (ESI time-of-flight mass spectrometry) methods.


Assuntos
Dinoflagelados/metabolismo , Macrolídeos/química , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Microalgas/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Dinoflagelados/química , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Macrolídeos/metabolismo , Microalgas/química , Análise Multivariada
3.
Mar Drugs ; 17(7)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340532

RESUMO

In vitro and in vivo studies have shown that phycotoxins can impact intestinal epithelial cells and can cross the intestinal barrier to some extent. Therefore, phycotoxins can reach cells underlying the epithelium, such as enteric glial cells (EGCs), which are involved in gut homeostasis, motility, and barrier integrity. This study compared the toxicological effects of pectenotoxin-2 (PTX2), yessotoxin (YTX), okadaic acid (OA), azaspiracid-1 (AZA1), 13-desmethyl-spirolide C (SPX), and palytoxin (PlTX) on the rat EGC cell line CRL2690. Cell viability, morphology, oxidative stress, inflammation, cell cycle, and specific glial markers were evaluated using RT-qPCR and high content analysis (HCA) approaches. PTX2, YTX, OA, AZA1, and PlTX induced neurite alterations, oxidative stress, cell cycle disturbance, and increase of specific EGC markers. An inflammatory response for YTX, OA, and AZA1 was suggested by the nuclear translocation of NF-κB. Caspase-3-dependent apoptosis and induction of DNA double strand breaks (γH2AX) were also observed with PTX2, YTX, OA, and AZA1. These findings suggest that PTX2, YTX, OA, AZA1, and PlTX may affect intestinal barrier integrity through alterations of the human enteric glial system. Our results provide novel insight into the toxicological effects of phycotoxins on the gut.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Neuroglia/efeitos dos fármacos , Intoxicação por Frutos do Mar/etiologia , Frutos do Mar/toxicidade , Animais , Bivalves/parasitologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dinoflagelados/química , Humanos , Mucosa Intestinal/inervação , Mucosa Intestinal/patologia , Neuroglia/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Frutos do Mar/parasitologia
4.
Mar Drugs ; 17(7)2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31252576

RESUMO

Due to the unique biodiversity and the physical-chemical properties of their environment, marine microorganisms have evolved defense and signaling compounds that often have no equivalent in terrestrial habitats. The aim of this study was to screen extracts of the dinoflagellate Amphidinium carterae for possible bioactivities (i.e., anticancer, anti-inflammatory, anti-diabetes, antibacterial and antifungal properties) and identify bioactive compounds. Anticancer activity was evaluated on human lung adenocarcinoma (A549), human skin melanoma (A2058), human hepatocellular carcinoma (HepG2), human breast adenocarcinoma (MCF7) and human pancreas carcinoma (MiaPaca-2) cell lines. Antimicrobial activities were evaluated against Gram-positive bacteria (Staphylococcus aureus MRSA and MSSA), Gram-negative bacteria (i.e., Escherichia coli and Klebsiella pneumoniae), Mycobacterium tuberculosis and the fungus Aspergillus fumigatus. The results indicated moderate biological activities against all the cancer cells lines and microorganisms tested. Bioassay-guided fractionation assisted by HRMS analysis allowed the detection of one new and two known amphidinols that are potentially responsible for the antifungal and cytotoxic activities observed. Further isolation, purification and structural elucidation led to a new amphidinol, named amphidinol 22. The planar structure of the new compound was determined by analysis of its HRMS and 1D and 2D NMR spectra. Its biological activity was evaluated, and it displayed both anticancer and antifungal activities.


Assuntos
Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Dinoflagelados/química , Policetídeos/farmacologia , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Aspergillus fumigatus/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Policetídeos/química , Policetídeos/isolamento & purificação , Relação Estrutura-Atividade
5.
Mar Drugs ; 17(6)2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31200525

RESUMO

On our quest for new bioactive molecules from marine sources, two cyclic imines (1, 2) were isolated from a dinoflagellate extract, inhibiting the growth of the respiratory syncytial virus (RSV). Compound 1 was identified as a known molecule portimine, while 2 was elucidated to be a new cyclic imine, named kabirimine. The absolute stereochemistry of 1 was determined by crystallographic work and chiral derivatization, whereas the structure of 2 was elucidated by means of spectroscopic analysis and computational study on all the possible isomers. Compound 1 showed potent cytotoxicity (CC50 < 0.097 µM) against HEp2 cells, while 2 exhibited moderate antiviral activity against RSV with IC50 = 4.20 µM (95% CI 3.31-5.33).


Assuntos
Dinoflagelados/química , Iminas/química , Antivirais/química , Antivirais/farmacologia , Organismos Aquáticos/química , Linhagem Celular Tumoral , Humanos , Iminas/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos
6.
ISME J ; 13(9): 2334-2345, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31118473

RESUMO

The acquisition of thermally tolerant algal symbionts by corals has been proposed as a natural or assisted mechanism of increasing coral reef resilience to anthropogenic climate change, but the cell-level processes determining the performance of new symbiotic associations are poorly understood. We used liquid chromatography-mass spectrometry to investigate the effects of an experimentally induced symbiosis on the host proteome of the model sea anemone Exaiptasia pallida. Aposymbiotic specimens were colonised by either the homologous dinoflagellate symbiont (Breviolum minutum) or a thermally tolerant, ecologically invasive heterologous symbiont (Durusdinium trenchii). Anemones containing D. trenchii exhibited minimal expression of Niemann-Pick C2 proteins, which have predicted biochemical roles in sterol transport and cell recognition, and glutamine synthetases, which are thought to be involved in nitrogen assimilation and recycling between partners. D. trenchii-colonised anemones had higher expression of methionine-synthesising betaine-homocysteine S-methyltransferases and proteins with predicted oxidative stress response functions. Multiple lysosome-associated proteins were less abundant in both symbiotic treatments compared with the aposymbiotic treatment. The differentially abundant proteins are predicted to represent pathways that may be involved in nutrient transport or resource allocation between partners. These results provide targets for specific experiments to elucidate the mechanisms underpinning compensatory physiology in the coral-dinoflagellate symbiosis.


Assuntos
Dinoflagelados/fisiologia , Proteoma/genética , Anêmonas-do-Mar/microbiologia , Simbiose , Animais , Recifes de Corais , Dinoflagelados/química , Dinoflagelados/genética , Temperatura Alta , Estresse Oxidativo , Proteínas/genética , Proteínas/metabolismo , Proteoma/metabolismo , Proteômica , Anêmonas-do-Mar/genética , Anêmonas-do-Mar/fisiologia
7.
Toxins (Basel) ; 11(5)2019 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-31130661

RESUMO

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kastela Bay. In order to taxonomically identify the Ostreopsis species responsible for this event and determine their toxin profile, cells collected in seawater and from benthic macroalgae were analyzed. Conservative taxonomic methods (light microscopy and SEM) and molecular methods (PCR-based assay) allowed the identification of the species Ostreopsis cf. ovata associated with Coolia monotis. The abundance of O. cf. ovata reached 2.9 × 104 cells L-1 in seawater, while on macroalgae, it was estimated to be up to 2.67 × 106 cells g-1 of macroalgae fresh weight and 14.4 × 106 cells g-1 of macroalgae dry weight. An indirect sandwich immunoenzymatic assay (ELISA) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) were used to determine the toxin profile. The ELISA assay revealed the presence of 5.6 pg palytoxin (PLTX) equivalents per O. cf. ovata cell. LC-HRMS was used for further characterization of the toxin profile, which showed that there were 6.3 pg of the sum of ovatoxins (OVTXs) and isobaric PLTX per O. cf. ovata cell, with a prevalence of OVTXs (6.2 pg cell-1), while the isobaric PLTX concentration was very low (0.1 pg cell-1). Among OVTXs, the highest concentration was recorded for OVTX-a (3.6 pg cell-1), followed by OVTX-b (1.3 pg cell-1), OVTX-d (1.1 pg cell-1), and OVTX-c (0.2 pg cell-1).


Assuntos
Dinoflagelados , Toxinas Marinhas/análise , Água do Mar/microbiologia , Dinoflagelados/química , Dinoflagelados/genética , Monitoramento Ambiental , Oceanos e Mares
8.
Environ Sci Pollut Res Int ; 26(17): 17512-17519, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025277

RESUMO

Phytoplankton assemblages were investigated in 2015 along the seasonal changes of the Genhe River in the Greater Hinggan Mountains. The survey was performed in June (spring), August (summer), and October (autumn) at nine sampling stations to study the community composition, abundance, and biodiversity. The results showed that 61 species belonging to 16 genera were identified, including Bacillariophyta of 31 species, Dinophyta 2 species, Cyanophyta 2 species, Chlorophyta 20 species, Chrysophyta 2 species, and Cryptophyta 1 species; Besides, Bacillariophyta are dominant species. Shannon-Wiener (H') and Pielou (J') indices indicated that phytoplankton community was stable. And these two indices were significantly lower in summer than in spring and autumn. Phytoplankton abundance and biomass show significant differences in each season. The total phytoplankton abundance (1122.3 × 104 ind/L) and biomass (6.5709 mg/L) in summer are much higher than that in spring and autumn. There were few species and low abundance and biomass in the upper reaches of Genhe River; this fact can be explained by the cold climate in the Greater Higgnan Mountains region. Canonical correspondence analysis (CCA) was used to analyze the data. It revealed that Fe3+, Cu2+, pH, and water temperature (WT) were responsible for most of the variation in space in the phytoplankton community. These environmental parameters play an essential role in the community structure variation of phytoplankton in the upper reaches of Genhe River, the strong association between phytoplankton community structure and ecological factors is varied in each season.


Assuntos
Clorófitas/química , Cianobactérias/química , Diatomáceas/classificação , Dinoflagelados/química , Fitoplâncton/classificação , Rios/química , Biodiversidade , Biomassa , China , Diatomáceas/química , Ecologia , Fitoplâncton/química , Estações do Ano , Temperatura Ambiente , Qualidade da Água
9.
Toxicon ; 164: 44-50, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30954452

RESUMO

Compounds similar to maitotoxin (MTX) have been isolated from several laboratory strains of the dinoflagellate Gambierdiscus spp. from the Caribbean. Mass spectral results suggest that these compounds differ from MTX by the loss of one sulfate group and, in some cases, the loss of one methyl group with the addition of one degree of unsaturation. NMR experiments, using approximately 50 nmol of one of these compounds, have demonstrated that the 9-sulfo group of MTX is still present, suggesting that these compounds are 40-desulfo congeners of MTX.


Assuntos
Dinoflagelados/química , Toxinas Marinhas/química , Oxocinas/química , Região do Caribe , Cromatografia Líquida , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular
10.
Mar Drugs ; 17(4)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010188

RESUMO

Recent studies have clearly shown the importance of omega-3 (ω-3) and omega-6 (ω-6) polyunsaturated fatty acids (PUFAs) for human and animal health. The long-chain eicosapentaenoic acid (EPA; 20:5ω-3) and docosahexaenoic acid (DHA; 22:6ω-3) are especially recognized for their nutritional value, and ability to alleviate many diseases in humans. So far, fish oil has been the main human source of EPA and DHA, but alternative sources are needed to satisfy the growing need for them. Therefore, we compared a fatty acid profile and content of 10 diatoms and seven dinoflagellates originating from marine, brackish and freshwater habitats. These two phytoplankton groups were chosen since they are excellent producers of EPA and DHA in aquatic food webs. Multivariate analysis revealed that, whereas the phytoplankton group (46%) explained most of the differences in the fatty acid profiles, habitat (31%) together with phytoplankton group (24%) explained differences in the fatty acid contents. In both diatoms and dinoflagellates, the total fatty acid concentrations and the ω-3 and ω-6 PUFAs were markedly higher in freshwater than in brackish or marine strains. Our results show that, even though the fatty acid profiles are genetically ordered, the fatty acid contents may vary greatly by habitat and affect the ω-3 and ω-6 availability in food webs.


Assuntos
Diatomáceas/metabolismo , Dinoflagelados/metabolismo , Ácidos Graxos Ômega-3/química , Ácidos Graxos/análise , Diatomáceas/química , Dinoflagelados/química , Ecossistema , Ácidos Graxos Ômega-3/metabolismo , Óleos de Peixe , Água Doce , Águas Salinas
11.
Mar Drugs ; 17(3)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897777

RESUMO

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.


Assuntos
Anti-Inflamatórios/farmacologia , Organismos Aquáticos/química , Dinoflagelados/química , Éteres/farmacologia , Fatores Imunológicos/farmacologia , Polímeros/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Linhagem Celular Tumoral , Doença Crônica/terapia , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/efeitos dos fármacos , Éteres/uso terapêutico , Humanos , Fatores Imunológicos/uso terapêutico , Pulmão/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Polímeros/uso terapêutico , Mucosa Respiratória/citologia , Doenças Respiratórias/tratamento farmacológico , Doenças Respiratórias/imunologia
12.
Chemistry ; 25(36): 8528-8542, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-30882926

RESUMO

Iriomoteolide-2a is a marine macrolide metabolite isolated from a cultured broth of the benthic dinoflagellate Amphidinium sp. HYA024 strain. This naturally occurring substance was reported to show remarkable cytotoxic activity against human cancer cell lines HeLa and DG-75 and in vivo antitumor activity against murine leukemia P388 cell line. Herein, the total synthesis, stereochemical revision, and biological assessment of iriomoteolide-2a are reported in detail. Total synthesis of the proposed structure 1 of iriomoteolide-2a featured a late-stage convergent assembly of three components by a Suzuki-Miyaura coupling, an esterification, and a ring-closing metathesis. However, the NMR data of synthetic 1 were not identical to those of the natural product. Careful analysis of the NMR data of the authentic material and synthesis/NMR analysis of appropriately designed model compounds led to consideration of four possible stereoisomers 2-5 as candidates for the correct structure. Accordingly, total syntheses of 2-5 were achieved by taking advantage of the convergent strategy, and comparison of the NMR spectra of synthetic 2-5 with those of the natural product led to the conclusion that 5 shows the correct relative configuration of iriomoteolide-2a. The absolute configuration of this natural product was finally established through chiral HPLC analysis of synthetic 5/ent-5 with the authentic sample. The antiproliferative activity of the synthetic compounds was assessed against HeLa and A549 cells to show that, in contrast to expectation, synthetic 5 and ent-5 were only marginally active in these cell lines. This work clearly underscores the vital role of total synthesis in the establishment of the structure and biological activity of natural products.


Assuntos
Produtos Biológicos/síntese química , Macrolídeos/química , Produtos Biológicos/química , Produtos Biológicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dinoflagelados/química , Dinoflagelados/metabolismo , Esterificação , Humanos , Macrolídeos/síntese química , Macrolídeos/toxicidade , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estereoisomerismo
13.
Toxicon ; 160: 1-7, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30639658

RESUMO

Okadaic acid (OA), a potent polyether marine toxin, accumulates in the digestive glands of marine mollusks and therefore can severely threaten the health of humans after ingestion of contaminated shellfish. In vivo and in vitro studies have revealed that exposure of various cells, including human embryonic amniotic cells, hepatocytes, neuroblastoma cells, to OA induces morphological and functional modifications as well as the death of cells. As the number of reports on OA poisoning has increased, this toxin has gradually attracted the public's attention, and researchers are trying to study it. This review summarizes the current literature on the toxicity effects of OA, in addition to its detection and detoxification.


Assuntos
Dinoflagelados/química , Ácido Okadáico/toxicidade , Animais , Humanos , Inativação Metabólica , Toxinas Marinhas/toxicidade , Moluscos/química , Ácido Okadáico/análise , Ácido Okadáico/metabolismo , Intoxicação por Frutos do Mar
14.
Mar Drugs ; 17(1)2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30654557

RESUMO

Amphidinolides are cytotoxic macrolides produced by symbiotic unicellular microalgae of the genus Amphidinium. Here we describe the identification of four related molecules belonging to this macrolide family isolated from the invertebrate Stragulum bicolor. The new molecules, named amphidinolide PX1-PX3 and stragulin A (1⁻4), show an unprecedented carbon skeleton whose complete stereochemistry has been determined by spectroscopic and computational methods. Differences in the structures of these molecules modulate their biological activity in a panel of tumor cell lines, but the opened derivative stragulin (4) shows a very potent and specific cytotoxic activity (IC50 0.18 µM) against the aggressive human melanoma cell A2058.


Assuntos
Antozoários/parasitologia , Antibióticos Antineoplásicos/farmacologia , Organismos Aquáticos/química , Dinoflagelados/química , Macrolídeos/farmacologia , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Macrolídeos/química , Macrolídeos/isolamento & purificação , Estrutura Molecular
15.
Protoplasma ; 256(2): 459-469, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30225813

RESUMO

Trichocyst-enriched fractions were isolated from the dinoflagellate Oxyrrhis marina and subjected to protein staining and lectin-binding studies, to dissociation experiments using heat, and to solubilization/reconstitution experiments using 6 M guanidine hydrochloride. The trichocysts could be stained by Alcian Blue and became labeled by the lectin Concanavalin A, but not by wheat germ agglutinin. The trichocysts did not dissociate when the fractions were heated for 5 min at 40 or 50 °C. Heating at 60 °C resulted in the dissociation of trichocysts into irregular filamentous structures. These filaments were still present when the fractions were incubated for 5 min at temperatures of 70 and 80 °C. Reassembly was not achieved by subsequent cooling steps. The disintegration of trichocysts was also achieved in 6 M guanidine hydrochloride, and reassembly into filamentous structures, similar to those obtained by heat, occurred after dialysis against distilled water. Electron microscopy revealed that the filaments created either by heat or using guanidine hydrochloride by far did not resemble native trichocysts. They were much thinner (5-7 nm in width), missed the characteristic striation of electron-dense and -transparent lines along the longitudinal axes, and showed much more bending. Furthermore, they tend to merge to thicker shapeless structures and blob-like aggregates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that fractions enriched either in trichocysts or in reconstituted filamentous structures obtained in the guanidine hydrochloride solubilization experiments were dominated by proteins with relative molecular weights in the range of approximately 15 to 29 kDa. Minor amounts of larger proteins were also detected. Tryptic in gel digestion followed by mass spectrometry confirmed the presence of almost the same set of proteins within the both, the trichocyst-enriched fractions, and the fractions of filaments reconstituted thereafter. These proteins were previously proposed to represent the matrix polypeptides of the trichocysts of Oxyrrhis marina (Rhiel et al., Protoplasma 255: 217-230, 2018).


Assuntos
Dinoflagelados/química , Lectinas/química , Microscopia Eletrônica de Transmissão/métodos , Animais
16.
Anal Sci ; 35(3): 301-305, 2019 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-30416167

RESUMO

Cypridina luciferase is a bioluminescent enzyme that has been used as a reporter either in gene expression reporter assays or in immunoassays accompanied by an antibody. To develop a novel bioluminescent assay for the detection of 5-hydroxymethylcytosine, we first conjugated Cypridina luciferase to the antibody against 5-hydroxymethylcytosine. Next, we performed modifications of guanine bases in the genome DNA samples with 4-azidophenylglyoxal and the biotinylation via the azide-Staudinger ligation, which allowed streptavidin to capture and immobilize the genome DNA samples under mild conditions. The detection of 5-hydroxymethylcytosine in the genome DNA samples was performed with the conjugates between Cypridina luciferase and the reduced antibody, which was also confirmed by a surface plasmon resonance assay with the antibody alone. The results obtained from the bioluminescent assay were in good agreement with that of the surface plasmon resonance assay. We succeeded in the detection of 5hmC in the genome DNA samples from the dinoflagellate Pyrocystis Lunula by using this method.


Assuntos
5-Metilcitosina/análogos & derivados , DNA/genética , Dinoflagelados/química , Técnicas Imunoenzimáticas/métodos , Medições Luminescentes/métodos , 5-Metilcitosina/análise , Anticorpos Imobilizados/química , Biotinilação , Dinoflagelados/genética , Epigênese Genética , Luciferases/química , Estreptavidina/química
17.
Sci Total Environ ; 653: 455-464, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30412890

RESUMO

High latitudes are considered particularly vulnerable to ocean acidification, since they are naturally low in carbonate ions. The edible mussel Mytilus chilensis is a common calcifier inhabiting marine ecosystems of the southern Chile, where culturing of this species is concentrated and where algal blooms produced by the toxic dinoflagellate A. catenella are becoming more frequent. Juvenile Mytilus chilensis were exposed to experimental conditions simulating two environmental phenomena: pCO2 increase and the presence of paralytic shellfish toxins (PST) produced by the dinoflagellate Alexandrium catenella. Individuals were exposed to two levels of pCO2: 380 µatm (control condition) and 1000 µatm (future conditions) over a period of 39 days (acclimation), followed by another period of 40 days exposure to a combination of pCO2 and PST. Both factors significantly affected most of the physiological variables measured (feeding, metabolism and scope for growth). However, these effects greatly varied over time, which can be explained by the high individual variability described for mussels exposed to different environmental conditions. Absorption efficiency was not affected by the independent effect of the toxic diet; however, the diet and pCO2 interaction affected it significantly. The inhibition of the physiological processes related with energy acquisition by diets containing PST, may negatively impact mussel fitness, which could have important consequences for both wild and cultured mussel populations, and thus, for socioeconomic development in southern Chile.


Assuntos
Dióxido de Carbono/efeitos adversos , Mudança Climática , Toxinas Marinhas/efeitos adversos , Mytilus/fisiologia , Água do Mar/química , Animais , Dinoflagelados/química , Metabolismo Energético , Aptidão Genética , Concentração de Íons de Hidrogênio , Mytilus/genética , Mytilus/crescimento & desenvolvimento
18.
J Photochem Photobiol B ; 190: 110-117, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30508759

RESUMO

The attribution of quantum beats observed in the time-resolved spectroscopy of photosynthetic light-harvesting antennae to nontrivial quantum coherences has sparked a flurry of research activity beginning a decade ago. Even though investigations into the functional aspects of photosynthetic light-harvesting were supported by X-ray crystal structures, the non-covalent interactions between pigments and their local protein environment that drive such function has yet to be comprehensively explored. Using symmetry-adapted perturbation theory (SAPT), we have comprehensively determined the magnitude and compositions of these non-covalent interactions involving light-harvesting chromophores in two quintessential photosynthetic pigment-protein complexes - peridinin chlorophyll-a protein (PCP) from dinoflagellate Amphidinium carterae and phycocyanin 645 (PC645) from cryptophyte Chroomonas mesostigmatica. In PCP, the chlorophylls are dispersion-bound to the peridinins, which in turn are electrostatically anchored to the protein scaffold via their polar terminal rings. This might be an evolutionary design principle in which the relative orientation of the carotenoids towards the aqueous environment determines the arrangement of the other chromophores in carotenoid-based antennas. On the other hand, electrostatics dominate the non-covalent interactions in PC645. Our ab initio simulations also suggest full protonation of the PC645 chromophores in physiological conditions, and that changes to their protonation states result in their participation as switches between folded and unfolded conformations.


Assuntos
Criptófitas/química , Dinoflagelados/química , Complexos de Proteínas Captadores de Luz/química , Modelos Teóricos , Carotenoides/química , Clorofila , Conformação Molecular , Ficocianina/química , Proteínas/metabolismo , Proteínas de Protozoários/química , Eletricidade Estática
19.
Harmful Algae ; 80: 15-34, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30502808

RESUMO

Azaspiracids (AZA) are a group of lipophilic polyether compounds which have been implicated in shellfish poisoning incidents around Europe. They are produced by a few species of the dinophycean genera Azadinium and Amphidoma (Amphidomataceae). The presence of AZA toxins in Norway is well documented, but knowledge of the distribution and diversity of Azadinium and other Amphidomataceae along the Norwegian coast is rather limited and poorly documented. On a research survey along the Norwegian coast in 2015 from the Skagerrak in the South to Trondheimsfjorden in the North, plankton samples from 67 stations were analysed for the presence of Azadinium and Amphidoma and their respective AZA by on-board live microscopy, real-time PCR assays specific for Amphidomataceae, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Microscopy using live samples and positive real-time PCR assays using a general family probe and two species specific probes revealed the presence of Amphidomataceae distributed throughout the sampling area. Overall abundance was low, however, and was in agreement with a lack of detectable AZA in plankton samples. Single cell isolation and morphological and molecular characterisation of established strains revealed the presence of 7 amphidomatacean species (Azadiniun spinosum, Az. poporum, Az. obesum, Az. dalianense, Az. trinitatum, Az. polongum, Amphidoma languida) in the area. Azaspiracids were produced by the known AZA producing species Az. spinosum, Az. poporum and Am. languida only. LC-MS/MS analysis further revealed that Norwegian strains produce previously unreported AZA for Norway (AZA-11 by Az. spinosum, AZA-37 by Az. poporum, AZA-38 and AZA-39 by Am. languida), and also four novel compounds (AZA-50, -51 by Az. spinosum, AZA-52, -53 by Am. languida), whose structural properties are described and which now can be included in existing analytical protocols. A maximum likelihood analysis of concatenated rDNA regions (SSU, ITS1-ITS2, partial LSU) showed that the strains of Az. spinosum fell in two well supported clades, where most but not all new Norwegian strains formed the new Ribotype B. Ribotype differentiation was supported by a minor morphological difference with respect to the presence/absence of a rim around the pore plate, and was consistently reflected by different AZA profiles. Strains of Az. spinosum from ribotype A produce AZA-1, -2 and -33, whereas the new strains of ribotype B produce mainly AZA-11 and AZA-51. Significant sequence differences between both Az. spinosum ribotypes underline the need to redesign the currently used qPCR probes in order to detect all AZA producing Az. spinosum. The results generally underline the conclusion that for the Norwegian coast area it is important that amphidomatacean species are taken into account in future studies and monitoring programs.


Assuntos
Dinoflagelados/química , Monitoramento Ambiental , Toxinas Marinhas/análise , Plâncton/química , Compostos de Espiro/análise , Biodiversidade , Cromatografia Líquida , Demografia , Noruega , Densidade Demográfica , Espectrometria de Massas em Tandem
20.
Mar Drugs ; 16(12)2018 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-30545093

RESUMO

Marine dinoflagellates are a valuable source of bioactive molecules. Many species produce cytotoxic compounds and some of these compounds have also been investigated for their anticancer potential. Here, we report the first investigation of the toxic dinoflagellate Alexandrium minutum as source of water-soluble compounds with antiproliferative activity against human lung cancer cells. A multi-step enrichment of the phenol⁻water extract yielded a bioactive fraction with specific antiproliferative effect (IC50 = 0.4 µg·mL-1) against the human lung adenocarcinoma cells (A549 cell line). Preliminary characterization of this material suggested the presence of glycoprotein with molecular weight above 20 kDa. Interestingly, this fraction did not exhibit any cytotoxicity against human normal lung fibroblasts (WI38). Differential gene expression analysis in A549 cancer cells suggested that the active fraction induces specific cell death, triggered by mitochondrial autophagy (mitophagy). In agreement with the cell viability results, gene expression data also showed that no mitophagic event was activated in normal cells WI38.


Assuntos
Antineoplásicos/farmacologia , Organismos Aquáticos/química , Dinoflagelados/química , Toxinas Marinhas/farmacologia , /efeitos dos fármacos , Células A549 , Antineoplásicos/isolamento & purificação , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/uso terapêutico
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