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1.
Chemosphere ; 228: 139-148, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31029959

RESUMO

Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filtering shellfish and can cause human intoxications. Humans can be exposed to combinations of several phycotoxins. The toxicological effects of phycotoxin mixtures on human health are largely unknown. Published data on phycotoxin co-exposure show that okadaic acid (OA) is simultaneously found with pectenetoxin-2 (PTX-2), 13-desmethylspirolide C (also known as SPX-1), or yessotoxin (YTX). Therefore, the aim of this study was to examine the effects of three binary mixtures, OA/PTX-2, OA/SPX-1 and OA/YTX on human intestinal Caco-2 cells. A multi-parametric approach for cytotoxicity determination was applied using a high-content analysis platform, including markers for cell viability, oxidative stress, inflammation, and DNA damage. Mixtures effects were analyzed using two additivity mathematical models. Our assays revealed that OA induced cytotoxicity, DNA strand breaks and interleukin 8 release. PTX-2 slightly induced DNA strand breaks, whereas SPX-1 and YTX did not affect the investigated endpoints. The combination of OA with another toxin resulted in reduced toxicity at low concentrations, suggesting antagonistic effects, but in increased effects at higher concentrations, suggesting additive or synergistic effects. Taken together, our results demonstrated that the cytotoxic effects of binary mixtures of lipophilic phycotoxins could not be predicted by additivity mathematical models. In conclusion, the present data suggest that combined effects of phycotoxins may occur which might have the potential to impact on risk assessment of these compounds.


Assuntos
Células CACO-2/efeitos dos fármacos , Combinação de Medicamentos , Interações Medicamentosas , Toxinas Marinhas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Furanos/farmacologia , Humanos , Inflamação , Intestinos/citologia , Toxinas Marinhas/análise , Ácido Okadáico/análise , Ácido Okadáico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Oxocinas/farmacologia , Piranos/farmacologia , Frutos do Mar/análise , Frutos do Mar/toxicidade , Compostos de Espiro/farmacologia
2.
Toxins (Basel) ; 11(2)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717108

RESUMO

Gambierdiscus species are the producers of the marine toxins ciguatoxins and maitotoxins which cause worldwide human intoxications recognized as Ciguatera Fish Poisoning. A deep chemical investigation of a cultured strain of G. belizeanus, collected in the Caribbean Sea, led to the identification of a structural homologue of the recently described gambierone isolated from the same strain. The structure was elucidated mainly by comparison of NMR and MS data with those of gambierone and ascertained by 2D NMR data analyses. Gratifyingly, a close inspection of the MS data of the new 44-methylgambierone suggests that this toxin would actually correspond to the structure of maitotoxin-3 (MTX3, m/z 1039.4957 for the protonated adduct) detected in 1994 in a Pacific strain of Gambierdiscus and recently shown in routine monitoring programs. Therefore, this work provides for the first time the chemical identification of the MTX3 molecule by NMR. Furthermore, biological data confirmed the similar activities of both gambierone and 44-methylgambierone. Both gambierone and MTX3 induced a small increase in the cytosolic calcium concentration but only MTX3 caused cell cytotoxicity at micromolar concentrations. Moreover, chronic exposure of human cortical neurons to either gambierone or MTX3 altered the expression of ionotropic glutamate receptors, an effect already described before for the synthetic ciguatoxin CTX3C. However, even when gambierone and MTX3 affected glutamate receptor expression in a similar manner their effect on receptor expression differed from that of CTX3C, since both toxins decreased AMPA receptor levels while increasing N-methyl-d-aspartate (NMDA) receptor protein. Thus, further studies should be pursued to clarify the similarities and differences in the biological activity between the known ciguatoxins and the new identified molecule as well as its contribution to the neurological symptoms of ciguatera.


Assuntos
Toxinas Marinhas/química , Toxinas Marinhas/farmacologia , Oxocinas/química , Oxocinas/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciguatoxinas/farmacologia , Dinoflagelados , Éteres/química , Éteres/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de Glutamato/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-30476593

RESUMO

The activities of two effectors, brevetoxin (PbTx) and manumycin-A (Man-A), of thioredoxin reductase (TrxR) have been evaluated against a series of fourteen TrxR orthologs originating from mammals, insects and protists and several mutants. Man-A, a molecule with numerous electrophilic sites, forms a covalent adduct with most selenocystine (Sec)-containing TrxR enzymes. The evidence also demonstrates that Man-A can form covalent adducts with some non-Sec-containing enzymes. The activities of TrxR enzymes towards various substrates are moderated by Man-A either positively or negatively depending on the enzyme. In general, the reduction of substrates by Sec-containing TrxR is inhibited and NADPH oxidase activity is activated. For non-Sec-containing TrxR the effect of Man-A on the reduction of substrates is variable, but NADPH oxidase activity can be activated even in the absence of covalent modification of TrxR. The effect of PbTx is less pronounced. A smaller subset of enzymes is affected by PbTx. With a single exception, the activities of most of this subset are activated. Although both PbTx variants can react with selenocysteine, a stable covalent adduct is not formed with any of the TrxR enzymes. The key findings from this work are (i) the identification of an alternate mechanism of toxicity for the algal toxin brevetoxin (ii) the demonstration that covalent modification of TrxR is not a prerequisite for the activation of NADPH oxidase activity of TrxR and (iii) the identification of an inhibitor which can discriminate between cytosolic and mitochondrial TrxR.


Assuntos
Toxinas Marinhas/farmacologia , Oxocinas/farmacologia , Polienos/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Tiorredoxina Dissulfeto Redutase/metabolismo , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Insetos , Mamíferos , Toxinas Marinhas/química , NADPH Oxidases/metabolismo , Oxocinas/química , Polienos/química , Alcamidas Poli-Insaturadas/química , Especificidade da Espécie , Tiorredoxinas/metabolismo
4.
J Org Chem ; 82(18): 9595-9618, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28840731

RESUMO

Structure-activity relationship studies of maitotoxin (MTX), a marine natural product produced by an epiphytic dinoflagellate, were conducted using chemically synthesized model compounds corresponding to the partial structures of MTX. Both enantiomers of the LMNO ring system were synthesized via aldol reaction of the LM ring aldehyde and the NO ring ketone. These fragments were derived from a common cis-fused pyranopyran intermediate prepared through a sequence involving Nozaki-Hiyama-Kishi reaction, intramolecular oxa-Michael addition, and Pummerer rearrangement. The NOPQR(S) ring system, in which the original seven-membered S ring was substituted with a six-membered ring, was also synthesized through the coupling of the QR(S) ring alkyne and the NO ring aldehyde and the construction of the P ring via 1,4-reduction, dehydration, and hydroboration. The inhibitory activities of the synthetic specimens against MTX-induced Ca2+ influx were evaluated. The LMNO ring system and its enantiomer induced 36 and 18% inhibition, respectively, at 300 µM, whereas the NOPQR(S) ring system elicited no inhibitory activity.


Assuntos
Aldeídos/farmacologia , Cálcio/metabolismo , Glioma/metabolismo , Cetonas/farmacologia , Toxinas Marinhas/antagonistas & inibidores , Óxido Nítrico/farmacologia , Oxocinas/antagonistas & inibidores , Piranos/farmacologia , Aldeídos/química , Animais , Relação Dose-Resposta a Droga , Cetonas/química , Toxinas Marinhas/química , Toxinas Marinhas/farmacologia , Conformação Molecular , Óxido Nítrico/química , Oxocinas/química , Oxocinas/farmacologia , Piranos/síntese química , Piranos/química , Ratos , Estereoisomerismo
5.
Mar Drugs ; 15(7)2017 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-28672825

RESUMO

Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.


Assuntos
Toxinas Marinhas/farmacologia , Oócitos/efeitos dos fármacos , Oxocinas/farmacologia , Canais de Cátion TRPC/metabolismo , Xenopus , Animais , Estimulação Elétrica , Eletrofisiologia , Potenciais da Membrana/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Cátion TRPC/genética
6.
PLoS One ; 11(12): e0167572, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27973568

RESUMO

Yessotoxins (YTXs) are a group of marine toxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. They may have medical interest due to their potential role as anti-allergic but also anti-cancer compounds. However, their biological activities remain poorly characterized. Here, we show that the small molecular compound YTX causes a slight but significant reduction of the ability of mast cells to degranulate. Strikingly, further examination revealed that YTX had a marked and selective cytotoxicity for the RBL-2H3 mast cell line inducing apoptosis, while primary bone marrow derived mast cells were highly resistant. In addition, YTX exhibited strong cytotoxicity against the human B-chronic lymphocytic leukaemia cell line MEC1 and the murine melanoma cell line B16F10. To analyse the potential role of YTX as an anti-cancer drug in vivo we used the well-established B16F10 melanoma preclinical mouse model. Our results demonstrate that a few local application of YTX around established tumours dramatically diminished tumour growth in the absence of any significant toxicity as determined by the absence of weight loss and haematological alterations. Our data support that YTX may have a minor role as an anti-allergic drug, but reveals an important potential for its use as an anti-cancer drug.


Assuntos
Antialérgicos/farmacologia , Antineoplásicos/farmacologia , Oxocinas/farmacologia , Animais , Antialérgicos/efeitos adversos , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dinoflagelados/química , Humanos , Toxinas Marinhas/efeitos adversos , Toxinas Marinhas/farmacologia , Camundongos , Oxocinas/efeitos adversos
7.
Mar Drugs ; 14(2)2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26828502

RESUMO

Yessotoxin (YTX) is a polyether compound produced by dinoflagellates and accumulated in filter feeding shellfish. No records about human intoxications induced by this compound have been published, however it is considered a toxin. Modifications in second messenger levels, protein levels, immune cells, cytoskeleton or activation of different cellular death types have been published as consequence of YTX exposure. This review summarizes the main intracellular pathways modulated by YTX and their pharmacological and therapeutic implications.


Assuntos
Dinoflagelados/metabolismo , Oxocinas/isolamento & purificação , Frutos do Mar/análise , Animais , Morte Celular/efeitos dos fármacos , Humanos , Oxocinas/farmacologia , Oxocinas/toxicidade , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
8.
Rev Biol Trop ; 64(2): 805-16, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29451969

RESUMO

The increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins.


Assuntos
Dinoflagelados/química , Oxocinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Vibrio vulnificus/efeitos dos fármacos , Dinoflagelados/genética , Reação em Cadeia da Polimerase
9.
Integr Cancer Ther ; 15(1): 87-95, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26036624

RESUMO

HYPOTHESIS: To assess the antitumor effects of protosappanin B extracted from Lignum Sappan. STUDY DESIGN: Lignum Sappan was sequentially extracted by boiling water and ethyl acetate. The resulting extract was separated by column chromatography, to yield protosappanin B. The compound was then identified by thin-layer chromatography, high-performance liquid chromatography, elemental analysis, and spectrometry (infrared and ultraviolet). The effects on tumor cell viability and growth of purified protosappanin B were evaluated in vitro by trypan blue exclusion and MTT assays, respectively. And the effects of protosappanin B were assessed in vivo, on H22 mouse liver cancer cell invasion and the survival of tumor-bearing mice. RESULTS: Protosappanin B (2 mg/mL) reduced the viability of human bladder cancer T24 cells and mouse bladder cancer BTT cells in a time-dependent manner (P < .05) and significantly inhibited the growth of the human colon cancer cell lines HCT-116 and SW-480. IC50 values of 21.32, 26.73, and 76.53 µg/mL were obtained for SW-480, HCT-116, and BTT cells, respectively, after 48 hours of treatment with protosappanin B. In addition, pretreatment of H22 cells with protosappanin B (final concentration = 6.25 mg/mL) resulted in complete inhibition of tumor formation in KM mice. Furthermore, protosappanin B (200 and 300 mg/kg) significantly increased the survival of BTT tumor-bearing T739 mice, at a rate comparable to that of 1 mg/kg mitomycin. CONCLUSION: Protosappanin B extracted from Lignum Sappan exerts marked antitumor effects both in vitro and in vivo.


Assuntos
Antineoplásicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Oxocinas/farmacologia , Extratos Vegetais/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HCT116 , Humanos , Camundongos
10.
Toxicol In Vitro ; 29(7): 1545-54, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26025416

RESUMO

Protein Kinase C (PKC) is a group of enzymes involved in pro-survival or pro-apoptotic events depending on the cellular model. Moreover, Yessotoxin (YTX) modulates its expression and activates different cell death pathways. In K-562 tumor cell line, YTX induces apoptosis and autophagy after 24 and 48 h of incubation, respectively, and the toxin carries out its action through the phosphodiesterase 4A (PDE4A). Therefore, the levels of two subtypes of PKC, conventional (cPKC) and δ isotype of novel PKC (PKCδ) were studied at these times after YTX incubation. Also their involvement in the cell death activated by the toxin and their relationship with PDE4A was checked. The expression of cPKC and PKCδ in cytosol, plasma membrane and nucleus was studied in normal and PDE4A-silenced cells. Furthermore, cell viability of normal cells, as well as cPKC-, PKCδ- and PDE4A-silenced cells was tested by Lactate Dehydrogenase (LDH) assay. As a result, PKCδ showed a key role in K-562 cell survive, since without this protein, K-562 cell decreased their viability. Furthermore, modulation of PKCs by YTX treatment was observed, however, the changes in the expression of these proteins are independent of cell death activated by the toxin. In addition, the modulation of PKCs detected is PDE4A-dependent, since the silencing of this protein change PKC expression pattern.


Assuntos
Oxocinas/farmacologia , Proteína Quinase C/metabolismo , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Citosol/metabolismo , Humanos , Isoenzimas/metabolismo , Células K562 , Leucemia/metabolismo
11.
J Pharm Pharmacol ; 67(10): 1439-47, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25920539

RESUMO

OBJECTIVES: This study aims to investigate antimicrobial ingredients from Sappan Lignum and to evaluate their synergy on methicillin-resistant Staphylococcus aureus strains with antibiotics. METHODS: Bioactivity-guided phytochemical procedures were used to screen the active compounds. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were assayed by broth microdilution. The synergy was evaluated through checkerboard microdilution and loss of viability assays. KEY FINDINGS: Protosappanins A (PsA) and B (PsB) were identified from Sappan Lignum extracts. They showed active against both S. aureus and MRSA with MIC or MIC50 at 64 (PsA) and 128 (PsB) mg/L alone. When they were used in combination with antibiotics, they showed best synergy with amikacin and gentamicin with MIC50 (mg/L) of amikacin reduced more significantly from 32 to four (with PsA) and eight (with PsB), and the fractional inhibitory concentration index (FICI) ranged between 0.078 and 0.500 (FICI50 = 0.375). Moreover, the resistance of MRSA towards amikacin and gentamicin could be reversed by the Clinical and Laboratory Standards Institute criteria. The combined bactericidal mode could as well be synergy. PsA and PsB showed very low cytotoxicity in comparison with their promising activity against MRSA. CONCLUSIONS: Protosappanins A and B showed both alone activities and resistance reversal effects of amikacin and gentamicin against MRSA, which warrant further investigations for potential combinatory therapy of MRSA infection.


Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxocinas/farmacologia , Fenóis/farmacologia , Amicacina/farmacologia , Antibacterianos/administração & dosagem , Antibacterianos/isolamento & purificação , Caesalpinia/química , Linhagem Celular , Linhagem Celular Tumoral , Farmacorresistência Bacteriana/efeitos dos fármacos , Sinergismo Farmacológico , Gentamicinas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Oxocinas/administração & dosagem , Oxocinas/isolamento & purificação , Fenóis/administração & dosagem , Fenóis/isolamento & purificação
12.
Eur J Pharmacol ; 751: 13-23, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25657114

RESUMO

Protosappanin B (PTB) is a bioactive dibenzoxocin derivative isolated from Caesalpinia sappan L. Here, we investigated the neuroprotective effects and the potential mechanisms of PTB on oxygen-glucose deprivation (OGD)-injured PC12 cells. Results showed that PTB significantly increased cell viability, inhibited cell apoptosis and up-regulated the expression of growth-associated protein 43 (a marker of neural outgrowth). Moreover, our study revealed that PTB effectively maintained mitochondrial homeostasis by up-regulation of mitochondrial membrane potential (MMP), inhibition of cytochrome c release from mitochondria and inactivation of mitochondrial caspase-9/3 apoptosis pathway. Further study showed that PTB significantly promoted cytoplasmic component degradation of p53 protein, a key negative regulator for mitochondrial function, resulting in a release of Bcl-2 from p53-Bcl-2 complex and an enhancing translocation of Bcl-2 to mitochondrial outer membrane. Finally, we found the degradation of p53 protein was induced by PTB via activation of a MDM2-dependent ubiquitination process. Taken together, our findings provided a new viewpoint of neuronal protection strategy for anoxia and ischemic injury with natural small molecular dibenzoxocin derivative by activating ubiquitin-dependent p53 protein degradation as well as increasing mitochondrial function.


Assuntos
Glucose/deficiência , Homeostase/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oxocinas/farmacologia , Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinas/metabolismo , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Morte Celular/efeitos dos fármacos , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Células PC12 , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ratos , Ubiquitinação/efeitos dos fármacos
13.
ACS Chem Neurosci ; 4(7): 1062-70, 2013 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-23527608

RESUMO

Yessotoxin is a marine phycotoxin that induces motor alterations in mice after intraperitoneal injection. In primary cortical neurons, yessotoxin treatment induced a caspase-independent cell death with an IC50 of 4.27 nM. This neurotoxicity was enhanced by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and partially blocked by amiloride. Unlike previous studies, yessotoxin did not increase cyclic adenosine monophosphate levels or produce any change in phosphodiesterase 4 steady state expression in triple transgenic neurons. Since phosphodiesterases (PDEs) are engaged in learning and memory, we studied the in vitro effect of the toxin against Alzheimer's disease hallmarks and observed that pretreatment of cortical 3xTg-AD neurons with a low nanomolar concentration of yessotoxin showed a decrease expression of hyperphosphorylated tau isoforms and intracellular accumulation of amyloid-beta. These effects were accompanied with an increase in the level of the inactive isoform of the glycogen synthase kinase 3 and also by a translocation of protein kinase C from cytosol to membrane, pointing to its activation. In fact, inhibition of protein kinase C with GF109203X blocked the effect of yessotoxin over tau protein. The data presented here shows that 1 nM yessotoxin activates protein kinase C with beneficial effects over the main Alzheimer's disease hallmarks, tau and Aß, in a cellular model obtained from 3xTg-AD fetuses.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Oxocinas/farmacologia , Proteína Quinase C/metabolismo , Proteínas tau/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Quinase 3 da Glicogênio Sintase/metabolismo , Indóis/farmacologia , Maleimidas/farmacologia , Camundongos , Camundongos Transgênicos , Proteína Quinase C/antagonistas & inibidores
14.
Environ Sci Pollut Res Int ; 20(2): 1189-92, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23054782

RESUMO

Yessotoxins (YTXs) are polycyclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter-feeding shellfish. Mouse bioassay is still the official method to detect these toxins, even if it is lacking of specificity and sensitivity. Moreover, there is growing resistance against the use of animal experiments. Many efforts have been made to determine YTXs with other methods. The detection of YTX using a functional assay allows its quantification with an automated and repetitive technique at concentrations in the range of the 1 mg of YTX equivalent/kg European regulatory limit. In this study, an in vitro functional assay based on YTX treatment of MCF-7 cells and resulting in the accumulation of a 100-kDa fragment of E-cadherin was developed on samples of Mytilus galloprovincialis collected from the Adriatic Sea, Italy, along the coasts of Abruzzo, Molise, and Emilia Romagna regions. The YTX concentrations ranged from 0.2 to 1.8 mg of YTX equivalent/kg. The occurrence of levels exceeding the above mentioned limit was observed only in samples of Emilia Romagna region. This last result could represent a risk for human health, but these shellfish were not intended to consumers, because they belonged to a preventive monitoring program.


Assuntos
Ecotoxicologia/métodos , Mytilus/química , Oxocinas/análise , Animais , Caderinas/análise , Caderinas/metabolismo , Monitoramento Ambiental/métodos , Humanos , Itália , Células MCF-7/efeitos dos fármacos , Oxocinas/farmacologia , Sensibilidade e Especificidade
15.
Electrophoresis ; 33(24): 3786-97, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23161537

RESUMO

MS-based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal-glial culture were subjected to excitotoxin N-methyl-D-aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH-L1, were first detected and assessed in the culture media by Western blot. The cell-conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data-dependent scanning but not UCH-L1. As a proof-of-principle study, rat glia-enriched cell cultures treated with MTX were used to investigate the time-dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial-enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS.


Assuntos
Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Espectrometria de Massas/métodos , Neuroglia/metabolismo , Neurônios/metabolismo , Proteômica/métodos , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/análise , Biomarcadores/metabolismo , Lesões Encefálicas/patologia , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/citologia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/química , Proteína Glial Fibrilar Ácida/metabolismo , Toxinas Marinhas/farmacologia , N-Metilaspartato/farmacologia , Necrose/metabolismo , Neuroglia/química , Neuroglia/citologia , Neurônios/química , Neurônios/citologia , Oxocinas/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Ubiquitina Tiolesterase/análise , Ubiquitina Tiolesterase/metabolismo
16.
J Cell Biochem ; 113(12): 3752-61, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22807343

RESUMO

Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3',5'-cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca(2+) ), whereas the contrary effect has been observed in a Ca(2+) -free medium. In the present article, the effect of YTX in K-562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K-562 cell line are completely opposite than in fresh human lymphocytes, since in K-562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K-562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K-562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca(2+) increase in fresh human lymphocytes but no effect was observed on Ca(2+) pools depletion in these cells. However, our results show that, in K-562 cells, YTX has no effect on cytosolic Ca(2+) levels in a medium with Ca(2+) and induces an increase on Ca(2+) pools depletion followed by a Ca(2+) influx. As far as Ca(2+) modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca(2+) reservoirs affected by YTX are different than thapsigargin-sensible pools. Furthermore, YTX-dependent Ca(2+) pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase-4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p-(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca(2+) , YTX and cAMP pathways. Also, results obtain demonstrate that YTX-dependent Ca(2+) influx was only abolished by FCCP pre-treatment, which indicates a link between YTX and mitochondria in K-562 cell line. Cytosolic expression of A-kinase anchor proteins (AKAPs), the proteins which integrates phosphodiesterases (PDEs) and PKA to the mitochondria, was determined in both cell models. On the one hand, in human fresh lymphocytes, YTX increases AKAP149 cytosolic expression. This fact is accompanied with a decrease in cAMP levels, and therefore PDEs activation, which finally leads to cell survival. On the other hand, in tumoural lymphocytes, YTX has an opposite effect since decreases AKAP149 cytosolic expression and increase cAMP levels which leads to cell death. This is the first time that YTX and mitochondrial AKAPs proteins relationship is characterised.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Linfócitos/efeitos dos fármacos , Oxocinas/farmacologia , Antineoplásicos/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Sobrevivência Celular , Meios de Cultura/metabolismo , AMP Cíclico/análogos & derivados , Citosol/metabolismo , Ativação Enzimática , Humanos , Isoquinolinas/farmacologia , Células K562 , Linfócitos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Rolipram/farmacologia , Sulfonamidas/farmacologia , Tapsigargina/farmacologia
17.
Mar Drugs ; 10(3): 583-97, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22611355

RESUMO

Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2). Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining) remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01) and CYP356A1 (p < 0.05) transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx) remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP) and stress (Hsp70), but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD) related genes.


Assuntos
Crassostrea/fisiologia , Hemócitos/fisiologia , Toxinas Marinhas/farmacologia , Oxocinas/farmacologia , Transcrição Genética/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Corantes , DNA Complementar/biossíntese , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Hemolinfa/citologia , Inativação Metabólica , Vermelho Neutro , RNA/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Estresse Fisiológico/genética , Sais de Tetrazólio , Tiazóis
18.
Bioorg Med Chem Lett ; 22(4): 1527-32, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22284816

RESUMO

A series of 3,6-epoxy [1,5]dioxocines were synthesized and evaluated for their antifilarial activity against adult parasites of human lymphatic filarial parasite Brugia malayi (sub-periodic strain) in vitro. Out of these, six compounds (4a-f) possessed improved in vitro anti-filarial activity and examples 4d and 4f were also found to be active in the in vivo experiments. These results demonstrate that 3,6-epoxy [1,5]dioxocines exhibits potent antifilarial activity and might be developed into a new class of antifilarial drug.


Assuntos
Brugia Malayi/efeitos dos fármacos , Compostos de Epóxi/síntese química , Filaricidas/farmacologia , Oxocinas/síntese química , Animais , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Feminino , Filaricidas/síntese química , Filaricidas/química , Concentração Inibidora 50 , Estrutura Molecular , Oxocinas/química , Oxocinas/farmacologia
19.
PLoS One ; 6(12): e28015, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22174763

RESUMO

Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one) was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus.


Assuntos
Celulose/antagonistas & inibidores , Chalconas/farmacologia , Técnicas de Química Combinatória/métodos , Gluconacetobacter xylinus/efeitos dos fármacos , Oxocinas/farmacologia , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Celulose/biossíntese , Chalconas/química , Cristalização , Meios de Cultura/farmacologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Gluconacetobacter xylinus/citologia , Gluconacetobacter xylinus/crescimento & desenvolvimento , Gluconacetobacter xylinus/ultraestrutura , Glucosiltransferases/metabolismo , Oxocinas/química , Bibliotecas de Moléculas Pequenas/química , Difração de Raios X
20.
Toxicol Lett ; 207(2): 167-72, 2011 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-21925578

RESUMO

We have studied the effects of the marine algal toxins yessotoxin (YTX) and okadaic acid (OA) on the T cell receptor complex (TCR) expression, an important mechanism by which T cell responsiveness is controlled. Immune system cells are relevant targets to study the immunoregulatory potential of marine toxins since the immune system has been reported as one of the targets of marine algal toxins. This study reports results from exposing the mouse T lymphocyte cell line EL-4 to increasing concentrations of YTX and OA for 72h. We found that both YTX and OA affected TCR recycling kinetics and induced a specific and reversible TCR down-regulation in T lymphocyte EL-4 cells that was time and concentration dependent. Experiments using the potent protein kinase C (PKC) inhibitor stausporine indicated that YTX-induced TCR down-regulation was partially mediated by PKC activation. In contrast, OA-induced TCR down-regulation was mediated by the serine/threonine protein phophatase 2A (PP2A) inhibition. In summary, the results suggest that OA and YTX concentrations in a similar range than those detected in mice bloodstream after oral administration have the potential to adjust the T cell responsiveness during the initiation of T cell activation by affecting the TCR expression levels via PKC and PP2A activities.


Assuntos
Adjuvantes Imunológicos/farmacologia , Ácido Okadáico/farmacologia , Oxocinas/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Complexo CD3/biossíntese , Linhagem Celular , Citometria de Fluxo , Camundongos , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/fisiologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/biossíntese , Complexo Receptor-CD3 de Antígeno de Linfócitos T/efeitos dos fármacos , Estaurosporina/farmacologia
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