RESUMO
Phorbas is a widely studied genus of marine sponge and produce structurally rich cytotoxic metabolites. Still, only few studies have assessed metabolites present in Brazilian species. To circumvent redundancy, in this work, we applied and herein report the use of a scouting liquid chromatographic system associate to the design of experiment produced by the DryLab® software to obtain a fast and efficient chromatographic separation of the active hexane fraction, further enabling untargeted high-resolution mass spectrometry (HRMS) data. To this end, a crude hydroalcoholic extract of the sponge Phorbas amaranthus collected in Brazilian coast was prepared and partitioned. The cytotoxicity of the crude extract and the fractions was evaluated using tumor cell culture models. Fragmentation pathways assembled from HRMS data allowed the annotation of 18 known Phorbas metabolites, while 17 metabolites were inferred based on Global Natural Product Social Molecular Networking (GNPS), matching with a further 29 metabolites annotated through molecular subnetwork. The workflow employed demonstrates that chromatographic method development can be accelerated by the use of automated scouting systems and DryLab®, which is useful for profiling natural product libraries, as well as data curation by molecular clusters and should be incorporated to the tools of natural product chemists.
Assuntos
Cromatografia Líquida/métodos , Poríferos , Extratos de Tecidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células HCT116 , Humanos , Lisofosfolipídeos/química , Poríferos/química , Poríferos/metabolismo , Esteroides/análise , Esteroides/química , Terpenos/análise , Terpenos/química , Extratos de Tecidos/análise , Extratos de Tecidos/metabolismo , Extratos de Tecidos/toxicidadeRESUMO
Extracts made from the skin of dead Lithodytes lineatus frog individuals with the application of the benzocaine-based anesthetic gel, introduced into the oral cavity, were analyzed by 1H Nuclear Magnetic Resonance to investigate whether the application of this product (oral) can make studies that use extracts from the skins of these animals unfeasible. For comparison, we used skins of another species of anuran following the same death protocol. No trace of the benzocaine substance was found in the 1H-NMR spectra of the skin extracts from any of the tested anuran species. Still, using the hierarchical clustering model, it was possible to observe the formation of well-defined groups between the skin extracts of anurans and the anesthetic used to kill these animals. Our results suggest that the lethal dose of benzocaine in gel used inside the mouth of frogs may have no influence on potential results regarding the chemical composition or even bioassays using extracts made from the skin of these animals killed under this protocol since there was no detection of this substance for the analyzed samples.
Assuntos
Anestésicos/análise , Anuros , Benzocaína/análise , Pele/química , Extratos de Tecidos/análise , Anestésicos/administração & dosagem , Animais , Benzocaína/administração & dosagem , Colágeno , Espectroscopia de Prótons por Ressonância Magnética , Manejo de Espécimes/métodos , Extratos de Tecidos/químicaRESUMO
The biological activities observed upon envenomation by Scorpaena plumieri could be linked to both the venom and the skin mucus. Through a proteomic/functional approach we analyzed protein composition and biological activities of the venom and skin mucus. We identified 885 proteins: 722 in the Venomous Apparatus extracts (Sp-VAe) and 391 in the Skin Mucus extract (Sp-SMe), with 494 found exclusively in Sp-VAe, being named S. plumieri Venom Proteins (Sp-VP), while 228 were found in both extracts. The majority of the many proteins identified were not directly related to the biological activities reported here. Nevertheless, some were classified as toxins/potentially interesting molecules: lectins, proteases and protease inhibitors were detected in both extracts, while the pore-forming toxin and hyaluronidase were associated with Sp-VP. Proteolytic and anti-microbial activities were linked to both extracts, while the main toxic activities - cardiovascular, inflammatory, hemolytic and nociceptive - were elicited only by Sp-VAe. Our study provided a clear picture on the composition of the skin mucus and the venom. We also show that the classic effects observed upon envenomation are produced by molecules from the venomous gland. Our results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins. SIGNIFICANCE: In this study a large number of proteins - including classical and non-classical toxins - were identified in the venomous apparatus and the skin mucus extracts of the Scorpaena plumieri fish through shotgun proteomic approach. It was shown that the toxic effects observed upon envenomation are elicited by molecules originated from the venomous gland. These results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins - so scarcely explored when compared to the venoms and bioactive components of terrestrial animals. Data are available via ProteomeXchange with identifier PXD009983.
Assuntos
Proteínas de Peixes/análise , Proteínas de Peixes/fisiologia , Venenos de Peixe/análise , Muco/química , Perciformes/metabolismo , Proteômica/métodos , Pele/química , Animais , Proteínas de Peixes/metabolismo , Venenos de Peixe/metabolismo , Venenos de Peixe/fisiologia , Masculino , Camundongos , Muco/metabolismo , Ratos , Ratos Wistar , Pele/metabolismo , Extratos de Tecidos/análise , Extratos de Tecidos/metabolismoRESUMO
Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.
Assuntos
Bivalves/química , Matriz Extracelular/química , Contaminação de Alimentos , Inspeção de Alimentos/métodos , Toxinas Marinhas/análise , Neurônios/efeitos dos fármacos , Frutos do Mar/análise , Alternativas aos Testes com Animais , Animais , Bivalves/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chile , Matriz Extracelular/metabolismo , Contaminação de Alimentos/prevenção & controle , Ensaios de Triagem em Larga Escala , Extração Líquido-Líquido , Toxinas Marinhas/biossíntese , Toxinas Marinhas/toxicidade , Camundongos , Neurônios/patologia , Reprodutibilidade dos Testes , Saxitoxina/análise , Saxitoxina/biossíntese , Saxitoxina/toxicidade , Frutos do Mar/efeitos adversos , Intoxicação por Frutos do Mar/etiologia , Intoxicação por Frutos do Mar/patologia , Intoxicação por Frutos do Mar/prevenção & controle , Especificidade da Espécie , Extratos de Tecidos/análise , Extratos de Tecidos/isolamento & purificação , Extratos de Tecidos/toxicidadeRESUMO
Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.
Assuntos
Flavivirus/química , RNA Viral/isolamento & purificação , Rhipicephalus/virologia , Proteínas não Estruturais Virais/química , Animais , Brasil , Bovinos , Sequência Conservada/genética , Flavivirus/classificação , Flavivirus/isolamento & purificação , Biblioteca Gênica , Interações Hidrofóbicas e Hidrofílicas , Filogenia , Reação em Cadeia da Polimerase , RNA Helicases/química , Alinhamento de Sequência/estatística & dados numéricos , Análise de Sequência de Proteína/métodos , Serina Endopeptidases/química , Extratos de Tecidos/análise , Transcriptoma/genéticaRESUMO
Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.
Assuntos
Animais , Bovinos , Flavivirus/química , RNA Viral/isolamento & purificação , Rhipicephalus/virologia , Proteínas não Estruturais Virais/química , Brasil , Sequência Conservada/genética , Flavivirus/classificação , Flavivirus/isolamento & purificação , Biblioteca Gênica , Interações Hidrofóbicas e Hidrofílicas , Filogenia , Reação em Cadeia da Polimerase , RNA Helicases/química , Alinhamento de Sequência/estatística & dados numéricos , Análise de Sequência de Proteína/métodos , Serina Endopeptidases/química , Extratos de Tecidos/análise , Transcriptoma/genéticaRESUMO
INTRODUCTION: Previous studies showed that placental extracts (PE) alleviates arthritic symptoms in animal models of arthritis. METHODS: To evaluate whether murine PEs obtained at embryonic days 7.5 (PE7) and 17.5 (PE18) regulate RANKL-induced osteoclast differentiation, RAW 264.7 cells were cultured with RANKL and MCSF in presence or not of PEs. Tartrate-resistant acid phosphatase (TRAP) was stained and multinucleated TRAP positive cells were visualized under a light microscope. Cathepsin K and metalloprotease expression was assessed by RT-PCR and gelatin zymography respectively. NFATc1 expression was determined by immunoblot. To analyze NFAT-dependent transcription, macrophages were transfected with a luciferase reporter plasmid. Cytokines were determined in PEs by ELISA and immunoblot. Transforming growth factor (TGF)- beta and Interleukin (IL)-10 receptor were inhibited in cell cultures with specific antibodies. RESULTS: PE7 and PE18 inhibited RANKL-induced multinucleated TRAP positive cells, Cathepsin K expression and metalloprotease activity, as well as NFATc1 expression and activity, thereby inhibiting osteoclast differentiation of RAW cells. Inflammatory/Regulatory cytokine ratio was higher in PE7 than in PE18. Blocking TGF-beta abolished the effect of both, PE7 and PE18, on multinucleated TRAP positive cells and metalloprotease expression, whereas blocking IL-10 receptor reverted the effect of PE18 but not of PE7. DISCUSSION: Inhibition of osteoclast differentiation by PEs was not unexpected, since cytokines detected in extracts were previously found to regulate osteoclast differentiation. CONCLUSIONS: PEs inhibited osteoclast differentiation of macrophages in vitro. Downregulation of NFATc1 might be involved in this effect. Regulatory/Th2 cytokines play a role in the effect of PEs on osteoclast differentiation.
Assuntos
Citocinas/farmacologia , Macrófagos/citologia , Osteoclastos/citologia , Placenta/metabolismo , Fosfatase Ácida/metabolismo , Animais , Catepsina K/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/análise , Embrião de Mamíferos , Feminino , Isoenzimas/metabolismo , Macrófagos/efeitos dos fármacos , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Placenta/química , Gravidez , Ligante RANK/farmacologia , Fosfatase Ácida Resistente a Tartarato , Extratos de Tecidos/análise , Extratos de Tecidos/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
O ácido retinóico (AR) tem sido utilizado para o tratamento de acne severa, rugas, estrias e celulite, no entanto, provoca irritação na pele e sofre rápida degradação quando exposto à luz e ao calor. Métodos analíticos rápidos para quantificação do AR são, portanto, necessários para ensaios de cinética de liberação in vitro. Nesse contexto, o objetivo deste trabalho foi desenvolver e validar um método rápido e sensível para o doseamento do AR em microcápsulas de alginato/quitosana contendo óleo de babaçu dispersas em gel natrosol® por cromatografia líquida de alta eficiência associada à espectroscopia UV e aplicá-lo na avaliação do perfil de liberação in vitro dessas formulações. As análises foram realizadas em modo isocrático utilizando coluna C18 de fase reversa 150 x 4,6 mm (5 μm) com detecção a 350 nm. A fase móvel foi constituída de metanol e ácido acético 1 por cento (85:15 v/v) com vazão de 1,8 mL/minuto. A faixa de linearidade do método foi de 0,5 a 60 μg/mL (r² = 0,999). O método validado mostrou-se sensível, específico, exato, preciso, de baixo custo e o tempo de retenção do AR foi de 5,8 ± 0,4 minutos sendo, desta forma, mais rápido do que os relatados na literatura.
Retinoic acid (RA) has been used in the treatment of severe acne, wrinkles and cellulite. However, it induces skin irritation and rapidly suffers degradation under light and high temperate exposure. Rapid analytical methods to quantify retinoic acid are therefore mandatory for in vitro drug release studies. In this framework, the aim of this study was to develop and validate a rapid and responsive method to quantify the RA in microcapsules of chitosan and alginate containing babassu oil dispersed in natrosol® hydrogel using high performance liquid chromatography (HPLC). Furthermore this method was used to quantify in vitro release kinetics of RA from microcapsules. The analyses have been carried through an isocratic HPLC-UV method using a reversed phase 150 x 4.6 mm C18 (5μm) column, a mobile phase constituted of methanol and 1 percent acetic acid (85:15) at a flow rate of 1.8 mL/min and detection at 350 nm. The linearity range was 0.5-60 μg/mL (r² = 0.999). The validated HPLC-UV method was responsive, specific, accurate, precise, and economic and the RA retention time was 5.8 ± 0.4 minutes, being therefore, faster than that previously reported.
Assuntos
Alginatos , Quitosana , Cromatografia Líquida/instrumentação , Tretinoína/farmacologia , Extratos de Tecidos/análise , Preparações Farmacêuticas/análise , Reprodutibilidade dos Testes/métodosRESUMO
Protein extraction methods [urea, trichloroacetic acid (TCA), and acetic acid] were compared for protein recovery from rat incisor developing enamel in the S phase (intermediate/late secretion), M1 phase (early maturation), M2 phase (intermediate maturation), and M3 phase (final maturation). We compared the protein recoveries with the percentage of enamel matrix dry weight burnt off by incineration. Our results indicate that TCA and urea were equally efficient for the extraction of S-stage proteins (85% and 90% recovery, respectively), while urea was the best for M1-stage proteins (92% recovery), and TCA the best for M2-stage (99% recovery) and M3-stage (60% recovery) proteins. The other methods yielded less than 30% recovery in comparison to incineration for M2 and M3 stages. The fact that urea extraction works well in the S and M1 stages and not thereafter is probably related to the changes in the proteins during enamel development and the amount of mineral that needs to be dissolved. TCA is the single method that effectively recovered proteins from all developmental stages of the rat incisor enamel.
Assuntos
Amelogênese , Proteínas do Esmalte Dentário/análise , Esmalte Dentário/química , Ácido Acético/química , Animais , Centrifugação , Esmalte Dentário/metabolismo , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Incisivo , Indicadores e Reagentes , Masculino , Inibidores de Proteases/química , Ratos , Ratos Wistar , Extratos de Tecidos/análise , Ácido Tricloroacético/química , Ultrafiltração , Ureia/químicaRESUMO
An extract of Triatoma infestans has previously been demonstrated to produce specific IgG and IgE both in animals and in atopic humans with rhinitis/asthma as well as hypersensitivity pneumonitis in guinea pigs aerosolized with T. infestans. We attempted to determine whether the antigen or antigens responsible belonged to the protease group, as occurs with other allergens such as house dust mites and cockroaches. To do this, T. infestans was studied by SDS-PAGE, Western blots and gelatinolysis with and without the use of specific protease inhibitors such as E-64, TLCK, TPCK, PMSF, leupeptin, o-phenanthrolene and pepstatin-A. These assays revealed serine-like proteolytic and gelatinolytic activities. The presence of 10 to 12 bands of between 14 and 100 kDa was detected. The proteolytic activity pattern of T. infestans was greatest at pH 8.5 and gelatinolytic activity was highly sensitive to PMSF, suggesting that this enzyme could be characterized as a serine protease. Western blots revealed that two bands of 17 and 58 kDA reacted with the sera of atopic humans with respiratory diseases and anti-IgE. However, whether these bands correlated with allergenicity is unclear since the presence of several proteins in each of these bands does not rule out the possibility that this correlation could exist, especially because cross-reactions with antigens from the cockroach Periplaneta americana and its specific antiserum in animals and atopic humans have been demonstrated. The role of proteases in the etiopathogenesis of perennial rhinitis and bronchial asthma in inhabitants of the area of Argentina infested by T. infestans requires further investigation.
Assuntos
Alérgenos/isolamento & purificação , Gelatinases/isolamento & purificação , Proteínas de Insetos/isolamento & purificação , Serina Endopeptidases/isolamento & purificação , Triatoma/enzimologia , Alérgenos/imunologia , Animais , Western Blotting , Baratas/imunologia , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Gelatinases/imunologia , Humanos , Soros Imunes , Proteínas de Insetos/imunologia , Coelhos , Serina Endopeptidases/imunologia , Especificidade da Espécie , Extratos de Tecidos/análise , Extratos de Tecidos/imunologia , Triatoma/imunologiaRESUMO
Se estudió el efecto angiogénico y la composición de proteínas y lípidos del omentum humano a partir de la inoculación intraestomal en la córnea de conejo de los extractos obteniodos tras homogeneizar el material biológico. Se encontraron valores muy bajos de proteínas y muy elevados de triglicéridos fundamentalmente en el extracto 2 de origen lípidico. En relación con la vascularización, ésta se logró con mayor intensidad en la córnea de los animales que se inocularon con el extracto 2
Assuntos
Animais , Coelhos , Colesterol/análise , Substância Própria/irrigação sanguínea , Inibidores da Angiogênese/análise , Neovascularização Fisiológica , Omento , Proteínas/análise , Coelhos , Extratos de Tecidos/análise , Triglicerídeos/análiseRESUMO
The methanol extract (mol. wt lower than 3,000 Da) of the sea squirt Phallusia nigra has stimulatory activity on guinea-pig ileum preparations. This effect was inhibited by cyproheptadine and mepyramine, but not be atropine. Mepyramine antagonized competitively the extract activity with a pA2 of 10.09 +/- 1.12, suggesting a direct activity on H1 histamine receptors. The extract was also assayed on guinea-pig right atria, however, only a mild increase in spontaneous contractions was observed compared to histamine, showing that the extract was a rather poor activator of cardiac H2 receptors. Histamine was not detected upon TLC analysis of the extract by comparison with an authentic standard.
Assuntos
Histamina/fisiologia , Extratos de Tecidos/farmacologia , Urocordados/química , Animais , Atropina/farmacologia , Ciproeptadina/farmacologia , Relação Dose-Resposta a Droga , Cobaias , Histamina/farmacologia , Íleo/efeitos dos fármacos , Íleo/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Pirilamina/farmacologia , Receptores Histamínicos H1/efeitos dos fármacos , Receptores Histamínicos H1/metabolismo , Extratos de Tecidos/análise , Extratos de Tecidos/química , Tubocurarina/farmacologiaRESUMO
We present a modification of a previously described radioenzymatic technique (Snyder et al., Journal of Pharmacology and Experimental Therapeutics, 153: 544-549, 1966) for the determination of histamine. This assay is based on the incubation of tissue histamine with a partially purified preparation of histamine-N-methyltransferase (S-adenosyl-L-methionine: histamine N-methyltransferase, E.C. 2.1.1.8; HMT) in the presence of the natural donor of methyl groups, [3H]-methyl-S-adenosyl-L-methionine ([3H]-SAMe). We have found that HMT retains its histamine N-methylating activity at 4 degrees C. Incubation at low temperature results in a considerable increase in the sample to blank ratio. The reduction of the total amount of [3H]-SAMe used and addition of anhydrous sodium sulfate before the last organic extraction step further reduced the blank levels. With these modifications, the sensitivity of the method was increased to the lower femtomole range. The present assay can be used for the determination of as little as 1 to 2 pg of histamine in samples from individual rat brain nuclei containing 2 to 10 micrograms of protein.
Assuntos
Química Encefálica , Histamina/análise , Extratos de Tecidos/análise , Animais , Histamina/biossíntese , Histamina N-Metiltransferase/metabolismo , Hipotálamo/metabolismo , Masculino , Microquímica , Ratos , Ratos Endogâmicos , S-Adenosilmetionina/metabolismoRESUMO
An opossum pancreatic insulin-like protein fraction was obtained at pH 5.2 which has high electrophoretic mobility. At pH 8.2, another pancreatic insulin-like protein fraction was obtained with low electrophoretic mobility. Isoelectric focusing studies showed that the high molecular weight protein has a pI value 8.35 and the low molecular weight protein has pI value of 3.9 when used at pH range between 3.5-10.0. The purified high and low molecular weight positive aldehyde-fuchsin proteins induced convulsions when administered subcutaneously to mice.
Assuntos
Convulsivantes/isolamento & purificação , Gambás/metabolismo , Pâncreas/análise , Proteínas/isolamento & purificação , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Masculino , Peso Molecular , Proteínas/análise , Especificidade da Espécie , Extratos de Tecidos/análiseRESUMO
The potent neurotoxin tetrodotoxin, which has previously been found in puffer fish of the order Tetraordontiformes, a goby (Gobius criniger), and the California newt (Taricha torosa), has now been identified in the skins of frogs of the genus Atelopus from Costa Rica.