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1.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500646

RESUMO

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A2 (cPLA2) functions as an N-acyltransferase to form NAPE. Since the cPLA2 family consists of six isoforms (α, ß, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA2 isoforms were labeled with [14C]ethanolamine, the increase in the production of [14C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [14C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [14C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA2γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA2γ is involved in the biosynthesis of NAE by its phospholipase A1/A2 and lysophospholipase activities.


Assuntos
Etanolaminas/metabolismo , Fosfolipases A2/metabolismo , Isoformas de Proteínas/metabolismo , Aciltransferases/metabolismo , Amidas/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Endocanabinoides/metabolismo , Etanolamina/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Oleicos/metabolismo , Ácidos Palmíticos/metabolismo , Fosfatidiletanolaminas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Alcamidas Poli-Insaturadas/metabolismo
2.
J Immunol ; 207(5): 1275-1287, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34389624

RESUMO

The airway epithelial cells (AECs) lining the conducting passageways of the lung secrete a variety of immunomodulatory factors. Among these, PGE2 limits lung inflammation and promotes bronchodilation. By contrast, IL-6 drives intense airway inflammation, remodeling, and fibrosis. The signaling that differentiates the production of these opposing mediators is not understood. In this study, we find that the production of PGE2 and IL-6 following stimulation of human AECs by the damage-associated molecular pattern extracellular ATP shares a common requirement for Ca2+ release-activated Ca2+ (CRAC) channels. ATP-mediated synthesis of PGE2 required activation of metabotropic P2Y2 receptors and CRAC channel-mediated cytosolic phospholipase A2 signaling. By contrast, ATP-evoked synthesis of IL-6 occurred via activation of ionotropic P2X receptors and CRAC channel-mediated calcineurin/NFAT signaling. In contrast to ATP, which elicited the production of both PGE2 and IL-6, the uridine nucleotide, UTP, stimulated PGE2 but not IL-6 production. These results reveal that human AECs employ unique receptor-specific signaling mechanisms with CRAC channels as a signaling nexus to regulate release of opposing immunomodulatory mediators. Collectively, our results identify P2Y2 receptors, CRAC channels, and P2X receptors as potential intervention targets for airway diseases.


Assuntos
Dinoprostona/metabolismo , Inflamação/imunologia , Interleucina-6/metabolismo , Mucosa Respiratória/metabolismo , Trifosfato de Adenosina/farmacocinética , Alarminas/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Células Cultivadas , Humanos , Imunomodulação , Interleucina-6/genética , Fatores de Transcrição NFATC/metabolismo , Fosfolipases A2/metabolismo , Receptores Purinérgicos P2X/metabolismo , Mucosa Respiratória/patologia , Transdução de Sinais , Nucleotídeos de Uracila/metabolismo
3.
Int J Biol Macromol ; 185: 494-512, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34197854

RESUMO

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a KD of 9.146 × 10-7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA2, thus proposing a new mechanism of PLA2 inhibition, and presenting more evidence of GA's potential as an antivenom compound.


Assuntos
Anacardium/química , Ácido Gálico/farmacologia , Miotoxicidade/tratamento farmacológico , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Venenos de Serpentes/enzimologia , Animais , Modelos Animais de Doenças , Ácido Gálico/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Miotoxicidade/enzimologia , Miotoxicidade/etiologia , Inibidores de Fosfolipase A2/química , Fosfolipases A2/química , Caules de Planta/química , Proteínas de Répteis/química , Proteínas de Répteis/metabolismo , Ressonância de Plasmônio de Superfície
4.
Toxicology ; 458: 152841, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34216699

RESUMO

The cardiotoxicity of various anticancer therapies, including radiotherapy, can lead to cardiovascular complications. These complications can range from damaging cardiac tissues within the irradiation field to increasing the long-term risks of developing heart failure, coronary artery disease, and myocardial infarction. We analyzed radiation-induced metabolites capable of mediating critical biological processes, such as inflammation, senescence, and apoptosis. Previously, by applying QTOF-MASS analysis to irradiated human fibroblasts, we identified that metabolite sets of lysophosphatidylcholine (LPC) were increased in these cells. In this study, radiation-induced LPC accumulation in human aortic endothelial cells (HAECs) increased reactive oxygen species (ROS) production and senescence-associated-beta-galactosidase staining, in addition to decreasing their tube-forming ability. Knockdown of lipoprotein-associated phospholipase A2 (Lp-PLA2) with small interfering RNA (siRNA) inhibited the increased LPC production induced by radiation, and reduced the radiation-induced cell damage produced by ROS and oxidized low-density lipoprotein (LDL). Lp-PLA2 depletion abolished the induction of proinflammatory factors, such as interleukin 1ß, tumor necrosis factor-alpha, matrix metalloproteinase 2, and matrix metalloproteinase 9, as well as adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and E-selection. Likewise, we showed that Lp-PLA2 expression was upregulated in the vasculature of irradiated rat, resulting in increased LPC production and LDL oxidation. Our data demonstrate that radiation-induced LPC production is a potential risk factor for cardiotoxicity that is mediated by Lp-PLA2 activity, suggesting that LPC and Lp-PLA2 offer potential diagnostic and therapeutic approaches to cardiovascular damage during radiotherapy.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/efeitos da radiação , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Lisofosfatidilcolinas/metabolismo , Fosfolipases A2/metabolismo , Fosfolipases A2/efeitos da radiação , Animais , Aorta/patologia , Aorta/efeitos da radiação , Citocinas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Inflamação/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/efeitos da radiação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/efeitos da radiação , Radiação Ionizante , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo
5.
Nat Metab ; 3(6): 762-773, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34140694

RESUMO

Chronic pain is the leading cause of disability worldwide1 and is commonly associated with comorbid disorders2. However, the role of diet in chronic pain is poorly understood. Of particular interest is the Western-style diet, enriched with ω-6 polyunsaturated fatty acids (PUFAs) that accumulate in membrane phospholipids and oxidise into pronociceptive oxylipins3,4. Here we report that mice administered an ω-6 PUFA-enriched diet develop persistent nociceptive hypersensitivities, spontaneously active and hyper-responsive glabrous afferent fibres and histologic markers of peripheral nerve damage reminiscent of a peripheral neuropathy. Linoleic and arachidonic acids accumulate in lumbar dorsal root ganglia, with increased liberation via elevated phospholipase (PLA)2 activity. Pharmacological and molecular inhibition of PLA2G7 or diet reversal with high levels of ω-3 PUFAs attenuate nociceptive behaviours, neurophysiologic abnormalities and afferent histopathology induced by high ω-6 intake. Additionally, ω-6 PUFA accumulation exacerbates allodynia observed in preclinical inflammatory and neuropathic pain models and is strongly correlated with multiple pain indices of clinical diabetic neuropathy. Collectively, these data reveal dietary enrichment with ω-6 PUFAs as a new aetiology of peripheral neuropathy and risk factor for chronic pain and implicate multiple therapeutic considerations for clinical pain management.


Assuntos
Biomarcadores , Dor Crônica/etiologia , Dor Crônica/metabolismo , Suscetibilidade a Doenças , Ácidos Graxos Ômega-6/metabolismo , Doenças do Sistema Nervoso Periférico/etiologia , Doenças do Sistema Nervoso Periférico/metabolismo , Animais , Dieta , Modelos Animais de Doenças , Ácidos Graxos Insaturados/metabolismo , Gânglios Espinais/metabolismo , Metabolismo dos Lipídeos , Camundongos , Fosfolipases A2/metabolismo , Fatores de Risco
6.
Front Immunol ; 12: 637778, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025647

RESUMO

Efferocytosis is critical for tissue homeostasis, as its deregulation is associated with several autoimmune pathologies. While engulfing apoptotic cells, phagocytes activate transcription factors, such as peroxisome proliferator-activated receptors (PPAR) or liver X receptors (LXR) that orchestrate metabolic, phagocytic, and inflammatory responses towards the ingested material. Coordination of these transcription factors in efferocytotic human macrophages is not fully understood. In this study, we evaluated the transcriptional profile of macrophages following the uptake of apoptotic Jurkat T cells using RNA-seq analysis. Results indicated upregulation of PPAR and LXR pathways but downregulation of sterol regulatory element-binding proteins (SREBP) target genes. Pharmacological inhibition and RNA interference pointed to LXR and PPARδ as relevant transcriptional regulators, while PPARγ did not substantially contribute to gene regulation. Mechanistically, lysosomal digestion and lysosomal acid lipase (LIPA) were required for PPAR and LXR activation, while PPARδ activation also demanded an active lysosomal phospholipase A2 (PLA2G15). Pharmacological interference with LXR signaling attenuated ABCA1-dependent cholesterol efflux from efferocytotic macrophages, but suppression of inflammatory responses following efferocytosis occurred independently of LXR and PPARδ. These data provide mechanistic details on LXR and PPARδ activation in efferocytotic human macrophages.


Assuntos
Apoptose/fisiologia , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , PPAR gama/metabolismo , Fagocitose/fisiologia , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Aciltransferases/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Células Jurkat , Receptores X do Fígado/genética , Lisossomos/metabolismo , PPAR gama/genética , Fosfolipases A2/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Transcrição Genética/genética , Transcriptoma/genética
7.
Int J Biol Macromol ; 182: 1602-1610, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34033823

RESUMO

Phospholipase A2 Bothropstoxin-I (PLA2 BthTX-I) is a myotoxic Lys49-PLA2 from Bothrops jararacussu snake venom. In order to evaluate the DNA damage caused by BthTX-I, we used the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster and Comet assay in HUVEC and DU-145 cells. For SMART, different concentrations of BthTX-I (6.72 to 430 µg/mL) were used and no significant changes in the survival rate were observed. Significant frequency of mutant spots was observed for the ST cross at the highest concentration of BthTX-I due to recombinogenic activity. In the HB cross, BthTX-I increased the number of mutant spots at intermediate concentrations, being 53.75 µg/mL highly mutagenic and 107.5 µg/mL predominantly recombinogenic. The highest concentrations were neither mutagenic nor recombinogenic, which could indicate cytotoxicity in the wing cells of D. melanogaster. In vitro, all BthTX-I concentrations (1 to 50 µg/mL) induced decrease in HUVEC cell viability, as well as in DU-145 cells at concentrations of 10, 25, and 50 µg/mL. The comet assay showed that in HUVEC and DU-145 cells, all BthTX-I concentrations promoted increase of DNA damage. Further studies should be performed to elucidate the mechanism of action of PLA2 BthTX-I and its possible use in therapeutic strategies against cancer.


Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/toxicidade , Fosfolipases A2/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Drosophila melanogaster , Células Endoteliais da Veia Umbilical Humana , Humanos , Mutação/genética
8.
Front Immunol ; 12: 618653, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968020

RESUMO

The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion and to identify the signaling pathways. The increase in [Ca2+]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca2+]i and mucin secretion that was blocked by the cell-permeant Ca2+ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca2+]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca2+]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca2+/ATPase that causes the intracellular Ca2+ stores to empty, and blockade of Ca2+ influx. Inhibition of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca2+]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca2+]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca2+]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca2+]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.


Assuntos
Anexina A1/metabolismo , Cálcio/metabolismo , Conjuntivite/metabolismo , Células Caliciformes/metabolismo , Mucinas/metabolismo , Animais , Anexina A1/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Conjuntivite/etiologia , Conjuntivite/patologia , Espaço Intracelular/metabolismo , Masculino , Fosfolipases A2/metabolismo , Ratos , Transdução de Sinais
9.
Toxins (Basel) ; 13(4)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33923919

RESUMO

Snake venom phospholipases A2 (PLA2s) have sequences and structures very similar to those of mammalian group I and II secretory PLA2s, but they possess many toxic properties, ranging from the inhibition of coagulation to the blockage of nerve transmission, and the induction of muscle necrosis. The biological properties of these proteins are not only due to their enzymatic activity, but also to protein-protein interactions which are still unidentified. Here, we compare sequence alignments of snake venom and mammalian PLA2s, grouped according to their structure and biological activity, looking for differences that can justify their different behavior. This bioinformatics analysis has evidenced three distinct regions, two central and one C-terminal, having amino acid compositions that distinguish the different categories of PLA2s. In these regions, we identified short linear motifs (SLiMs), peptide modules involved in protein-protein interactions, conserved in mammalian and not in snake venom PLA2s, or vice versa. The different content in the SLiMs of snake venom with respect to mammalian PLA2s may result in the formation of protein membrane complexes having a toxic activity, or in the formation of complexes whose activity cannot be blocked due to the lack of switches in the toxic PLA2s, as the motif recognized by the prolyl isomerase Pin1.


Assuntos
Fosfolipases A2/metabolismo , Venenos de Serpentes/enzimologia , Motivos de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Modelos Moleculares , Fosfolipases A2/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato
10.
Molecules ; 26(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924693

RESUMO

As a consequence of recent progression in biomedicine and nanotechnology, nanoparticle-based systems have evolved as a new method with extensive applications in responsive therapy, multimodal imaging, drug delivery and natural product separation. Meanwhile, the magnetic nanoparticulate system has aroused great interest for separation and purification because of its excellent magnetic properties. Phospholipase A2 (PLA2) is a highly expressed regulator to promote the growth of various cancers and is an ideal target to treat cancers. In this study, a novel strategy based on ligand-receptor interactions to discover novel PLA2 inhibitors was established, in which PLA2-functionalized Fe3O4@PLGA-PEG-NH2 magnetic nanoparticles were used as a supporting material combined with high-performance liquid chromatography-mass spectrometry, aiming to accelerate the discovery of novel PLA2 inhibitors from natural sources such as mangrove endophytic fungi. Under the optimized ligand fishing conditions, six target compounds were ultimately fished and identified to be cyclic peptides (1-3) and sterols (4-6), which compounds 1, 2 and 4-6 have well-documented cytotoxicities. Compound 3 exerted better inhibitory effect on A549 cells by experiment. In conclusion, PLA2-functionalized Fe3O4@PLGA-PEG-NH2 magnetic nanoparticles-based ligand fishing provided a feasible, selective and effective platform for the efficient screening and identification of antitumor components from natural products.


Assuntos
Enzimas Imobilizadas/química , Extratos Vegetais/química , Células A549 , Cromatografia Líquida de Alta Pressão , Humanos , Fosfolipases A2/metabolismo , Espectrometria de Massas em Tandem
11.
Artigo em Inglês | MEDLINE | ID: mdl-33676005

RESUMO

We investigated the antiophidic properties of isohemigossypolone (ISO), a naphthoquinone isolated from the outer bark of the Pachira aquatic Aubl. The inhibition of phospholipase A2, coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by Bothrops pauloensis venom (Pb) was investigated. For this, we use samples resulting from the incubation of Pb with ISO in different concentrations (1:1, 1:5 and 1:10 w/w), we also evaluated the condition of treatment using ISO after 15 min of venom inoculation. The activities of phospholipase A2, coagulant, fibrinogenolytic, hemorrhagic and myotoxic induced by the B. pauloensis venom were significantly inhibited when the ISO was pre-incubated with the crude venom. For in vivo neutralization tests, the results were observed even when the ISO was applied after 15 min of inoculation of the venom or metalloprotease (BthMP). Also, to identify the inhibition mechanism, we performed in silico assays, across simulations of molecular coupling and molecular dynamics, it was possible to identify the modes of interaction between ISO and bothropic toxins BmooMPα-I, Jararacussin-I and BNSP-7. The present study shows that naphthoquinone isohemigossypolone isolated from the P. aquatica plant inhibited part of the local and systemic damage caused by venom proteins, demonstrating the pharmacological potential of this compound in neutralizing the harmful effects caused by snakebites.


Assuntos
Bombacaceae/química , Venenos de Crotalídeos/antagonistas & inibidores , Naftoquinonas , Extratos Vegetais , Mordeduras de Serpentes/tratamento farmacológico , Animais , Masculino , Metaloproteases/metabolismo , Camundongos , Naftoquinonas/química , Naftoquinonas/farmacologia , Fosfolipases A2/metabolismo , Casca de Planta/química , Extratos Vegetais/farmacologia
12.
PLoS Pathog ; 17(3): e1009467, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33765093

RESUMO

Xenorhabdus hominickii, an entomopathogenic bacterium, inhibits eicosanoid biosynthesis of target insects to suppress their immune responses by inhibiting phospholipase A2 (PLA2) through binding to a damage-associated molecular pattern (DAMP) molecule called dorsal switch protein 1 (DSP1) from Spodoptera exigua, a lepidopteran insect. However, the signalling pathway between DSP1 and PLA2 remains unknown. The objective of this study was to determine whether DSP1 could activate Toll immune signalling pathway to activate PLA2 activation and whether X. hominickii metabolites could inhibit DSP1 to shutdown eicosanoid biosynthesis. Toll-Spätzle (Spz) signalling pathway includes two Spz (SeSpz1 and SeSpz2) and 10 Toll receptors (SeToll1-10) in S. exigua. Loss-of-function approach using RNA interference showed that SeSpz1 and SeToll9 played crucial roles in connecting DSP1 mediation to activate PLA2. Furthermore, a deletion mutant against SeToll9 using CRISPR/Cas9 abolished DSP1 mediation and induced significant immunosuppression. Organic extracts of X. hominickii culture broth could bind to DSP1 at a low micromolar range. Subsequent sequential fractionations along with binding assays led to the identification of seven potent compounds including 3-ethoxy-4-methoxyphenol (EMP). EMP could bind to DSP1 and prevent its translocation to plasma in response to bacterial challenge and suppress the up-regulation of PLA2 activity. These results suggest that X. hominickii inhibits DSP1 and prevents its DAMP role in activating Toll immune signalling pathway including PLA2 activation, leading to significant immunosuppression of target insects.


Assuntos
Alarminas/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Bactérias Gram-Negativas/metabolismo , Spodoptera/metabolismo , Xenorhabdus/metabolismo , Animais , Infecções por Bactérias Gram-Negativas/imunologia , Proteínas de Insetos/metabolismo , Fosfolipases A2/metabolismo , Salicilatos/metabolismo , Transdução de Sinais/fisiologia , Spodoptera/imunologia
13.
Dokl Biochem Biophys ; 496(1): 10-13, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33689066

RESUMO

It was established that the administration of an aqueous solution of bis(µ-tartrato)di(µ-hydroxy) germanate (IV) triethanolammonium to animals daily for 2 months at a dose of the active substance of 10 mg/kg of the animal's weight leads to inhibition of the total activity of the alkaline phospholipase A2 of mononuclear cells. The results of the study can be used to correct lipid metabolism in the development of disorders in hyperlipidemia. This makes it possible to expand the scope of use of the studied substance and create new pharmaceuticals based on bis(µ-tartrato)di(µ-hydroxy) germanate (IV) triethanolammonium prevent and inhibit the development of hyperlipidemia.


Assuntos
Etanolaminas/farmacologia , Hiperlipidemias/tratamento farmacológico , Compostos Organometálicos/farmacologia , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Animais , Colesterol/sangue , Germânio/química , Germânio/farmacologia , Hiperlipidemias/enzimologia , Hiperlipidemias/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Fosfolipases A2/sangue , Coelhos
14.
Toxicon ; 196: 44-55, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33781796

RESUMO

Based on the antimicrobial activity of bothropstoxin-I (BthTX-I) and on the premise that a C-terminal peptide of Lys49 myotoxin can reproduce the antimicrobial activity of the parent protein, we aimed to study the mechanism of action of a peptide derived from the C-terminal region of the myotoxin BthTX-I [(p-BthTX-I)2, sequence: KKYRYHLKPFCKK, disulfide-linked dimer] against Gram-positive and Gram-negative bacteria. Fluorescence quenching technique showed that the carboxyfluorescein labeled-peptide [CF-(p-BthTX-I)2] when incubated with E. coli displayed a superior penetration activity than when incubated with S. aureus. Cell death induced by the peptide (p-BthTX-I)2 showed a loss of membrane integrity in E. coli and S. aureus; however, the mechanisms of cell death were different, characterized by the presence of necrosis-like and apoptosis-like deaths, respectively. Scanning electron microscopy studies in E. coli and S. aureus showed morphological changes in the cells, with superficial deformities, appearance of wrinkles and bubbles, and formation of vesicles. Our results demonstrate that the mechanism of action of the peptide (p-BthTX-I)2 is different in Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. Knowledge of the mechanism of action of these peptides is important, since they are promising prototypes for new antimicrobial drugs.


Assuntos
Bothrops , Venenos de Crotalídeos/toxicidade , Fosfolipases A2/metabolismo , Animais , Antibacterianos/farmacologia , Escherichia coli , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Peptídeos/farmacologia , Poliésteres , Staphylococcus aureus
15.
PLoS Negl Trop Dis ; 15(2): e0009105, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33544727

RESUMO

The argasid tick Ornithodoros moubata is the main vector of human relapsing fever (HRF) and African swine fever (ASF) in Africa. Salivary proteins are part of the host-tick interface and play vital roles in the tick feeding process and the host infection by tick-borne pathogens; they represent interesting targets for immune interventions aimed at tick control. The present work describes the transcriptome profile of salivary glands of O. moubata and assesses the gene expression dynamics along the trophogonic cycle using Illumina sequencing. De novo transcriptome assembling resulted in 71,194 transcript clusters and 41,011 annotated transcripts, which represent 57.6% of the annotation success. Most salivary gene expression takes place during the first 7 days after feeding (6,287 upregulated transcripts), while a minority of genes (203 upregulated transcripts) are differentially expressed between 7 and 14 days after feeding. The functional protein groups more abundantly overrepresented after blood feeding were lipocalins, proteases (especially metalloproteases), protease inhibitors including the Kunitz/BPTI-family, proteins with phospholipase A2 activity, acid tail proteins, basic tail proteins, vitellogenins, the 7DB family and proteins involved in tick immunity and defence. The complexity and functional redundancy observed in the sialotranscriptome of O. moubata are comparable to those of the sialomes of other argasid and ixodid ticks. This transcriptome provides a valuable reference database for ongoing proteomics studies of the salivary glands and saliva of O. moubata aimed at confirming and expanding previous data on the O. moubata sialoproteome.


Assuntos
Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Ornithodoros/genética , Ornithodoros/metabolismo , Transcriptoma , África , Febre Suína Africana , Animais , Asfarviridae , Feminino , Expressão Gênica , Imunidade , Ixodidae/genética , Ixodidae/metabolismo , Redes e Vias Metabólicas/genética , Ornithodoros/imunologia , Ornithodoros/virologia , Fosfolipases A2/metabolismo , Proteômica/métodos , Saliva , Glândulas Salivares , Suínos
16.
Hum Exp Toxicol ; 40(2): 259-273, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33527851

RESUMO

Disorders of the endoplasmic reticulum (ER) lead to cellular damage but can cause cell death if ER dysfunction is prolonged. We aimed to examine liver/kidney functions, neutral sphingomyelinase (N-SMase) activity, sphingolipid levels, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) protein expression in rats under ER stress. ER stress was induced by tunicamycin (TM) and the ER stress inhibitor taurodeoxycholic acid (TUDCA) was injected before induction of ER stress. ER stress was confirmed by increased tissue levels of GRP78. Hematological and biochemical profiles were measured by autoanalyzers while hepatic and renal injury was evaluated via microscopy and histopathological scoring. Tissue levels of C16-C24 sphingomyelins (SM), C16-C24 ceramides (CERs) and sphingosine-1-phosphate (S1P) were determined by LC-MS/MS. Tissue cPLA2 and COX-2 were measured by western blot and activity assays. Tunicamycin treatment caused kidney and liver function test abnormalities, increased hematocrit and hemoglobin levels but decreased white blood cell counts. Histopathological findings showed hepatic necroinflammation and renal tubular damage in rats treated with TM. TUDCA administration attenuated WBC abnormalities and TM- induced hepatic/renal functional impairment in ER stress, as evident by significantly restored serum ALT, AST, creatinine, and total bilirubin levels. A significant increase was observed in N-SMase activity, tissue levels of C16-C24 CERs, cPLA2 and COX-2 expression in liver and kidney tissue under ER stress. TUDCA administration decreased tissue CER levels, cPLA2 and COX-2 expression as well as prostaglandin E2 (PGE2) formation. These results signify that ER stress causes hepatic and renal toxicity as well as CER-induced PGE2 formation in liver and kidney.


Assuntos
Estresse do Retículo Endoplasmático , Inflamação/metabolismo , Rim/metabolismo , Fígado/metabolismo , Esfingolipídeos/metabolismo , Animais , Ceramidas/metabolismo , Dinoprostona/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Fosfolipases A2/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos Wistar , Esfingomielina Fosfodiesterase/metabolismo , Tunicamicina
17.
Eur J Ophthalmol ; 31(2): 367-378, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31875691

RESUMO

BACKGROUND: The protective role of Prdx6 on rat corneal tissue against ultraviolet B injury in vivo has been confirmed previously. We further investigated the function and molecular mechanism of Prdx6 in human corneal epithelial cells under ultraviolet B radiation. METHODS: The experimental groups were designed as follows: (1) Prdx6 RNAi, (2) Prdx6 RNAi + ultraviolet B radiation, (3) normal human corneal epithelial cells, (4) normal human corneal epithelial cells + ultraviolet B radiation, (5) wild-type Prdx6 overexpression, (6) wild-type Prdx6 overexpression + ultraviolet B radiation, (7) mutant-type Prdx6 overexpression, and (8) mutant-type Prdx6 overexpression + ultraviolet B radiation. The cell survival rate was detected by a Thiazolyl Blue Tetrazolium Bromide assay. Apoptosis, reactive oxygen species, and malondialdehyde were detected with a commercial kit. Gene expression was detected by real-time polymerase chain reaction. RESULTS: We found the following results. (1) Compared to normal cells, the survival rates were 32%, 87%, and 58% under ultraviolet B radiation in the Prdx6 interference, wild-type overexpression, and mutant-type overexpression groups, respectively. The survival rates were decreased to 50% at 24 h and 31% at 48 h when the phospholipase A2 activity of Prdx6 was inhibited after ultraviolet B radiation. (2) Apoptosis, reactive oxygen species content, and malondialdehyde levels were increased when Prdx6 was downregulated. This phenomenon became more severe under ultraviolet B radiation. (3) The expression levels of apoptosis-related and antioxidant genes all changed along with the changes in expression of Prdx6. CONCLUSION: (1) Both peroxidase and phospholipase A2 activities of Prdx6 are crucial for its protective role in corneal tissue. (2) Downregulated expression of Prdx6 resulted in high endoplasmic reticulum stress. (3) Apoptosis in human corneal epithelial cells with downregulated Prdx6 coupled with ultraviolet B radiation was related to the pathways of DNA damage and the death receptor. (4) Low levels of antioxidants are sufficient for maintaining homeostasis in human corneal epithelial cells without external stimuli. Under the condition that Prdx6 was downregulated, human corneal epithelial cells were more sensitive to ultraviolet B radiation.


Assuntos
Epitélio Corneano/efeitos da radiação , Regulação da Expressão Gênica/fisiologia , Peroxirredoxina VI/genética , Lesões Experimentais por Radiação/prevenção & controle , Raios Ultravioleta/efeitos adversos , Animais , Apoptose , Sobrevivência Celular , Corantes/metabolismo , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Humanos , Malondialdeído/metabolismo , Peroxidase/metabolismo , Fosfolipases A2/metabolismo , Plasmídeos , RNA Mensageiro/genética , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , Transfecção
18.
Food Chem ; 343: 128428, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33131955

RESUMO

The antioxidant effect of porcine pancreatic phospholipase A2 (PLA2) was previously demonstrated. Understanding how PLA2 inhibits lipid oxidation promoted by hemoglobin (Hb) is important for its applications in muscle foods. Effects of enzyme dose, pH, and calcium ion on the ability of PLA2 to inhibit trout hemoglobin-mediated lipid oxidation were investigated in washed cod muscle (WCM). Results indicated that PLA2 required calcium ion for both the hydrolyzing activity and the antioxidant effect. The abilities of PLA2 to inhibit lipid oxidation and suppress oxidation of Hb to form methemoglobin and ferryl hemoglobin were pH-dependent. The lag phase before lipid oxidation enters the exponential phase reciprocally shortened as more hemin was bound to the insoluble matrix of WCM. However, PLA2 was able to inhibit lipid oxidation without preventing the interaction between hemin and the insoluble matrix of the washed muscle.


Assuntos
Hemina/metabolismo , Hemoglobinas/metabolismo , Fosfolipases A2/química , Truta/metabolismo , Animais , Antioxidantes/metabolismo , Produtos Pesqueiros , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Hemina/química , Hemoglobinas/química , Concentração de Íons de Hidrogênio , Metabolismo dos Lipídeos , Músculo Esquelético/metabolismo , Oxirredução , Fosfolipases A2/metabolismo , Suínos
19.
Mol Pharmacol ; 99(2): 125-132, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33239333

RESUMO

A first-in-class cannabinoid analog called lenabasum that is a CB2 agonist is being developed as an inflammation-resolving drug candidate. Thus far, specific therapeutic targets include scleroderma, cystic fibrosis, dermatomyositis, and lupus, all of which represent unmet medical needs. Two somewhat-independent molecular mechanisms for this type of action are here proposed. Both pathways initially involve the release of free arachidonic acid after activation of the CB2 receptor and phospholipase A2 by lenabasum. The pathways then diverge into a cyclooxygenase 2-mediated and a lipoxygenase-mediated route. The former leads to increased levels of the cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin-J2 that can activate the NLPR3 inflammasome, which in turn releases caspase-3, leading to apoptosis and the resolution of chronic inflammation. The lipoxygenase-mediated pathway stimulates the production of lipoxin A4 as well as other signaling molecules called specialized proresolving mediators. These also have inflammation-resolving actions. It is not well understood under which conditions each of these mechanisms operates and whether there is crosstalk between them. Thus, much remains to be learned about the mechanisms describing the actions of lenabasum. SIGNIFICANCE STATEMENT: The resolution of chronic inflammation is a major unmet medical need. The synthetic nonpsychoactive cannabinoid lenabasum could provide a safe and effective drug for this purpose. Two putative molecular mechanisms are suggested to better understand how lenabasum produces this action. In both, different metabolites of arachidonic acid act as mediators.


Assuntos
Anti-Inflamatórios/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Dronabinol/análogos & derivados , Animais , Ácido Araquidônico/metabolismo , Dronabinol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fosfolipases A2/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo
20.
Toxicon ; 190: 31-38, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33307108

RESUMO

The envenomation caused by the Bothrops pauloensis snake leads to severe local and systemic effects including acute kidney injury. In this study, we investigated the renal effects by phospholipases A2 (PLA2s), divided into two main subgroups, Asp-49 and Lys-49, isolated from the Bothrops pauloensis snake venom (BpV) in isolated rat kidney system. Both PLA2s (3 µg/mL), added alone to the perfusion system and analyzed for 120 min, had significant effects on isolated rat kidney. Asp-49 reduced Glomerular Filtration Rate (GFR) at 60, 90 and 120 min, and the percentage of total tubular sodium transport (%TNa+) and potassium transport (%TK+) at 120 min. Lys-49 increased Perfusion Pressure (PP) at 120 min and reduced GFR, %TNa+ and the percentage of total tubular chloride transport (%TCl-) at 60, 90 and 120 min. Cytokine release in the kidney tissues were increased with Asp-49 PLA2 (IL-10) and Lys-49 PLA2 (TNF-α, IL-1ß, IL-10). Both increased MPO activity. Asp-49 PLA2 decreased Glutathione (GSH) and increased nitrite levels, while Lys-49 PLA2 increased Malondialdehyde (MDA), GSH and nitrite levels. Histological analysis of the perfused kidneys revealed the presence of glomerular degeneration and atrophy, deposit of proteinaceous material in Bowman's space and intratubular with both PLA2s. These findings indicated that both PLA2s modified the functional parameters in an isolated perfused kidney model with increased oxidative stress and cytokine release. PLA2s are one of the components at high concentration in BpV and our results provide important knowledge about their involvement with the nephrotoxic mechanism.


Assuntos
Injúria Renal Aguda/metabolismo , Venenos de Crotalídeos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosfolipases A2/metabolismo , Animais , Bothrops , Citocinas , Rim , Glomérulos Renais , Ratos , Venenos de Serpentes
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