RESUMO
In Brazil, latex from Euphorbia umbellata (African milk tree) has been increasingly used in folk medicine to treat several types of cancer, including melanoma. The effect of lyophilized latex (LL), its hydroethanolic extract (E80), triterpene (F-TRI)- and diterpene (F-DIT)-enriched fractions, along with six isolated phorbol esters from LL and phorbol 12-myristate 13-acetate (PMA) on J774A.1, THP-1, SK-MEL-28, and B16-F10 cell line viability were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The compounds were identified by 2D-NMR and HRESIMS. The effect of the LL, extract and fractions on cell viability was also assessed through a resazurin reduction assay. At 100 µg/ml, LL, and its fractions moderately inhibited J774A.1 (37.5-59.5%) and THP-1 (12.6-43.6%) metabolism. LL (IC50 70 µg/ml) and F-TRI (IC50 68 µg/ml) were barely more effective against B16-F10 cells, and only F-TRI exerted an inhibitory effect on SK-MEL-28 cells (IC50 66-75 µg/ml). The samples did not effectively inhibit THP-1 growth (IC50 69-87 µg/ml, assessed by MTT). B16-F10 was susceptible to PMA (IC50 53 µM) and two 12-phenylacetate esters (IC50 56-60 µM), while SK-MEL-28 growth was inhibited (IC50 58 µM) by one of these kinds of esters with an additional 4ß-deoxy structure. Synagrantol A (IC50 39 µM) was as effective as PMA (IC50 47 µM) in inhibiting J774A.1 growth in a dose-dependent manner. Furthermore, an in silico study with target receptors indicated a high interaction of the compounds with the PKC proteins. These results provide useful knowledge on the effect of tigliane-type diterpenes on tumor cell from the perspective of medicinal chemistry.
Assuntos
Euphorbia , Látex , Ésteres de Forbol , Euphorbia/química , Látex/química , Ésteres de Forbol/farmacologia , Humanos , Camundongos , Animais , Linhagem Celular Tumoral , Estrutura Molecular , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Brasil , Monócitos/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Diterpenos/farmacologia , Diterpenos/isolamento & purificação , Terpenos/farmacologia , Terpenos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Acetato de Tetradecanoilforbol , Melanoma/tratamento farmacológicoRESUMO
The safety of Jatropha curcas L. cake (JCC) in animal feed remains under scrutiny, despite the advent of low phorbol ester (PE) variants. This study investigates the impact of low PE JCC on swine health when used as a protein feed. Pigs were fed a 5% JCC diet with a PE concentration of 0.98 mg/kg, which surprisingly still induced toxicity. Symptoms included depression, decreased food intake, increased diarrhea, along with hypothalamus and colon lesions. The toxicity was associated with a decrease in antioxidant enzymes, an increase in inflammatory cytokines in the hypothalamus, plasma, and colon, and a rise in pro-inflammatory colon microbes and metabolites. Disturbances in neurotransmitters further suggest that this toxicity is related to disruption of the microbiota-gut-brain axis, indicating that JCC's toxic elements are not solely due to PE. The sensitivity of pigs to JCC underscores the need for thorough detoxification prior to its use as feed. These findings significantly contribute to the discourse on the safety of low PE JCC in animal feed, highlighting implications for both the feed industry and public health.
Assuntos
Ração Animal , Eixo Encéfalo-Intestino , Microbioma Gastrointestinal , Jatropha , Ésteres de Forbol , Animais , Suínos , Ésteres de Forbol/efeitos adversos , Eixo Encéfalo-Intestino/fisiologia , Dieta/veterinária , Ingestão de Alimentos , Citocinas/metabolismo , Colo/metabolismo , Hipotálamo/metabolismo , Depressão/metabolismo , Neurotransmissores/metabolismoRESUMO
AIMS: Phorbol esters (PE) are toxic diterpenoids accumulated in physic nut (Jatropha curcas L.) seed tissues. Their biosynthetic pathway remains unknown, and the participation of roots in this process may be possible. Thus, we set out to study the deposition pattern of PE and other terpenoids in roots and leaves of genotypes with detected (DPE) and not detected (NPE) phorbol esters based on previous studies. OUTLINE OF DATA RESOURCES: We analyzed physic nut leaf and root organic extracts using LC-HRMS. By an untargeted metabolomics approach, it was possible to annotate 496 and 146 metabolites in the positive and negative electrospray ionization modes, respectively. KEY RESULTS: PE were detected only in samples of the DPE genotype. Remarkably, PE were found in both leaves and roots, making this study the first report of PE in J. curcas roots. Furthermore, untargeted metabolomic analysis revealed that diterpenoids and apocarotenoids are preferentially accumulated in the DPE genotype in comparison with NPE, which may be linked to the divergence between the genotypes concerning PE biosynthesis, since sesquiterpenoids showed greater abundance in the NPE. UTILITY OF THE RESOURCE: The LC-HRMS files, publicly available in the MassIVE database (identifier MSV000092920), are valuable as they expand our understanding of PE biosynthesis, which can assist in the development of molecular strategies to reduce PE levels in toxic genotypes, making possible the food use of the seedcake, as well as its potential to contain high-quality spectral information about several other metabolites that may possess biological activity.
Assuntos
Jatropha , Jatropha/genética , Jatropha/metabolismo , Ésteres de Forbol/análise , Ésteres de Forbol/metabolismo , Folhas de Planta/metabolismo , Sementes/genéticaRESUMO
Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C (PKC). The efficacy of phorbol esters in binding with PKC is attributed to the presence of oxygen groups at positions C20, C3/C4, and C9 of phorbol. Concurrently, the lipids located at positions C12/C13 are essential for both the anti-HIV-1 activity and the formation of the PKC-ligand complex. The influence of the cyclopropane ring at positions C13 and C14 in phorbol derivatives on their anti-HIV-1 activity requires further exploration. This research entailed the hydrolysis of phorbol, producing seco-cyclic phorbol derivatives. The anti-HIV-1 efficacy of these derivatives was assessed, and the affinity constant (Kd) for PKC-δ protein of selected seco-cyclic phorbol derivatives was determined through isothermal titration calorimetry. The findings suggest that the chemical modification of cyclopropanols could affect both the anti-HIV-1 activity and the PKC binding affinity. Remarkably, compound S11, with an EC50 of 0.27 µmol·L-1 and a CC50 of 153.92 µmol·L-1, demonstrated a potent inhibitory effect on the intermediate products of HIV-1 reverse transcription (ssDNA and 2LTR), likely acting at the viral entry stage, yet showed no affinity for the PKC-δ protein. These results position compound S11 as a potential candidate for further preclinical investigation and for studies aimed at elucidating the pharmacological mechanism underlying its anti-HIV-1 activity.
Assuntos
Fármacos Anti-HIV , HIV-1 , HIV-1/efeitos dos fármacos , Humanos , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/química , Ésteres de Forbol/farmacologia , Ésteres de Forbol/química , Estrutura Molecular , Proteína Quinase C/metabolismo , Proteína Quinase C/química , Relação Estrutura-AtividadeRESUMO
In this study, 37 derivatives of phorbol esters were synthesized and their anti-HIV-1 activities evaluated, building upon our previous synthesis of 51 phorbol derivatives. 12-Para-electron-acceptor-trans-cinnamoyl-13-decanoyl phorbol derivatives stood out, demonstrating remarkable anti-HIV-1 activities and inhibitory effects on syncytia formation. These derivatives exhibited a higher safety index compared with the positive control drug. Among them, 12-(trans-4-fluorocinnamoyl)-13-decanoyl phorbol, designated as compound 3c, exhibited the most potent anti-HIV-1 activity (EC50 2.9 nmol·L-1, CC50/EC50 11 117.24) and significantly inhibited the formation of syncytium (EC50 7.0 nmol·L-1, CC50/EC50 4891.43). Moreover, compound 3c is hypothesized to act both as an HIV-1 entry inhibitor and as an HIV-1 reverse transcriptase inhibitor. Isothermal titration calorimetry and molecular docking studies indicated that compound 3c may also function as a natural activator of protein kinase C (PKC). Therefore, compound 3c emerges as a potential candidate for developing new anti-HIV drugs.
Assuntos
Fármacos Anti-HIV , Forbóis , Simulação de Acoplamento Molecular , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/química , Forbóis/química , Forbóis/farmacologia , Ésteres de Forbol/farmacologia , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Relação Estrutura-AtividadeRESUMO
The affinity and selectivity of small molecules for proteins drive drug discovery and development. We report a fluorescent probe cellular binding assay (FPCBA) for determination of these values for native (untagged) proteins overexpressed in living cells. This method uses fluorophores such as Pacific Blue (PB) linked to cell-permeable protein ligands to generate probes that rapidly and reversibly equilibrate with intracellular targets, as established by kinetic assays of cellular uptake and efflux. To analyze binding to untagged proteins, an internal ribosomal entry site (IRES) vector was employed that allows a single mRNA to encode both the protein target and a separate orthogonal fluorescent protein (mVenus). This enabled cellular uptake of the probe to be correlated with protein expression by flow cytometry, allowing measurement of cellular dissociation constants (Kd) of the probe. This approach was validated by studies of the binding of allosteric activators to eight different Protein Kinase C (PKC) isozymes. Full-length PKCs expressed in transiently transfected HEK293T cells were used to measure cellular Kd values of a probe comprising PB linked to the natural product phorbol via a carbamate. These values were further used to determine competitive binding constants (cellular Ki values) of the nonfluorescent phorbol ester PDBu and the anticancer agent bryostatin 1 for each isozyme. For some PKC-small molecule pairs, these cellular Ki values matched known biochemical Ki values, but for others, altered selectivity was observed in cells. This approach can facilitate quantification of interactions of small molecules with physiologically relevant native proteins.
Assuntos
Ésteres de Forbol , Proteína Quinase C , Humanos , Células HEK293 , Proteína Quinase C/química , Ligação CompetitivaRESUMO
The function of the α1B-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with non-phosphorylatable amino acids. Substitution of the intracellular loop 3 (IL3) sites did not alter baseline or stimulated receptor phosphorylation, whereas substitution of phosphorylation sites in the carboxyl terminus (Ctail) or both domains (IL3/Ctail) markedly decreased receptor phosphorylation. Cells expressing the IL3 or Ctail receptor mutants exhibited a noradrenaline-induced calcium-maximal response similar to those expressing the wild-type receptor, and a shift to the left in the concentration-response curve to noradrenaline was also noticed. Cells expressing the IL3/Ctail mutant exhibited higher apparent potency and increased maximal response to noradrenaline than those expressing the wild-type receptor. Phorbol ester-induced desensitization of the calcium response to noradrenaline was reduced in cells expressing the IL3 mutant and abolished in cells in which the Ctail or the IL3/Ctail were modified. In contrast, desensitization in response to preincubation with noradrenaline was unaffected in cells expressing the distinct receptor mutants. Noradrenaline-induced ERK phosphorylation was surprisingly increased in cells expressing IL3-modified receptors but not in those expressing receptors with the Ctail or IL3/Ctail substitutions. Our data indicate that phosphorylation sites in the IL3 and Ctail domains mediate and regulate α1B-adrenergic receptor function. Phorbol ester-induced desensitization seems to be closely associated with receptor phosphorylation, whereas noradrenaline-induced desensitization likely involves other elements.
Assuntos
Cálcio , Norepinefrina , Fosforilação , Cálcio/metabolismo , Norepinefrina/farmacologia , Ésteres de Forbol , Receptores Adrenérgicos/metabolismoRESUMO
Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.
Assuntos
Carcinógenos , Neoplasias , Feminino , Gravidez , Humanos , Animais , Camundongos , Via de Sinalização Wnt , Quinase 3 da Glicogênio Sintase , Ésteres de Forbol , ÉsteresRESUMO
Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According to the different functional groups, they can be classified into types of phorbol esters, C-4 deoxyphorbol esters, C-12 deoxyphorbol esters, C-16 or C-17 substituted phorbol esters and others. Most of them present promising antiviral activities and cytotoxic activities and are expected to be developed as candidates for anti-AIDS, anti-tuberculosis, and anti-tumor clinical trials, demonstrating great potential for the application in healthcare. This paper reviews 115 novel tigliane-type diterpenoids discovered since 2013 and summarize their chemical structures and bioactivities, aiming to lay a foundation for further development and utilization of these compounds and provide new ideas for the development of clinical drugs.
Assuntos
Diterpenos , Forbóis , Estrutura Molecular , Diterpenos/farmacologia , Diterpenos/química , Antivirais , Ésteres de ForbolRESUMO
GPRC5A is the first member of a new class of orphan receptors coupled to G proteins, which also includes GPRC5B, GPRC5C, and GPRC5D. Since its cloning and identification in the 1990s, substantial progress has been made in understanding the possible functions of this receptor. GPRC5A has been implicated in a variety of cellular events, such as cytoskeleton reorganization, cell proliferation, cell cycle regulation, migration, and survival. It appears to be a central player in different pathological processes, including tumorigenesis, inflammation, immune response, and tissue damage. The levels of GPRC5A expression differ depending on the type of cancer, with increased expression in colon, pancreas, and prostate cancers; decreased expression in lung cancer; and varied results in breast cancer. In this review, we discuss the early discovery of GPRC5A as a phorbol ester-induced gene and later as a retinoic acid-induced gene, its regulation, and its participation in important canonical pathways related to numerous types of tumors and inflammatory processes. GPRC5A represents a potential new target for cancer, inflammation, and immunity therapies.
Assuntos
Neoplasias Pulmonares , Receptores do Ácido Retinoico , Masculino , Humanos , Ésteres de Forbol , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Pulmonares/patologia , Inflamação , TretinoínaRESUMO
Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung disorders. TRPV4 expresses in lung tissue and plays an important role in the maintenance of respiratory homeostatic function. TRPV4 is upregulated in life-threatening respiratory diseases like pulmonary hypertension, asthma, cystic fibrosis, and chronic obstructive pulmonary diseases. TRPV4 is linked to several proteins that have physiological functions and are sensitive to a wide variety of stimuli, such as mechanical stimulation, changes in temperature, and hypotonicity, and responds to a variety of proteins and lipid mediators, including anandamide (AA), the arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid (5,6-EET), a plant dimeric diterpenoid called bisandrographolide A (BAA), and the phorbol ester 4-alpha-phorbol-12,13-didecanoate (4α-PDD). This study focused on relevant research evidence of TRPV4 in lung disorders and its agonist and antagonist effects. TRPV4 can be a possible target of discovered molecules that exerts high therapeutic potential in the treatment of respiratory diseases by inhibiting TRPV4.
Assuntos
Hipertensão Pulmonar , Canais de Potencial de Receptor Transitório , Humanos , Canais de Cátion TRPV/metabolismo , Ésteres de Forbol/farmacologia , Hipertensão Pulmonar/metabolismoRESUMO
Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting activities can be modulated by two classes of ligands, phorbol esters and bryostatins. Phorbol esters are known tumor promoters, while bryostatins have anti-cancer properties. This is despite both ligands binding to the C1b domain of PKC-δ (δC1b) with a similar affinity. The molecular mechanism behind this discrepancy in cellular effects remains unknown. Here, we have used molecular dynamics simulations to investigate the structure and intermolecular interactions of these ligands bound to δC1b with heterogeneous membranes. We observed clear interactions between the δC1b-phorbol complex and membrane cholesterol, primarily through the backbone amide of L250 and through the K256 side-chain amine. In contrast, the δC1b-bryostatin complex did not exhibit interactions with cholesterol. Topological maps of the membrane insertion depth of the δC1b-ligand complexes suggest that insertion depth can modulate δC1b interactions with cholesterol. The lack of cholesterol interactions suggests that bryostatin-bound δC1b may not readily translocate to cholesterol-rich domains within the plasma membrane, which could significantly alter the substrate specificity of PKC-δ compared to δC1b-phorbol complexes.
Assuntos
Forbóis , Proteína Quinase C-delta , Humanos , Briostatinas , Isoenzimas/metabolismo , Ésteres de Forbol/química , Lactonas/químicaRESUMO
Phorbol is a tetracyclic diterpenoid found in Euphorbia tirucalli, Croton tiglium, and Rehmannia glutinosa, and is nuclear of various phorbol esters. The rapid obtaining of phorbol with high purity highly contributes to its application, such as synthesizing phorbol esters with designable side chains and particular therapeutic efficacy. This study introduced a biphasic alcoholysis method for obtaining phorbol from croton oil by using polarity imparity organic solvents in both phases and established a high-speed countercurrent chromatography method for simultaneous separation and purification of phorbol. The optimized operation conditions of biphasic alcoholysis were a reaction time of 91 min, a temperature of 14°C, and a croton oil-methanol ratio of 1:30 (g:ml). The phorbol during the biphasic alcoholysis was 3.2-fold higher in content than that obtained in conventional monophasic alcoholysis. The optimized high-speed countercurrent chromatography method was using the ethyl acetate/n-butyl alcohol/water at 4.7:0.3:5 (v:v:v) with Na2 SO4 at 0.36 g/10 ml as the solvent system, using the mobile phase flow rate of 2 ml/min, the revolution of 800 r/min, under which the retention of the stationary phase was achieved at 72.83%. The crystallized phorbol following high-speed countercurrent chromatography was obtained as high purity of 94%.
Assuntos
Distribuição Contracorrente , Forbóis , Distribuição Contracorrente/métodos , Óleo de Cróton , Solventes/química , Extratos Vegetais/química , Ésteres de Forbol , Cromatografia Líquida de Alta PressãoRESUMO
Physic nut Jatropha curcas cake/meal obtained after oil extraction has a high protein content, however, the presence of antinutrients (trypsin inhibitor, lectin and phytate) and toxic compounds (phorbol esters) limit their use as an alternative feedstuff. Thus, the detoxification process in cake/meal is necessary to allow their inclusion in fish diets. The present study aimed to evaluate the effects of solvent and extrusion-treated jatropha cake (SETJC) in Nile tilapia (Oreochromis niloticus) diets on growth, body composition, nutrient utilization, metabolic and hematological responses, and digestibility of experimental diets. Five experimental diets were formulated to be isonitrogenous (28.50% digestible protein) and isoenergetic (13.39 MJ/kg digestible energy) with graded levels of SETJC (0, 3, 6, 9, and 12%). The experimental design was completely randomized with five treatments and four replicates. The detoxification treatments reduced the phorbol esters (PE) of jatropha cake by 96% (0.58 mg/g of PE before and 0.023 mg/g of PE after treatments). Increased levels of SETJC depressed growth, feed efficiency, and protein digestibility. A similar trend was observed for hematological and biochemistry parameters. Aspartate and alanine aminotransferase, as well as phosphorus and magnesium concentrations in the fillets, increased at the highest levels of SETJC. Thus, the data of the present study suggests that the residual content, different structural forms of phorbol ester and its biological activity, as well as some antinutritional factors, can influence negatively the growth, metabolism and digestibility of experimental diets for Nile tilapia.
Assuntos
Ciclídeos , Jatropha , Animais , Jatropha/química , Jatropha/metabolismo , Ração Animal/análise , Solventes/análise , Dieta/veterinária , Ésteres de Forbol/farmacologia , Ésteres de Forbol/análise , Ésteres de Forbol/metabolismo , Sementes/química , Sementes/metabolismoRESUMO
C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during the activation of the proteins containing these domains. Although the overall structure of the C1 domains are similar, there are differences in their primary sequence and in the orientation of the ligand/lipid binding residues. To gain structural insights into the ligand/lipid binding, we performed molecular docking of phorbol 13-acetate into the C1 domain and 1.0 µs molecular dynamics simulation on the C1 domain-ligand-lipid ternary system for PKCθ C1A, PKCδ C1B, PKCßII C1B, PKCθ C1B, Munc13-1 C1, and ßII-Chimaerin C1. We divided these C1 domains into three types based on the orientations of Gln-27 and Trp/Tyr-22. In type 1, Trp/Tyr-22 is outside and Gln-27 is inside the ligand binding pocket. In type 2, both Trp/Tyr-22 and Gln-27 are outside the ligand binding pocket, and in type 3, Trp/Tyr-22 is inside and Gln-27 is outside the pocket. The type 1 C1 domains showed higher ligand binding and higher membrane binding with a shorter distance between the C1 domain and the membrane than the type 2 and type 3. For ligand binding, Pro-11 plays a major role in the type 1 and 2, and Gly-23 in the type 1 and type 3 C1 domains. This study elucidates the role of Gln-27, Trp-22, Pro-11 and Gly-23 in ligand/lipid binding in typical C1 domains and bears significance in developing selective modulators of C1 domain-containing proteins.Communicated by Ramaswamy H. Sarma.
Assuntos
Simulação de Dinâmica Molecular , Ésteres de Forbol , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Ligantes , Ligação Proteica , Sítios de Ligação , Ésteres de Forbol/química , Ésteres de Forbol/metabolismo , LipídeosRESUMO
Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According to the different functional groups, they can be classified into types of phorbol esters, C-4 deoxyphorbol esters, C-12 deoxyphorbol esters, C-16 or C-17 substituted phorbol esters and others. Most of them present promising antiviral activities and cytotoxic activities and are expected to be developed as candidates for anti-AIDS, anti-tuberculosis, and anti-tumor clinical trials, demonstrating great potential for the application in healthcare. This paper reviews 115 novel tigliane-type diterpenoids discovered since 2013 and summarize their chemical structures and bioactivities, aiming to lay a foundation for further development and utilization of these compounds and provide new ideas for the development of clinical drugs.
Assuntos
Forbóis , Estrutura Molecular , Diterpenos/química , Antivirais , Ésteres de ForbolRESUMO
Four new diterpene esters, shirakindicans A-D (1-4), along with eight related known diterpene esters (5-12), were isolated from the fruits of the Bangladeshi medicinal plant Shirakiopsis indica. The structures of 1-4 were elucidated by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Shirakindican A (1) was assigned as a tigliane-type diterpene ester possessing an unusual 6ß-hydroxy-1,7-dien-3-one structure, while shirakindican B (2) exhibits a tiglia-1,5-dien-3,7-dione structure. The anti-HIV activities of the isolated diterpene esters were evaluated and showed significant activities for sapintoxins A (5) and D (11), with EC50 values of 0.0074 and 0.044 µM, respectively, and TI values of 1â¯100 and 5â¯290. Sapatoxin A (12) also exhibited anti-HIV activity with an EC50 value of 0.13 µM and a TI value of 161.
Assuntos
Fármacos Anti-HIV , Euphorbiaceae , HIV , Ésteres de Forbol , Euphorbiaceae/química , Frutas/química , Estrutura Molecular , HIV/efeitos dos fármacos , Ésteres de Forbol/química , Ésteres de Forbol/isolamento & purificação , Ésteres de Forbol/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Linhagem Celular , HumanosRESUMO
We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.
Assuntos
Neurônios do Núcleo Supraquiasmático , Peptídeo Intestinal Vasoativo , Camundongos , Animais , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/farmacologia , Peptídeo Intestinal Vasoativo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neurônios do Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Luciferases/metabolismo , Ésteres de ForbolRESUMO
Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs exist, but their differential roles are not well understood. We studied the interdependence of phorbolesters (DAG mimics) with Munc13-1 and ubMunc13-2 in mouse adrenal chromaffin cells. Although expression of either Munc13-1 or ubMunc13-2 stimulated secretion, phorbolester was only stimulatory for secretion when ubMunc13-2 expression dominated, but inhibitory when Munc13-1 dominated. Accordingly, phorbolester stimulated secretion in wildtype cells, or cells overexpressing ubMunc13-2, but inhibited secretion in Munc13-2/Unc13b knockout (KO) cells or in cells overexpressing Munc13-1. Phorbolester was more stimulatory in the Munc13-1/Unc13a KO than in WT littermates, showing that endogenous Munc13-1 limits the effects of phorbolester. Imaging showed that ubMunc13-2 traffics to the plasma membrane with a time-course matching Ca2+-dependent secretion, and trafficking is independent of Synaptotagmin-7 (Syt7). However, in the absence of Syt7, phorbolester became inhibitory for both Munc13-1 and ubMunc13-2-driven secretion, indicating that stimulatory phorbolester x Munc13-2 interaction depends on functional pairing with Syt7. Overall, DAG/phorbolester, ubMunc13-2 and Syt7 form a stimulatory triad for dense-core vesicle priming.
Assuntos
Diglicerídeos , Ésteres de Forbol , Animais , Camundongos , Vesículas de Núcleo Denso , Exocitose , Proteínas SNARE/metabolismo , SinaptotagminasRESUMO
The excessive amount of reactive species under chronic inflammation, which are accompanied by an increase body temperature, lead to diabetic complications. Phagocyte NADPH oxidase is the key enzyme in these processes. The role of high temperature in its regulation in diabetes is not clear. The aim was to investigate the effect of high temperature on NADPH-oxidase-dependent generation of reactive species in diabetic patients. Chemiluminescent method was applied to assess respiratory burst kinetics initiated by opsonized zymosan in blood or phorbol ester in isolated granulocytes. Analyzing ROC curves, the main predictors and changes in stages of activation of NADPH oxidase were determined. Phosphoisoforms of p47phox and p67phox were quantified by immunoblotting. Response to opsonized zymosan was lower in all subjects at 40 °C vs 37 °C, its kinetic parameters (except Tmax) were higher in blood of patients vs controls. Response rate was the main significant predictor to distinguish groups of subjects at 40 °C indicating NADPH oxidase upregulation in diabetes. Ca2+-dependent generation of reactive species by cells increased in both groups at 40 °C vs 37 °C, kinetic parameters were higher in patients. Initial phospho-p47phox level was higher in patient cells vs ones in controls. It was increased by ionomycin, phorbol ester, or 40 °C in control cells and unchanged in patient ones. Phospho-p67phox level was unchangeable in intact cells of healthy donors and patients at both temperatures. Excessive amounts of reactive species in patient cells were the consequence of granulocyte priming due to p47phox phosphorylation. Thus, high temperature decreased phagocytosis- and enhanced Ca2+-dependent generation of reactive species making the differences between controls and patients less pronounced. The effect of temperature on the generation of reactive species in blood granulocytes is associated with activity of NADPH oxidase that can be a prospective therapeutic target for pathologies accompanied by inflammation including type 2 diabetes.