RESUMEN
Objective: Cordycepin has been shown to exhibit multiple pharmacological activities, such as antitumor, antifungi, antivirus, and immune-regulation activities, and is involved in the regulation of T cells. However, cordycepin that affects T cell activity is still not clear, and the molecular mechanism of cordycepin in regulation of TCR signaling has not yet been elucidated. In this study, the potential effect of cordycepin on T cells was observed in CFA-induced inflammation mice model, and the function of cordycepin in regulating TCR signaling cascade was investigated.Methods: A CFA-induced inflammation mice model was established for observing the effect of cordycepin on the thymus and spleen swellings, and T cell infiltration in paw tissue was detected by immunohistochemistry. The protein expression or phosphorilation was detected by western blotting, and the NFAT1 nuclear translocation was determined by fluorescence imaging. The cell proliferation, apoptosis, and IL-2 production were analyzed by CCK-8 method, flow cytometry, and ELISA.Results: In the mice model, the thymus and spleen swellings were suppressed and the T cell infiltration in paw tissue was inhibited by cordycepin at a concentration of 10 mg/kg. Although the expressions of ZAP70 and PLCγ1 were not significantly changed in the human T cell line Jurkat with cordycepin pretreatment, the CD3-antibody-induced phosphorylations of ZAP70 and PLCγ1 were markedly blocked. The protein level of p85 decreased when Jurkat cells were pretreated with cordycepin, and cordycepin blocked TCR downstream molecule Erk phosphorylation and NFAT1 nuclear translocation. Further investigation revealed that cordycepin inhibited T cell proliferation, reduced IL-2 production, and induced T cell apoptosis. Conclusions: These findings suggest that cordycepin regulates TCR signaling to inhibit excessive T cell activation in inflammation. Thus, cordycepin may be a potential therapeutic application in inflammation-associated diseases.
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Antiinflamatorios/farmacología , Desoxiadenosinas/farmacología , Adyuvante de Freund/inmunología , Inflamación/prevención & control , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones Endogámicos , Transducción de Señal , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/inmunología , Timo/efectos de los fármacos , Timo/inmunologíaRESUMEN
Eicosanoids play an important role in inducing complex and crucial physiological processes in animals. Eicosanoid biosynthesis in animals is widely reported; however, eicosanoid production in invertebrate tissue is remarkably different to vertebrates and in certain respects remains elusive. We, for the first time, compared the orthologs involved in arachidonic acid (AA) metabolism in 14 species of invertebrates and 3 species of vertebrates. Based on parsimony, a complex AA-metabolic system may have existed in the common ancestor of the Metazoa, and then expanded and diversified through invertebrate lineages. A primary vertebrate-like AA-metabolic system via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways was further identified in the basal chordate, amphioxus. The expression profiling of AA-metabolic enzymes and lipidomic analysis of eicosanoid production in the tissues of amphioxus supported our supposition. Thus, we proposed that the ancestral complexity of AA-metabolic network diversified with the different lineages of invertebrates, adapting with the diversity of body plans and ecological opportunity, and arriving at the vertebrate-like pattern in the basal chordate, amphioxus.
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Ácido Araquidónico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Anfioxos/metabolismo , Metabolismo de los Lípidos/genética , Lipooxigenasa/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Evolución Biológica , Sistema Enzimático del Citocromo P-450/clasificación , Sistema Enzimático del Citocromo P-450/genética , Ácido Eicosapentaenoico/metabolismo , Regulación de la Expresión Génica , Anfioxos/genética , Lipoxinas/metabolismo , Lipooxigenasa/clasificación , Lipooxigenasa/genética , Anotación de Secuencia Molecular , Filogenia , Prostaglandina-Endoperóxido Sintasas/clasificación , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandinas/metabolismo , Tromboxanos/metabolismoRESUMEN
Recently, amphioxus has served as a model for studying the origin and evolution of vertebrate immunity. However, little is known about how microRNAs (miRNAs) are involved in the immune defense in amphioxus. In this article, we present a systematic study of amphioxus miRNAs in the acute-phase response to bacterial infection; miR-92d was found to regulate the complement pathway in this basal chordate. We identified all 155 possible miRNAs present in the amphioxus Branchiostoma belcheri genome by bioinformatics analyses, including 57 newly identified miRNAs (called bbe-miRNAs), and characterized the miRNA expression pattern. Four miRNAs (bbe-miR-7, bbe-miR-4868a, bbe-miR-2065, and bbe-miR-34b) were upregulated and bbe-miR-92d was downregulated under the challenge of both Vibrio anguillarum and Staphylococcus aureus bacteria. We further predicted miRNA targets and identified mRNA targets of immune-related miRNA using the hybrid PCR method. We propose that miR-92d regulates the complement pathway through targeting C3 for controlling the acute immune response to bacterial infections. This study provides evidence for the complex immune regulation of miRNAs in the acute-phase response in basal chordates.
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Cordados no Vertebrados/genética , Cordados no Vertebrados/inmunología , Complemento C3/metabolismo , Estudio de Asociación del Genoma Completo/métodos , MicroARNs/metabolismo , Inmunidad Adaptativa/genética , Animales , Cordados no Vertebrados/microbiología , Complemento C3/genética , Modelos Animales de Enfermedad , Evolución Molecular , Marcación de Gen/métodos , Células HEK293 , Humanos , MicroARNs/biosíntesis , MicroARNs/genética , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunología , Vibriosis/genética , Vibriosis/inmunologíaRESUMEN
H9N2 subtype avian influenza viruses are widespread in domestic poultry, and vaccination remains the most effective way to protect the chicken population from avian influenza pandemics. Currently, egg-based H9N2 influenza vaccine production has several disadvantages and mammalian MDCK cells are being investigated as candidates for influenza vaccine production. However, little research has been conducted on low pathogenic avian influenza viruses (LPAIV) such as H9N2 replicating in mammalian cells using microcarrier beads in a bioreactor. In this study, we present a systematic analysis of a safe H9N2 influenza vaccine derived from MDCK cells for protecting chickens against influenza virus infection. In 2008, we isolated two novel H9N2 influenza viruses from chickens raised in southern China, and these H9N2 viruses were adapted to MDCK cells. The H9N2 virus was produced in MDCK cells in a scalable bioreactor, purified, inactivated, and investigated for use as a vaccine. The MDCK-derived H9N2 vaccine was able to induce high titers of neutralizing antibodies in chickens of different ages. Histopathological examination, direct immunofluorescence, HI assay, CD4(+)/CD8(+) ratio test, and cytokine evaluation indicated that the MDCK-derived H9N2 vaccine evoked a rapid and effective immune response to protect chickens from influenza infection. High titers of H9N2-specific antibodies were maintained in chickens for 5 months, and the MDCK-derived H9N2 vaccine had no effects on chicken growth. The use of MDCK cells in bioreactors for LPAIV vaccine production is an attractive option to prevent outbreaks of LPAIV in poultry.
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Biotecnología/métodos , Subtipo H9N2 del Virus de la Influenza A/patogenicidad , Vacunas contra la Influenza/inmunología , Gripe Aviar/prevención & control , Animales , Anticuerpos Neutralizantes , Reactores Biológicos , Pollos/inmunología , Pollos/virología , Medio de Cultivo Libre de Suero , Perros , Técnica del Anticuerpo Fluorescente Directa , Subtipo H9N2 del Virus de la Influenza A/genética , Subtipo H9N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/inmunología , Células de Riñón Canino Madin Darby/virología , Filogenia , VacunaciónRESUMEN
BACKGROUND: The mechanisms responsible for cervical cancer radioresistance are still largely unexplored. The present study aimed to identify miRNAs associated with radioresistance of cervical cancer cells. METHODS: The radioresistant cervical cancer cell variants were established by repeated selection with irradiation. The miRNA profiles of radioresistant cells and their corresponding controls were analyzed and compared using microarray. Differentially expressed miRNAs were confirmed by quantitative real-time PCR. Cervical cancer cells were transfected with miRNA-specific mimics or inhibitors. Radiosensitivity of cervical cancer cells were determined using colony-forming assay. RESULTS: Among the differentially expressed miRNAs, 20 miRNAs showed the similar pattern of alteration (14 miRNAs were overexpressed whilst 6 were suppressed) in all three radioresistant cervical cancer cell variants compared to their controls. A miRNA signature consisting of 4 miRNAs (miR-630, miR-1246, miR-1290 and miR-3138) exhibited more than 5 folds of increase in radioresistant cells. Subsequent analysis revealed that these four miRNAs could be up-regulated in cervical cancer cells by radiation treatment in both time-dependent and dose-dependent manners. Ectopic expression of each of these 4 miRNAs can dramatically increase the survival fraction of irradiated cervical cancer cells. Moreover, inhibition of miR-630, one miRNA of the specific signature, could reverse radioresistance of cervical cancer cells. CONCLUSIONS: The present study indicated that miRNA is involved in radioresistance of human cervical cancer cells and that a specific miRNA signature consisting of miR-630, miR-1246, miR-1290 and miR-3138 could promote radioresistance of cervical cancer cells.
RESUMEN
Resveratrol has been shown to possess multiple pharmacological activities including anti-tumor, anti-inflammation and immunomodulation, and participates in the regulation of the NF-kappaB signaling pathway. However, the mechanism of the NF-kappaB signaling pathway inhibited by resveratrol remains obscure. In this study, we first examined the effect of resveratrol on endogenous and TNF-alpha-induced NF-kappaB activation, and found that resveratrol suppressed NF-kappaB activation in a dose dependent manner. Resveratrol reduced the transcriptional activity of p65, but neither affected the DNA-binding activity of NF-kappaB nor blocked the nuclear translocation of p65. Moreover, resveratrol had no effect on the expression level of IkappaBalpha protein and inhibited IkappaBalpha degradation. Further investigation revealed that resveratrol blocked the ubiquitination of NEMO and inhibited IkappaB kinase(beta)-mediated NF-kappaB activation. These results demonstrated that resveratrol effectively suppressed NF-kappaB signaling through inhibiting the activities of NF-kappaB and IkappaB kinase. Therefore, resveratrol may provide a novel approach to treating inflammation-associated diseases and cancer.
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Antioxidantes/farmacología , Quinasa I-kappa B/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Estilbenos/farmacología , Factor de Transcripción ReIA/antagonistas & inhibidores , Western Blotting , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Células HEK293 , Humanos , Inmunoprecipitación , Luciferasas/genética , Luciferasas/metabolismo , Microscopía Fluorescente , Plásmidos/genética , Resveratrol , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Translocación Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Objective: The prognostic factors for elderly patients with cervical cancer differ from those of younger patients. Competitive risk events could cause biases in the Cox proportional hazards (PH) model. This study aimed to construct a competitive risk model (CRM) nomogram for patients aged > 65 years with nonmetastatic cervical cancer. Methods: We retrospectively analyzed data extracted from the Surveillance, Epidemiology, and End Results (SEER) database and a total of 1856 patients from 18 cancer registries across the United States diagnosed between 2010 and 2015 were included. Kaplan-Meier analysis and log-rank tests were used to compare intergroup survival. Univariate and multivariate Cox proportional regression analyses were performed to identify independent prognostic factors. The cumulative incidence function (CIF) and Fine and Gray's test were used to determine the impact of competitive risk events on prognosis. The CRM nomogram was internally and externally validated using time-dependent receiver operator characteristic (ROC) curve (time-AUC), Brier scores, Harrell's concordance index (C-index), calibration curve, and decision curve analysis (DCA). Results: Analyses revealed that histology, age, the International Federation of Gynaecologists and Obstetricians (FIGO) stage, number of in situ malignancies, chemotherapy, radiotherapy (RT), and surgery were independent prognostic factors. The CRM nomogram accurately predicted 1-year, 3-year, and 5-year disease-specific survival (DSS). The C-indexes and Brier scores of the CRM nomogram were 0.641 and 0.094, respectively, at the 1-year cut-off in the training set. The time-AUC of the CRM nomogram at the 1-year, 3-year, and 5-year intervals in the training set were 77.6%, 77.3%, and 74.5%, respectively. The calibration curve demonstrated a favorable concordance. DCA suggested that the nomogram had a good net benefit. Therefore, the Cox model underestimated the weight of risk factors compared to CRM. Conclusions: This study presents the CRM nomogram to predict DSS in patients aged > 65 years with nonmetastatic cervical cancer. It can help clinicians implement more accurate personalized diagnostic and treatment modalities for elderly patients with cervical cancer.
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Nomogramas , Neoplasias del Cuello Uterino , Anciano , Femenino , Humanos , Neoplasias del Cuello Uterino/epidemiología , Neoplasias del Cuello Uterino/terapia , Estudios Retrospectivos , Pronóstico , Calibración , Programa de VERFRESUMEN
BACKGROUND: The caspase family, which plays a central role in apoptosis in metazoans, has undergone an expansion in amphioxus, increasing to 45 members through domain recombination and shuffling. RESULTS: In order to shed light on the conservation and uniqueness of this family in amphioxus, we cloned three representative caspase genes, designated as bbtCaspase-8, bbtCaspase-1/2 and bbtCaspase3-like, from the amphioxus Branchiostoma belcheri tsingtauense. We found that bbtCaspase-8 with conserved protein architecture is involved in the Fas-associated death domain-Caspase-8 mediated pro-apoptotic extrinsic pathway, while bbtCaspase3-like may mediate a nuclear apoptotic pathway in amphioxus. Also, bbtCaspase-1/2 can co-localize with bbtFADD2 in the nucleus, and be recruited to the cytoplasm by amphioxus apoptosis associated speck-like proteins containing a caspase recruitment domain, indicating that bbtCaspase-1/2 may serve as a switch between apoptosis and caspase-dependent innate immune response in invertebrates. Finally, amphioxus extrinsic apoptotic pathway related caspases played important roles in early embryogenesis. CONCLUSIONS: Our study not only demonstrates the conservation of bbtCaspase-8 in apoptosis, but also reveals the unique features of several amphioxus caspases with novel domain architectures arose some 500 million years ago.
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Caspasas/genética , Cordados/genética , Secuencia Conservada/genética , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Caspasas/química , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Cordados/embriología , Desarrollo Embrionario/efectos de los fármacos , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Filogenia , Inhibidores de Proteasas/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Alineación de SecuenciaRESUMEN
Purpose: Hypoxia has crucial functions in the development and metastasis of cervical cancer by inducing the expression of numerous genes, including microRNA genes. But we know little about how the hypoxia factors and microRNAs orchestrate to regulate hallmarks of cervical cancer cells. Methods: We conducted RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) experiments to investigate the targets of HIF-3α or miR-630. ChIP-qPCR and RT-qPCR were carried out to validate the results of ChIP-seq and RNA-seq. Cellular, molecular, and radiation experiments were conducted to explore the functions of miR-630. Results: In this study, we showed that hypoxia-induced overexpression of HIF-3α increased the expression of dozens of miRNAs, including miR-630. Hypoxia could also directly induce miR-630 expression. ChIP-seq data showed that HIF-3α activates miR-630 expression by directly binding to the promoter of its host gene. Meanwhile, stable overexpression of miR-630 increased the expression of HIF-3α, but repressed the expression of HIF-1α, indicating a positive feedback loop between HIF-3α and miR-630. Consequently, stable overexpression of miR-630 in HeLa cells promotes cancer hallmarks, including radioresistance, inhibition of apoptosis, increased migration and invasion, and EMT-mediated metastasis. Meanwhile, inhibition of miR-630 showed opposite features. Conclusion: Taken together, our findings indicate a novel hypoxia-induced HIF-3α and miR-630 regulatory feedback loop contributing to metastasis and progression of cervical cancer cells and suggest that HIF-3α and miR-630 might act as potential biomarkers and therapeutic targets for cervical cancer in the future.
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Proteínas Reguladoras de la Apoptosis , MicroARNs , Neoplasias del Cuello Uterino , Femenino , Humanos , Células HeLa , Hipoxia/genética , MicroARNs/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Proteínas Reguladoras de la Apoptosis/genética , Retroalimentación FisiológicaRESUMEN
OBJECTIVE: To study the effects of osthole on sciatica induced by lumber disc herniation and its mechanisms. METHODS: 54 male SD rats were randomly divided into 6 groups. Model (M) group (n = 12): Autologous nucleus pulposus was harvested from the tail and applied to the L5 dorsal root ganglion (DRG) and epidural space. Epidural catheterization was performed. Control(C) group (n = 12): On the basis of nucleus pulposus group, 50 microL tween-80 was administered epidurally on the day 6th after surgery. T2 (n = 6), T6 (n = 12), T13 (n = 6) and T20 (n = 6) group: 50 microL 2% osthole was administered epidurally on the 2th, 6th, 13th day and 20th after surgery respectively. General behaviors were observed and 50% paw withdrawal threshold (50% PWT) was measured 1 day before surgery, on the 1st, 3th, 7th,14th, 21th, 28th day after surgery, immediately before and 1 hour after osthole or tween-80 administration in each group. On the 7th day after surgery, the left L5 DRGs were obtained for detecting the expression of NOS and COX-2 in M, C and T6 group with 6 rats. RESULTS: No lameness or autophagy was oberserved. 50% PWT decreased after surgery (P < 0.05). In T2 and T6 group, 50% PWT after osthole administration were significantly higher than those of M group and C group (P < 0.05), which recovered to the same level as 1 day before surgery (P > 0.05). In T13 and T20 group, 50% PWT 1 hour after osthole administration were significantly higher than those of M group and C group (P < 0.05), which recovered to the same level as 1 day before surgery (P > 0.05), but on days after 1 hour after administration, there was no significant difference when 50% PWT compared with M group or nucleus C group (P > 0.05). NOS positive cells and COX-2 positive cells were no significant difference when M group compared with C group (P > 0.05). But these positive cells in T6 group were significantly lower than those of M group and C group (P < 0.05). CONCLUSION: 50 microL 2% osthole could completely inhibit the mechanical allodynia in the rat model of sciatica induced by lumbar disc herniation when it was administered epidurally on 2 or 6 day after surgery. But when administered on 13 or 20 day after surgery, its analgesic effect was transient. The effect of 50 microL 2% osthole epidural administration on day 6 after surgery on the rat model of sciatica induced by lumbar disc herniation may relate to inhibition of the expression of COX-2 and NOS in DRG.
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Analgésicos/farmacología , Cumarinas/farmacología , Ganglios Espinales/metabolismo , Desplazamiento del Disco Intervertebral/complicaciones , Ciática/tratamiento farmacológico , Analgésicos/administración & dosificación , Animales , Conducta Animal , Cumarinas/administración & dosificación , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Ganglios Espinales/cirugía , Inyecciones Epidurales , Vértebras Lumbares/cirugía , Masculino , Óxido Nítrico Sintasa/metabolismo , Dimensión del Dolor/métodos , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Ciática/etiología , Ciática/metabolismo , Raíces Nerviosas Espinales/lesionesRESUMEN
Cordycepin, a natural derivative of adenosine, has been shown to exert pharmacological properties including anti-oxidation, antitumor, and immune regulation. It is reported that cordycepin is involved in the regulation of macrophage function. However, the effect of cordycepin on inflammatory cell infiltration in inflammation remains ambiguous. In this study, we investigated the potential role of cordycepin playing in macrophage function in CFA-induced inflammation mice model. In this model, we found that cordycepin prevented against macrophage infiltration in paw tissue and reduced interferon-γ (IFN-γ) production in both serum and paw tissue. Using luciferase reporter assay, we found that cordycepin suppressed IFN-γ-induced activators of transcription-1 (STAT1) transcriptional activity in a dose-dependent manner. Moreover, western blotting data demonstrated that cordycepin inhibited IFN-γ-induced STAT1 activation through attenuating STAT1 phosphorylation. Further investigations revealed that cordycepin inhibited the expressions of IFN-γ-inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig), which were the effector genes in IFN-γ-induced STAT1 signaling. Meanwhile, the excessive inflammatory cell infiltration in paw tissue was reduced by cordycepin. These findings demonstrate that cordycepin alleviates excessive inflammatory cell infiltration through down-regulation of macrophage IP-10 and Mig expressions via suppressing STAT1 phosphorylation. Thus, cordycepin may be a potential therapeutic approach to prevent and treat inflammation-associated diseases.
Asunto(s)
Quimiocina CXCL10/antagonistas & inhibidores , Quimiocina CXCL9/antagonistas & inhibidores , Desoxiadenosinas/uso terapéutico , Interferón gamma/toxicidad , Macrófagos/efectos de los fármacos , Factor de Transcripción STAT1/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Quimiocina CXCL10/biosíntesis , Quimiocina CXCL9/biosíntesis , Desoxiadenosinas/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Adyuvante de Freund/toxicidad , Expresión Génica , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Macrófagos/metabolismo , Ratones , Células RAW 264.7 , Distribución Aleatoria , Factor de Transcripción STAT1/metabolismoRESUMEN
In this work, a novel M-superfamily conotoxin, designated lt3a, was purified from the crude venom of Conus litteratus. Combined with peptide sequencing, MALDI-TOF mass spectrometry and cDNA cloning techniques, the amino acid sequence of lt3a was supposed to be DgammaCCgamma OQWCDGACDCCS, where O is hydroxyproline and gamma is carboxyglutamate. The Cys framework of lt3a (-CC-C-C-CC-) is similar to that of psi-, mu-, kappaM-conotoxins, which are representatives of M-conotoxins. Peptide lt3a is categorized into M1 branch based on the number of residues in the last Cys loop. Whole cell patch-clamp study on adult rat dorsal root ganglion neurons indicated that lt3a could enhance tetrodotoxin-sensitive sodium currents. This is a previously unknown function of M-superfamily conotoxins.
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Conotoxinas/química , Conotoxinas/toxicidad , Canales de Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Conotoxinas/aislamiento & purificación , Caracol Conus/metabolismo , Conductividad Eléctrica , Etiquetas de Secuencia Expresada/química , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tetrodotoxina/farmacologíaRESUMEN
In this work, two P-superfamily conotoxins, lt9a and lt9b, were purified and characterized from the crude venom of Conus litteratus. The amino acid sequences of lt9a and lt9b were determined by the Edman degradation method. It has been suggested that both lt9a and lt9b are produced from the precursor encoded by the gene Lt9.1. During the conotoxin maturation process, different post-translational modifications occurred between lt9a and lt9b. Conotoxin lt9b was predicted to have two prolines that underwent hydroxylation and one glutamate that underwent carboxylation, while lt9a had no hydroxyproline and carboxyglutamate residue. The calculated mass weights of two P-superfamily conotoxins with three proposed disulfide bonds were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry after considering corresponding post-translational modifications. These two conotoxins showed different effects on tetrodotoxin-sensitive sodium currents. Conotoxin lt9a (300 nM) resulted in marked slowing of the tetrodotoxin-sensitive sodium current decay, a notable increase in the peak current, and an alteration in reversal potential. However, lt9b inhibits tetrodotoxin-sensitive sodium currents, and the inhibition showed a concentration-dependent with the half maximal inhibitory concentration (IC50) value of 504.04 nM, but there were no change in the activation and inactivation kinetics of currents. To the best of our knowledge, this is the first investigation of two P-superfamily conotoxins identified to act on voltage-sensitive sodium channels with different modifications.
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Conotoxinas/química , Conotoxinas/aislamiento & purificación , Canales de Sodio/efectos de los fármacos , Animales , Caracol Conus , Procesamiento Proteico-Postraduccional , Ratas Sprague-Dawley , Análisis de Secuencia de Proteína , Transducción de Señal/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis-trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis-trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.
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Conotoxinas/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Conotoxinas/química , Caracol Conus , Estabilidad de Enzimas , Escherichia coli , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Conotoxin lt14a is a small peptide consisting of 13 amino acids. It was originally identified from the cDNA of Conus litteratus in the South China Sea. Previous reports showed lt14a exhibited antinociceptive activity using a hot plate-induced pain mouse model and acted as an antagonist of neuronal nicotinic acetylcholine receptors. We confirmed that conotoxin lt14a administration resulted in antinociception activity using a mouse inflammatory pain model and a rat model of mechanically-induced pain. The mRNA expression of c-fos and NOS in the spinal cord of rats was suppressed by lt14a. Labeling of lt14a with an Alexa Fluor 488 ester showed that lt14a was bound to the surface of PC12 cells and that this binding was inhibited by pre-application of the nicotinic acetylcholine receptor (nAChR) antagonist tubocurarine chloride (TUB) and the nAChR blocker hexamethonium bromide (HB). These data confirm previous reports that showed lt14a binds to the surface of PC12 cells via nAChRs with patch clamp whole-cell recordings. Additional results showed that lt14a suppressed extracellular signal-regulated kinase (ERK1/2) phosphorylation in PC12 cells activated by Ach. Our results showed that lt14a did not induce drug dependence but rather suppressed morphine withdrawal symptoms. Our work suggests that lt14a is a novel antinociceptive agent that targets the nAChR receptor without inducing drug dependence.
Asunto(s)
Analgésicos/farmacología , Conotoxinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Cartilla de ADN/genética , Formaldehído , Ratones , Óxido Nítrico Sintasa/metabolismo , Células PC12 , Dimensión del Dolor , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cone snails are incredibly rich sources of bioactive conopeptides with potential for use in neuroscience research and novel drug development. In order to investigate the synthesis of diversified conopeptides in venom glands, the proteome and peptidome profiles of conus venom were analyzed using HPLC and mass spectrometry. The peptidome profile of the venom components with a molecular weight under 10 kDa showed that the peptides with unique mass values from the venom glands of Conus caracteristicus, Conus lividus and Conus textile are 188, 413 and 265, respectively, and there are 39 overlapping peptides among the three species. Proteome profiling of the components with molecular weights above 10 kDa showed that the most abundant proteins (38.6%) are involved in metabolism and that approximately 6.8% of proteins are involved in protein synthesis, folding and post-translational modification. Among these proteins, PPIase is one protein identified from C. textile based on proteomic analysis. Conus PPIase was successfully expressed as a fusion protein with TRX in an Escherichia coli expression system for further function study. In-vitro enzyme activity assays showed that cone snail PPIase could induce the cis-trans isomerization of the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The HPLC mapping analyses of linear lt14a, a conotoxin with 3 prolines, showed that different lt14a isoforms appear after incubation with PPIase. Our results suggest that PPIase may modify conotoxins containing prolines and play an important role in the process of peptide folding and modification in venom glands and contribute to conotoxin diversity.
Asunto(s)
Conotoxinas/análisis , Conotoxinas/química , Caracol Conus/química , Isomerasa de Peptidilprolil/química , Proteoma/análisis , Proteoma/química , Animales , Cromatografía Líquida de Alta Presión , Espectrometría de MasasRESUMEN
The conotoxin cysteine framework XV (-C-C-CC-C-C-C-C-), which was named Lt15a, was firstly identified from the cDNA library of Conus litteratus. After that, 18 new framework XV conotoxin sequences were cloned from nine Conus species. Like other conopeptides, the XV-conotoxins have the conserved signal peptide and propeptide, and there are also some conserved residues in their mature peptide. All the framework XV conotoxins were apparently converged into two branches, because of the indel and point mutations occurred in their mature peptides. By fused with thioredoxin and 6×His tag, six XV-conotoxins were successfully expressed in Escherichia coli and purified. Different framework XV conotoxins have distinct biological activities on mice and frogs, and that may be related to the diversity of the toxin sequences. All the six XV-conotoxins had no obvious effects on the sodium currents of DRG neuron cells of Sprague-Dawley (SD) rats. The identification of this framework of conotoxins enriches our understanding of the structural and functional diversity of conotoxin.
Asunto(s)
Conotoxinas/metabolismo , Secuencia de Aminoácidos , Animales , Anuros , Secuencia de Bases , Células Cultivadas , Conotoxinas/química , Conotoxinas/genética , Conotoxinas/farmacología , Caracol Conus , Ganglios Espinales/citología , Ratones , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Alineación de SecuenciaRESUMEN
Cone snails, which are predatory marine gastropods, produce a cocktail of venoms used for predation, defense and competition. The major venom component, conotoxin, has received significant attention because it is useful in neuroscience research, drug development and molecular diversity studies. In this study, we report the genomic characterization of nine conotoxin gene superfamilies from 18 Conus species and investigate the relationships among conotoxin gene structure, molecular evolution and diversity. The I1, I2, M, O2, O3, P, S, and T superfamily precursors all contain three exons and two introns, while A superfamily members contain two exons and one intron. The introns are conserved within a certain gene superfamily, and also conserved across different Conus species, but divergent among different superfamilies. The intronic sequences contain many simple repeat sequences and regulatory elements that may influence conotoxin gene expression. Furthermore, due to the unique gene structure of conotoxins, the base substitution rates and the number of positively selected sites vary greatly among exons. Many more point mutations and trinucleotide indels were observed in the mature peptide exon than in the other exons. In addition, the first example of alternative splicing in conotoxin genes was found. These results suggest that the diversity of conotoxin genes has been shaped by point mutations and indels, as well as rare gene recombination or alternative splicing events, and that the unique gene structures could have made a contribution to the evolution of conotoxin genes.
Asunto(s)
Caracol Conus/genética , Evolución Molecular , Intrones , Péptidos/genética , Venenos de Serpiente/genética , Empalme Alternativo , Animales , Composición de Base , Caracol Conus/metabolismo , Exones , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Orden Génico , Familia de Multigenes , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Venenos de Serpiente/químicaRESUMEN
OBJECTIVE: To evaluate whether saw palmetto extract (SPE) relaxes corpus cavernosum and explore the underlying mechanisms. METHODS: Forty Sprague-Dawley rats and 30 New Zealand rabbits were randomly allocated into 3 SPE-treated groups (low-, middle-, and high-dose) and 1 saline-treated control group. SPE was administered intragastrically for 7 consecutive days. Another 23 rats treated with sildenafil were used to appraise the erectile response to electrical stimulation of nerves in the corpus cavernosum. The erectile functions of rats and rabbits were evaluated 24 hours after the last SPE administration or 15 minutes after intragastric sildenafil. Outcome measures included corpus cavernosum electrical activity recording, phosphodiesterase 5 (PDE5) activity detected by the colorimetric quantitative method, and messenger ribonucleic acid (mRNA) expression level for PDE5 and inducible nitric oxide synthase (iNOS) determined using real-time polymerase chain reaction. RESULTS: In the SPE-treated animals, the relaxant response to electrical stimulation of nerves in the corpus cavernosum, reflected by the amplitude of the electrical activity within the cavernosum, was significantly and dose-dependently augmented. Similar effects were observed in the sildenafil-treated rats. PDE5 activity in rat and rabbit corpus cavernosum tissues was significantly and dose-dependently inhibited in SPE-treated animals, whereas the iNOS mRNA level increased compared with the saline group. PDE5 mRNA, however, was only significantly enhanced in the rats treated with the middle dose of SPE. CONCLUSION: The results suggest that SPE may have potential application value for the prevention or treatment of erectile dysfunction through an increase in iNOS mRNA expression and inhibition of PDE5 activity in corpus cavernosum smooth muscles.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Activación Enzimática/efectos de los fármacos , Erección Peniana/efectos de los fármacos , Pene/metabolismo , Inhibidores de Fosfodiesterasa 5/farmacología , Extractos Vegetales/farmacología , ARN Mensajero/metabolismo , Análisis de Varianza , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Estimulación Eléctrica , Masculino , Óxido Nítrico Sintasa de Tipo II/genética , Pene/efectos de los fármacos , Pene/fisiología , Piperazinas/farmacología , Purinas/farmacología , Conejos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Serenoa , Citrato de Sildenafil , Sulfonas/farmacologíaRESUMEN
A series of novel N(2)-alkylated quaternary ß-carbolines was synthesized by modification of position-1, 2, 7 and 9 of ß-carboline nucleus with various alkyl and arylated alkyl substituents, and their cytotoxic activities in vitro and antitumor potencies in mice were evaluated. Compound 3m was found to be the most potent antitumor agent. SARs analysis revealed that (1) the substituents in position-2 and 9 of ß-carboline nucleus played a vital role in modulation of antitumor activity; (2) the benzyl and 3-phenylpropyl substituents in position-2 and 9 of ß-carboline ring were the optimal substituents giving rise to significant antitumor agent. These compounds might be a novel promising class of antitumor agents with clinical development potential.