RESUMO
This study aims to interpret the energetic basis of complex DNA-peptide interactions according to a novel allosteric interaction network approach. In common with other designed peptides, five new conjugates incorporating the XPRK or XHypRK motif (Hyp = hydroxyproline) attached to a N-methylpyrrole (Py) tract with a basic tail have been found to display cooperative binding to DNA involving multiple monodentate as well as interstrand bidentate interactions. Using quantitative DNase I footprinting it appears that allosteric communication via cooperative binding to multiple sites on complementary DNA strands corresponds to two different types of DNA-peptide interaction network. Temperature variation experiments using a dodecapeptide RY-12 show that lower temperature (25 °C) favor a circuit type of allosteric interaction network, whereas higher temperatures (31 and 37 °C) afford only a partial-circuit type of network. Circular dichroism studies show that our five peptides induce significant local conformational changes in DNA via the minor groove, with apparently dimeric binding stoichiometry. Isothermal titration calorimetry reveals that these peptides, together with another seven for comparison, are strongly exothermic upon binding to a model 13-mer DNA duplex, characterized by ΔH ranging from -14.7 to -74.4 kcal mol(-1), and also high TΔS ranging from -6.5 to -65.9 kcal mol(-1). Multiple monodentate and bidentate interactions, as well as ionic forces that mediate positive cooperativity in sequence recognition, are consistent with a dramatic decrease in entropy and a 'tightening' effect of DNA conformation. Distinctive enthalpy-entropy compensation (EEC) relationships are demonstrated for the interaction of all twelve designed peptides with DNA, affording a straight line of slope close to unity when ΔH is plotted versus TΔS, with a y-axis intercept (average ΔG) corresponding to -8.5 kcal mol(-1), while the observed ΔG ranges from -8.2 to -9.1 kcal mol(-1) for the peptides. The EEC seen with peptide RY-12 binding to the model duplex persists throughout various incubation temperatures. The net compensation of energy between the favorable negative ΔH and unfavorable negative ΔS components thus constrains the value of net binding free energy ΔG within a remarkably constant range, as is clearly visible in a 3-dimensional energetic plot. We conclude that the preservation of a rather narrowly-defined ΔG value is central to the EEC in DNA-peptide interactions, illuminating the universal EEC paradox commonly found in diverse biochemical reactions.
Assuntos
DNA/química , Peptídeos/química , TermodinâmicaRESUMO
Allostery in the binding of peptides to DNA has been studied by quantitative DNase I footprinting using four newly designed peptides containing the XP(Hyp)RK motif and N-methylpyrrole (Py) moieties. Apparent binding constants in the micromolar range as well as Hill coefficients were determined for each peptide. The results, together with previous studies on five other peptides support the proposal that interaction network cooperativity is highly preferred in DNA-peptide interactions that involve multiple recognition sites. It is envisaged that interstrand bidentate interactions participate in the relay of conformational changes between recognition sites on the complementary strands. Models for interpreting DNA allostery based upon interaction networks are outlined. Circular dichroism experiments involving the titration of peptides against a short oligonucleotide duplex indicate that some of these peptides bind in a dimeric manner to DNA via the minor groove, inducing characteristic conformational changes. These insights should prompt the design of new DNA-binding peptides for investigating allosteric interactions between peptides and DNA, as well as novel interaction networks, and ultimately may shed light upon the fundamental chemical rules that govern allostery in more complex biological process such as DNA-protein interaction networks.
Assuntos
DNA/química , Peptídeos/química , Autorradiografia , Dicroísmo Circular , Pegada de DNA , Desoxirribonuclease I/química , Ligantes , Oligonucleotídeos/química , Ligação Proteica , Conformação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-AtividadeRESUMO
Quantitative DNase I footprinting shows that three designed peptides containing N-methylpyrrole (Py) moieties display different types of network-based allosteric communication in binding to DNA: circuit type, incomplete-circuit type, and non-circuit type characterized by interstrand bidentate interactions. Positive cooperative binding of all three peptides to individual DNA binding sites is commonly observed. CD spectral characterization of the interaction between peptides and model undecanucleotide duplexes is consistent with the footprinting results and supports the allosteric model. This study provides insights relating to the interaction network nature of allostery in complex DNA-small molecule interactions.
Assuntos
Pegada de DNA , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , DNA/química , Peptídeos/químicaRESUMO
This study was to investigate the anti-inflammatory activities in vitro of various carrageenans (Car) fractions (κ-, ι-, and λ-types) with well characterized molecular properties, using murine microglia BV-2 cell line treated with lipopolysaccharide (LPS) as model. It is indicated that pretreatments with the oligosaccharide fractions from κ- or ι-carrageenan acid hydrolysates (κ- and ι-CarAOS, respectively) at 125-500⯵g/mL significantly and dose-dependently decreased the levels of tumor necrosis factor α (TNF-α) secreted from LPS-treated BV-2 cells, showing promisingly anti-inflammatory effects. Differently, pretreatments of most of polymeric carrageenans at 250-500⯵g/mL significantly increased the TNF-α level, implying the co-inflammatory effects with LPS. The co-inflammatory effectiveness of pure carrageenans at 125⯵g/mL was notable for λ-Car, followed by ι-Car, and insignificantly for κ-Car. Generally, cytokine TNF-α was a more sensitive biomarker to the presence of carrageenans than was the IL-6. The TNF-α level varied greatly at a low carrageenan concentration (125⯵g/mL) and high polymer percentage (e.g. purified κ- and ι-Car). Conclusively, the anti-inflammatory effects on LPS-treated BV-2 cells could be attenuated by pretreatments with κ- and ι-CarAOS at 125-500⯵g/mL.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Carragenina/química , Lipopolissacarídeos/antagonistas & inibidores , Microglia/efeitos dos fármacos , Oligossacarídeos/farmacologia , Polissacarídeos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/isolamento & purificação , Linhagem Celular Transformada , Fracionamento Químico/métodos , Relação Dose-Resposta Imunológica , Hidrólise , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Interleucina-6/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/citologia , Microglia/imunologia , Oligossacarídeos/isolamento & purificação , Polissacarídeos/isolamento & purificação , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Cordyceps species have been traditionally used for the enhancement of sexual function, however, there is few direct evidence to prove this. We investigated the spermatogenic effect of Cordyceps militaris (CM) by supplementation with CM mycelium to subfertile boars. Seventeen Duroc and 12 Landrace boars (29 to 40 months old) were selected to feed with regular diet (control groups, n = 8 and 6, respectively) or diet supplemented with CM mycelium (treatment groups, n = 9 and 6, respectively) for 2 months. Semen was collected once a week. The quality of fertile sperm (normally greater than 62% of motility and 70% of normal morphology) and the quantity (semen volume, and total sperm number) were compared in these boars. The result showed that sperm production was enhanced significantly at the end of first month (p < 0.05), peaked at the second month (p < 0.01) of supplementation with CM and was maintained for 2 weeks after stopping the treatment (p < 0.01). Plasma cordycepin concentration was detected in boars supplemented with CM but not in the controls. More importantly, the percentages of motile sperm cells and sperm morphology were also improved significantly in most of treated boars during the second month of supplementation (p < 0.01) and 2 weeks after the treatment (p < 0.05) as compared to their initial values. These results indicate that supplementation with CM mycelium improves sperm quality and quantity in subfertile boars and may partly support the role of Cordyceps in sexual enhancement.
Assuntos
Cordyceps , Medicamentos de Ervas Chinesas/uso terapêutico , Infertilidade Masculina/tratamento farmacológico , Espermatogênese/efeitos dos fármacos , Animais , Suplementos Nutricionais , Medicamentos de Ervas Chinesas/farmacologia , Infertilidade Masculina/fisiopatologia , Infertilidade Masculina/veterinária , Masculino , Fitoterapia/métodos , Contagem de Espermatozoides , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Espermatogênese/fisiologia , Espermatozoides/patologia , Sus scrofaRESUMO
Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. It has been cross-linked with organic acids and polysaccharides by gamma irradiation. The cross-linked (CL)-bromelain preparation resisted an acidic environment of pH 3 for 2 h and preserved 80% of its enzyme activity. Pretreatment of rats with CL-bromelain intragastrically for 7 days significantly reduced serum cytokine production induced by injected i.p. with 2.5 mg/kg of lipopolysaccharide (LPS). Bromelain significantly reduced serum glutamate-oxalacetate transaminase induced by LPS. The anti-inflammatory effect of CL-bromelain was correlated with reduced LPS-induced NF-kappaB activity and cyclooxygenase 2 (COX-2) mRNA expression in rat livers. In addition, CL-bromelain dose-dependently inhibited LPS-induced COX-2 mRNA and prostaglandin E2 (PGE2) in BV-2 microglial cells. CL-Bromelain also suppressed the LPS-activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). In conclusion, the anti-inflammatory effects of the CL-bromelain preparation in vivo and in vitro suggest its therapeutic potentials.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Bromelaínas/química , Bromelaínas/farmacologia , Citocinas/biossíntese , Lipopolissacarídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Reagentes de Ligações Cruzadas , Ciclo-Oxigenase 2/genética , Citocinas/sangue , Dinoprostona/metabolismo , Ativação Enzimática/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Varredura , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Sesame seed oil increases the survival after cecal ligation and puncture in mice and the increased IL-10 levels with non-lethal lipopolysaccharides (LPS) challenge. We showed that sesamin and sesamolin, major lignans of sesame oil, regulated LPS-induced nitric oxide production in the murine microglia and BV-2 cell line. In this study, we studied the effect of sesamin on cytokine production by LPS stimulation. The result showed that sesamin significantly inhibited LPS-stimulated IL-6 mRNA and protein, and to a lesser degree TNF-alpha, in BV-2 microglia. Sesamin and sesamolin also reduced LPS-activated p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB activations. Furthermore, SB203580, a specific inhibitor of p38 MAP kinase, specifically inhibited LPS-induced IL-6 production. These results suggest that sesamin inhibited LPS-induced IL-6 production by suppression of p38 MAPK signal pathway and NF-kappaB activation.
Assuntos
Antioxidantes/farmacologia , Citocinas/metabolismo , Dioxóis/farmacologia , Lignanas/farmacologia , NF-kappa B/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The alpha-ketoglutarate dehydrogenase complex (KGDHC) is a mitochondrial enzyme in the TCA cycle. Inhibition of KGDHC activity by alpha-keto-beta-methyl-n-valeric acid (KMV) is associated with neuron death. However, the effect of KMV in microglia is unclear. Therefore, we investigated the effect of KMV on BV-2 microglial cells exposed to hypoxia or oxidative stress. The results showed that KMV (1-20 mM) enhanced the cell viability under hypoxia. KMV dose-dependently reduced ROS and LDH releases from hypoxic BV-2 cells. KMV also reduced ROS production and enhanced the cell viability under H2O2 but failed to reduce the SIN-1 and sodium nitroprusside (SNP) toxicity. KMV also reduced caspase-3 and -9 activation under stress. These results suggest that KMV protects BV-2 cells from stress and acts by reducing ROS production through inhibition of KDGHC.
Assuntos
Hipóxia Celular/fisiologia , Citoproteção/efeitos dos fármacos , Cetoácidos/farmacologia , Microglia/efeitos dos fármacos , Estresse Oxidativo , Apoptose/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Peróxido de Hidrogênio/farmacologia , Microglia/citologia , Microglia/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Reactive oxygen species (ROS) has been proposed to play a pathogenic role in neuronal injury. Sesame antioxidants that inhibit lipid peroxidation and regulate cytokine production may suppress ROS generation. In this study, we focused on the effect of sesamolin on H2O2-induced neurotoxicity and ROS production in the murine microglial cell line BV-2. Results indicate that the H2O2 elicited BV-2 cell death in a concentration- and time-dependent manner. ROS generation in BV-2 cells was time-dependently increased by the H2O2 treatment. Sesamolin reduced ROS generation in BV-2 cells. p38 mitogen-activated protein kinase (MAPK) and caspase-3 were also activated in BV-2 cells under H2O2 stress. Sesamolin was able to inhibit H2O2-induced p38 MAPK and caspase-3 activation and cell death. In addition, sesamolin preserved superoxide dismutase and catalase activities in BV-2 cells under H2O2 stress. In conclusion, sesamolin protects microglia against H2O2-induced cell injury and this protective effect was accompanied by its inhibition of p38 MAPK and caspase-3 activation and ROS production.
Assuntos
Dioxóis/farmacologia , Peróxido de Hidrogênio/toxicidade , Microglia/efeitos dos fármacos , Microglia/enzimologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antioxidantes/metabolismo , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Oxirredução/efeitos dos fármacos , Fosforilação , Espécies Reativas de Oxigênio/metabolismoRESUMO
In the present study, experiments were performed to explore the action of quercetin, the most widely distributed flavonoids, and its major metabolite, quercetin-3'-sulfate, on lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) production in BV-2 microglia. Quercetin could suppress LPS- and IFN-gamma-induced NO production and inducible nitric oxide synthase (iNOS) gene transcription, while quercetin-3'-sulfate had no effect. LPS-induced IkappaB kinase (IKK), nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1) activation, and IFN-gamma-induced NF-kappaB, signal transducer and activator of transcription-1 (STAT1) and interferon regulatory factor-1 (IRF-1) activation were reduced by quercetin. Moreover quercetin was able to induce heme oxygenase-1 expression. To address the involvement of heme oxygenase-1 induction in iNOS inhibition, heme oxygenase-1 antisense oligodeoxynucleotide was used. Quercetin-mediated inhibition of NO production and iNOS protein expression were partially reversed by heme oxygenase-1 antisense oligodeoxynucleotide, but was mimicked by hemin, a heme oxygenase-1 inducer. The involvement of signal pathways in quercetin-induced heme oxygenase-1 gene expression was associated with tyrosine kinase and mitogen-activated protein kinases activation. All these results suggest quercetin should provide therapeutic benefits for suppression of inflammatory-related neuronal injury in neurodegenerative diseases.
Assuntos
Heme Oxigenase-1/genética , Quinase I-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Quercetina/farmacologia , Fator de Transcrição STAT1/metabolismo , Amidas/farmacologia , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Butadienos/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Hemina/farmacologia , Quinase I-kappa B/metabolismo , Immunoblotting , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Luciferases/genética , Luciferases/metabolismo , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , TransfecçãoRESUMO
Lower molecular weight of hyaluronan (HA) fragments are capable of activating macrophages to express a number of inflammatory mediators through the interaction with the HA receptor CD44. Recent evidence has demonstrated that concomitant induction of CD44 and HA synthase 2 (HAS-2) mRNA in microglia of the ischemic brain. However, the influence of HA fragments on the activation of microglia is poorly understood. In this study, we demonstrated that HA fragments induced inducible NO synthase (iNOS) expression in BV-2 microglia in a dose-dependent manner and was synergized with interferon-gamma (IFN-gamma). Moreover, HA fragments could induce the activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/ERK2), and c-Jun N-terminal kinase (JNK) in a time and dose-dependent fashion. The HA fragments-induced iNOS expression was suppressed by the selective inhibitors of JNK and, to a lesser extent, p38 MAPK. These results suggest that the induction of iNOS by HA fragments is significantly dependent on JNK than on p38 MAPK signaling pathways and support the hypothesis that HA fragments may be an important regulator in the activation of microglia at sites of ischemic brain.
Assuntos
Ácido Hialurônico/farmacologia , Microglia/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Óxido Nítrico Sintase/biossíntese , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Ácido Hialurônico/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno , Microglia/enzimologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Sesame antioxidants have been shown to inhibit lipid peroxidation and regulate cytokine production. In this study, we focused on the effect of sesamin and sesamolin, on nitric oxide (NO) induction by lipopolysaccharides (LPS) in the murine microglial cell line BV-2 and rat primary microglia. The results showed that sesamin and sesamolin significantly inhibited NO production, iNOS mRNA and protein expression in LPS-stimulated BV-2 cells. Sesamin or sesamolin significantly reduced LPS-activated p38 MAPK of BV-2 cells. Furthermore, SB203580, a specific inhibitor of p38 MAP kinase, dose-dependently inhibited NO production in LPS-stimulated BV-2 cells. Taken together, the inhibition of NO production might be due to the reduction of LPS-induced p38 MAPK signal pathway by sesamin and sesamolin.
Assuntos
Antioxidantes/farmacologia , Microglia/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óleo de Gergelim/química , Animais , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Lipopolissacarídeos/farmacologia , Microglia/enzimologia , Microglia/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Gravidez , Piridinas/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , gama-Tocoferol/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Sesamin and sesamolin were tested for their ability to protect BV-2 microglia from hypoxia-induced cell death. These antioxidants dose-dependently reduced hypoxia-induced lactate dehydrogenase (LDH) release and dichlorofluorescein (DCF)-sensitive reactive oxygen species (ROS) production. Their effects on signaling pathway mitogen-activated protein kinases (MAPKs) and caspase-3 in hypoxia-induced cell death were further examined. Extracellular signal-regulated protein kinases (ERK1/2), c-jun NH(2)-terminal kinase (JNK), and p38 MAPKs were activated during hypoxia. The sesamin or sesamolin reduced caspase-3 and MAPK activation correlated well with diminished LDH release in BV-2 cells under hypoxia. Furthermore, they preserved superoxide dismutase (SOD) and catalase activities in BV-2 cells under hypoxia. Taken together, these results indicate that the mechanism of sesame antioxidants involves inhibition of MAPK pathways and apoptosis through scavenging of ROS in hypoxia-stressed BV-2 cells.
Assuntos
Antioxidantes/farmacologia , Dioxóis/farmacologia , Lignanas/farmacologia , Microglia/efeitos dos fármacos , Animais , Western Blotting/métodos , Caspase 3 , Caspases/metabolismo , Catalase/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , L-Lactato Desidrogenase/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sais de Tetrazólio , TiazóisRESUMO
Dehydroepiandrosterone (DHEA), a major steroid secreted by the adrenal gland, is known to have antiproliferative effects but the mechanism is unclear. We recently reported that DHEA induces growth inhibition and apoptosis in BV-2 cells and these effects are inversely associated with glucose concentrations in the medium. Here, we further showed that incubation of BV-2 cells with DHEA under glucose deprivation (G0) led to dose- and time-dependent decrease in cellular ATP levels. The decrease in ATP preceded growth inhibition and apoptosis induced by DHEA and all these effects of DHEA (i.e., loss of ATP, antiproliferation and apoptosis) were prevented by glucose added at 4.5 mg/ml (G4.5) during incubation. In addition, two ATP-depleting agents, iodoacetic acid (IAA) and 2,4-dinitrophenylhydrazine (2,4-DNP), potentiated the antiproliferative and apoptotic effects of DHEA. We also determined whether decrease in nucleic acid synthesis (due to glucose-6-phosphate dehydrogenase inhibition by DHEA) contributes to DHEA-induced antiproliferation and apoptosis. Using a mixture of deoxyribonucleotides (DN) and ribonucleotides (RN), we showed that DNRN had little or no effect on DHEA-induced growth inhibition and apoptosis. We also showed that mevalonic acid (MVA) did not affect DHEA-induced antiproliferation and apoptotic effects, indicating that protein isoprenylation and membrane association are not affected by DHEA in BV-2 cells. Taken together, the present results demonstrate that depletion of ATP by DHEA plays an important role in DHEA-induced antiproliferation and apoptosis of BV-2 cells.
Assuntos
Trifosfato de Adenosina/fisiologia , Apoptose/efeitos dos fármacos , Desidroepiandrosterona/farmacologia , 2,4-Dinitrofenol/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , DNA/química , DNA/genética , Fragmentação do DNA , Inibidores Enzimáticos/farmacologia , Glucose/farmacologia , Histonas , Ácido Iodoacético/farmacologia , Camundongos , OxirreduçãoRESUMO
Green tea is one of the most-consumed beverages due to its taste and antioxidative polyphenols. However, the protective effects of green tea and its constituent, gallic acid (GA), against kainic acid (KA)-induced seizure have not been studied. We investigated the effect of fresh green tea leaf (GTL) and GA on KA-induced neuronal injury in vivo and in vitro. The results showed that GTL and GA reduced the maximal seizure classes, predominant behavioral seizure patterns, and lipid peroxidation in male FVB mice with status epilepticus (SE). GTL extract and GA provided effective protection against KA-stressed PC12 cells in a dose-dependent manner. In the protective mechanism study, GTL and GA decreased Ca(2+) release, ROS, and lipid peroxidation from KA-stressed PC12 cells. Western blot results revealed that mitogen-activated protein kinases (MAPKs), RhoA, and COX-2 expression were increased in PC12 cells under KA stress, and expression of COX-2 and p38 MAPK, but not RhoA, was significantly reduced by GTL and GA. Furthermore, GTL and GA were able to reduce PGE(2) production from KA-stressed PC12 cells. Taken together, the results showed that GTL and GA provided neuroprotective effects against excitotoxins and may have a clinical application in epilepsy.
Assuntos
Ácido Gálico/farmacologia , Ácido Caínico , Estresse Oxidativo/efeitos dos fármacos , Estado Epiléptico/metabolismo , Chá/química , Animais , Antioxidantes , Cálcio/metabolismo , Ácido Caínico/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Fármacos Neuroprotetores , Células PC12 , Extratos Vegetais/farmacologia , Ratos , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológicoRESUMO
The growth factor receptor-bound protein Src homology 2 (Grb2-SH2) plays an important role in the oncogenic Ras signaling pathway, which involves in cell proliferation and differentiation. Therefore, the antagonist of Grb2-SH2 has become a potential target for developing anticancer agents. Recently, we discovered a peptide 1 (Fmoc-Glu-Tyr-Aib-Asn-NH(2)) with high affinity for the Grb2-SH2 domain by using surface plasmon resonance (SPR)-biosensor technology. Herein, we report the further design of the lead peptide 1 by addition of an Arg-Gly-Asp sequence to 1 to enhance binding to Grb2-SH2 and inducing apoptosis in cancer cells. Both the linear and cyclic analogs of the newly designed compound were prepared along with an analog in which the N(alpha)-Fmoc group was removed. These peptide analogs were assayed for their affinity for the Grb2-SH2, their antiproliferative effect on human breast cancer cells, their specificity for cancer cells, and their effects on cytotoxicity and the cell cycle. MCF-7 and MDA-MB-453 breast cancer cells were treated with various concentrations of each peptide. The cell viability and cytotoxicity of peptide-treated cells were determined by using the cell proliferation kit (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-tetrazolium bromide, MTT) and cytotoxicity kit (lactate dehydrogenase, LDH), respectively. Effects of peptides on the cell cycle progression of cancer cells and apoptosis were analyzed by using flow cytometry. Results demonstrated that the peptide analog 2 (H-Arg-Gly-Asp-Glu-Tyr-Aib-Asn-Arg-Gly-Asp-NH(2)) had anti-proliferative effects on MCF-7 and MDA-MB-453 cells with an IC(50) of 45.7 microM and 47.4 microM, respectively. The cytotoxicity and percentage of sub-G1 in the cell cycle were increased in these cancer cells when cells were treated with higher concentration of the Arg-Gly-Asp-containing peptide 2. These results provide important information for the development of anti-cancer agents.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteína Adaptadora GRB2/antagonistas & inibidores , Peptídeos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Proteína Adaptadora GRB2/metabolismo , Humanos , Proteínas Imobilizadas/química , Cinética , Peptídeos/química , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Estrutura Terciária de ProteínaRESUMO
Substance P (SP), a neurotransmitter, may play an important role in neurogenic inflammation. Ginseng has been used extensively in traditional medicine; however, few studies were focused on their anti-allergic effect. Therefore, the effect and mechanism of ginsenoside Rb1 on the SP enhancement of allergic mediators were explored. In this study, SP and dinitrophenyl-bovine serum albumin (DNP-BSA) were used to activate rat basophilic leukemia (RBL)-2H3 cells. The cultured supernatants were assayed for histamine, leukotriene C(4)(LTC(4)) and interleulin-4 (IL-4) production. The mitogen-activated protein kinases (MAPKs) signaling pathway was determined by Western blotting analysis. We found that IgE/DNP-BSA, SP, ginsenoside Rb1, or MAPK specific inhibitors had no effect on cell viability and cytotoxicity. SP (30 microM) alone, did not induce histamine and LTC(4) release, but it enhanced allergen-induced histamine and LTC(4) release. In addition, SP significantly induced and enhanced allergen-activated IL-4. Ginsenoside Rb1 dose-dependently inhibited these effects. SP enhanced the allergen-activated ERK pathway in RBL-2H3 cells, and Rb1 effectively inhibited the ERK pathway activation. Although MAPK specific inhibitors suppressed LTC(4) and IL-4, only U0126 inhibited the SP enhanced histamine release. These results demonstrate that Rb1 dose-dependently inhibited SP enhanced allergen-induced mediator release and its mechanism was through the inhibition of the ERK pathway.
Assuntos
Ginsenosídeos/uso terapêutico , Mediadores da Inflamação/metabolismo , Inflamação Neurogênica/tratamento farmacológico , Animais , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Linhagem Celular , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ratos , Soroalbumina Bovina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Substância P/farmacologiaRESUMO
Hydroxyhydroquinone or 1,2,4-benzenetriol (BT) detected in the beverages has a structure that coincides with the water-soluble form of a sesame lignan, sesamol. We previously showed that sesame antioxidants had neuroprotective abilities due to their antioxidant properties and/or inducible nitric oxide synthase (iNOS) inhibition. However, studies show that BT can induce DNA damage through the generation of reactive oxygen species (ROS). Therefore, we were interested to investigate the neuroprotective effect of BT in vitro and in vivo. The results showed that instead of enhancing free radical generation, BT dose-dependently (10-100 microM) attenuated nitrite production, iNOS mRNA and protein expression in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. BT significantly reduced LPS-induced NF-kappaB and p38 MAPK activation. It also significantly reduced the generation of ROS in H2O2-induced BV-2 cells and in H2O2-cellfree conditions. The neuroprotective effect of BT was further demonstrated in the focal cerebral ischemia model of Sprague-Dawley rat. Taken together, the inhibition of LPS-induced nitrite production might be due to the suppression of NF-kappaB, p38 MAPK signal pathway and the ROS scavenging effect. These effects might help to protect neurons from the ischemic injury.
Assuntos
Hidroquinonas/farmacologia , Lipopolissacarídeos/farmacologia , Microglia , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico/metabolismo , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Peróxido de Hidrogênio/farmacologia , Hidroquinonas/química , Hidroquinonas/uso terapêutico , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Estrutura Molecular , NF-kappa B/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/uso terapêutico , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Oxidantes/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The DNA-binding preferences of two oligopeptide amides, (His-Pro-Arg-Lys)(3)NH(2) (HR-12) and (Ser-Pro-Arg-Lys)(3)NH(2) (SP-12), have been examined by quantitative DNase I footprinting studies. Two different DNA fragments were investigated: a pair of 5'-(32)P-labeled duplexes from pBR322 with one or other of the complementary strands labeled and a corresponding pair of 5'-(32)P-labeled duplexes representing fragments of the latent membrane protein (LMP-1) gene from a pathogenic Epstein-Barr virus variant derived from nasopharyngeal carcinoma. The major objective was to examine molecular recognition and cooperative features associated with sequence-selective binding of synthetic peptides to the LMP-1 fragments. At various binding sites on the pBR322 fragments, Hill coefficients (n(H)) ranging from 1.9-2.2 were observed; these results indicate modest positive cooperativity between binding sites for both peptides. By contrast, unusually high values of n(H), ranging from 4.0-9.3, were observed at various binding sites on the LMP-1 fragments. Allosteric models can be constructed to interpret the observed cooperative interactions between different DNA recognition sites in the LMP-1 gene upon binding of the peptide ligands. It is noteworthy that these models feature a novel network of cooperativity interconnecting multiple DNA allosteric sites. The evidence of sequence selectivity and strong cooperativity discovered in this work may prove to be a general feature of peptide interactions with some nucleic acids.
Assuntos
DNA Viral/genética , DNA Viral/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Peptídeos/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Sequência de Bases , Fenômenos Químicos , Físico-Química , Pegada de DNA , Desoxirribonuclease I/metabolismo , Dados de Sequência MolecularRESUMO
Reactive oxygen species (ROS) are important mediators of a variety of pathological processes, including inflammation and ischemic injury. The neuroprotective effects of sesame antioxidants, sesamin and sesamolin, against hypoxia or H2O2-induced cell injury were evaluated by cell viability or lactate dehydrogenase (LDH) activity. Sesamin and sesamolin reduced LDH release of PC12 cells under hypoxia or H2O2-stress in a dose-dependent manner. Dichlorofluorescein (DCF)-sensitive ROS production was induced in PC12 cells by hypoxia or H2O2-stress but was diminished in the presence of sesamin and sesamolin. We evaluated further the role of mitogen-activated protein kinases (MAPKs) and caspase-3 in hypoxia-induced PC12 cell death. Extracellular signal-regulated protein kinase (ERK) 1, c-jun N-terminal kinase (JNK), and p38 MAPKs of signaling pathways were activated during hypoxia. We found that the inhibition of MAPKs and caspase-3 by sesamin and sesamolin correlated well with the reduction in LDH release under hypoxia. Furthermore, the hypoxia-induced apoptotic-like cell death in cultured cortical cells as detected by a fluorescent DNA binding dye was reduced significantly by sesamin and sesamolin. Taken together, these results suggest that the protective effect of sesamin and sesamolin on hypoxic neuronal and PC12 cells might be related to suppression of ROS generation and MAPK activation.