RESUMO
The sensitivity of two commercial metal oxide (MOx) sensors to ethylene is tested at different relative humidities. One sensor (MiCS-5914) is based on tungsten oxide, the other (MQ-3) on tin oxide. Both sensors were found to be sensitive to ethylene concentrations down to 10 ppm. Both sensors have significant response times; however, the tungsten sensor is the faster one. Sensor models are developed that predict the concentration of ethylene given the sensor output and the relative humidity. The MQ-3 sensor model achieves an accuracy of ±9.2 ppm and the MiCS-5914 sensor model predicts concentration to ±7.0 ppm. Both sensors are more accurate for concentrations below 50 ppm, achieving ±6.7 ppm (MQ-3) and 5.7 ppm (MiCS-5914).
Assuntos
Técnicas de Química Analítica/instrumentação , Etilenos/análise , Óxidos/química , Compostos de Estanho/química , Tungstênio/química , Técnicas de Química Analítica/métodos , Desenho de Equipamento , Análise de Alimentos , Umidade , Modelos LinearesRESUMO
BACKGROUND: Lavender remedies have been used in traditional medicine because of antimicrobial, anti-inflammatory and mood alleviating effects, but underlying molecular mechanisms are not yet fully elucidated. Recently, studies investigating the effects of lavender oil in the context of psychiatric disorders have indicated potent pharmacological properties. Metabolism of tryptophan by indoleamine 2,3-dioxygenase (IDO) was found to provide a biochemical link between immunology and neuroendocrinology and to be a frequent target of natural products. METHODS: In this in vitro study, interferences of lavender oil and constituents (-)-linalool, (+)-α-pinene and (+)-limonene with tryptophan catabolism by IDO and formation of neopterin via guanosine triphosphate (GTP)-cyclohydrolase-I and of interferon-γ have been investigated using unstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). RESULTS: Treatment with lavender oil dose-dependently suppressed PHA-induced tryptophan breakdown and kynurenine formation. Similar effects were observed for the three constituents. In parallel, formation of neopterin and interferon-γ was diminished upon lavender oil treatment. In unstimulated PBMC, effect of lavender oil treatment was similar, but less pronounced. CONCLUSION: Data from this in vitro study suggest that lavender oil treatment might contribute to the modulation of the immune and neuroendocrine system by interfering with activation-induced tryptophan breakdown and IDO activity.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Lavandula/química , Leucócitos Mononucleares/efeitos dos fármacos , Óleos Voláteis/farmacologia , Extratos Vegetais/farmacologia , Óleos de Plantas/farmacologia , Terpenos/farmacologia , Triptofano/metabolismo , Monoterpenos Acíclicos , Monoterpenos Bicíclicos , Células Cultivadas , Cicloexenos/farmacologia , Humanos , Interferon gama/metabolismo , Cinurenina/metabolismo , Leucócitos Mononucleares/enzimologia , Limoneno , Monoterpenos/farmacologia , Neopterina/metabolismo , Óleos Voláteis/química , Fito-Hemaglutininas/farmacologia , Óleos de Plantas/químicaRESUMO
BACKGROUND: Transcriptome analysis in combination with pathway-focused bioassays is suggested to be a helpful approach for gaining deeper insights into the complex mechanisms of action of herbal multicomponent preparations in living cells. The polyherbalism based concept of Tibetan and Ayurvedic medicine considers therapeutic efficacy through multi-target effects. A polyherbal Indo-Tibetan preparation, Padma 28, approved by the Swiss drug authorities (Swissmedic Nr. 58436), was applied to a more detailed dissection of mechanism of action in human hepatoma HepG2 cells. Cell-free and cell-based assays were employed to evaluate the antioxidant capacity. Genome-wide expression profiling was done by applying Human Genome U133 Plus 2.0 Affymetrix arrays. Pathway- and network-oriented analysis elucidated the affected biological processes. The results were validated using reporter gene assays and quantitative real-time PCR. RESULTS: To reveal the direct radical scavenging effects of the ethanolic extract of the Indo-Tibetan polyherbal remedy Padma 28, an in vitro oxygen radical absorbance capacity assay (ORAC) was employed, which resulted in a peroxyl-radical scavenging activity of 2006 ± 235 µmol TE/g. Furthermore, the antioxidant capacity of Padma 28 was analysed in living HepG2 cells, by measuring its scavenging potential against radical induced ROS. This formulation showed a considerable antioxidant capacity by significantly reducing ROS levels in a dose-dependent manner.Integrated transcriptome analysis revealed a major influence on phase I and phase II detoxification and the oxidative stress response. Selected target genes, such as heme oxygenase 1, were validated in qPCR experiments. Network analysis showed 18 interrelated networks involved in important biological functions such as drug and bio-molecule metabolism, molecular transport and cellular communication. Some molecules are part of signaling cascades that are active during development and morphogenesis or are involved in pathological conditions and inflammatory response. CONCLUSIONS: The identified molecular targets and pathways suggest several mechanisms that underlie the biological activity of the preparation. Although extrapolation of these findings to the in vivo situation is not possible, the results obtained might be the basis for further investigations and new hypotheses to be tested. This study demonstrates the potential of the combination of focused and unbiased research strategies in the mode of action analysis of multicomponent herbal mixtures.
Assuntos
Antioxidantes/farmacologia , Bioensaio , Perfilação da Expressão Gênica , Medicina Herbária , Extratos Vegetais/farmacologia , Proliferação de Células/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Hep G2 , Humanos , Medicina Tradicional Tibetana , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
One of the shortcomings of vaccinia virus (VACV) as immunization vector is the down-regulation of HLA and costimulatory molecules in antigen presenting cells. To overcome this problem we investigated the use of protein kinase C (PKC) as immune stimulatory agent. Thus several classical and atypical PKCs were inserted into wild-type or attenuated VACV using recombination into the hemagglutinin gene and the expression driven by the VACV 7,5K-IE gene promoter. Recombinant constructs expressing PKC-alpha, -beta, -theta as well as wild-type, constitutive active or dominant negative PKC-zeta constructs were generated. Additional constructs expressing PKB/Akt1 and ICAM-1 were used for comparison. Immature and mature peripheral blood derived-dendritic cells (DC) as well as lymphoid cell lines capable of obtaining a DC-like phenotype upon mitogen stimulation were infected. Disappointingly, VACV-driven PKC overexpression did not significantly enhance expression of various activation markers or costimulatory molecules tested. Neither CD86 nor HLA-DR expression was upregulated and also no influence on the maturation of DC, as measured by DC-SIGN and CD83, was observed. However, VACV did not interfere with LPS induced up-regulation of CD83 and did not lead to substantial apoptosis of infected DC within the first 24 hours.
Assuntos
Antígeno B7-2/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/virologia , Antígenos HLA-DR/metabolismo , Proteína Quinase C/metabolismo , Regulação para Cima , Animais , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Células Dendríticas/metabolismo , Vetores Genéticos , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/imunologia , Recombinação Genética , Vaccinia virus/enzimologia , Vaccinia virus/genéticaRESUMO
Neopterin production is induced in human monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-gamma (IFN-gamma). In parallel, IFN-gamma induces the tryptophan-(trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and triggers the formation of reactive oxygen species (ROS). Translocation of the signal transduction element nuclear factor-kappaB (NF-kappaB) is induced by ROS and accelerates the pro-inflammatory response by activation of other pro-inflammatory pathways. Therefore, a close relationship between NF-kappaB expression, the production of neopterin and the degradation of trp can be assumed, although this has not been demonstrated so far. In the present in vitro study we compared the influence of lipopolysaccharide (LPS) on NF-kappaB activation, neopterin formation and the degradation of trp in THP-1Blue cells, which represent the human myelomonocytic cell line THP-1 stably transfected with an NF-kappaB inducible reporter system. In cells stimulated with LPS, a significant induction of NF-kappaB was observed, and this was paralleled by an increase of kynureunine (kyn) and neopterin concentrations and a decline of trp. The increase of the kyn to trp quotient indicates accelerated IDO activity. Higher LPS concentrations and longer incubation of cells were associated with higher activities of all three biochemical pathways and significant correlations existed between NF-kappaB activation, neopterin release and trp degradation (all p<0.001). We conclude that there is a parallel induction of NF-kappaB, neopterin formation and trp degradation in monocytic THP-1 cells, which is elicited by pro-inflammatory triggers like LPS during innate immune responses.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Lipopolissacarídeos/imunologia , NF-kappa B/metabolismo , Neopterina/biossíntese , Triptofano/metabolismo , Linhagem Celular Tumoral , Humanos , Monócitos/imunologia , Transporte ProteicoRESUMO
BACKGROUND: Androgen receptor (AR) signaling is implicated in prostate cancer progression. Therefore, identification of AR downstream genes is potentially important for selection of novel markers and therapy targets in prostate cancer. METHODS: Expression of a thyroid hormone T3-binding protein mu-crystallin (CRYM) mRNA and protein in cell lines was evaluated by real-time PCR and Western blot, respectively. CRYM expression in vivo was analyzed in patients' samples by immunohistochemistry. The effects of androgen and T3 on proliferation of MDA PCa 2b cells were assessed by (3)H-thymidine uptake assay. RESULTS: CRYM expression was detected in AR-positive LNCaP and MDA PCa 2b cells. In MDA PCA 2b cells, CRYM was regulated by androgens. Androgen-induced CRYM expression was diminished by antiandrogens or AR siRNA. Inhibition of transcription by alpha-amanitin caused a reduction in CRYM mRNA. The lack of CRYM expression was noted in LAPC-4 cells and in AR-negative prostate cancer cell lines PC-3 and DU-145. CRYM protein was increased in cancer tissue and decreased in samples from patients after hormonal therapy. In samples from patients with therapy-refractory cancer CRYM was not detectable. We also found that androgens and T3 have additive effects on stimulation of MDA PCa 2b cells proliferation. CONCLUSION: CRYM is a novel androgen-regulated gene whose expression is elevated in prostate cancer but down-regulated in castration therapy-resistant tumors.
Assuntos
Androgênios/fisiologia , Cristalinas/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias da Próstata/genética , Antagonistas de Receptores de Andrógenos , Biomarcadores Tumorais/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Cristalinas/biossíntese , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Orquiectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Hormônios Tireóideos/biossíntese , Hormônios Tireóideos/genética , Regulação para Cima/genética , Cristalinas mu , Proteínas de Ligação a Hormônio da TireoideRESUMO
Using yeast two-hybrid, we isolated atypical PKCzeta as a PKCtheta-interacting kinase and demonstrated that it selectively interacted with, and was phosphorylated by, PKCtheta. Importantly, however, both atypical PKCzeta and PKCiota were functionally required in TCR/CD28-mediated activation of NF-kappaB downstream of PKCtheta in Jurkat T cells albeit, activation responses of PKCzeta-deficient CD3+ T cells were comparable with wildtype controls. This normal activation thresholds of PKCzeta-/- T cells suggested that PKCiota, the closest structural relative, might play a compensatory role in TCR/CD28-induced signalling. Consistently, both PKCzeta and PKCiota resided in the plasma membrane lipid raft microdomains of Jurkat as well as primary mouse CD3+ T cells. Thus, PKCtheta, the established constituent of the immunological synapse, physically and functionally interacted with PKCzeta and PKCiota. Together, these data demonstrate that atypical PKCzeta/iota isotypes serve as direct downstream targets of PKCtheta in the signalling pathway leading to NF-kappaB activation in T lymphocytes.
Assuntos
NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Linfócitos T/enzimologia , Ativação Transcricional/genética , Animais , Complexo CD3 , Domínio Catalítico , Proliferação de Células , Genes Dominantes , Humanos , Interleucina-2/metabolismo , Isoenzimas/metabolismo , Células Jurkat , Microdomínios da Membrana/enzimologia , Camundongos , Ligação Proteica , Proteína Quinase C-theta , Transporte Proteico , Ratos , Especificidade por Substrato , Linfócitos T/metabolismo , TransfecçãoRESUMO
Investigations of cellular processes initiated by volatile organic compounds (VOCs) are limited when modelling realistic long-term exposure scenarios at low concentrations. Exposure to indoor VOCs is associated with a range of adverse effects, but data on molecular changes at regulatory threshold limits are lacking. Activity analysis of VOC in vitro can be a valuable complement to inhalation toxicological evaluations. We developed an exposure platform that generates a stable VOC atmosphere and allows the exposure of cells for longer periods. Using formaldehyde as a model analyte, air-liquid interface cultured A549 lung epithelial cells were exposed to critical concentrations of 0.1 and 0.5 ppm for 3 days. Owing to the lack of known exposure biomarkers, we applied a genome-wide transcriptional analysis to investigate cellular responses at these sublethal concentrations. We demonstrate a minor overlap of differentially expressed transcripts for both treatment concentrations, which can be further analyzed for their use as exposure biomarkers. Moreover, distinct expression patterns emerge for 0.1 and 0.5 ppm formaldehyde exposure, which is reflected in significant enrichment of distinct biological processes. More specifically, metabolism of specific compound classes, lipid biosynthesis and lung-associated functions are affected by lower exposure levels and processes affecting proliferation and apoptosis dominate the higher exposure levels.
Assuntos
Poluentes Atmosféricos/análise , Formaldeído/efeitos adversos , Pulmão/efeitos dos fármacos , Hipersensibilidade Respiratória , Compostos Orgânicos Voláteis/análise , Células A549/efeitos dos fármacos , Poluição do Ar em Ambientes Fechados , Biomarcadores/metabolismo , Sobrevivência Celular , Exposição Ambiental , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Lipídeos/química , Pulmão/citologia , Material Particulado/análise , Transcrição GênicaRESUMO
INTRODUCTION: Degradation of the essential amino acid tryptophan via indoleamine 2,3-dioxygenase (IDO1) represents an important antiproliferative strategy of the cellular immune response. Tryptophan shortage and accumulation of kynurenine downstream products also affect T-cell responses, providing a negative feedback control of immune activation. IDO1 activity can promote a regulatory phenotype in both T cells and dendritic cells. These phenomena can support tumor immune escape. AREAS COVERED: IDO1 activity reflects the course of several malignancies, and determination of kynurenine to tryptophan ratio in serum/plasma can be used to assess immune activation. Moreover, the accelerated breakdown of tryptophan has been correlated with the development of cancer-associated disturbances such as anemia, weight loss and depression. Tumoral IDO1 expression was correlated with a poor prognosis in several types of tumors, which makes it to an interesting target for immunotherapy. In addition, according to recent data, a role of trytptophan 2,3-dioxygenase (TDO) in tumorigenesis cannot be excluded. EXPERT OPINION: Tryptophan metabolism is critical for cell proliferation, inflammation and immunoregulation. Accelerated tryptophan breakdown favors tumor immune escape. Accordingly, targeting IDO1 by immunotherapy may represent a favorable approach; however, blocking crucial immunoregulatory pathways could also introduce the risk of immune system overactivation, finally leading to unresponsiveness.
Assuntos
Imunoterapia/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Neoplasias/terapia , Animais , Proliferação de Células , Células Dendríticas/imunologia , Humanos , Imunidade Celular , Inflamação/imunologia , Cinurenina/sangue , Cinurenina/metabolismo , Neoplasias/imunologia , Linfócitos T/imunologia , Triptofano/sangue , Triptofano/metabolismoRESUMO
Bisphenol A (BPA) is a widely used plasticizer, which came into focus because of its genotoxic and sensitizing potential. Besides its toxic properties, BPA is also well-known for its antioxidant chemical properties. This in vitro study investigated the interference of BPA with interferon-γ (IFN-γ)-induced tryptophan breakdown and neopterin production in human peripheral blood mononuclear cells (PBMC). The pro-inflammatory cytokine IFN-γ induces the conversion of the essential amino acid tryptophan into kynurenine via the enzyme indoleamine-2,3-dioxygenase (IDO-1). In parallel, GTP-cyclohydrolase produces neopterin, a marker of immune activation. A model system of phytohaemagglutinin (PHA)-stimulated PBMC was used to assess potential immunomodulatory properties of BPA. Treatment of cells with BPA [12.5-200µM] resulted in a significant and dose-dependent suppression of mitogen-induced tryptophan breakdown and neopterin formation along with a decrease of IFN-γ levels. Similar but less pronounced effects were observed in unstimulated cells. We postulate that the inhibitory effects of BPA on both T-cell activation and IDO-1 activity that we describe here may be critical for immune surveillance and is likely to influence T helper (Th) type 1/Th2 balance. Such immunosuppressive effects likely contribute to counteract inflammation. Further studies are required to address the in vivo relevance our in vitro findings.
Assuntos
Compostos Benzidrílicos/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Fenóis/farmacologia , Células Th1/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Neopterina/imunologia , Neopterina/metabolismo , Fito-Hemaglutininas/farmacologia , Células Th1/imunologia , Células Th1/metabolismo , Triptofano/imunologia , Triptofano/metabolismoRESUMO
Protein kinase (PK) Ctheta and Akt/PKBalpha cooperate in T cell receptor/CD28-induced T cell signaling. We here demonstrate the recruitment of endogenous Akt1 and PKCtheta to lipid rafts in CD3-stimulated T cells. Further we show that Myr-PKCtheta mediates translocation of endogenous Akt1 to the plasma membrane as well as to lipid rafts, most likely explained by the observed complex formation of both protein kinases. In addition, in peripheral mouse T cells, the PKC inhibitor Gö6850 could partially block Akt1 activation in CD3-induced signaling, placing PKC isotype(s) upstream of Akt1. However, T cells derived from PKCtheta knockout mice were not impaired in CD3- or phorbol ester-induced Akt1 activity. Taken together, the results of this study give new insights into the functional link of Akt1 and PKCtheta in T cell signaling, demonstrating the co-recruitment of the two kinases and showing a novel pathway leading to Akt1 transactivation where PKC isotype(s) are involved but PKCtheta is not essential.
Assuntos
Complexo CD3/metabolismo , Isoenzimas/metabolismo , Microdomínios da Membrana/enzimologia , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Linfócitos T/enzimologia , Animais , Antígenos CD28/metabolismo , Membrana Celular/enzimologia , Ativação Enzimática , Humanos , Isoenzimas/análise , Isoenzimas/genética , Células Jurkat , Camundongos , Camundongos Knockout , Mutação , Proteína Quinase C/análise , Proteína Quinase C/genética , Proteína Quinase C-theta , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
The novel lead bis(1H-2-indolyl)methanone inhibits autophosphorylation of platelet-derived growth factor (PDGF) receptor tyrosine kinase in intact cells. Various substituents in the 5- or 6-position of one indole ring increase or preserve potency, whereas most modifications of the ring structures and of the methanone group as well as substitution at both indoles result in weak or no activity. An ATP binding site model, derived by homology from the FGFR-1 tyrosine kinase crystal structure suggesting hydrogen bonds of one indole NH and the methanone oxygen with the backbone carbonyl and amide, respectively, of Cys684, explains why only one indole moiety is open for substitution and locates groups in the 5- or 6-position outside the pocket. The hitherto most active derivatives, 39, 53 and 67, inhibit both isoforms of the PDGF receptor kinase in intact cells, with IC(50) of 0.1-0.3 microM, and purified PDGFbeta-receptor in vitro, with IC(50) of 0.09, 0.1, or 0.02 microM, respectively. PDGF-stimulated DNA synthesis is inhibited by these derivatives with IC(50) values of 1-3 microM. Kinetic analysis of 53 showed an ATP-competitive mode of inhibition. The compounds are inactive or weakly active toward a number of other tyrosine kinases, including the FGF receptor 1, EGF receptor, and c-Src kinase, as well as toward serine-threonine kinases, including different PKC isoforms and GRK2, and appear therefore selective for PDGF receptor inhibition.
Assuntos
Inibidores Enzimáticos/síntese química , Glicina/síntese química , Indóis/síntese química , Cetonas/síntese química , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Animais , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Glicina/química , Glicina/farmacologia , Humanos , Indóis/química , Indóis/farmacologia , Cetonas/química , Cetonas/farmacologia , Cinética , Modelos Moleculares , Estrutura Molecular , Fosforilação , Receptores Proteína Tirosina Quinases/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/química , Relação Estrutura-AtividadeRESUMO
Replicative senescence of human fibroblasts is a widely used cellular model for human aging. While it is clear that telomere erosion contributes to the development of replicative senescence, it is assumed that additional factors contribute to the senescent phenotype. The free radical theory of aging suggests that oxidative damage is a major cause of aging; furthermore, the expression of activated oncogenes, such as oncogenic Ras, can induce premature senescence in primary cells. The functional relation between the various inducers of senescence is not known. The present study was guided by the hypothesis that constitutive activation of normal, unmutated Ras may contribute to senescence-induced growth arrest in senescent human fibroblasts. When various branches of Ras-dependent signaling were investigated, constitutive activation of the Ras/Raf/MEK/ERK pathway was not observed. To evaluate the role of oxidative stress for the senescent phenotype, we also investigated stress-related protein kinases. While we found no evidence for alterations in the activity of p38, we could detect an increased activity of Jun kinase in senescent fibroblasts. We also found higher levels of reactive oxygen species (ROS) in senescent fibroblasts compared to their younger counterparts. The accumulation of ROS in senescent cells may be related to the constitutive activation of Jun kinase.
Assuntos
Senescência Celular/fisiologia , Fibroblastos/citologia , Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases/fisiologia , Estresse Oxidativo/fisiologia , Proteínas ras/metabolismo , Western Blotting , Comunicação Celular/fisiologia , Células Cultivadas , Humanos , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , MAP Quinase Quinase 4 , Proteína Quinase 8 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Phorbol esters (PEs) and their derivatives are potent tumor-promoting agents. The best known receptors for these substances are the novel and classical isotypes of protein kinase C (PKC), which bind PE and the physiological second messenger diacylglycerol (DAG) by cysteine-rich domains, the C1 domains. However, PKC is not the sole receptor of PE, a concept that has been largely ignored in the past. PE (in addition to DAG) also targets C1-containing receptors unrelated to PKC. In order to get a better insight into DAG/PE-mediated signaling and the pathways involved, it is necessary to first determine all ligand-interacting proteins. Employing various sources of data, 66 different C1-containing human proteins are presented and predictions of their DAG/PE-binding potential are attempted. Defining the entire set of key mediators for the physiological DAG responses and for PE-induced tumorigenesis may aid our understanding of signal integration and can also help to design new strategies for therapeutic cancer intervention.
Assuntos
Carcinógenos/metabolismo , Diglicerídeos/metabolismo , Ésteres de Forbol/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Bases de Dados de Proteínas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas/química , Alinhamento de SequênciaRESUMO
Oxidative stress is considered to be critically involved in the normal aging process but also in the development and progression of various human pathologies like cardiovascular and neurodegenerative diseases, as well as of infections and malignant tumors. These pathological conditions involve an overwhelming production of reactive oxygen species (ROS), which are released as part of an anti-proliferative strategy during pro-inflammatory immune responses. Moreover, ROS themselves are autocrine forward regulators of the immune response. Most of the beneficial effects of antioxidants are considered to derive from their influence on the immune system. Due to their antioxidant and/or radical scavenging nature, phytochemicals, botanicals and herbal preparations can be of great importance to prevent oxidation processes and to counteract the activation of redox-regulated signaling pathways. Antioxidants can antagonize the activation of T-cells and macrophages during the immune response and this anti-inflammatory activity could be of utmost importance for the treatment of above-mentioned disorders and for the development of immunotolerance. Herein, we provide an overview of in vitro assays for the measurement of antioxidant and anti-inflammatory activities of plant-derived substances and extracts, by discussing possibilities and limitations of these methods. To determine the capacity of antioxidants, the oxygen radical absorbance capacity (ORAC) assay and the cell-based antioxidant activity (CAA) assay are widely applied. To examine the influence of compounds on the human immune response more closely, the model of mitogen stimulated human peripheral blood mononuclear (PBMC) cells can be applied, and the production of the inflammatory marker neopterin as well as the breakdown of the amino acid tryptophan in culture supernatants can be used as readout to indicate an immunomodulatory potential of the tested compound. These two biomarkers of immune system activation are robust and correlate with the course of cardiovascular, neurodegenerative and malignant tumor diseases, but also with the normal aging process, and they are strongly predictive. Thus, while the simpler ORAC and CAA assays provide insight into one peculiar chemical aspect, namely the neutralization of peroxyl radicals, the more complex PBMC assay is closer to the in vivo conditions as the assay comprehensively enlights several properties of immunomodulatory test compounds.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Fatores Imunológicos/farmacologia , Inflamação/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lipopolissacarídeos , Mitógenos , Modelos Biológicos , Neopterina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Triptofano/metabolismoRESUMO
Although the major activities of traditional remedies have mostly been known since ancient times, their molecular mechanisms of action have usually not been investigated in much detail. The pharmaceutically relevant activities of botanical therapeutics frequently result from additive or synergistic effects of a multitude of components. Several studies have been published that analyze the effects of complex preparations on selected in vitro model cell systems by using gene expression analysis. Herein, the examples referred to include transcriptional studies with extracts from Ginkgo biloba and Echinacea as well as the Tibetan Formula Padma 28. Transcriptional profiles can be used to deduce key molecules and pathways affected upon treatment. Differentially expressed gene sets can further be integrated with information derived from interaction databases, thus giving a more comprehensive view of activated biological processes. Transcriptomics, by using microarray technology, is used as a tool in different fields of natural product research, ranging from activity monitoring to toxicity profiling.
Assuntos
Medicina Tradicional Tibetana , Fitoterapia/métodos , Extratos Vegetais/uso terapêutico , Sinergismo Farmacológico , Echinacea , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Ginkgo biloba , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fitoterapia/efeitos adversos , Extratos Vegetais/efeitos adversos , Transcrição Gênica/efeitos dos fármacosRESUMO
Antimalarial chloroquine is also used for the treatment of immune-mediated diseases. The interference of chloroquine with interferon-γ-induced tryptophan breakdown and neopterin production has been investigated in human peripheral blood mononuclear cells (PBMC) in vitro. Micromolar concentrations (2-50 µM) of chloroquine dose-dependently suppressed mitogen-induced tryptophan breakdown in PBMC but not in the myelomonocytic THP-1-Blue cell line, after 48 h of treatment. In stimulated PBMC, neopterin production was super-induced by 10 µM chloroquine, while it was significantly suppressed at a concentration of 50 µM. These anti-inflammatory effects may relate to the therapeutic benefit of chloroquine in inflammatory conditions and may widen the spectrum of its clinical applications.
RESUMO
Phytocannabinoids present in Cannabis plants are well known to exert potent anti-inflammatory and immunomodulatory effects. Previously, we have demonstrated that the psychoactive D9-tetrahydrocannabinol (THC) and the non-psychotropic cannabidiol (CBD) modulate mitogen-induced Th1-type immune responses in peripheral blood mononuclear cells (PBMC). The suppressive effect of both cannabinoids on mitogen-induced tryptophan degradation mediated by indoleamine-2,3-dioxygenase (IDO), suggests an additional mechanism by which antidepressive effects of cannabinoids might be linked to the serotonergic system. Here, we will review the role of tryptophan metabolism in the course of cell mediated immune responses and the relevance of cannabinoids in serotonergic signaling. We conclude that in particular the non-psychotropic CBD might be useful for the treatment of mood disorders in patients with inflammatory diseases, since this cannabinoid seems to be safe and its effects on activation-induced tryptophan degradation by CBD were more potent as compared to THC.
RESUMO
Diffuse-type gastric and lobular breast cancers are characterized by frequent mutations in the cell adhesion molecule E-cadherin. Here we report that tumor-associated mutations of E-cadherin enhanced random cell movement of transfected MDA-MB-435S mammary carcinoma cells as compared to wild-type (wt) E-cadherin-expressing cells. The mutations included in frame deletions of exons 8 or 9 and a point mutation in exon 8 which all affect putative calcium-binding sites within the linker region of the second and third extracellular domain. Motility enhancement by mutant E-cadherin was investigated by time-lapse laser scanning microscopy. Increased cell motility stimulated by mutant E-cadherin was influenced by cell-matrix interactions. The motility-increasing activity of mutant E-cadherin was blocked by application of pharmacological inhibitors of epidermal growth factor receptor and phosphatidylinositol (PI) 3-kinase. Investigation of the activation status of PI 3-kinase and the downstream signaling molecules Akt/protein kinase B and MAP kinase p44/42 showed that these kinases are not more strongly activated in mutant E-cadherin-expressing cells than in wt E-cadherin-expressing cells. Instead, the basal level of PI 3-kinase is necessary for mutant E-cadherin-enhanced cell motility. Our data suggest a critical role of E-cadherin mutations for the fine tuning of tumor cell motility.
Assuntos
Neoplasias da Mama/genética , Caderinas/genética , Carcinoma/genética , Movimento Celular/genética , Receptores ErbB/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Neoplasias da Mama/enzimologia , Neoplasias da Mama/fisiopatologia , Caderinas/metabolismo , Carcinoma/enzimologia , Carcinoma/fisiopatologia , Adesão Celular/genética , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/genética , Invasividade Neoplásica/genética , Inibidores de Fosfoinositídeo-3 Quinase , Mutação Puntual/genética , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Células Tumorais CultivadasRESUMO
Activation of Raf-1 suppresses integrin activation, potentially through the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). However, bulk ERK1/2 activation does not correlate with suppression. PEA-15 reverses suppression of integrin activation and binds ERK1/2. Here we report that PEA-15 reversal of integrin suppression depends on its capacity to bind ERK1/2, indicating that ERK1/2 function is indeed required for suppression. Mutations in either the death effector domain or C-terminal tail of PEA-15 that block ERK1/2 binding abrogated the reversal of integrin suppression. Furthermore, we used ERK/p38 chimeras and site-directed mutagenesis to identify ERK1/2 residues required for binding PEA-15. Mutations of residues that precede the alphaG helix and within the mitogen-activated protein kinase insert blocked ERK2 binding to PEA-15, but not activation of ERK2. These ERK2 mutants blocked the ability of PEA-15 to reverse suppression of integrin activation. Thus, PEA-15 regulation of integrin activation depends on its binding to ERK1/2. To directly test the role of ERK1/2 localization in suppression, we enforced membrane association of ERK1 and 2 by joining a membrane-targeting CAAX box sequence to them. Both ERK1-CAAX and ERK2-CAAX were membrane-localized and suppressed integrin activation. In contrast to suppression by membrane-targeted Raf-CAAX, suppression by ERK1/2-CAAX was not reversed by PEA-15. Thus, ERK1/2 are the Raf effectors for suppression of integrin activation, and PEA-15 reverses suppression by binding ERK1/2.