RESUMEN
Nitrogen (N) and phosphorus (P) are two important nutrient elements that limit the growth of plants and microorganisms. The effect of the N supply on soil P cycling and its mechanism remain poorly known. Here, we characterized the effects of different N application rates on soil P availability, the abundances of P-cycling functional genes, and microbial communities involved in P-cycling following the application of N for 13 years in a tea plantation. Soil available P (AP) decreased significantly under N application. The opposite pattern was observed for the activity of soil phosphatases including alkaline (ALP) and acid phosphatase (ACP). Furthermore, N addition increased the abundance of ppa but decreased the abundance of phoD in soil. Both ppa- and phoD-harboring communities varied with N application levels. Redundancy analysis (RDA) showed that soil pH was a key variable modulating ppa-harboring and phoD-harboring microbial communities. Partial least squares path modeling (PLS-PM) revealed that long-term N application indirectly reduced soil P availability by altering the abundances of phoD-harboring biomarker taxa. Overall, our findings indicated that N-induced reductions in AP increased microbial competition for P by selecting microbes with P uptake and starvation response genes or those with phosphatases in tea plantation system. This suggests that tea plantations should be periodically supplemented with P under N application, especially under high N application levels.
Asunto(s)
Camellia sinensis , Microbiota , Suelo/química , Fósforo/análisis , Nitrógeno/análisis , Microbiología del Suelo , Monoéster Fosfórico Hidrolasas/farmacología , TéRESUMEN
BACKGROUND AND AIMS: Aberrant activation of fatty acid synthase (FASN) is a major metabolic event during the development of HCC. We evaluated the therapeutic efficacy of TVB3664, a FASN inhibitor, either alone or in combination, for HCC treatment. APPROACH AND RESULTS: The therapeutic efficacy and the molecular pathways targeted by TVB3664, either alone or with tyrosine kinase inhibitors or the checkpoint inhibitor anti-programmed death ligand 1 antibody, were assessed in human HCC cell lines and multiple oncogene-driven HCC mouse models. RNA sequencing was performed to elucidate the effects of TVB3664 on global gene expression and tumor metabolism. TVB3664 significantly ameliorated the fatty liver phenotype in the aged mice and AKT-induced hepatic steatosis. TVB3664 monotherapy showed moderate efficacy in NASH-related murine HCCs, induced by loss of phosphatase and tensin homolog and MET proto-oncogene, receptor tyrosine kinase (c-MET) overexpression. TVB3664, in combination with cabozantinib, triggered tumor regression in this murine model but did not improve the responsiveness to immunotherapy. Global gene expression revealed that TVB3664 predominantly modulated metabolic processes, whereas TVB3664 synergized with cabozantinib to down-regulate multiple cancer-related pathways, especially the AKT/mammalian target of rapamycin pathway and cell proliferation genes. TVB3664 also improved the therapeutic efficacy of sorafenib and cabozantinib in the FASN-dependent c-MYC-driven HCC model. However, TVB3664 had no efficacy nor synergistic effects in FASN-independent murine HCC models. CONCLUSIONS: This preclinical study suggests the limited efficacy of targeting FASN as monotherapy for HCC treatment. However, FASN inhibitors could be combined with other drugs for improved effectiveness. These combination therapies could be developed based on the driver oncogenes, supporting precision medicine approaches for HCC treatment.
Asunto(s)
Carcinoma Hepatocelular , Acido Graso Sintasa Tipo I , Neoplasias Hepáticas , Anilidas , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mamíferos/metabolismo , Ratones , Monoéster Fosfórico Hidrolasas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Piridinas , Sorafenib/farmacología , Serina-Treonina Quinasas TOR , TensinasRESUMEN
In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147-155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75%) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2, a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3'-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/metabolismo , Mandíbula/enzimología , Nephropidae/enzimología , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos/genética , Animales , Northern Blotting/métodos , Southern Blotting/métodos , Línea Celular , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/genética , Insectos/citología , Proteínas de la Membrana/metabolismo , Membranas/enzimología , Datos de Secuencia Molecular , Nephropidae/genética , Especificidad de Órganos , Monoéster Fosfórico Hidrolasas/farmacología , ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN/métodos , Solubilidad , Transcripción Genética/genéticaRESUMEN
Mistletoe lectin has been reported to induce apoptosis in different cancer cell lines in vitro and to show antitumor activity against a variety of tumors in animal models. We previously demonstrated the Korean mistletoe lectin (Viscum album var. coloratum, VCA)-induced apoptosis by down-regulation of Bcl-2 and telomerase activity and by up-regulation of Bax through p53- and p21-independent pathway in hepatoma cells. In the present study, we observed the induction of apoptotic cell death through activation of caspase-3 and the inhibition of telomerase activity through transcriptional down-regulation of hTERT in the VCA-treated A253 cells. We also observed the inhibition of telomerase activity and induction of apoptosis resulted from dephosphorylation of Akt in the survival signaling pathways. In addition, combining VCA with the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) upstream of Akt, wortmannin and LY294002 showed an additive inhibitory effect of telomerase activity. In contrast, the inhibitor of protein phosphatase 2A (PP2A), okadaic acid inhibited VCA-induced dephosphorylation of Akt and inhibition of telomerase activity. Taken together, VCA induces apoptotic cell death through Akt signaling pathway in correlated with the inhibition of telomerase activity and the activation of caspase-3. From these results, together with our previous studies, we suggest that VCA triggers molecular changes that resulting in the inhibition of cell growth and the induction of apoptotic cell death of cancer cells, which suggest that VCA may be useful as chemotherapeutic agent for cancer cells.
Asunto(s)
Apoptosis , Línea Celular Tumoral , Lectinas/efectos adversos , Lectinas/química , Muérdago/química , Fosfatidilinositol 3-Quinasas/metabolismo , Telomerasa/antagonistas & inhibidores , Caspasa 3 , Caspasas/efectos adversos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Sinergismo Farmacológico , Humanos , Corea (Geográfico) , Lectinas/antagonistas & inhibidores , Lectinas/aislamiento & purificación , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/farmacología , Fosforilación/efectos de los fármacos , Extractos Vegetales/química , Preparaciones de Plantas/efectos adversos , Preparaciones de Plantas/antagonistas & inhibidores , Preparaciones de Plantas/química , Proteínas de Plantas/efectos adversos , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas Inactivadoras de Ribosomas Tipo 2 , Telomerasa/genética , Toxinas Biológicas/efectos adversos , Toxinas Biológicas/química , Regulación hacia ArribaRESUMEN
Activation of G(q)-coupled alpha(1)-adrenergic receptors leads to hypertrophic growth of neonatal rat ventricular cardiomyocytes that is associated with increased expression of hypertrophy-related genes, including atrial natriuretic peptide (ANP) and myosin light chain-2 (MLC), as well as increased ribosome synthesis. The role of inositol phosphates in signaling pathways involved in these changes in gene expression was examined by overexpressing inositol phosphate-metabolizing enzymes and determining effects on ANP, MLC, and 45 S ribosomal gene expression following co-transfection of appropriate reporter gene constructs. Overexpression of enzymes that metabolize inositol 1,4,5-trisphosphate did not reduce ANP or MLC responses, but overexpression of the enzyme primarily responsible for metabolism of inositol 4,5-bisphosphate (Ins(1,4)P(2)), inositol polyphosphate 1-phosphatase (INPP), reduced ANP and MLC responses associated with alpha(1)-adrenergic receptor-mediated hypertrophy. Similarly overexpressed INPP reduced ANP and MLC responses associated with contraction-induced hypertrophy. In addition, overexpression of INPP reduced the increase in ribosomal DNA transcription associated with both hypertrophic models. Hypertrophied cells from both cell models as well as ventricular tissue from mouse hearts hypertrophied by pressure overload in vivo contained heightened levels of Ins(1,4)P(2), suggesting reduced INPP activity in three different models of hypertrophy. These studies provide evidence for an involvement of Ins(1,4)P(2) in hypertrophic signaling pathways in ventricular myocytes.
Asunto(s)
Hipertrofia/tratamiento farmacológico , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/farmacología , Animales , Factor Natriurético Atrial/metabolismo , Western Blotting , Células CHO , Miosinas Cardíacas/metabolismo , Células Cultivadas , Clonación Molecular , Cricetinae , ADN Complementario/metabolismo , ADN Ribosómico/metabolismo , Electroforesis en Gel de Poliacrilamida , Biblioteca de Genes , Genes Reporteros , Humanos , Fosfatos de Inositol/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Químicos , Miocardio/metabolismo , Miocardio/patología , Cadenas Ligeras de Miosina/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteína Quinasa C/metabolismo , Ratas , Transducción de Señal , Factores de Tiempo , Transcripción Genética , TransfecciónRESUMEN
Bioactive compound(s) extracted from cinnamon potentiate insulin activity, as measured by glucose oxidation in the rat epididymal fat cell assay. Wortmannin, a potent PI 3'-kinase inhibitor, decreases the biological response to insulin and bioactive compound(s) from cinnamon similarly, indicating that cinnamon is affecting an element(s) upstream of PI 3'-kinase. Enzyme studies done in vitro show that the bioactive compound(s) can stimulate autophosphorylation of a truncated form of the insulin receptor and can inhibit PTP-1, a rat homolog of a tyrosine phosphatase (PTP-1B) that inactivates the insulin receptor. No inhibition was found with alkaline phosphate or calcineurin suggesting that the active material is not a general phosphatase inhibitor. It is suggested, then, that a cinnamon compound(s), like insulin, affects protein phosphorylation-dephosphorylation reactions in the intact adipocyte. Bioactive cinnamon compounds may find further use in studies of insulin resistance in adult-onset diabetes.
Asunto(s)
Cinnamomum zeylanicum/análisis , Insulina/fisiología , Extractos Vegetales/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Androstadienos/farmacología , Animales , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Monoéster Fosfórico Hidrolasas/farmacología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Ratas , Receptor de Insulina/efectos de los fármacos , WortmaninaRESUMEN
1. Single channel current recordings were used to study the characteristics of a large conductance Ca(2+)-activated K+ (BKCa) channel present in neurones acutely dissociated from the rat motor cortex. Application of ATP to the intracellular surface of excised inside-out patches produced a large, concentration-dependent increase in BKCa channel activity. 2. This ATP-mediated activation was dependent upon the presence of Mg2+ in the intracellular bathing solution and was diminished by the phosphatases 2,3-butanedione monoxime (BDM) or alkaline phosphatase and by the protein kinase inhibitors staurosporine, H-7 and PKI. 3. ADP stimulated BKCa channel activity in a Mg(2+)-dependent manner, an action also inhibited by the concomitant application of PKI or BDM. The effect of ADP was reduced by application of hexokinase and glucose or by application of the adenylate kinase inhibitor Ap5A. 4. Of other nucleotides tested, only CTP consistently activated BKCa channel activity. 5. Using the cell-attached configuration, bath application of forskolin or dibutyryl cAMP stimulated BKCa channel activity. 6. It is concluded that BKCa channel activity in the rat motor cortex is subject to modulation by the activity of a closely associated kinase. The ability of cAMP activators to stimulate BKCa channel activity in the intact cell suggests that this system may be of physiological importance.
Asunto(s)
Adenosina Trifosfato/fisiología , Calcio/fisiología , Corteza Motora/metabolismo , Neuronas/metabolismo , Canales de Potasio/metabolismo , Adenosina Trifosfato/farmacología , Animales , Electrofisiología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Magnesio/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Membranas/efectos de los fármacos , Membranas/metabolismo , Corteza Motora/citología , Corteza Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Nucleótidos/farmacología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/farmacología , Fosforilación , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Inhibidores de Proteínas Quinasas , Ratas , Ratas Sprague-DawleyRESUMEN
In order to determine which drugs would be most effective as inhibitors of platelet release and aggregation, in vitro release reactions and platelet aggregometry were used to evaluate aspirin, dipyridamole, sulfinpyrazone, flurbiprofen, low molecular weight dextran (dextran 40), prostaglandin E1 (PGE1), apyrase, and adenosine. Adenosine diphosphate-induced aggregation was most effectively inhibited by PGF1, sulfinpyrazone, and dipyridamole. The latter had to be used in large doses. Collagen and epinephrine-induced release and aggregation were inhibited by the same drugs as well as by aspirin and apyrase. Antihynocyte globulin (ATG)-induced release and aggregation could only be partially blocked by these agents. In vitro studies suggest that sulfinpyrazone is one of the most effective of platelet inhibitors currently available for clinical testing.