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1.
Biotechnol Bioeng ; 2023 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-37691165

RESUMEN

A continuous downstream process of monoclonal antibody was developed based on the process characterization. Periodic-counter current chromatography (PCCC) with two protein A columns was used for the capture step. For low pH virus inactivation (VI), a batch reactor was employed, which can work as a surge (buffer) tank. Flow-through chromatography (FTC) with two connected columns of different separation modes (anion-mixed mode and cation exchange) was designed as a polish step. After 24 h PCCC run, the collected pool was processed for VI. After adjusting pH and electric conductivity, the solution was fed to the two connected FTC columns for 24 h. Virus filter was also connected to the exit of the connected-column. PCCC and FTC were run in parallel. Six runs of different feed rates (0.5-10 L/day) and feed concentrations (1-3.2 g/L) were performed with protein A columns of 1-5 mL and FTC columns of 3-10 mL. The largest run (feed rate 10 L/day, feed concentration 2 g/L) was carried out at a GMP facility with 15 mL protein A columns and 100 mL FTC columns. Good recovery and purity values were obtained for all runs. The process was found to be flexible and stable for feed fluctuations. Only three surge or pool tanks were needed in addition to the final product pool tank.

2.
J Am Chem Soc ; 143(1): 132-136, 2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33372776

RESUMEN

We report characterization of the biosynthetic pathway of the potent immunosuppressant (-)-FR901483 (1) through heterologous expression and enzymatic assays. The biosynthetic logic to form the azatricyclic alkaloid is consistent with those proposed in biomimetic syntheses and involves aza-spiro annulation of dityrosyl-piperazine to form a ketoaldehyde intermediate, followed by regioselective aldol condensation, stereoselective ketoreduction, and phosphorylation. A possible target of 1 is proposed based on the biosynthetic studies.


Asunto(s)
Inmunosupresores/metabolismo , Compuestos Organofosforados/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Familia de Multigenes
3.
Plant J ; 100(1): 187-198, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31148337

RESUMEN

The phytopathogen Pseudomonas syringae delivers into host cells type III secreted effectors (T3SEs) that promote virulence. One virulence mechanism employed by T3SEs is to target hormone signaling pathways to perturb hormone homeostasis. The phytohormone abscisic acid (ABA) influences interactions between various phytopathogens and their plant hosts, and has been shown to be a target of P. syringae T3SEs. In order to provide insight into how T3SEs manipulate ABA responses, we generated an ABA-T3SE interactome network (ATIN) between P. syringae T3SEs and Arabidopsis proteins encoded by ABA-regulated genes. ATIN consists of 476 yeast-two-hybrid interactions between 97 Arabidopsis ABA-regulated proteins and 56 T3SEs from four pathovars of P. syringae. We demonstrate that T3SE interacting proteins are significantly enriched for proteins associated with transcription. In particular, the ETHYLENE RESPONSIVE FACTOR (ERF) family of transcription factors is highly represented. We show that ERF105 and ERF8 displayed a role in defense against P. syringae, supporting our overall observation that T3SEs of ATIN converge on proteins that influence plant immunity. In addition, we demonstrate that T3SEs that interact with a large number of ABA-regulated proteins can influence ABA responses. One of these T3SEs, HopF3Pph6 , inhibits the function of ERF8, which influences both ABA-responses and plant immunity. These results provide a potential mechanism for how HopF3Pph6 manipulates ABA-responses to promote P. syringae virulence, and also demonstrate the utility of ATIN as a resource to study the ABA-T3SE interface.


Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Pseudomonas syringae/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Mapas de Interacción de Proteínas/genética , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidad , Virulencia/genética
4.
Plant Cell ; 29(8): 1984-1999, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28765510

RESUMEN

During gravitropism, the directional signal of gravity is perceived by gravity-sensing cells called statocytes, leading to asymmetric distribution of auxin in the responding organs. To identify the genes involved in gravity signaling in statocytes, we performed transcriptome analyses of statocyte-deficient Arabidopsis thaliana mutants and found two candidates from the LAZY1 family, AtLAZY1/LAZY1-LIKE1 (LZY1) and AtDRO3/AtNGR1/LZY2 We showed that LZY1, LZY2, and a paralog AtDRO1/AtNGR2/LZY3 are redundantly involved in gravitropism of the inflorescence stem, hypocotyl, and root. Mutations of LZY genes affected early processes in gravity signal transduction without affecting amyloplast sedimentation. Statocyte-specific expression of LZY genes rescued the mutant phenotype, suggesting that LZY genes mediate gravity signaling in statocytes downstream of amyloplast displacement, leading to the generation of asymmetric auxin distribution in gravity-responding organs. We also found that lzy mutations reversed the growth angle of lateral branches and roots. Moreover, expression of the conserved C-terminal region of LZY proteins also reversed the growth direction of primary roots in the lzy mutant background. In lateral root tips of lzy multiple mutants, asymmetric distribution of PIN3 and auxin response were reversed, suggesting that LZY genes regulate the direction of polar auxin transport in response to gravity through the control of asymmetric PIN3 expression in the root cap columella.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Gravitación , Familia de Multigenes , Raíces de Plantas/fisiología , Brotes de la Planta/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Transporte Biológico , Regulación hacia Abajo/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Gravitropismo , Ácidos Indolacéticos/metabolismo , Mutación/genética
5.
BMC Plant Biol ; 18(1): 211, 2018 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-30261844

RESUMEN

BACKGROUND: ETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses. RESULTS: In this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively. CONCLUSIONS: Together these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.


Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Inmunidad de la Planta/fisiología , Proteínas Represoras/metabolismo , Secuencias de Aminoácidos , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Muerte Celular , Regulación de la Expresión Génica de las Plantas , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Enfermedades de las Plantas , Plantas Modificadas Genéticamente , Pseudomonas syringae/patogenicidad , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Ácido Salicílico/metabolismo , Serina/genética , Transducción de Señal , Nicotiana/genética
6.
Mol Cell Proteomics ; 11(12): 1741-57, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22964224

RESUMEN

Lapatinib is a clinically potent kinase inhibitor for breast cancer patients because of its outstanding selectivity for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). However, there is only limited information about the in vivo effects of lapatinib on EGFR/HER2 and downstream signaling targets. Here, we profiled the lapatinib-induced time- and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics. Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5-7%) was regulated at least twofold by 1-10 µm lapatinib. We obtained a comprehensive phosphorylation map of 21 sites on EGFR/HER2, including nine novel sites on HER2. Among them, serine/threonine phosphosites located in a small region of HER2 (amino acid residues 1049-1083) were up-regulated by the drug, whereas all other sites were down-regulated. We show that cAMP-dependent protein kinase is involved in phosphorylation of this particular region of HER2 and regulates HER2 tyrosine kinase activity. Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation, such as RNP complexes (i.e. hnRNP, snRNP, telomerase, ribosome), are linked to EGFR/HER2 signaling networks. To our knowledge, this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib.


Asunto(s)
Receptores ErbB/metabolismo , Quinazolinas/farmacología , Receptor ErbB-2/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Lapatinib , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos
7.
Proc Natl Acad Sci U S A ; 107(31): 13666-71, 2010 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-20639466

RESUMEN

Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered Gq-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of alpha subunit of Gq protein (Galphaq) by inhibiting the GDP release from Galphaq. X-ray crystal structure analysis of the Galphaqbetagamma-YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Galphaq. The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Galpha subunit and an insight into the molecular mechanism of G protein activation.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia
8.
Planta ; 231(2): 491-7, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19915862

RESUMEN

Root hydrotropism is the phenomenon of directional root growth toward moisture under water-deficient conditions. Although physiological and genetic studies have revealed the involvement of the root cap in the sensing of moisture gradients, and those of auxin and abscisic acid (ABA) in the signal transduction for asymmetric root elongation, the overall mechanism of root hydrotropism is still unclear. We found that the promoter activity of the Arabidopsis phospholipase Dzeta2 gene (PLDzeta2) was localized to epidermal cells in the distal root elongation zone and lateral root cap cells adjacent to them, and that exogenous ABA enhanced the activity and extended its area to the entire root cap. Although pldzeta2 mutant root caps did not exhibit a morphological phenotype in either the absence or presence of exogenous ABA, the inhibitory effect of ABA on gravitropism, which was significant in wild-type roots, was not observed in pldzeta2 mutant roots. In root hydrotropism experiments, pldzeta2 mutations significantly retarded or disturbed root hydrotropic responses. A drought condition similar to that used in a hydrotropism experiment enhanced the PLDzeta2 promoter activity in the root cap, as did exogenous ABA. These results suggest that PLDzeta2 responds to drought through ABA signaling in the root cap and accelerates root hydrotropism through the suppression of root gravitropism.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/fisiología , Gravitropismo/fisiología , Fosfolipasa D/metabolismo , Raíces de Plantas/fisiología , Agua/fisiología , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Gravitropismo/efectos de los fármacos , Mutación/genética , Fosfolipasa D/genética , Cápsula de Raíz de Planta/efectos de los fármacos , Cápsula de Raíz de Planta/enzimología , Cápsula de Raíz de Planta/fisiología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/enzimología , Regiones Promotoras Genéticas/genética
9.
Bioorg Med Chem ; 18(8): 2964-75, 2010 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-20363142

RESUMEN

Forty-one derivatives of papyriferic acid were prepared based on our previous finding that methyl papyriferate (3) showed potent reversing effect on cytotoxicity of colchicine against multidrug resistance (MDR) human cancer cells (KB-C2), and evaluated for their cytotoxicity and effect on reversing P-gp-mediated MDR against KB-C2 cells. 3-O-(Morpholino-beta-oxopropanoyl)-12beta-acetoxy-3alpha,25-dihydroxy-(20S,24R)-epoxydammarane (37) significantly increased the sensitivity of colchicine against KB-C2 cells by 185-fold at 5microg/mL (7.4microM), and the cytotoxicity of colchicine was recovered to nearly that of sensitive (KB) cells. The other several new amide derivatives also exhibited potent reversal activity comparable to or more potent than methyl papyriferate and verapamil.


Asunto(s)
Antineoplásicos/química , Malonatos/química , Morfolinas/química , Triterpenos/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Malonatos/síntesis química , Malonatos/toxicidad , Morfolinas/síntesis química , Morfolinas/farmacología , Triterpenos/síntesis química , Triterpenos/farmacología , Triterpenos/toxicidad
10.
J Nat Prod ; 73(3): 393-8, 2010 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-19839606

RESUMEN

Bioassay-guided fractionation of the extract of a consortium of a marine cyanobacterium and a red alga (Rhodophyta) led to the discovery of a novel compound, palmyramide A, along with the known compounds curacin D and malyngamide C. The planar structure of palmyramide A was determined by one- and two-dimensional NMR studies and mass spectrometry. Palmyramide A is a cyclic depsipeptide that features an unusual arrangement of three amino acids and three hydroxy acids; one of the hydroxy acids is the rare 2,2-dimethyl-3-hydroxyhexanoic acid unit (Dmhha). The absolute configurations of the six residues were determined by Marfey's analysis, chiral HPLC analysis, and GC/MS analysis of the hydrolysate. Morphological and phylogenetic studies revealed the sample to be composed of a Lyngbya majuscula-Centroceras sp. association. MALDI-imaging analysis of the cultured L. majuscula indicated that it was the true producer of this new depsipeptide. Pure palmyramide A showed sodium channel blocking activity in neuro-2a cells and cytotoxic activity in H-460 human lung carcinoma cells.


Asunto(s)
Cianobacterias/química , Depsipéptidos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Depsipéptidos/química , Ensayos de Selección de Medicamentos Antitumorales , Cromatografía de Gases y Espectrometría de Masas , Humanos , Biología Marina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estereoisomerismo
11.
Nat Commun ; 11(1): 76, 2020 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-31900388

RESUMEN

In many plant species, roots maintain specific growth angles relative to the direction of gravity, known as gravitropic set point angles (GSAs). These contribute to the efficient acquisition of water and nutrients. AtLAZY1/LAZY1-LIKE (LZY) genes are involved in GSA control by regulating auxin flow toward the direction of gravity in Arabidopsis. Here, we demonstrate that RCC1-like domain (RLD) proteins, identified as LZY interactors, are essential regulators of polar auxin transport. We show that interaction of the CCL domain of LZY with the BRX domain of RLD is important for the recruitment of RLD from the cytoplasm to the plasma membrane by LZY. A structural analysis reveals the mode of the interaction as an intermolecular ß-sheet in addition to the structure of the BRX domain. Our results offer a molecular framework in which gravity signal first emerges as polarized LZY3 localization in gravity-sensing cells, followed by polar RLD1 localization and PIN3 relocalization to modulate auxin flow.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Transporte Biológico , Gravitropismo , Sensación de Gravedad , Ácidos Indolacéticos/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Brotes de la Planta , Unión Proteica
12.
Int J Oncol ; 32(3): 545-55, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18292931

RESUMEN

Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in vitro and in vivo. Various studies related to their antitumor activity and mechanism of action have been reported for HDAC inhibitors, but the relationship of their antitumor effects to their pharmacodynamic and pharmacokinetic properties in vivo has not ever fully characterized. We report here the discovery of a novel cyclic-peptide-based HDAC inhibitor, YM753. YM753 is a bacteria-derived natural product containing a disulfide bond. It potently inhibited HDAC enzyme with an IC50 of 2.0 nM in the presence of dithiothreitol. YM753 was rapidly converted to a reduced form in tumor cells, and then induced accumulation of acetylated histones, followed by p21WAF1/Cip1 expression, tumor cell growth inhibition and tumor-selective cell death. In an in vitro washout study, YM753 showed prolonged accumulation of acetylated histones in WiDr human colon carcinoma cells. In vivo YM753 dosing of mice harboring WiDr colon tumor xenografts significantly inhibited the tumor growth via sustained accumulation of acetylated histones in the tumor tissue. In a pharmacokinetic study, YM753 rapidly disappeared from the plasma, but its reduced form remained in the tumor tissue. Moreover, the accumulation of acetylated histones induced by YM753 was tumor tissue selective compared to several normal tissues. This study provides evidence that YM753 has antitumor activity that is the result of selective, sustained accumulation of acetylated histones in tumor tissues despite rapid disappearance of the drug from the plasma. These results suggest that the novel HDAC inhibitor, YM753 has attractive pharmacodynamic and pharmacokinetic properties giving it potential as an antitumor agent.


Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Inhibidores de Histona Desacetilasas , Histonas/metabolismo , Péptidos Cíclicos/uso terapéutico , Acetilación/efectos de los fármacos , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Células HL-60 , Humanos , Células K562 , Masculino , Ratones , Ratones Desnudos , Modelos Biológicos , Modelos Moleculares , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Profármacos/metabolismo , Especificidad por Sustrato , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Br J Pharmacol ; 148(1): 61-9, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16520742

RESUMEN

1 The effects of YM-254890, a specific Galpha(q/11) inhibitor, on platelet functions, thrombus formation under high-shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM-254890 concentration dependently inhibited ADP-induced intracellular Ca(2+) elevation, with an IC(50) value of 0.92+/-0.28 microM. 3 P-selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM-254890, with IC(50) values of 0.51+/-0.02 and 0.16+/-0.08 microM, respectively. 4 YM-254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP-stimulated washed platelets. 5 YM-254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM-254890 attenuated ADP-, collagen-, TRAP-, arachidonic acid- and U46619-induced platelet aggregation with IC(50) values of <1 microM, whereas it had no effect on phorbol 12-myristate 13-acetate-, ristocetin-, thapsigargin- or A23187-induced platelet aggregation. 7 High-shear stress-induced platelet aggregation and platelet-rich thrombus formation on a collagen surface under high-shear flow conditions were concentration dependently inhibited by YM-254890. 8 The antithrombotic effect of YM-254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM-254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM-254890 is a useful tool for investigating Galpha(q/11)-coupled receptor signaling and the physiological roles of Galpha(q/11).


Asunto(s)
Plaquetas/efectos de los fármacos , Arteria Femoral/efectos de los fármacos , Fibrinolíticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/prevención & control , Adenosina Difosfato , Animales , Plaquetas/citología , Plaquetas/metabolismo , Calcio/metabolismo , Forma de la Célula , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Arteria Femoral/fisiopatología , Arteria Femoral/cirugía , Fibrinógeno/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Ligadura , Macaca fascicularis , Selectina-P/metabolismo , Agregación Plaquetaria , Receptores de Trombina , Flujo Sanguíneo Regional , Estrés Mecánico , Trombosis/metabolismo , Trombosis/fisiopatología
14.
Eur J Pharmacol ; 536(1-2): 154-61, 2006 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-16566917

RESUMEN

The protective effect of YM-254890, a specific Galphaq/11 inhibitor, on laurate-induced peripheral arterial disease in rats was compared with those of prostaglandin E1 (PGE1), beraprost, and clopidogrel. YM-254890 inhibited ADP-induced ex vivo rat platelet aggregation at a dose of 3 microg/kg. Furthermore, YM-254890 strongly inhibited phenylephrine-, serotonin- and endothelin-1-induced contractions in the rat aorta, and improved dermal blood flow after the laurate injection. The intra-arterial single bolus administration of YM-254890 15 min after the laurate injection dose-dependently inhibited the progression of the lesion, with significance, at 3 microg/kg without affecting systemic blood pressure. PGE1 and beraprost, when administered before the laurate injection, were effective, but their potencies were less than that of YM-254890. Clopidogrel significantly suppressed lesion progression when administered at 30 mg/kg twice a day for 3 days, which completely inhibited platelet aggregation. These results suggest that the local administration of YM-254890 may be useful for treating peripheral arterial disease.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Enfermedades Vasculares Periféricas/prevención & control , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Clopidogrel , Dermis/irrigación sanguínea , Dermis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiología , Epoprostenol/análogos & derivados , Epoprostenol/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Técnicas In Vitro , Ácidos Láuricos , Masculino , Enfermedades Vasculares Periféricas/inducido químicamente , Enfermedades Vasculares Periféricas/patología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Ratas , Ratas Sprague-Dawley , Ticlopidina/análogos & derivados , Ticlopidina/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología
15.
Thromb Haemost ; 94(1): 184-92, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16113802

RESUMEN

The pharmacological properties of YM-254890, a specific G(alpha)q/11 inhibitor, on acute thrombosis and chronic neointima formation after vascular injury have been investigated. FeCl3 was used to induce vascular injury in the carotid artery of mice. For the thrombosis studies, the test drug was either intravenously or orally administered before vascular injury. For the neointima studies, the test drug was orally administered 1 h before and twice daily for 1 week after vascular injury. Histological analysis was then performed 3 weeks later. YM-254890 significantly inhibited ex vivo platelet aggregation 5 min after intravenous bolus injection at 0.03 mg/kg or more, and 1 h after oral administration at 1 mg/kg. YM-254890 significantly inhibited thrombus formation after intravenous bolus injection at 0.03 mg/kg as well as after oral administration at 1 mg/kg, but tail transection bleeding time was significantly prolonged at 0.1 mg/kg for intravenous injection and 3 mg/kg for oral administration. Furthermore, oral administration of YM-254890 dose-dependently inhibited neointima formation 3 weeks after vascular injury with significant effects at 1 mg/kg twice daily for 1 week. Clopidogrel also significantly inhibited neointima formation at its antithrombotic dose, but its inhibitory potency was less than that of YM-254890. However, YM-254890 significantly reduced systemic blood pressure at doses 3 times higher than those that produced significant inhibitory effects on thrombosis and neointima formation. Though the systemic use of YM-254890 may be limited, owing to its narrow therapeutic window, this unique compound is a useful research tool for investigating the physiological roles of G(alpha)q/11 .


Asunto(s)
Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Trombosis/tratamiento farmacológico , Administración Oral , Animales , Presión Sanguínea , Arterias Carótidas/patología , Proliferación Celular , Cloruros , Cromonas/farmacología , Clopidogrel , Relación Dosis-Respuesta a Droga , Compuestos Férricos/farmacología , Frecuencia Cardíaca , Masculino , Ratones , Ratones Endogámicos ICR , Modelos Químicos , Morfolinas/farmacología , Músculo Liso/citología , Músculo Liso Vascular/patología , Agregación Plaquetaria , Trombosis/patología , Ticlopidina/análogos & derivados , Ticlopidina/farmacología , Factores de Tiempo , Túnica Íntima/patología
16.
Thromb Haemost ; 90(3): 406-13, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12958608

RESUMEN

We examined the antithrombotic and thrombolytic effects of the G(q/11) inhibitor YM-254890 in an electrically-induced carotid artery thrombosis model in rats. YM-254890 dose-dependently inhibited ex vivo ADP-induced platelet aggregation after i.v. bolus injection. In the thrombosis study, YM-254890 dosedependently prolonged time to occlusion at doses of 3 and 10 g/kg i.v. and decreased occlusion rate at 10 g/kg i.v. In the thrombolysis study, YM-254890 at 30 micro g/kg i.v. shortened the time to reperfusion and prevented reocclusion after thrombolysis with a modified tissue-type plasminogen activator. YM-254890, at 10 micro g/kg and more, significantly improved carotid patency status after thrombolysis. However, at 30 micro g/kg and more, YM-254890 decreased systemic blood pressure. These results suggest that YM-254890 may be effective for treating G(q)-mediated diseases, and that YM-254890 is a useful tool for investigating the biological roles of G(q/11).


Asunto(s)
Fibrinolíticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Péptidos Cíclicos/uso terapéutico , Terapia Trombolítica/métodos , Trombosis/tratamiento farmacológico , Animales , Presión Sanguínea/efectos de los fármacos , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Enfermedades de las Arterias Carótidas/prevención & control , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Fibrinolíticos/administración & dosificación , Péptidos Cíclicos/administración & dosificación , Agregación Plaquetaria/efectos de los fármacos , Ratas , Trombosis/prevención & control , Activador de Tejido Plasminógeno/farmacología , Resultado del Tratamiento , Grado de Desobstrucción Vascular/efectos de los fármacos
17.
J Antibiot (Tokyo) ; 55(1): 30-5, 2002 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11918062

RESUMEN

A novel benz[a]anthraquinone, YM-181741, was isolated from the culture broth of actinomycete strain Q57219. The strain was identified as Streptomyces sp. by morphological and chemotaxonomic characterization. YM-181741 was purified from the culture supernatant by serial column chromatography. The structure of YM-181741 was determined by spectroscopic analysis including one- and two-dimensional NMR experiments. YM-181741 showed selective anti-Helicobacterpylori activity with a MIC value of 0.2 microg/ml.


Asunto(s)
Antraquinonas/química , Antibacterianos/química , Antraquinonas/aislamiento & purificación , Antraquinonas/farmacología , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Fermentación , Helicobacter pylori/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Streptomyces
18.
J Antibiot (Tokyo) ; 57(6): 379-82, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15323126

RESUMEN

Through our screening for novel antifungal compounds, YM-215343 was found in the culture extract of Phoma sp. QN04621. The structure of YM-215343 was determined by several spectroscopic experiments as a novel compound closely related to apiosporamide and fischerin. YM-215343 exhibited antifungal activity against the pathogenic fungi, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus with MIC values of 2 to approximately 16 microg/ml. It also showed cytotoxicity against HeLa S3 cells with an IC50 of 3.4 microg/ml.


Asunto(s)
Antifúngicos/química , Antifúngicos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Hongos Mitospóricos/metabolismo , Naftalenos/química , Naftalenos/farmacología , Piridonas/química , Piridonas/farmacología , Antifúngicos/aislamiento & purificación , Cromatografía en Gel , Fermentación , Células HeLa , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Hongos Mitospóricos/química , Naftalenos/aislamiento & purificación , Resonancia Magnética Nuclear Biomolecular , Plantas/microbiología , Piridonas/aislamiento & purificación , Espectrometría de Masa Bombardeada por Átomos Veloces
19.
J Antibiot (Tokyo) ; 56(4): 358-63, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12817809

RESUMEN

A novel platelet aggregation inhibitor, YM-254890, was isolated from the culture broth of strain QS3666. This strain was isolated from a soil sample collected at Okutama, Tokyo, Japan, and was identified as Chromobacterium sp. by morphological and physiological criteria. YM-254890 was purified from the culture supernatant by solvent extraction, ODS and silica gel flash chromatography, followed by preparative HPLC. YM-254890 inhibited ADP-induced platelet aggregation in human platelet-rich plasma with an IC50 value below 0.6 microM by blocking the P2Y1 receptor-signal transduction pathway.


Asunto(s)
Chromobacterium/metabolismo , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , Adenosina Difosfato/antagonistas & inhibidores , Adenosina Difosfato/farmacología , Animales , Calcio/metabolismo , Chromobacterium/química , Chromobacterium/clasificación , Fermentación , Humanos , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Antagonistas del Receptor Purinérgico P2 , Ratas , Receptores Purinérgicos P2Y1 , Microbiología del Suelo , Células Tumorales Cultivadas
20.
Plant Signal Behav ; 9(9): e29570, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25763694

RESUMEN

Differential organ growth during gravitropic response is caused by differential accumulation of auxin, that is, relative higher auxin concentration in lower flanks than in upper flanks of responding organs. Auxin responsive reporter systems such as DR5::GUS and DR5::GFP have usually been used as indicators of gravitropic response in roots and hypocotyls of Arabidopsis. However, in the inflorescence stems, the reporter systems don't work well to monitor gravitropic response. Here, we aim to certify appropriate gravitropic response indicators (GRIs) in inflorescence stems. We performed microarray analysis comparing gene expression profiles between upper and lower flanks of Arabidopsis inflorescence stems after gravistimulation. Thirty genes showed > 2-fold differentially increased expression in lower flanks at 30 min, of which 19 were auxin response genes. We focused on IAA5 and IAA2 and verified whether they are appropriate GRIs by real-time qRT-PCR analyses. Transcript levels of IAA5 and IAA2 were remarkably higher in lower flanks than in upper flanks after gravistimulation. The biased IAA5 or IAA2 expression is disappeared in sgr2-1 mutant which is defective in gravity perception, indicating that gravity perception process is essential for formation of the biased gene expression during gravitropism. IAA5 expression was remarkably increased in lower flanks at 30 min after gravistimulation, whereas IAA2 expression was gradually decreased in upper flanks in a time-dependent manner. Therefore, we conclude that IAA5 is a sensitive GRI to monitor asymmetric auxin signaling caused by gravistimulation in Arabidopsis inflorescence stems.


Asunto(s)
Arabidopsis/genética , Arabidopsis/fisiología , Genes de Plantas , Gravitropismo/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Gravitropismo/fisiología , Inflorescencia/genética , Inflorescencia/crecimiento & desarrollo , Inflorescencia/fisiología , Mutación , Proteínas Nucleares/genética , Fosfolipasas/genética , Plantas Modificadas Genéticamente , Transducción de Señal
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