RESUMO
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.
RESUMO
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.
Assuntos
Imunossupressores/metabolismo , Compostos Organofosforados/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Família MultigênicaRESUMO
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.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Pseudomonas syringae/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Mapas de Interação de Proteínas/genética , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidade , Virulência/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Gravitação , Família Multigênica , Raízes de Plantas/fisiologia , Brotos de Planta/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Transporte Biológico , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Gravitropismo , Ácidos Indolacéticos/metabolismo , Mutação/genéticaRESUMO
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.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Imunidade Vegetal/fisiologia , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Morte Celular , Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Doenças das Plantas , Plantas Geneticamente Modificadas , Pseudomonas syringae/patogenicidade , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Ácido Salicílico/metabolismo , Serina/genética , Transdução de Sinais , Nicotiana/genéticaRESUMO
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.
Assuntos
Receptores ErbB/metabolismo , Quinazolinas/farmacologia , Receptor ErbB-2/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Lapatinib , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Gravitropismo/fisiologia , Fosfolipase D/metabolismo , Raízes de Plantas/fisiologia , Água/fisiologia , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Gravitropismo/efeitos dos fármacos , Mutação/genética , Fosfolipase D/genética , Coifa/efeitos dos fármacos , Coifa/enzimologia , Coifa/fisiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Regiões Promotoras Genéticas/genéticaRESUMO
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.
Assuntos
Antineoplásicos/química , Malonatos/química , Morfolinas/química , Triterpenos/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Humanos , Malonatos/síntese química , Malonatos/toxicidade , Morfolinas/síntese química , Morfolinas/farmacologia , Triterpenos/síntese química , Triterpenos/farmacologia , Triterpenos/toxicidadeRESUMO
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.
Assuntos
Cianobactérias/química , Depsipeptídeos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Depsipeptídeos/química , Ensaios de Seleção de Medicamentos Antitumorais , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Biologia Marinha , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , EstereoisomerismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Transporte Biológico , Gravitropismo , Sensação Gravitacional , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Brotos de Planta , Ligação ProteicaRESUMO
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.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Inibidores de Histona Desacetilases , Histonas/metabolismo , Peptídeos Cíclicos/uso terapêutico , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Células HL-60 , Humanos , Células K562 , Masculino , Camundongos , Camundongos Nus , Modelos Biológicos , Modelos Moleculares , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Pró-Fármacos/metabolismo , Especificidade por Substrato , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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).
Assuntos
Plaquetas/efeitos dos fármacos , Artéria Femoral/efeitos dos fármacos , Fibrinolíticos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Trombose/prevenção & controle , Difosfato de Adenosina , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Cálcio/metabolismo , Forma Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Artéria Femoral/fisiopatologia , Artéria Femoral/cirurgia , Fibrinogênio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Ligadura , Macaca fascicularis , Selectina-P/metabolismo , Agregação Plaquetária , Receptores de Trombina , Fluxo Sanguíneo Regional , Estresse Mecânico , Trombose/metabolismo , Trombose/fisiopatologiaRESUMO
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.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Doenças Vasculares Periféricas/prevenção & controle , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Clopidogrel , Derme/irrigação sanguínea , Derme/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotélio Vascular/fisiologia , Epoprostenol/análogos & derivados , Epoprostenol/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Técnicas In Vitro , Ácidos Láuricos , Masculino , Doenças Vasculares Periféricas/induzido quimicamente , Doenças Vasculares Periféricas/patologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Ratos Sprague-Dawley , Ticlopidina/análogos & derivados , Ticlopidina/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
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 .
Assuntos
Inibidores Enzimáticos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Trombose/tratamento farmacológico , Administração Oral , Animais , Pressão Sanguínea , Artérias Carótidas/patologia , Proliferação de Células , Cloretos , Cromonas/farmacologia , Clopidogrel , Relação Dose-Resposta a Droga , Compostos Férricos/farmacologia , Frequência Cardíaca , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Químicos , Morfolinas/farmacologia , Músculo Liso/citologia , Músculo Liso Vascular/patologia , Agregação Plaquetária , Trombose/patologia , Ticlopidina/análogos & derivados , Ticlopidina/farmacologia , Fatores de Tempo , Túnica Íntima/patologiaRESUMO
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).
Assuntos
Fibrinolíticos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Peptídeos Cíclicos/uso terapêutico , Terapia Trombolítica/métodos , Trombose/tratamento farmacológico , Animais , Pressão Sanguínea/efeitos dos fármacos , Doenças das Artérias Carótidas/tratamento farmacológico , Doenças das Artérias Carótidas/prevenção & controle , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Fibrinolíticos/administração & dosagem , Peptídeos Cíclicos/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Ratos , Trombose/prevenção & controle , Ativador de Plasminogênio Tecidual/farmacologia , Resultado do Tratamento , Grau de Desobstrução Vascular/efeitos dos fármacosRESUMO
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.
Assuntos
Antraquinonas/química , Antibacterianos/química , Antraquinonas/isolamento & purificação , Antraquinonas/farmacologia , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Fermentação , Helicobacter pylori/efeitos dos fármacos , Testes de Sensibilidade Microbiana , StreptomycesRESUMO
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.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Fungos Mitospóricos/metabolismo , Naftalenos/química , Naftalenos/farmacologia , Piridonas/química , Piridonas/farmacologia , Antifúngicos/isolamento & purificação , Cromatografia em Gel , Fermentação , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/isolamento & purificação , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Fungos Mitospóricos/química , Naftalenos/isolamento & purificação , Ressonância Magnética Nuclear Biomolecular , Plantas/microbiologia , Piridonas/isolamento & purificação , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
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.
Assuntos
Chromobacterium/metabolismo , Inibidores da Agregação Plaquetária/isolamento & purificação , Difosfato de Adenosina/antagonistas & inibidores , Difosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Chromobacterium/química , Chromobacterium/classificação , Fermentação , Humanos , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Antagonistas do Receptor Purinérgico P2 , Ratos , Receptores Purinérgicos P2Y1 , Microbiologia do Solo , Células Tumorais CultivadasRESUMO
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.