RESUMEN
We synthesized a new silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose (SGlc). Conjugation with SGlc improved A549 cellular uptake without significant changes in the photophysical and photochemical properties and subcellular localization. This improved cellular uptake led to enhanced photodynamic activity. Furthermore, conjugation with SGlc suppressed dark toxicity. These advantages were not observed for a conjugate with a glucose molecule. These results indicated that the conjugation with SGlc is a promising strategy for enhancing photodynamic efficacy.
Asunto(s)
Fotoquimioterapia , Porfirinas , Humanos , Fármacos Fotosensibilizantes/farmacología , Fotoquimioterapia/métodos , Células A549 , Glucosa , Porfirinas/farmacologíaRESUMEN
α-Sulfoquinovosylacyl-1,3-propanediol (SQAP) is a semi-synthetic derivative of natural sulfoglycolipid that sensitizes tumors to external-beam radiotherapy. How SQAP affects internal radiotherapy, however, is not known. Here, we investigated the effects of SQAP for radioimmunotherapy (RIT) targeting tissue factor (TF) in a stroma-rich refractory pancreatic cancer mouse model, BxPC-3. A low dose of SQAP (2 mg/kg) increased tumor uptake of the 111In-labeled anti-TF antibody 1849, indicating increased tumor perfusion. The addition of SQAP enhanced the growth-inhibitory effect of 90Y-labeled 1849 without leading to severe body weight changes, allowing for the dose of 90Y-labeled 1849 to be reduced to half that when used alone. Histologic analysis revealed few necrotic and apoptotic cells, but Ki-67-positive proliferating cells and increased vascular formation were detected. These results suggest that the addition of a low dose of SQAP may improve the therapeutic efficacy of TF-targeted RIT by increasing tumor perfusion, even for stroma-rich refractory pancreatic cancer.
RESUMEN
Malignant pleural mesothelioma (MPM) is a highly malignant disease that develops after asbestos exposure. Although the number of MPM cases is predicted to increase, no effective standard therapies have been established. The novel radiosensitizer α-sulfoquinovosyl-acylpropanediol (SQAP) enhances the effects of γ-radiation in human lung and prostate cancer cell lines and in animal models. In this study, we explored the radiosensitizing effect of SQAP and its mechanisms in MPM. The human MPM cell lines MSTO-211H and MESO-4 were implanted subcutaneously into the backs and thoracic cavities of immunodeficient KSN/Slc mice, then 2 mg/kg SQAP was intravenously administered with or without irradiation with a total body dose of 8 Gy. In both the orthotopic and ectopic xenograft murine models, the combination of irradiation plus SQAP delayed the implanted human MSTO-211H tumor growth. The analysis of the changes in the relative tumor volume of the MSTO-211H indicated a statistically significant difference after 8 Gy total body combined with 2 mg/kg SQAP, compared to both the untreated control (P = 0.0127) and the radiation treatment alone (P = 0.0171). After the treatment in each case, immunostaining of the harvested tumors revealed decreased cell proliferation, increased apoptosis and normalization of tumor blood vessels in the SQAP- and irradiation-treated group. Furthermore, hypoxia-inducible factor (HIF) 1 mRNA and protein expression were decreased, indicating reoxygenation in this group. In conclusion, SQAP improved hypoxic conditions in tumor tissue and may elicit a radiosensitizing effect in malignant mesothelioma models.
Asunto(s)
Antineoplásicos , Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurales , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Humanos , Masculino , Mesotelioma/tratamiento farmacológico , Mesotelioma/metabolismo , Mesotelioma/radioterapia , Ratones , Ratones Desnudos , Neoplasias Pleurales/tratamiento farmacológico , Neoplasias Pleurales/metabolismo , Neoplasias Pleurales/radioterapia , Tolerancia a RadiaciónRESUMEN
Sulfoquynovosylacyl propanediol (SQAP; 1) has been developed as a radiosensitizer (anti-cancer agent) for solid tumors, but it was easily cleaved in vivo and had a problem of short residence time. We synthesized a novel compound of a SQAP derivative (3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside: ODSG; 2) to solve these problems not easily cleaved by lipase. ODSG (2) cytotoxicity was investigated in vitro, resulting in low toxicity like SQAP (1).
Asunto(s)
Lipasa/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Células A549 , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Fármacos Sensibilizantes a Radiaciones/química , Fármacos Sensibilizantes a Radiaciones/metabolismo , Relación Estructura-ActividadRESUMEN
Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys52 and Cys83 in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Peroxirredoxinas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Línea Celular Tumoral , Inhibidores Enzimáticos , Compuestos Epoxi/química , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Estructura Molecular , Mutación , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Polienos/químicaRESUMEN
Sulfoglycolipid, SQAP, is a radiosensitizing agent that makes tumor cells more sensitive to radiation therapy. A previous study revealed that SQAP induced the degradation of hypoxia-inducible factor-1α (HIF-1α) and inhibited angiogenesis in a hepatoma model mouse. Herein, we examined the biological activities of SQAP against hepatocarcinoma cells under low oxygen conditions. Cell growth inhibition of SQAP under hypoxic conditions was significantly higher than that under normoxic conditions. In addition, SQAP was found to impair the expression of histone deacetylase (HDAC) under low oxygen conditions. Our present data suggested that SQAP induced the degradation of HIF-1α and then decreased the expression of HDAC1. Unlike known HDAC inhibitors, SQAP increased the acetylation level of histone in cells without inhibition of enzymatic activity of HDACs. Our data demonstrated hypoxia-specific unique properties of SQAP.
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Muerte Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucolípidos/química , Glucolípidos/farmacología , Histona Desacetilasa 1/metabolismo , Hipoxia Tumoral/efectos de los fármacos , Acetilación/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Histonas/metabolismo , HumanosRESUMEN
INTRODUCTION: Our understanding of the mechanism of action of bioactive small molecules contributes to the research and development of new medical drugs, as well as elucidating the pathological mechanisms underlying various diseases. Researchers in this field are committed to a very ambitious goal: the discovery of novel therapeutic compounds along with their molecular targets. To achieve this goal, new methodological developments are indispensable. AREAS COVERED: This review gives an update on the advancements of phage display (PD) technology in the past decade (2011-2020) for determining the targets of the small molecule therapeutics. In particular, other than providing a brief overview of this field of research, we focus on reporting the research trends and the results solely obtained using this strategy. EXPERT OPINION: Despite the development of bioinformatics tools and artificial intelligence (AI)-mediated methods, affinity-guided information obtained experimentally are still indispensable to identify drug-protein interactions. By taking advantage of small-molecule-oriented PD methods and their improvements, the extension of the druggable proteome will be further expanded, providing new opportunities to generate small-molecule therapeutics.
Asunto(s)
Técnicas de Visualización de Superficie Celular , Desarrollo de Medicamentos/métodos , Terapia Molecular Dirigida , Inteligencia Artificial , Biología Computacional , Descubrimiento de Drogas/métodos , Humanos , Bibliotecas de Moléculas PequeñasRESUMEN
Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65. For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-14C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body. The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-14C]-SQAP (CP-3635) was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice. The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney. Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.
Asunto(s)
Glucolípidos/farmacocinética , Fármacos Sensibilizantes a Radiaciones/farmacocinética , Administración Intravenosa , Animales , Autorradiografía , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Glucolípidos/administración & dosificación , Glucolípidos/uso terapéutico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones Desnudos , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Espectrometría de Masas en TándemRESUMEN
: Hypoxic zones in solid tumors contribute to radioresistance, and pharmacologic agents that increase tumor oxygenation prior to radiation, including antiangiogenic drugs, can enhance treatment response to radiotherapy. Although such strategies have been applied, imaging assessments of tumor oxygenation to identify an optimum time window for radiotherapy have not been fully explored. In this study, we investigated the effects of α-sulfoquinovosylacyl-1,3-propanediol (SQAP or CG-0321; a synthetic derivative of an antiangiogenic agent) on the tumor microenvironment in terms of oxygen partial pressure (pO2), oxyhemoglobin saturation (sO2), blood perfusion, and microvessel density using electron paramagnetic resonance imaging, photoacoustic imaging, dynamic contrast-enhanced MRI with Gd-DTPA injection, and T2*-weighted imaging with ultrasmall superparamagnetic iron oxide (USPIO) contrast. SCCVII and A549 tumors were grown by injecting tumor cells into the hind legs of mice. Five days of daily radiation (2 Gy) combined with intravenous injection of SQAP (2 mg/kg) 30 minutes prior to irradiation significantly delayed growth of tumor xenografts. Three days of daily treatment improved tumor oxygenation and decreased tumor microvascular density on T2*-weighted images with USPIO, suggesting vascular normalization. Acute effects of SQAP on tumor oxygenation were examined by pO2, sO2, and Gd-DTPA contrast-enhanced imaging. SQAP treatment improved perfusion and tumor pO2 (ΔpO2: 3.1 ± 1.0 mmHg) and was accompanied by decreased sO2 (20%-30% decrease) in SCCVII implants 20-30 minutes after SQAP administration. These results provide evidence that SQAP transiently enhanced tumor oxygenation by facilitating oxygen dissociation from oxyhemoglobin and improving tumor perfusion. Therefore, SQAP-mediated sensitization to radiation in vivo can be attributed to increased tumor oxygenation. SIGNIFICANCE: A multimodal molecular imaging study evaluates pharmacological alteration of the tumor microenvironment to improve radiation response.
Asunto(s)
Imagen Molecular/métodos , Imagen Multimodal/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Microambiente Tumoral , Células A549 , Acústica , Inhibidores de la Angiogénesis/farmacología , Animales , Espectroscopía de Resonancia por Spin del Electrón , Gadolinio/química , Gadolinio DTPA/química , Glucolípidos/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hipoxia , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C3H , Microcirculación , Trasplante de Neoplasias , Neoplasias/metabolismo , Oxígeno/química , Oxígeno/metabolismo , Fotoquímica , Tolerancia a Radiación , RadioterapiaRESUMEN
Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.
Asunto(s)
Bacteriófago T7/genética , Técnicas Biosensibles , Técnicas de Visualización de Superficie Celular , Biología Computacional , Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Secuencia de Aminoácidos , Biología Computacional/métodos , Descubrimiento de Drogas/métodos , Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Análisis de Secuencia de ADN , Programas InformáticosRESUMEN
We developed a high-power abiotic direct glucose fuel cell system using a Au-Pt bimetallic anode catalyst. The high power generation (95.7 mW cm-2) was attained by optimizing operating conditions such as the composition of a bimetallic anode catalyst, loading amount of the metal catalyst on a carbon support, ionomer/carbon weight ratio when the catalyst was applied to the anode, glucose and KOH concentrations in the fuel solution, and operating temperature and flow rate of the fuel solution. It was found that poly(N-vinyl-2-pyrrolidone)-stabilized Au80Pt20 nanoparticles (mean diameter 1.5 nm) on a carbon (Ketjen Black 600) support function as a highly active anode catalyst for the glucose electrooxidation. The ionomer/carbon weight ratio also greatly affects the cell properties, which was found to be optimal at 0.2. As for the glucose concentration, a maximum cell power was derived at 0.4-0.6 mol dm-3. A high KOH concentration (4.0 mol dm-3) was preferable for deriving the maximum power. The cell power increased with the increasing flow rate of the glucose solution up to 50 cm3 min-1 and leveled off thereafter. At the optimal condition, the maximum power density and corresponding cell voltage of 58.2 mW cm-2 (0.36 V) and 95.7 mW cm-2 (0.34 V) were recorded at 298 and 328 K, respectively.
RESUMEN
SQAP is a novel and promising anticancer agent that was obtained by structural modifications from a natural compound. SQAP inhibits angiogenesis in vivo resulting in increased hypoxia and reduced tumor volume. In this study, the mechanism by which SQAP modifies the tumor microenvironment was revealed through the application of a T7 phage display screening. This approach identified five SQAP-binding proteins including sterol carrier protein 2, multifunctional enzyme type 2, proteasomal ubiquitin receptor, UV excision repair protein and focal adhesion kinase (FAK). All the interactions were confirmed by surface plasmon resonance analysis. Since FAK plays an important role in cell turnover and angiogenesis, the influence of SQAP on FAK was the principal goal of this study. SQAP decreased FAK phosphorylation and cell migration in human umbilical vein endothelial cells and A549 cancer cells. These findings suggest that inhibition of FAK phosphorylation works as the mechanism for the anti-angiogenesis activity of SQAP.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Glucolípidos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Antineoplásicos/síntesis química , Sitios de Unión , Carcinoma de Células Pequeñas/enzimología , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Pequeñas/patología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Quinasa 1 de Adhesión Focal/química , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Glucolípidos/síntesis química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Desnudos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteína-2 Multifuncional Peroxisomal/química , Proteína-2 Multifuncional Peroxisomal/genética , Proteína-2 Multifuncional Peroxisomal/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Saccharification of cellulose is a promising technique for producing alternative source of energy. However, the efficiency of conversion of cellulose into soluble sugar using any currently available methodology is too low for industrial application. Many additives, such as surfactants, have been shown to enhance the efficiency of cellulose-to-sugar conversion. In this study, we have examined first whether cattle saliva, as an additive, would enhance the cellulase-catalyzed hydrolysis of cellulose, and subsequently elucidated the mechanism by which cattle saliva enhanced this conversion. Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose. Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva. We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect. Third, the mechanism of cattle saliva mediated enhancement of cellulase activity was probably similar to that of the canonical surfactants. Cattle saliva is available in large amounts easily and cheaply, and it can be used without further purification. Thus, cattle saliva could be a promising additive for efficient saccharification of cellulose on an industrial scale.
Asunto(s)
Celulosa/metabolismo , Saliva/metabolismo , Adsorción , Animales , Biomasa , Bovinos , Celulasa/metabolismo , Fraccionamiento Químico , Cristalografía por Rayos X , Proteínas/aislamiento & purificaciónRESUMEN
OBJECTIVES: To examine the effects of combined treatment with sulfoquinovosylacylpropanediol and X-ray irradiation on the remodeling of the prostate cancer microenvironment, including angiogenic and hypoxic characteristics. METHODS: Human prostate cancer cells (DU145 and PC3) were implanted subcutaneously into the right hind legs of athymic nude mice. After the tumor volume reached 100-300mm(3) , 2mg/kg/day sulfoquinovosylacylpropanediol was given intravenously from day0 to day4, and cells were exposed to 4Gy X-ray irradiation on days0 and 3 (for a total of 8Gy). Tumors were fixed and stained for pathological analyses and immunohistochemical evaluations. To analyze vascular normalization, 60mg/kg pimonidazole dissolved in saline was injected intraperitoneally. RESULTS: Combined treatment with sulfoquinovosylacylpropanediol plus X-ray irradiation enhanced growth inhibition in DU145 xenografts. The tumor vessel density in DU145 cells significantly decreased after the combined treatment. Staining for αsmooth muscle actin in vessels was significantly increased. Pimonidazole staining, showing hypoxic lesions, was negative from 72h, but positive at 6 and 24h after the first combined treatment. In contrast, no enhancement of the microenvironment in PC3 xenografts was observed with sulfoquinovosylacylpropanediol plus X-ray irradiation. CONCLUSION: Sulfoquinovosylacylpropanediol could be a novel potent radiosensitizing agent targeting angiogenesis in prostate cancer.
Asunto(s)
Vasos Sanguíneos/efectos de la radiación , Glucolípidos/administración & dosificación , Neovascularización Patológica/radioterapia , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/radioterapia , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Actinas/análisis , Animales , Vasos Sanguíneos/química , Vasos Sanguíneos/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Neoplasias de la Próstata/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/efectos de la radiaciónRESUMEN
Roxithromycin (RXM) is a semi-synthetic fourteen-membered macrolide antibiotic that shows anti-angiogenic activity in solid tumors. In the present study, we conducted biopanning of T7 phage-displayed peptides either on a 96-well formatted microplate, a flow injection-type quartz-crystal microbalance (QCM) biosensor, or a cuvette-type QCM. RXM-selected peptides of different sequence, length and number were obtained from each mode of screening. Subsequent bioinformatics analysis of the RXM-selected peptides consistently gave positive scores for the extracellular domain (E458-T596) of angiomotin (Amot), indicating that this may comprise a binding region for RXM. Bead pull down assay and QCM analysis confirmed that RXM directly interacts with Amot via the screen-guided region, which also corresponds to the binding site for the endogenous anti-angiogenic inhibitor angiostatin (Anst). Thus, multimodal biopanning of T7PD revealed that RXM binds to the extracellular domain on Amot as a common binding site with Anst, leading to inhibition of angiogenesis-dependent tumor growth and metastasis. These data might explain the molecular basis underlying the mechanism of action for the anti-angiogenic activity of RXM.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Bacteriófago T7/química , Proteínas de la Membrana/antagonistas & inhibidores , Péptidos/química , Roxitromicina/farmacología , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/química , Angiomotinas , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células MCF-7 , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos , Estructura Molecular , Biblioteca de Péptidos , Tecnicas de Microbalanza del Cristal de Cuarzo , Roxitromicina/síntesis química , Roxitromicina/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
MAIN CONCLUSION: Enzymatic activities of Oryza sativa expansins, which were heterologously overexpressed in Escherichia coli , were analyzed. Results suggested that expansins promote degradation of cellulose by cellulase in a synergistic manner. Sustainable production of future biofuels is dependent on efficient saccharification of lignocelluloses. Expansins have received a lot of attention as proteins promoting biological degradation of cellulose using cellulase. The expansins are a class of plant cell wall proteins that induce cell wall loosening without hydrolysis. In this study, the expansins from Oryza sativa were classified using phylogenetic analysis and five proteins were selected for functional evaluation. At low cellulose loading, the cellulase in expansin mixtures was up to 2.4 times more active than in mixtures containing only cellulase, but at high cellulose loading the activity of cellulase in expansin mixtures and cellulase only mixtures did not differ. Furthermore, expansin activity was greater in cellulase mixtures compared with cellulase-deficient mixtures. Therefore, the expansins showed significant synergistic activity with cellulase. Expansin may play an important role in efficient saccharification of cellulose.
Asunto(s)
Celulasa/metabolismo , Celulosa/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Pared Celular/metabolismo , Celulosa/química , Cristalización , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Modelos Biológicos , Oryza/genética , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Unión Proteica , Difracción de Rayos XRESUMEN
BACKGROUND: Methotrexate (MTX) is an agent used in chemotherapy of tumors and autoimmune disease including rheumatoid arthritis (RA). In addition, MTX has some anti-inflammatory activity. Although dihydrofolate reductase (DHFR) is a well-known target for the anti-tumor effect of MTX, the mode of action for the anti-inflammatory activity of MTX is not fully understood. METHODOLOGY/RESULT: Here, we performed a screening of MTX-binding proteins using T7 phage display with a synthetic biotinylated MTX derivative. We then characterized the interactions using surface plasmon resonance (SPR) analysis and electrophoretic mobility shift assay (EMSA). Using a T7 phage display screen, we identified T7 phages that displayed part of high-mobility group box 1 (HMGB1) protein (K86-V175). Binding affinities as well as likely binding sites were characterized using genetically engineered truncated versions of HMGB1 protein (Al G1-K87, Bj: F88-K181), indicating that MTX binds to HMGB1 via two independent sites with a dissociation constants (KD) of 0.50±0.03 µM for Al and 0.24 ± 0.01 µM for Bj. Although MTX did not inhibit the binding of HMGB1 to DNA via these domains, HMGB1/RAGE association was impeded in the presence of MTX. These data suggested that binding of MTX to part of the RAGE-binding region (K149-V175) in HMGB1 might be significant for the anti-inflammatory effect of MTX. Indeed, in murine macrophage-like cells (RAW 264.7), TNF-α release and mitogenic activity elicited by specific RAGE stimulation with a truncated monomeric HMGB1 were inhibited in the presence of MTX. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that HMGB1 is a direct binding protein of MTX. Moreover, binding of MTX to RAGE-binding region in HMGB1 inhibited the HMGB1/RAGE interaction at the molecular and cellular levels. These data might explain the molecular basis underlying the mechanism of action for the anti-inflammatory effect of MTX.
Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Antimetabolitos Antineoplásicos/metabolismo , Proteína HMGB1/metabolismo , Metotrexato/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/farmacología , Bacteriófago T7/genética , Sitios de Unión , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Proteína HMGB1/química , Proteína HMGB1/farmacología , Humanos , Cinética , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metotrexato/química , Metotrexato/farmacología , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Péptidos/farmacología , Unión Proteica , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Nowadays, chemically synthesized proteins and peptides are attractive building blocks and have potential in many important applications as biomaterials. In this review, applications of biomaterials to thermotropic liquid crystals are discussed. The review covers the improvement of the performance of liquid crystal displays using liquid crystal physical gels consisting of a liquid crystal and amino acid-based gelators, and also new functionalization of liquid crystals. Moreover, the influence of DNA, which is one of the more attractive biomaterials, dispersed in thermotropic liquid crystals and its potential use in the liquid crystal industry is described. In addition, we found interesting results during electrooptical measurements of liquid crystals doped with DNA, and explain them from the point of view of biological applications. These recent approaches suggest that these biomaterials may be applicable in the electronic device industry and should be considered as an interesting material with their physical properties having the potential to create or refine an industrial product.
Asunto(s)
Materiales Biocompatibles/química , Cristales Líquidos/química , Biotecnología , ADN/química , Electrónica , Péptidos/químicaRESUMEN
In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Bacteriófago T7/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Triazoles/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/efectos de los fármacos , Sitios de Unión , Técnicas Biosensibles , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , ADN Viral/genética , Indicadores y Reactivos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Programas Informáticos , Ensayo de Placa ViralRESUMEN
The genome of an organism is under constant attack from endogenous and exogenous DNA damaging factors, such as reactive radicals, radiation, and genotoxins. Therefore, DNA damage response systems to sense DNA damage, arrest cell cycle, repair DNA lesions, and/or induce programmed cell death are crucial for maintenance of genomic integrity and survival of the organism. Genome sequences revealed that, although plants possess many of the DNA damage response factors that are present in the animal systems, they are missing some of the important regulators, such as the p53 tumor suppressor. These observations suggest differences in the DNA damage response mechanisms between plants and animals. In this review the DNA damage responses in plants and animals are compared and contrasted. In addition, the function of SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a plant-specific transcription factor that governs the robust response to DNA damage, is discussed.