RESUMO
Pectin methylesterases (PMEs) catalyze pectin demethylation and facilitate the determination of the degree of methyl esterification of cell wall in higher plants. The regulation of PME activity through endogenous proteinaceous PME inhibitors (PMEIs) alters the status of pectin methylation and influences plant growth and development. In this study, we performed a PMEI screening assay using a chemical library and identified a strong inhibitor, phenylephrine (PE). PE, a small molecule, competitively inhibited plant PMEs, including orange PME and Arabidopsis PME. Physiologically, cultivation of Brassica campestris seedlings in the presence of PE showed root growth inhibition. Microscopic observation revealed that PE inhibits elongation and development of root hairs. Molecular studies demonstrated that Root Hair Specific 12 (RHS12) encoding a PME, which plays a role in root hair development, was inhibited by PE with a Ki value of 44.1⯵M. The biochemical mechanism of PE-mediated PME inhibition as well as a molecular docking model between PE and RHS12 revealed that PE interacts within the catalytic cleft of RHS12 and interferes with PME catalytic activity. Taken together, these findings suggest that PE is a novel and non-proteinaceous PME inhibitor. Furthermore, PE could be a lead compound for developing a potent plant growth regulator in agriculture.
Assuntos
Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fenilefrina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Brassica/efeitos dos fármacos , Brassica/crescimento & desenvolvimento , Brassica/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Fenilefrina/química , Plântula/efeitos dos fármacos , Plântula/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Rubus coreanus fruit (RF) has been used as a traditional medicine formulation to treat various diseases including diarrhea, asthma, and cancer in East Asia (Korea, China, and Japan). RF, which is native to Korea, has a larger fruit size than that of exotic species. In this study, we aimed to compare the anti-inflammatory activities of immature and mature RF extracted with different solvents. METHODS: Mature and immature RF (MRF and IRF) were extracted with 30% ethanol, 70% ethanol and water at room temperature. The antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assays. Anti-inflammatory activity was determined by measuring nitric oxide (NO) production, expression of inflammatory proteins (inducible NO synthase [iNOS], cyclooxygenase [COX]-2, nuclear factor [NF]-κB, and inhibitor of NF-κB [IκB]), and inflammatory cytokines using polymerase chain reaction in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. RESULTS: The IRF 30% ethanol extract showed higher radical scavenging activity in DPPH and ABTS assays (half-maximal inhibitory concentration [IC50] 16.0 ± 0.5 and 15.9 ± 0.4) than MRF did. In addition, the IRF 30% ethanol extract (200 µg/mL) significantly reduced the production of the inflammatory mediator NO by approximately 80% and inhibited iNOS, COX-2, phosphorylated (p)-IκB, and p-NF-κB activation compared with MRF. Moreover, IRF extract decreased the inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 compared with the MRF extract. CONCLUSIONS: This study revealed that IRF showed more beneficial effects than MRF did in LPS-stimulated RAW 264.7 macrophages, suggesting that IRF may be a useful anti-inflammatory agent.
Assuntos
Anti-Inflamatórios/farmacologia , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Rubus/química , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Sequestradores de Radicais Livres , Frutas/química , Lipopolissacarídeos/efeitos adversos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Extratos Vegetais/química , Células RAW 264.7RESUMO
Small heat shock proteins are well-known to function as chaperone in the protection of proteins and subcellular structures against stress-induced denaturation in many cell compartments. Irrespective of such general functional assignment, a proof of function in a living organism is missing. Here, we used heat-induced orchardgrass small Hsp17.2 (DgHsp17.2). Its function in in vitro chaperone properties has shown in protecting the model substrate, malate dehydrogenase (MDH) and citrate synthase (CS). Overexpression of DgHsp17.2 triggering strong chaperone activity enhanced in vivo thermotolerance of yeast cells. To identify the functional domain on DgHsp17.2 and correlationship between in vitro chaperone property and in vivo thermotolerance, we generated truncation mutants of DgHsp17.2 and showed essentiality of the N-terminal arm of DgHsp17.2 for the chaperone function. In addition, beyond for acquisition of thermotolerance irrespective of sequences are diverse among the small Hsps. However, any truncation mutants of DgHsp17.2 did not exhibit strong interaction with orchardgrass heat shock protein 70 (DgHsp70) different from mature DgHsp17.2, indicating that full-length DgHsp17.2 is necessary for cooperating with Hsp70 protein. Our study indicates that the N-terminal arm of DgHsp17.2 is an important region for chaperone activity and thermotolerance.
Assuntos
Dactylis/enzimologia , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Simulação por Computador , Ativação Enzimática , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Mangosenone F (MSF), a natural xanthone, was isolated form Carcinia mangotana, and a few studies have reported its glycosidase inhibitor effect. In this study we investigated the anti lung cancer effect of MSF both in vitro and in vivo. MSF inhibited cancer cell cytotoxicity and induced and induced apoptosis via reactive oxygen species (ROS) generation in NCI-H460. MSF treatment also showed in pronounced release of apoptogenic cytochrome c from the mitochondria to the cytosol, downregulation of Bcl-2 and Bcl-xL, and upregulation of Bax, suggesting that caspase-mediated pathways were involved in MSF-induced apoptosis. ROS activation of the mitogen-activated protein kinase signaling pathway was shown to play a predominant role in the apoptosis mechanism of MSF. Compared with cisplatin treatment, MSF treatment showed significantly increased inhibition of the growth of NCI-H460 cells xenografted in nude mice. Together, these results indicate the potential of MSF as a candidate natural anticancer drug by promoting ROS production.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Garcinia mangostana , Neoplasias Pulmonares/tratamento farmacológico , Xantonas/farmacologia , Animais , Caspases/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Citocromos c/metabolismo , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro compared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 Å resolution, respectively. The structures are highly conserved at the active site and the Zn(2+) binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site-directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues.
Assuntos
Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Mamíferos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/química , Fósforo-Oxigênio Liases/genética , Filogenia , Conformação Proteica , Pterinas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro) is a key enzyme that plays an important role in SARS virus replication. The ethanol extract of the seeds of Psoralea corylifolia showed high activity against the SARS-CoV PLpro with an IC50 of value of 15 µg/ml. Due to its potency, subsequent bioactivity-guided fractionation of the ethanol extract led to six aromatic compounds (1-6), which were identified as bavachinin (1), neobavaisoflavone (2), isobavachalcone (3), 4'-O-methylbavachalcone (4), psoralidin (5) and corylifol A (6). All isolated flavonoids (1-6) inhibited PLpro in a dose-dependent manner with IC50 ranging between 4.2 and 38.4 µM. Lineweaver-Burk and Dixon plots and their secondary replots indicated that inhibitors (1-6) were mixed inhibitors of PLpro. The analysis of KI and KIS values proved that the two most promising compounds (3 and 5) had reversible mixed type I mechanisms.
Assuntos
Fenóis/farmacologia , Psoralea/embriologia , Sementes/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacosRESUMO
Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32â M MgCl2, 0.08â M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a=97.11, b=135.92, c=46.03â Å. Diffraction data were collected to 2.08â Å resolution.
Assuntos
Methanosarcina/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/metabolismo , Catálise , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trometamina/análogos & derivados , Difração de Raios XRESUMO
A DJ-1 homologue protein from Arabidopsis thaliana (AtDJ-1D) belongs to the DJ-1/ThiJ/Pfpl superfamily and contains two tandem arrays of DJ-1-like sequences, but no structural information is available to date for this protein. AtDJ-1D was expressed in Escherichia coli, purified and crystallized for structural analysis. A crystal of AtDJ-1D was obtained by the hanging-drop vapour-diffusion method using 0.22 M NaCl, 0.1 M bis-tris pH 6.5, 21% polyethylene glycol 3350. AtDJ-1D crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 56.78, b = 75.21, c = 141.68 Å, ß = 96.87°, and contained a trimer in the asymmetric unit. Diffraction data were collected to 2.05 Å resolution. The structure of AtDJ-1D has been determined using the multiple-wavelength anomalous dispersion (MAD) method.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Cristalização , Cristalografia por Raios XRESUMO
Salt stress is a major constraint of crop productivity because it reduces yield and limits the expansion of agriculture. This study investigated salt tolerance in 26 cultivars of cut lilies (Lilium hybrids) by examining the effect of salt stress on the growth and morphological characteristics of flowers and leaves and their physiological properties (chlorophyll a fluorescence). Salt stress significantly affected the growth and development of cut lilies. Canonical discriminant analysis indicates that the middle leaf width, number of flowers, first flower diameter, petal width, and chlorophyll a fluorescence were correlated with salt stress, whereas plant height, the middle leaf length, days to flowering, and sepal width were less affected by the stress. The cultivars examined were divided into three groups: Group 1 included the salt-sensitive cultivars, which failed to develop normal flowers; Group 2 included cultivars sensitive to salt stress but tolerant to osmotic stress; and Group 3 was the salt-tolerant group, which developed commercially valuable flowers. In conclusion, the cultivars contained a variable range of cut flower characteristics and growth traits that can be employed for lily breeding programs and as material for molecular mechanisms and signaling networks under salt stress.
RESUMO
Coreopsis lanceolata L. is a perennial plant of the family Asteraceae, and its flower is known to contain flavonoids with various bioactivities. We evaluated the effect of Coreopsis lanceolata L. flower (CLF) extracts on H2O2-induced oxidative stress (OS) in neuronal cells and mouse neurons. The flowering part of CL was used as CLF1 (70% ethanol extract) and CLF2 (water extract), and 10 types of phenolic compounds were quantified using high-performance liquid chromatography. To evaluate the neuroprotective effects of CLF, the antioxidant activities of the extracts were measured, and the expression levels of antioxidant enzymes and proteins related to OS-induced apoptosis in neuronal cells and mouse neurons treated with the extracts were investigated. In the in vitro study, CLF ameliorated H2O2-induced oxidative stress and induced the expression of antioxidant enzymes in PC12 cells. Furthermore, CLF1 enhanced the expression of the Bcl-xL protein but reduced the expression of Bax and the cleavage of caspase-3. In the same manner, CLF1 showed neuroprotective effects against OS in vivo. Pretreatment with CLF1 (200 mg/kg) increased the Bcl-2 protein and decreased Bax compared with the 1-methyl-4-phenylpyridinium ion (MPP+)-treated C57BL/6 mice model group. Our results suggest that the protective effects of CLF1 on MPP+-induced apoptosis may be due to its anti-apoptotic activity, through regulating the expression of the Bcl-2 family. CLF1 exerts neuroprotective effects against OS-induced apoptosis in PC12 cells in a Parkinson's disease model mouse. This effect may be attributable to the upregulation of Bcl-2 protein expression, downregulation of Bax expression, and inhibition of caspase-3 activation. These data indicate that CLF may provide therapeutic value for the treatment of progressive neurodegenerative diseases.
RESUMO
Veterinary antibiotics such as sulfonamides are widely used to increase feed efficiency and to protect against disease in livestock production. The sulfonamide antimicrobial mechanism involves the blocking of folate biosynthesis by inhibiting bacterial dihydropteroate synthase (DHPS) activity competitively. Interestingly, most treatment antibiotics can be released into the environment via manure and result in significant diffuse pollution in the environment. However, the physiological effects of sulfonamide during plant growth and development remain elusive because the plant response is dependent on folate biosynthesis and the concentration of antibiotics. Here, we present a chemical interaction docking model between Napa cabbage (Brassica campestris) DHPS and sulfamethoxazole and sulfamethazine, which are the most abundant sulfonamides detected in the environment. Furthermore, seedling growth inhibition was observed in lentil bean (Lens culinaris), rice (Oryza sativa), and Napa cabbage plants upon sulfonamide exposure. The results revealed that sulfonamide antibiotics target plant DHPS in a module similar to bacterial DHPS and affect early growth and the development of crop seedlings. Taking these results together, we suggest that sulfonamides act as pollutants in crop fields.
RESUMO
Reactive oxygen species (ROS), associated with oxidative stress, are involved in many biological processes such as apoptosis, necrosis, and autophagy. Oxidative stress might induce neuronal damage via ROS generation, causing neurodegenerative diseases. Erigeron annuus (EA) has antioxidant properties and could protect neurons from oxidative stress. In this study, we investigated the protective effect of the aerial parts (EAA) and flowers (EAF) from EA on ROS-mediated apoptosis in pheochromocytoma 12 cells. We quantified 18 types of phenolic compounds using high-performance liquid chromatography. Pretreatment of the cells with EAA and EAF attenuated ROS generation and induced the expression of antioxidant enzymes such as superoxide dismutase 2, catalase, and glutathione peroxidase. In addition, EAF reduced the expression of apoptotic proteins such as Bax/Bcl-xL, caspase-3, and caspase-8 to a greater extent than that with EAA. These results suggested that the protective effect of EAF against oxidative stress-induced apoptosis might be due to the prevention of ROS generation mediated by oxidative enzymes.
RESUMO
Scent is one of the most important economic traits in Freesia hybrida. "Shiny Gold", a popular cultivar in South Korea, is widely cultivated for its scent. The relative scent intensity of "Shiny Gold" was approximately 16% higher in full-bloomed flower when compared to the yellow bud stage, while tissue-specifically, tepals showed higher intensity in electronic-nose (e-nose) analysis. E-nose analysis also showed that the scent intensity of "Shiny Gold" was higher and lower than "10C3-424" and "10C3-894", respectively, and was similar to "Yvonne". These results correlated to those of the olfactory tests. In total, 19 volatile compounds, including linalool, ß-ocimene, D-limonene, trans-ß-ionone were detected in gas chromatography-mass spectrometry analysis. Among these, linalool was the major volatile compound, accounting for 38.7% in "Shiny Gold". Linalool synthase and TPS gene expression corresponded to the scent intensity of the four cultivars, with the lowest expression in the "10C3-424". TPS 2, TPS 3, TPS 5, TPS 6 and TPS 8 were highly expressed in both bud and flower in "Shiny Gold", while the expression of TPS 4 was lower, relative to other TPS genes in both the flowering stages. These results may aid in enhancing scent composition in Freesia cultivars using marker-assisted selection.
RESUMO
6-Pyruvoyltetrahydropterin synthase from E. coli (ePTPS) has been crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular-shaped crystals were obtained. Diffraction data were collected from the hexagonal and rectangular crystals to 3.0 and 2.3 A resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 A , and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 A , and contained six molecules in the asymmetric unit. The structure of ePTPS in both crystal forms has been determined by molecular replacement.
Assuntos
Escherichia coli/enzimologia , Fósforo-Oxigênio Liases/química , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce D-threo-BH(4) [6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of L-erythro-BH(4), in the last step of tetrahydrobiopterin (BH(4)) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH(4). To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR-NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 x 0.6 x 0.1 mm. The crystal belonged to space group P2(1), with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 A, beta = 100.00 degrees , and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR-NAD dimers. Diffraction data were collected to 2.16 A resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method.
Assuntos
Dictyostelium/enzimologia , Di-Hidropteridina Redutase/química , Proteínas de Protozoários/química , Animais , Cristalização/métodos , Cristalografia por Raios X , Di-Hidropteridina Redutase/isolamento & purificação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas de Protozoários/isolamento & purificaçãoRESUMO
Cys49Ser mutant Helicobacter pylori alkyl hydroperoxide reductase (C49S HpAhpC) was purified under reducing conditions in monomeric and decameric forms. The monomeric form was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.25 A resolution and belonged to space group C2, with unit-cell parameters a = 245.8, b = 140.7, c = 189.5 A, beta = 127 degrees , and contained 20 molecules in the asymmetric unit. A crystal of the decameric form was obtained by the microbatch crystallization method and diffracted to 2.8 A resolution. It belonged to space group C222, with unit-cell parameters a = 257.5, b = 417.5, c = 95.6 A. The structure of the monomeric form of C49S HpAhpC has been solved by the molecular-replacement method.
Assuntos
Proteínas de Bactérias/química , Helicobacter pylori/enzimologia , Peroxidases/química , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Peroxidases/genética , Peroxidases/metabolismo , Mutação Puntual , Difração de Raios XRESUMO
Sepiapterin reductase from Chlorobium tepidum (cSR) catalyzes the synthesis of a distinct tetrahydrobiopterin (BH4), L-threo-BH4, different from the mammalian enzyme product. The 3-D crystal structure of cSR has revealed that the product configuration is determined solely by the substrate binding mode within the well-conserved catalytic triads. In cSR, the sepiapterin is stacked between two aromatic side chains of Phe-99 and Trp-196 and rotated approximately 180 degrees C around the active site from the position in mouse sepiapterin reductase. To confirm their roles in substrate binding, we mutated Phe-99 and/or Trp-196 to alanine (F99A, W196A) by site-directed mutagenesis and comparatively examined substrate binding of the purified proteins by kinetics analysis and differential scanning calorimetry. These mutants had higher Km values than the wild type. Remarkably, the W196A mutation resulted in a higher Km increase compared with the F99A mutation. Consistent with the results, the melting temperature (Tm) in the presence of sepiapterin was lower in the mutant proteins and the worst was W196A. These findings indicate that the two residues are indispensable for substrate binding in cSR, and Trp-196 is more important than Phe-99 for different stereoisomer production.
Assuntos
Oxirredutases do Álcool/química , Biopterinas/análogos & derivados , Chlorobium/enzimologia , Modelos Químicos , Modelos Moleculares , Fenilalanina/química , Triptofano/química , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Biopterinas/síntese química , Simulação por Computador , Ativação Enzimática , Estabilidade Enzimática , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Saponarin (SA), a natural flavonoid, is known for its antioxidant and hepatoprotective activities. SA is the predominant compound (1142.7 ± 0.9 mg per 100 g) in barley sprouts, constituting 72% of the total polyphenol content. We investigated, for the first time, the effects of SA from barley sprouts on cellular anti-inflammatory responses. In lipopolysaccharide (LPS)-induced RAW 264.7 macrophages, SA suppressed the activation of NF-κB, as evidenced by the inhibition of NF-κB DNA binding, nuclear translocation, IκBα phosphorylation, and reporter gene expression, and it downregulated the expression of the pro-inflammatory mediator IL-6. Furthermore, SA reduced the transcription of NF-κB target molecules COX2 and FLIP inhibited the phosphorylation of mitogen-activated protein kinases ERK and p38. These results suggest that SA isolated from barley sprouts exerts anti-inflammatory effects in LPS-induced RAW 264.7 macrophages via inhibition of NF-κB, ERK and p38 signaling. Thus, SA may be a promising natural anti-inflammatory agent.
Assuntos
Apigenina/farmacologia , Glucosídeos/farmacologia , Hordeum/química , Lipopolissacarídeos/efeitos adversos , Proteínas Quinases Ativadas por Mitógeno/genética , NF-kappa B/genética , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Apigenina/análise , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo , Glucosídeos/análise , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Espectrometria de Massas em Tandem , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Neuraminidase (NA) is one of the key enzymes responsible for bacterial infection and pathogenesis. This study aimed to gain deeper insights into the inhibitory effects of flavone-glucosides (1-9) isolated from barley sprouts (BS) on neuraminidase activity. The isolated compounds were identified as, lutonarin (1), saponarin (2), isoorientin (3), orientin (4), isovitexin (5), isoscoparin-7-O-[6-sinapoyl]-glucoside (6), isoscoparin-7-O-[6-feruloyl]-glucoside (7), isovitexin-7-O-[6-sinapoyl]-glucoside (8), and isovitexin-7-O-[6-feruloyl]-glucoside (9). Among them, compounds 1-5 exhibited neuraminidase-inhibitory activities in a dose-dependent manner, with IC50 values ranging from 20.1 to 32.7 µM, in a non-competitive inhibition mode according to kinetic studies. Moreover, the individual flavone-glucoside levels differed notably, in particular, lutonarin (1) and saponarin (2) were shown to be present in the greatest amounts, according to UPLC analysis. Consequently, our results suggest that BS may be utilized as an effective NA inhibitor in human health food, additives, and feed.
Assuntos
Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Flavonas/química , Flavonas/farmacologia , Glucosídeos/química , Glucosídeos/farmacologia , Hordeum/química , Neuraminidase/metabolismo , Ativação Enzimática/efeitos dos fármacosRESUMO
High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and circular dichroism (CD). These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.