RESUMEN
BACKGROUND: Identification of bioactive standard chemicals is a major challenge in the study of the Chinese medicinal formula. In particular, the chemical components may interact differently depending on the preparative methods, therefore affecting the amounts of bioactive components and their pharmacological properties in the medicinal formula. With the use of Erxian decoction (EXD) as a study model-a well-known Chinese medicinal formula for treating menopausal symptoms, a novel and rapid approach in seeking standard chemicals has been established by differentially comparing the HPLC profiles and the menopause-related biochemical parameters of combined decoction of EXD (EXD-C) and mixtures of the decoctions of its individual herbs (EXD-S). METHODS: The levels of six chemicals, which exerted actions on the HPO axis, have been measured in EXD-C and EXD-S by HPLC. Twelve-month-old female Sprague-Dawley rats were employed and treated with EXD-C and EXD-S. Their endocrine functions after treatment were evaluated by determining the ovarian mRNA levels of aromatase, a key enzyme for estradiol biosynthesis. The effect of the antioxidant regimen was determined by the hepatic superoxide dismutase-1 (SOD), catalase (CAT) and glutathione peroxidase (GPx-1) mRNA levels. RESULTS: The amounts of mangiferine, ferulic acid, jatrorrhizine and palmatine in EXD-S were twofold higher than those in EXD-C. EXD-S was more effective in stimulating ovarian aromatase and the expression of the antioxidant enzymes compared with EXD-C. CONCLUSION: Mangiferine, ferulic acid, jatrorrhizine and palmatine are suitable for use as standard chemicals for quality evaluation of EXD according to our approach. EXD-S could be more effective than EXD-C.
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Carcinoma Hepatocelular/virología , Hepatitis B/complicaciones , Neoplasias Hepáticas/virología , MicroARNs/genética , Animales , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Hepatitis B/virología , Virus de la Hepatitis B/genética , Hepatocitos/patología , Hepatocitos/virología , Humanos , Neoplasias Hepáticas/genética , Ratones , Ratones Desnudos , Transactivadores/metabolismo , Regulación hacia Arriba , Proteínas Reguladoras y Accesorias Virales , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rhizomes of Dioscorea species are traditionally used for relieving menopausal syndromes in Chinese medicine. The estrogen-stimulating bioactive principles have been demonstrated in our previous study. In this study, the estrogen-stimulating effects of proteins isolated from four Dioscorea species [D. alata L. (DA), D. zingiberensis C.H. Wright (DH), D. collettii var. hypoglauca (Palib.) S.J. Pei & C.T. Ting (DH), and D. oppositifolia L. (DO)] have been investigated and compared. Microscopic authentication of four Dioscorea species was performed by using paraffin and powder sections of the rhizomes. The potential bioactive proteins of four Dioscorea species have been rapidly isolated by using a DOI-antibody affinity column chromatography on immobilized antibodies against on estradiol-stimulating protein from DO (DOI), and their bioactivity has been rapidly confirmed and compared by phenotypic (i.e., estradiol-stimulating effect) and target-based (i.e., STAR, aromatase, estrogen receptors) screening approaches. The estrogen-stimulating activity of bioactive proteins from DO is the highest. In addition, bioactive proteins from DO upregulated the estradiol-metabolizing enzymes (aromatase and steroidogenic acute regulatory protein). Meanwhile, bioactive proteins from DA, DH and DO upregulated estrogen receptor ß (ERß). All bioactive proteins did not change the expression of estrogen receptor ß (ERα). The estrogen-stimulating bioactive proteins isolated from DO increased biosynthesis of estradiol and upregulated the protein expression of aromatase, steroidogenic acute regulatory protein, and ERß. The results scientifically support the traditional use of DO in Chinese medicine for relieving menopausal syndrome. Besides, proteins from DA and DZ could also upregulate the translational levels of ERß, and potentially reducing the risk of ovarian cancer, which also support the clinical use of them for treating female aging disorder. Graphical Abstract Comparative Analysis of DOI-like Proteins with Stimulating Activity on Ovarian Estradiol Biosynthesis from Four Different Dioscorea Species in vitro.
Asunto(s)
Dioscorea/metabolismo , Estradiol/biosíntesis , Menopausia/efectos de los fármacos , Menopausia/fisiología , Ovario/metabolismo , Proteínas de Plantas/farmacología , Animales , Aromatasa/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos , Ovario/citología , Ovario/efectos de los fármacos , Adhesión en Parafina , Fenotipo , Fosfoproteínas/metabolismo , Polvos , Ratas Sprague-Dawley , Receptores de Estrógenos/metabolismo , Rizoma/químicaRESUMEN
Alzheimer's disease (AD) is the most common type of dementia that leads to increasing death and mental disability among humans. Current therapy of AD mainly relies on the use of acetylcholinesterase inhibitors (AChEIs) or antagonists of N-methyl-D-aspartate receptors (NMDARs), which only relieve the symptoms of the disease but not halt its progression. Nevertheless, Traditional Chinese medicines (TCM) are highly prized as many bioactive components isolated from TCM are beneficial for treating AD. In this review, we summarize the latest information on TCM and the bioactive components according to their mechanistic role in alleviating AD. They act as modulators of α- and ß-secretases, and inhibitors of betaamyloid (Aß) aggregation. Some of them suppress Aß-induced neuronal cytotoxicity and inflammation. Hence, this work has demonstrated the feasibility of applying TCM in AD therapy and the possibility of screening of constituents in TCM in the near future.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Medicamentos Herbarios Chinos/uso terapéutico , Medicina Tradicional China , Animales , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/aislamiento & purificación , HumanosRESUMEN
Lactoferricin and lactoferrampin, peptides derived from the whey protein lactoferrin, are antimicrobial agents with a promising prospect and are currently one of the research focuses. In this review, a basic introduction including location and solution structures of these two peptides is given. Their biological activities encompassing antiviral, antibacterial, antifungal and anti-inflammatory activities with possible mechanisms are mentioned. In terms of modification studies, research about identification of their active derivatives and crucial amino acid residues is also discussed. Various attempts at modification of lactoferricin and lactoferrampin such as introducing big hydrophobic side-chains; employing special amino acids for synthesis; N-acetylization, amidation, cyclization and peptide chimera are summarized. The studies on lactoferricin-lactoferrampin chimera are discussed in detail. Future prospects of lactoferricin and lactoferrampin are covered.
Asunto(s)
Antiinfecciosos/farmacología , Antiinflamatorios/farmacología , Lactoferrina/farmacología , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Antiinflamatorios/química , Humanos , Lactoferrina/química , Lactoferrina/fisiología , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Conformación ProteicaRESUMEN
Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p ≤ 0.001 in BT474; p ≤ 0.01 in SKBR3) and in vivo (p ≤ 0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p ≤ 0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p ≤ 0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p ≤ 0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Proteínas de la Membrana/metabolismo , Receptor ErbB-2/metabolismo , Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Resistencia a Antineoplásicos , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Trastuzumab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40 degrees C. The enzyme activity was enhanced by Mn2+ and Fe3+ ions, but inhibited by Hg2+ ions.
Asunto(s)
Agaricales/enzimología , Amilasas/química , Amilasas/aislamiento & purificación , Cuerpos Fructíferos de los Hongos/enzimología , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Concentración de Iones de HidrógenoRESUMEN
A 28-kDa ribonuclease, with an optimum pH of 4.0 and an optimum temperature at 58 °C, was isolated from fruiting bodies of the edible mushroom Hygrophorus russula. It was purified by ion exchange chromatography on carboxymethyl-cellulose, dithyaminoethyl-cellulose, quaternary amine-sepharose and sulphopropyl-sepharose, followed by fast protein liquid chromatography gel filtration on Superdex 75. The N-terminal amino acid sequence was ASAGG which showed homology to those of other fungal RNases to some degree. It exerted the highest RNase activity on poly C and poly U. The Michaelis constant (K(m)) value of the RNase on yeast tRNA was 3.6 µM, and the maximal velocity (V(max)) was 0.6 µM. The RNase activity was suppressed by some ions including Fe(2+) and Zn(2+) ions. The RNase inhibited the activity of HIV-1 reverse transcriptase with an IC(50) of 4.64 µM.
Asunto(s)
Agaricales/química , Fármacos Anti-VIH/química , Cuerpos Fructíferos de los Hongos/química , Proteínas Fúngicas/química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Ribonucleasas/química , Agaricales/enzimología , Fármacos Anti-VIH/aislamiento & purificación , Cationes Bivalentes , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cuerpos Fructíferos de los Hongos/enzimología , Proteínas Fúngicas/aislamiento & purificación , Transcriptasa Inversa del VIH/química , Concentración de Iones de Hidrógeno , Hierro/química , Cinética , Poli C/química , Poli U/química , Ribonucleasas/aislamiento & purificación , Temperatura , Zinc/químicaRESUMEN
Fungi produce a variety of proteins with antiproliferative activity toward tumor cells and anticancer activity in tumor bearing mice. The aforementioned fungal proteins include ribonucleases, antifungal proteins, ubiquitin-like peptides, ribosome inactivating proteins, hemolysins, hemagglutinins/lectins, laccases, and protein-bound polysaccharopeptides.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Fúngicas/farmacología , Animales , Hemaglutininas/farmacología , Proteínas Hemolisinas/farmacología , Humanos , Ratones , Ribonucleasas/farmacología , Proteínas Inactivadoras de Ribosomas/farmacología , Ubiquitinas/farmacologíaRESUMEN
OBJECTIVE: Our previous study demonstrated that polysaccharides of Dendrobium officinale Kimura et Migo (DP) were capable of enhancing immunomodulation in an experimental model of Sjögren's syndrome, a chronic autoimmune disease mainly affecting the salivary glands. In the present study, we further investigated the protective effect of DP on a human salivary gland cell line A-253 against tumor necrosis factor (TNF)-α-induced apoptosis. MATERIALS: TNF-α (100 U/ml) was used as the stimulus for treating the A-253 cells to induce cellular apoptosis. Nuclear factor-kappa B (NF-κB, p65), phosphorylation of mitogen-activated protein kinases (MAPK), reactive oxygen species (ROS) generation, mitochondrial membrane potential and proapoptotic proteins were examined. A-253 cells were pre-treated with DP for 12 h before TNF-α stimulation. RESULTS: We observed translocation of NF-κB into the nuclei, prolonged MAPK, excessive ROS generation and strongly decreased mitochondrial membrane potential, and subsequently cytochrome C release and caspase-3 activation. However, pre-treatment with DP significantly inhibited the TNF-α-induced apoptotic factors. CONCLUSIONS: Our data suggested the inhibitory effect of DP on TNF-α-induced apoptosis in a human salivary gland cell line. This inhibition indicated potential inference of DP in the initial plasma membrane-bound complex of TNF-α and its receptors.
Asunto(s)
Apoptosis/efectos de los fármacos , Dendrobium , Extractos Vegetales/farmacología , Polisacáridos/farmacología , Glándulas Salivales/citología , Glándulas Salivales/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Caspasa 3/metabolismo , Línea Celular , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Glándulas Salivales/metabolismoRESUMEN
A 5447 Da antifungal peptide with an N-terminal sequence highly homologous to plant defensins was purified from Phaseolus vulgaris cv. 'King Pole Bean' by anion-exchange chromatography on Q Sepharose and FPLC-gel filtration on Superdex 75. The isolated peptide inhibited growth of a number of fungal species, including Mycosphaerella arachidicola, Saccharomyces cerevisiae and Candida albicans, with IC(50) values of 3.9, 4.0 and 8.4 µM, respectively. Using the membrane non-permeable DNA-binding dye SYTOX green, it was found that the peptide increased the cell membrane permeability of M. arachidicola, S. cerevisiae and C. albicans.
Asunto(s)
Antifúngicos/metabolismo , Defensinas/metabolismo , Phaseolus/metabolismo , Secuencia de Aminoácidos , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Defensinas/química , Defensinas/aislamiento & purificación , Defensinas/farmacología , Hongos/efectos de los fármacos , Datos de Secuencia Molecular , Phaseolus/químicaRESUMEN
It is well known that legumes, especially soybeans, are beneficial to health due to the presence of saponins and an abundance of fiber and proteins. Defensins and other related defense proteins with antifungal activity in legumes act against fungi which are human pathogens and also against phytopathogenic fungi. These proteins exhibit HIV-1 reverse transcriptase inhibitory and antitumor activities. Some of these proteins have remarkable stability to proteases, and changes in pH and temperature and are promising therapeutic candidates.
Asunto(s)
Antiinfecciosos/farmacología , Defensinas/farmacología , Fabaceae/química , Proteínas de Plantas/farmacología , Secuencia de Aminoácidos , Antiinfecciosos/química , Defensinas/química , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/química , Semillas/químicaRESUMEN
Ribosome inactivating proteins (RIPs) are toxic RNA N-glycosidases that cleave an adenine-ribose glycosidic bond at position adenine(4324) with the conserved ricin/α-sarcin loop in the eukaryotic 28S ribosomal RNA. RIPs have captured the attention of botanists, biochemists, and drug discoverers, due to their diverse potent defensive activities, and inter alia, their antitumor and anti-HIV activities. Out of the 145 families of plants, Trichosanthes ranks among the top 5 genera with a good potential of use for discovery of anticancer drugs. Trichosanthin (TCS) is a famous type I RIP purified from T. kirilowii that has been known for around 30 years. Based on the results of voluminous in vitro and in vivo investigations, TCS is considered a good candidate for the treatment of HIV/AIDS and neoplasms. Here we integrate recent progress of the research on the different medicinal activities of TCS. In addition to TCS, other promising RIPs from the same species (such as TAP29 and trichoanguin), and from the same genus Trichosanthes are included. This review presents a brief panorama of the studies on Trichosanthes RIPs. Regarding the debilitating nature of AIDS and different tumors, further understanding of these multifunctional proteins is worthwhile since it may help to open a novel therapeutic window for these stubborn diseases.
Asunto(s)
VIH-1/efectos de los fármacos , Proteínas de Plantas/farmacología , Trichosanthes/metabolismo , Tricosantina/farmacología , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Humanos , Neoplasias/tratamiento farmacológico , Proteínas de Plantas/uso terapéutico , Plantas Medicinales/metabolismo , Tricosantina/uso terapéuticoRESUMEN
AIM OF STUDY: This study aimed to elucidate and compare the anti-metastatic mechanism of Tian-Xian liquid (TXL) and its bioactive components namely butanol (BU), ethyl-acetate (EA) and aqueous (WA) fractions on human colorectal cancer in vitro (HT-29 cancer cells) and in vivo (nude mouse xenografts). MATERIALS AND METHODS: The anti-proliferative effects of TXL and its bioactive components in HT-29 cells were determined by MTT assay. Their modulations on the potential angiogenic and metastatic marker expressions on HT-29 cells and xenografts were investigated by real-time PCR and Western blot at transcriptional and translational levels, respectively. For the in vitro study, migration abilities of HT-29 cells were determined using wound healing assay. For the in vivo study, daily measurements of the tumor size and volume of the xenografts were also performed. RESULTS: TXL, BU, EA and WA effectively inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner. The IC(50) value of TXL on HT-29 cells was obtained after incubation with 1% (v/v) TXL for 4h; whereas IC(50) values were obtained for the following bioactive components: BU at 1.25% (v/v); EA at 5% (v/v); and WA at 0.3125% (v/v). It was found that 1% (v/v) TXL significantly down-regulated MMP2 and MMP7 expression at both transcriptional and translational levels and it reduced MMP9 and VEGF protein expression in vitro. TXL decreased the metastatic ability of HT-29 cells as demonstrated by wound healing assay. TXL and its bioactive fractions caused no significant changes in the body weight indicating lack of toxicity to the xenografts. CONCLUSIONS: In summary, TXL multi-targeted to down-regulate the metastatic markers in both in vitro and in vivo models. However, the effects of its bioactive fractions were not obvious. This study profoundly elucidated the anti-proliferative mechanism of TXL, which is vital for the development of future anti-cancer regime in Chinese medicinal formulations.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Acetatos/química , Animales , Antineoplásicos Fitogénicos/química , Western Blotting , Butanoles/química , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fraccionamiento Químico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/secundario , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/química , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HT29 , Humanos , Concentración 50 Inhibidora , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solventes/química , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Agua/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bitter gourd (Momordica charantia, BG) is both a nutritious and healthy food with a distinctive bitter flavor, and it is also widely exploited in folklore medicine. This review focuses on the efficacies and molecular mechanisms of BG-induced anti-diabetic, anti-HIV, and antitumor activities contributed by over twenty active components. The intent of this review is to provide comprehensive and valuable information for medicinal researchers, drug investigators, clinicians, and even patients with an interest in BG. In conclusion, BG is a cornucopia of health and it deserves in-depth investigations for clinical application in the future.
Asunto(s)
Fármacos Anti-VIH/farmacología , Antineoplásicos/farmacología , Hipoglucemiantes/farmacología , Momordica charantia/química , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/aislamiento & purificación , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Citocinas/metabolismo , Citocinas/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , VIH/fisiología , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/aislamiento & purificación , Insulina/metabolismo , Insulina/farmacologíaRESUMEN
A 20-kDa Kunitz-type trypsin inhibitor was isolated from Gymnocladus chinensis (Yunnan bean) seeds. The isolation procedure involved ion exchange chromatography on diethylaminoethyl cellulose (DEAE-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on sulfopropyl sepharose (SP-sepharose), and gel filtration by FPLC on Superdex 75. The trypsin inhibitor was adsorbed on DEAE-cellulose, unadsorbed on Affi-gel blue gel, and adsorbed on SP-Sepharose. It dose-dependently inhibited trypsin with an IC(50) value of 0.4 µM. Dithiothreitol reduced its trypsin inhibitory activity, suggesting that an intact disulfide bond is indispensable to the activity. It suppressed [methyl-(3)H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC(50) value of 4.7 and 9.4 µM, respectively. There was no effect on human immunodeficiency virus(4)-1 reverse transcriptase activity and fungal growth when the trypsin inhibitor was tested up to 100 µM.
Asunto(s)
Antineoplásicos Fitogénicos/aislamiento & purificación , Fabaceae/química , Proteínas de Plantas/aislamiento & purificación , Inhibidores de Tripsina/aislamiento & purificación , Secuencia de Aminoácidos , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/farmacología , Plantas Medicinales/química , Semillas/química , Tripsina/metabolismo , Inhibidores de Tripsina/farmacologíaRESUMEN
A 20.9-kDa metalloprotease was isolated from dried fruiting bodies of the wild basidiomycete mushroom Lepista nuda. The N-terminal amino acid sequence of the protease was seen to be ATFVLTAATNTLFTA, thus displaying no similarity with the sequences of previously reported metalloproteases. The protease was purified using a procedure that entailed ion-exchange chromatography on CM-Cellulose, Q-Sepharose, and Mono S, and FPLC-gel filtration on Superdex 75. The protease functioned at an optimum pH of 7.0 and an optimum temperature of 50 degrees C. It was also noted that the protease demonstrated a proteolytic activity of 1,756 U/mg toward casein. The Km of the purified protease toward casein was 6.36 mg/ml at a pH of 7.0 and with a temperature of 37 degrees C, whereas the Vmax was 9.11 microgram ml(-1) min(-1). The activity of the protease was adversely affected by EDTA-2Na, suggesting that it is a metalloprotease. PMSF, EGTA, aprotinin, and leupeptin exerted no striking inhibitory effect. The activity of the protease was enhanced by Fe2+, but was curtailed by Cd2+, Cu2+, Hg2+, Pb2+, Zn2+, and Fe3+ ions. The protease also exhibited inhibitory activity against HIV-1 reverse transcriptase with an IC50 value of 4.00 micrometer. The IC50 values toward hepatoma Hep G2 and leukemia L1210 cells in vitro were 4.99 micrometer and 3.67 micrometer, respectively.
Asunto(s)
Agaricales/enzimología , Metaloproteasas/aislamiento & purificación , Metaloproteasas/metabolismo , Caseínas/metabolismo , Cationes Bivalentes/metabolismo , Línea Celular Tumoral , Cromatografía Liquida/métodos , Activadores de Enzimas/metabolismo , Inhibidores Enzimáticos/metabolismo , Estabilidad de Enzimas , Cuerpos Fructíferos de los Hongos/enzimología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Cinética , Metaloproteasas/química , Metaloproteasas/toxicidad , Metales/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
A thermostable novel lectin with a molecular weight of 30.4 kDa was isolated from dried fruiting bodies of Agaricus arvensis. It was a dimer made up of two 15.2 kDa subunits. The lectin was unadsorbed on DEAE-cellulose in 10 mM phosphate buffer (pH 7.5), subsequently adsorbed on CM-cellulose in 10 mM NaAc buffer (pH 4.6) and then on SP-Sepharose in 10 mM NaAc buffer (pH 4.6), and finally purified by fast protein liquid chromatography-gel filtration on Superdex 75. The hemagglutinating activity of the lectin was stable at temperatures up to 90 °C. The activity was preserved in concentrations of NaOH solution up to 50 mM, but was sensitive to HCl and declined to 12.5% in 12.5 mM HCl. The activity was unaffected by Ca(2+), Mn(2+), Zn(2+) and Mg(2+) ions, but was activated by Al(3+) and Fe(3+) ions. Among the carbohydrates tested, only inulin could inhibit the hemagglutinating activity of the lectin. It did not exhibit anti-HIV reverse transcriptase activity. Proliferation of HepG2 and MCF7 tumor cells was inhibited by the lectin with an IC(50) of 1.64 and 0.82 µM, respectively. The lectin was devoid of antifungal activity. The lectin has a remarkable thermostablity and a unique N-terminal amino acid sequence, TYAVLNFVYG. The present report is the first report on a lectin from wild mushroom Agaricus arvensis.