RESUMEN
Autocrine and paracrine factors play key roles in the process of Androgenetic alopecia (AGA), which are secreted by balding dermal papilla cells (DPCs) after dihydrotestosterone (DHT) induction. Camellia seed cake is an oriental oil extraction byproduct, and its extract has been traditionally used to wash hair in China. This study elucidated the hair growth-promoting effects of Camellia seed cake extract (CSCE) in DHT-treated cultured DPCs and its underlying mechanisms. The effect of CSCE on cell viability and release of inflammatory factors IL-6 and IL-1α was performed on human dermal papilla cells (DPCs) incubated with DHT. Relative expression of bax, bcl-2, p53, androgen receptor (AR) and 5α- reductase type II (SRD5A2) was determined by PCR. Senescence-associated was examined by ß-galactosidase (SA-ß-Gal) assays. CSCE restored DHT-induced cell damage in a dose-dependent manner, and effectively reduced the production of IL-6 and IL-1α in DHT-treated DPCs. CSCE exhibited an anti-apoptotic effect, which increased the expression of bcl-2, and decreased the expressions of bax and p53 in DHT-incubated DPCs. CSCE also showed an anti-androgenic effect reversing the increase in AR and SRD5A2 expressions in DPCs driven by DHT incubation. In addition, CSCE inhibited the ß-galactosidase enzyme activity and slowed down the cell senescence of DPCs which is crucial for AGA progression. In this study, we found that CSCE may have the potential to prevent and alleviate AGA by abrogating the effect of DHT in cultured DPCs.
Asunto(s)
Camellia , Dihidrotestosterona , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa , Alopecia/tratamiento farmacológico , Alopecia/metabolismo , Células Cultivadas , Dihidrotestosterona/farmacología , Cabello , Folículo Piloso , Humanos , Interleucina-6/metabolismo , Proteínas de la Membrana/metabolismo , Extractos Vegetales/metabolismo , Extractos Vegetales/farmacología , Receptores Androgénicos/metabolismo , Semillas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
The main step of classical desensitization of a receptor, by mean of its disappearance from the plasma membrane, is its internalization. This is a key factor in the regulation of agonist-mediated signaling pathways, as it most of the time stops the activation of the receptor. Internalization is thus important to evaluate, as a complementary information for a natural ligand or an alternative synthetic agonist. Enzyme fragment complementation is an elegant but delicate way to measure this phenomenon, by fusing two complementary parts of an enzyme to two partners, and to measure the activity of the reconstituted enzyme upon complexation of the partners. In the present chapter, using two parts of ß-galactosidase, one fused to the C-terminus of the MT1 receptor, the other to an endosomal protein, one can measure the formation of the complex; thus, the transfer of the receptor to the endosome from which MT1 will be recirculated.
Asunto(s)
Melatonina , Receptor de Melatonina MT1 , Membrana Celular/metabolismo , Ligandos , Melatonina/metabolismo , Receptor de Melatonina MT1/metabolismo , Transducción de Señal , beta-Galactosidasa/metabolismoRESUMEN
Munage grape (Vitis vinifera L. cv. Munage.) is a unique cultivar in southern Xinjiang, China. Spike stalk browning in this species has becomes more common in recent years, negatively impacting the shelf life, and causing severe economic losses during storage. This study investigated the changes in metabolisms of cell wall by Botrytis cinerea infection in association with spike stalk browning. Morphological and physiological observations showed that preharvest B. cinerea infection accelerates the spike stalk browning during storage in Munage grapes by promoting cell wall degradation. Accordingly, the cell structures in infected spike stalk showed severe collapse, while the cell structures in uninfected spike stalk remained relatively complete. Furthermore, the contents of CDTA-soluble pectin (CSP), Na2 CO3 -soluble pectin (NSP), cellulose, and hemicellulose were reduced, while the water-soluble pectin (WSP) content was increased during infection. In addition, the activities of polygalacturonase (PG), pectin methylesterase (PME), beta-galactosidase (ß-Gal), and cellulase (Cx) were highly promoted by B. cinerea. Correspondingly, the expression levels of VvPG were markedly upregulated after inoculation and played a major role in cell wall degradation. Additionally, the spike stalk inoculated by B. cinerea showed higher activities of PPO and POD, and content of total phenolics. These results contribute to elucidating the relationship between cell wall degradation induced by B. cinerea during spike stalk browning and provide a basis for future research on improving the ability of the host cell wall to resist degrading enzymes. PRACTICAL APPLICATIONS: Botrytis cinerea is the main fungal pathogen causing the gray mold of grapes. It usually enters the tissue early in crop development, has a long incubation period, and rapidly infects the tissue when the environment is favorable and the host physiology changes. Gray mold has been reported as one of the major postharvest diseases of grapes. However, there are relatively few reports on the pathways through which B. cinerea causes the browning of grape stalks. Controlling browning caused by B. cinerea may require clarification of the physiological and molecular mechanisms by which browning occurs. The elucidation of the role of B. cinerea in causing browning of grape stalks through the cell wall degradation pathway will help to provide scientific basis for further controlling browning, maintaining freshness of stalks, developing biological agents to prevent browning, improving grape quality, and extending storage period.
Asunto(s)
Celulasas , Vitis , Factores Biológicos/metabolismo , Botrytis , Pared Celular/metabolismo , Celulasas/metabolismo , Celulosa/metabolismo , Pectinas , Enfermedades de las Plantas/microbiología , Poligalacturonasa/genética , Vitis/microbiología , Agua , beta-Galactosidasa/metabolismoRESUMEN
Ageing is one of the major risk factors of human diseases, including cancer, diabetes, and cardiovascular disease. Mulberry exhibits a wide range of functions, such as anti-oxidant, anti-inflammation, and anti-diabetes. In this study, we investigated the role of mulberry polyphenol extract (MPE) in K-Ras-induced senescence of smooth muscle cells. Forced expression of K-Ras enhanced senescence of smooth muscle A7r5 cells as shown by the elevation of ß-galactosidase activity. Treatment with MPE significantly repressed the Ras, phosphorylated ERK, and ß-galactosidase level. MPE triggered the association of cyclins with their corresponding cyclin-dependent protein kinases and hyperphosphorylated retinoblastoma (RB). MPE also down-regulated the levels of K-Ras-induced CDK inhibitors. MPE enhanced the phosphorylated AMP-dependent protein kinase (AMPK) and inducible nitric oxide synthase (iNOS) level in the presence of K-Ras. Pretreatment with either L-NAME or AMPK inhibitor reversed the effects of MPE. In addition, L-NAME and AMPK inhibitor repressed the MPE-induced total and phosphorylated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-Co A) level. MPE repressed K-Ras-induced G0/G1 arrest, whereas L-NAME and AMPK inhibitor blocked the effects of MPE. Our results indicated that MPE recovered the K-Ras-induced senescence of vascular smooth muscle cells through iNOS and AMPK-dependent pathway. Our findings suggested that MPE may prevent ageing-induced atherosclerosis.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Morus/química , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Extractos Vegetales/farmacología , Polifenoles/farmacología , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Acilcoenzima A/metabolismo , Células Cultivadas , Expresión Génica , Humanos , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Proteolisis , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Aging is a risk factor for multiple retinal degeneration diseases. Entraining brain gamma oscillations with gamma-flicker light (γFL) has been confirmed to coordinate pathological changes in several Alzheimer's disease mouse models and aged mice. However, the direct effect of γFL on retinal aging remains unknown. We assessed retinal senescence-associated beta-galactosidase (ß-gal) and autofluorescence in 20-month-old mice and found reduced ß-gal-positive cells in the inner retina and diminished lipofuscin accumulation around retinal vessels after 6 days of γFL. In immunofluorescence, γFL was further demonstrated to ameliorate aging-related retinal changes, including a decline in microtubule-associated protein 1 light chain 3 beta expression, an increase in complement C3 activity, and an imbalance between the anti-oxidant factor catalase and pro-oxidant factor carboxymethyl lysine. Moreover, we found that γFL can increase the expression of activating transcription factor 4 (ATF4) in the inner retina, while revealing a decrease of ATF4 expression in the inner retina and positive expression in the outer segment of photoreceptor and RPE layer for aged mice. Western blotting was then used to confirm the immunofluorescence results. After mRNA sequencing (NCBI Sequence Read Archive database: PRJNA748184), we found several main mechanistic clues, including mitochondrial function and chaperone-mediated protein folding. Furthermore, we extended γFL to aged Apoe-/- mice and showed that 1-m γFL treatment even improved the structures of retinal-pigment-epithelium basal infolding and Bruch's membrane. Overall, γFL can orchestrate various pathological characteristics of retinal aging in mice and might be a noninvasive, convenient, and tissue-specific therapeutic strategy for retinal aging.
Asunto(s)
Complemento C3 , Lipofuscina , Factor de Transcripción Activador 4/metabolismo , Animales , Antioxidantes/metabolismo , Apolipoproteínas E/metabolismo , Catalasa/metabolismo , Complemento C3/metabolismo , Lipofuscina/metabolismo , Lisina/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Retina/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Effect of edible coatings of gum Arabic, carrageenan and xanthan gum containing lemon grass essential oil 1% w/v on postharvest quality of strawberry was studied under refrigeration for a period of 12 days. Results showed all the three coatings maintained fruit quality parameters during storage compared to control. Among all the coatings, carrageenan coated fruits showed delayed weight loss (10.1 to 8%), decay percentage (78.42 to 14.29%), retained ascorbic acid (0.15 to 0.27 g kg-1), antioxidant activity (18.17 to 25.85%), firmness (9.07 to 12.43 N), L* (32.38 to 40.42), a* (16.08 to 17.22) and b* (27.36 to 33.54). Carrageenan gum also showed lowest cellulase activity (0.03 units h-1 mg protein-1), pectin methylesterase activity (1.13 A620 min-1 mg protein-1) and ß-galactosidase activity (0.51 µmol min-1 mg protein-1), while showed maximum reduction in polygalacturonase activity (0.07 units h-1 mg protein-1) at the end of storage. Carrageenan gum was found effective in retention of anthocyanins and phenolic compounds during storage. Coatings loaded with antimicrobial agent inhibited psychrophilic bacteria, yeast and mold growth. It is concluded that carrageenan gum could better retain strawberry quality up to 12 days under refrigeration.
Asunto(s)
Antiinfecciosos/química , Carragenina/química , Películas Comestibles , Embalaje de Alimentos , Conservación de Alimentos , Fragaria/enzimología , Frutas/enzimología , Goma Arábiga/química , Aceites de Plantas/química , Polisacáridos Bacterianos/química , Antocianinas/metabolismo , Antiinfecciosos/farmacología , Antioxidantes/metabolismo , Ácido Ascórbico/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Celulasa/metabolismo , Cymbopogon , Microbiología de Alimentos , Almacenamiento de Alimentos , Fragaria/microbiología , Frutas/microbiología , Fenoles/química , Aceites de Plantas/aislamiento & purificación , Aceites de Plantas/farmacología , Poligalacturonasa/metabolismo , Refrigeración , Factores de Tiempo , beta-Galactosidasa/metabolismoRESUMEN
Targeting microRNAs (miRs) using small chemical molecules has become a promising strategy for disease treatment. miR216a has been reported to be a potential therapeutic target in endothelial senescence and atherosclerosis via the Smad3/NFκB signaling pathway. Ginsenoside Rb2 (Rb2) is the main bioactive component extracted from the plant Panax ginseng, and is a widely used traditional Chinese medicine. In the present study, Rb2 was identified to have a high score for miR216a via bioinformatics analysis based on its sequence and structural features. The microscale thermophoresis experiment further demonstrated that Rb2 had a specific binding affinity for miR216a and the dissociation constant was 17.6 µM. In both young and senescent human umbilical vein endothelial cells (HUVECs), as well as human aortic endothelial cells, Rb2 decreased the expression of endogenous miR216a. Next, a replicative endothelial senescence model of HUVECs was established by infection with premiR216a recombinant lentiviruses (LvmiR216a) and the number of populationdoubling level (PDL) was calculated. Stable overexpression of miR216a induced a premature senescentlike phenotype, whereas the senescent features and increased activity of senescenceassociated ßgalactosidase (SAßgal) were reversed after Rb2 treatment. The percentage of SAßgalpositive cells in senescent PDL25 cells transfected with LvmiR216a was decreased 76% by Rb2 treatment compared with the LvmiR216a group without Rb2 treatment (P=0.01). Mechanistically, miR216a inhibited Smad3 protein expression, promoted IκBα degradation and activated NFκBresponsive genes, such as vascular cell adhesion molecule 1 (VCAM1), which promoted the adhesiveness of endothelial cells to monocytes. These proinflammatory effects of miR216a were significantly suppressed by Rb2 treatment. When Smad3 was suppressed by small interfering RNA, the elevated expression levels of intercellular adhesion molecule 1 and VCAM1 induced by miR216a were significantly reversed. Collectively, to the best of our knowledge, the present study demonstrated for the first time that Rb2 exerted an antiinflammation effect on the process of endothelial cell senescence and could be a potential therapeutic drug by targeting miR216a.
Asunto(s)
Antiinflamatorios/farmacología , Senescencia Celular , Ginsenósidos/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , MicroARNs/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , MicroARNs/genética , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Advances in deep learning technology have enabled complex task solutions. The accuracy of image classification tasks has improved owing to the establishment of convolutional neural networks (CNN). Cellular senescence is a hallmark of ageing and is important for the pathogenesis of ageing-related diseases. Furthermore, it is a potential therapeutic target. Specific molecular markers are used to identify senescent cells. Moreover senescent cells show unique morphology, which can be identified. We develop a successful morphology-based CNN system to identify senescent cells and a quantitative scoring system to evaluate the state of endothelial cells by senescence probability output from pre-trained CNN optimised for the classification of cellular senescence, Deep Learning-Based Senescence Scoring System by Morphology (Deep-SeSMo). Deep-SeSMo correctly evaluates the effects of well-known anti-senescent reagents. We screen for drugs that control cellular senescence using a kinase inhibitor library by Deep-SeSMo-based drug screening and identify four anti-senescent drugs. RNA sequence analysis reveals that these compounds commonly suppress senescent phenotypes through inhibition of the inflammatory response pathway. Thus, morphology-based CNN system can be a powerful tool for anti-senescent drug screening.
Asunto(s)
Forma de la Célula , Senescencia Celular , Aprendizaje Profundo , Evaluación Preclínica de Medicamentos , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/toxicidad , Redes Neurales de la Computación , beta-Galactosidasa/metabolismoRESUMEN
The changes of texture and cell wall characteristics of apricot were investigated in ten clones at two maturity stages. Fruit firmness, cell wall composition and enzyme activity of three apricot flesh zones were analysed. The AIS (alcohol-insoluble solids) were characterised by high amounts of uronic acid (179-300 mg g-1 AIS) and relatively high amounts of cellulosic glucose (118-214 mg g-1 AIS). The methylesterification degree varied significantly among the different clones ranging from 58 to 97 in Ab 5 and Mans 15 respectively. Conversely to zones firmness, enzymatic activity was higher in pistil followed by equatorial and peduncle zones. The ripening effect has been observed in firmness evolution according to enzymatic activity. This correlation allowed a classification of clones depending on softening. Among studied clones, Ab 5, Marouch 16, Mans 15 and Cg 2 were less influenced by softening and have the advantage of a technological valorisation for the processing industry.
Asunto(s)
Pared Celular/química , Frutas/citología , Prunus armeniaca/química , Prunus armeniaca/citología , Azúcares/análisis , Hidrolasas de Éster Carboxílico/metabolismo , Frutas/química , Humanos , Pectinas/metabolismo , Proteínas de Plantas/metabolismo , Prunus armeniaca/crecimiento & desarrollo , Azúcares/química , beta-Galactosidasa/metabolismoRESUMEN
Vine tea (Ampelopsis grossedentata) has been approved as a new food ingredient in 2013. Both vine tea extract (VTE) and its active ingredient, 2R, 3R-Dihydromyricetin (DMY), showed good antibacterial activity. The mechanism of VTE and DMY against Staphylococcus aureus were evaluated by morphology observation, cell membrane and wall assay, protein assay, and DNA assay in this study. The results of SEM and TEM revealed that the VTE and DMY changed the morphology of S. aureus. The leakage of AKPase and ß-galactosidase in treated groups demonstrated that the membrane integrity of S. aureus was disrupted. Meanwhile, the results of protein assay showed that VTE and DMY inhibited the expression of total proteins, and decreased activities of a few energy metabolism enzymes, total ATPase. Moreover, spectral and competitive analysis revealed that VTE and DMY interacted with DNA by groove and intercalation binding. Finally, the suspension experiments of Chinese cabbage and barley showed that inhibitors had strong inhibitory effect on bacteria growth. Overall, the results suggested that VTE and DMY may be potential food preservatives for inhibiting pathogen.
Asunto(s)
Ampelopsis/química , Antibacterianos/farmacología , Flavonoles/farmacología , Staphylococcus aureus/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/efectos de los fármacos , Conservación de Alimentos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Extractos Vegetales/química , Staphylococcus aureus/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Activities of plant polyphenols (PPs), resveratrol and quercetin, alone or in combination with four conventional antibiotics against Escherichia coli have been investigated. In medium without antibiotics, both polyphenols caused a dose-dependent growth inhibition. However, pretreatment with resveratrol (40 and 100 µg ml-1) and quercetin (40 µg ml-1) reduced the bacteriostatic effect of kanamycin, streptomycin, cefotaxime and partially of ciprofloxacin. With few exceptions, both PPs also reduced the bactericidal effect of tested antibiotics. Paradoxically, low doses of PPs enhanced the bactericidal effect of kanamycin and partially ciprofloxacin. Compared to quercetin, resveratrol showed a weaker effect on the induction of antioxidant genes and the resistance of E. coli to the oxidative stress generated by hydrogen peroxide treatment. Both polyphenols at high doses reduced membrane potential. Altogether, these findings suggest that the decrease in the bactericidal effect of antibiotics by high doses of polyphenols is mostly due to bacteriostatic action of the latter. In the case of quercetin, the contribution of its antioxidant activity for antibiotic protection may be significant. There is a growing interest in the use of plant-derived compounds to enhance the toxicity of traditional antibiotics. This and other studies show that, under certain conditions, the use of polyphenols as adjuvants may not exert the expected therapeutic effect, but rather to decrease antimicrobial activity of antibiotics.
Asunto(s)
Antioxidantes/farmacología , Escherichia coli/efectos de los fármacos , Quercetina/farmacología , Resveratrol/farmacología , Antibacterianos/farmacología , Cefotaxima/farmacología , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/metabolismo , Kanamicina/farmacología , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Polifenoles/farmacología , Estreptomicina/farmacología , Estrés Fisiológico/efectos de los fármacos , beta-Galactosidasa/metabolismoRESUMEN
Whey, a byproduct of cheese production, is often treated as an industrial dairy waste. A large volume of this product is disposed of annually due to inadequate bioconversion approaches. With its high pollutant load, disposal without pretreatment has raised a lot of environmental concerns alerting the need to seek optimal methods for adequately extracting and utilizing its organic content. In recent years, several techniques for whey valorization have emerged which may serve as interventionary measures against its environmental effects after disposal. In this review, we discuss five major approaches, by which whey can be converted into eco-friendly products, to significantly cut whey wastage. The approaches to whey valorization are therefore examined under the following perspectives: whey as a raw material for the production of bioethanol and prebiotic oligosaccharides via ß-galactosidase and microbe catalyzed reactions, for the production of refined lactose as an excipient for pharmaceutical purposes, and the clinical significance of whey hydrolysates and their antifungal activity in food processing.
Asunto(s)
Queso , Conservación de los Recursos Naturales , Industria Lechera , Suero Lácteo , Biocombustibles , Productos Agrícolas , Suplementos Dietéticos , Etanol/metabolismo , Fermentación , Aditivos Alimentarios , Conservación de Alimentos , Hidrólisis , Residuos Industriales , Lactosa/aislamiento & purificación , Oligosacáridos/metabolismo , Prebióticos , Proteína de Suero de Leche , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: The tuber of Pinellia ternata has been used for a thousand years in China. P. ternata possessed the activities of anti-emetic, sedative-hypnotic, anti-cancer, anti-asthmatic, anti-tussive, and anti-inflammatory. It is the representative of expectorant medicines in Traditional Chinese Medicine (TCM). Phlegm is the pathological product and a new pathogenic factor of the metabolite, which is analogous to the damage of oxidative stress. PURPOSE: The objectives of the study were to investigate the protective activity and mechanism of ethanol extract of P. ternata tubers (PTE) and its main constituents on oxidative stress-induced cell senescence. METHODS: H2O2 and AAPH were used to establish cellular senescence models. The anti-aging effects of PTE and its components were evaluated by SA-ß-gal staining, flow cytometry, scanning electron microscope (SEM), and multiple microplate reader, the molecular mechanisms of them were investigated by qRT-PCR and Western Blot. RESULTS: We found PTE exhibited the apparent effect on cell senescence, evidenced by inhibiting senescence ß-Galactosidase (SA-ß-gal) expression, lipofuscin accumulation, cell cycle arrest at the G2/M phase, oxidative damage and apoptosis, and increasing telomerase activity. Their mechanisms were related to increase expressions of SIRT1, forkhead box 3a (Foxo3a), Bcl-2, active regulator of SIRT1, RPS19BP1 (AROS), and Hu antigen R (HuR), but decrease Bax, p53 and deleted in breast cancer 1 (DBC1) levels. Furthermore, adenosine, and succinic acid, as the critical substances in PTE, could also inhibit SA-ß-gal expression and cell cycle arrest, down-regulate the expression of Bax, and up-regulate Bcl-2, SirT1, and Foxo3a. CONCLUSIONS: We have demonstrated that PTE slows down oxidative stress-induced cell senescence, and adenosine and succinic acid are the key active components.
Asunto(s)
Estrés Oxidativo/efectos de los fármacos , Pinellia/química , Extractos Vegetales/farmacología , Animales , Senescencia Celular/efectos de los fármacos , Chlorocebus aethiops , Regulación hacia Abajo/efectos de los fármacos , Medicamentos Herbarios Chinos/química , Etanol/química , Humanos , Peróxido de Hidrógeno/farmacología , Lipofuscina/metabolismo , Células PC12 , Extractos Vegetales/química , Tubérculos de la Planta/química , Sustancias Protectoras/química , Sustancias Protectoras/farmacología , Proteínas/genética , Proteínas/metabolismo , Ratas , Telomerasa/metabolismo , Células Vero , beta-Galactosidasa/metabolismoRESUMEN
MAIN CONCLUSION: ß-(1,4)-galactan determines the interactions between different matrix polysaccharides and cellulose during the cessation of cell elongation. Despite recent advances regarding the role of pectic ß-(1,4)-galactan neutral side chains in primary cell wall remodelling during growth and cell elongation, little is known about the specific function of this polymer in other developmental processes. We have used transgenic Arabidopsis plants overproducing chickpea ßI-Gal ß-galactosidase under the 35S CaMV promoter (35S::ßI-Gal) with reduced galactan levels in the basal non-elongating floral stem internodes to gain insight into the role of ß-(1,4)-galactan in cell wall architecture during the cessation of elongation and the beginning of secondary growth. The loss of galactan mediated by ßI-Gal in 35S::ßI-Gal plants is accompanied by a reduction in the levels of KOH-extracted xyloglucan and an increase in the levels of xyloglucan released by a cellulose-specific endoglucanase. These variations in cellulose-xyloglucan interactions cause an altered xylan and mannan deposition in the cell wall that in turn results in a deficient lignin deposition. Considering these results, we can state that ß-(1,4)-galactan plays a key structural role in the correct organization of the different domains of the cell wall during the cessation of growth and the early events of secondary cell wall development. These findings reinforce the notion that there is a mutual dependence between the different polysaccharides and lignin polymers to form an organized and functional cell wall.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Pared Celular/química , Cicer/enzimología , Galactanos/análisis , Pectinas/química , beta-Galactosidasa/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Pared Celular/metabolismo , Celulosa/análisis , Cicer/genética , Galactanos/metabolismo , Lignina/análisis , Pectinas/metabolismo , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Xilanos/análisis , beta-Galactosidasa/genéticaRESUMEN
Parthenium argentatum (Gray), commonly known as guayule, has been used to obtain natural rubber since the beginning of the 20th century. Additionally, the so called "resin" is a waste product derived from the industrial process. The cycloartane-type triterpene Argentatin A (AA) is one of the main constituents of the industrial waste resin. In this study we evaluated the AA anticancer activity both in vitro and in vivo in the HCT116 colon cancer cells. The apoptosis promotion of AA was assessed by the annexin V/propidium iodide (PI) assay. The senescence was evaluated for SA-ß-galactosidase, and PCNA was used as a marker of proliferation. Its antitumor activity was evaluated using a xenograft mouse model. The results indicated that AA-induced apoptosis in HCT-116 cells and was positively stained for SA-ß-galactosidase. In the xenografted mice test, the administration of AA at the dose of 250 mg/kg three times a week for 21 days reduced tumor growth by 78.1%. A comparable tumor reduction was achieved with cisplatin at the dose of 2 mg/kg administered three times a week for 21 days. However, nude mice treated with AA did not lose weight, as they did remarkably when treated with cisplatin. Furthermore, the animals treated with AA showed similar blood profiles as the healthy control group. These data indicate the low toxicity of AA compared to that shown by cisplatin.
Asunto(s)
Antineoplásicos/administración & dosificación , Triterpenos/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Biomarcadores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Inmunohistoquímica , Ratones , Estructura Molecular , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , beta-Galactosidasa/metabolismoRESUMEN
Cellular senescence is a process of physiological growth arrest that can be induced by intrinsic or extrinsic stress signals. Some cancer therapies are associated with senescence of cancer cells with a typical cell cycle arrest. Doxorubicin (Dox) induces senescence by a p53-dependent pathway and telomere dysfunction of numerous cancers. However, cellular senescence induces suppression in proliferation activity, and these cells will remain metabolically active and play an important role in tumor relapse and development of drug resistance. In the current study, we investigated the apoptotic effect of curcumin (Cur), caffeine (Caff), and thymoquinone (TQ) on senescent colon cancer HCT116 and breast cancer MCF7 cell lines treated with Dox. Results showed typical senescence markers including decreased bromodeoxyuridine incorporation, increased accumulation of senescence-associated ß-galactosidase (SA-ß-gal), cell cycle arrest, and upregulation of p53, P-p53, and p21 proteins. Annexin-V analysis by flow cytometry revealed 2- to 6-fold increases in annexin-V-positive cells in Dox-treated MCF7 and HCT116 cells by Cur (15 µM), Caff (10 mM), and TQ (50 µM; P < .001). In comparison between proliferative and senescent of either HCT116 or MCF7 cells, Caff at 15 mM and TQ at 25 µM induced significant increases in apoptosis of Dox-treated cells compared with proliferative cells (P < .001). Data revealed that Cur, Caff, and TQ potentially induced apoptosis of both proliferative and senescent HCT116 and MCF7 cells. In vivo and clinical trials are of great importance to validate this result.
Asunto(s)
Benzoquinonas/farmacología , Neoplasias de la Mama , Cafeína/farmacología , Senescencia Celular , Neoplasias del Colon , Curcumina/farmacología , Doxorrubicina/farmacología , beta-Galactosidasa/metabolismo , Antimetabolitos Antineoplásicos/análisis , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Bromodesoxiuridina/análisis , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Senescencia Celular/fisiología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , HumanosRESUMEN
Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.
Asunto(s)
Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/análisis , Macrófagos/microbiología , Proteínas de la Membrana/fisiología , Animales , Pollos , Biología Computacional , Medios de Cultivo/química , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/crecimiento & desarrollo , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/fisiología , Eliminación de Gen , Expresión Génica , Concentración de Iones de Hidrógeno , Análisis por Micromatrices/veterinaria , Mutación , Nitrógeno/deficiencia , Enfermedades de las Aves de Corral/microbiología , ARN Bacteriano/química , ARN Bacteriano/aislamiento & purificación , ARN Complementario/química , ARN Complementario/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Organismos Libres de Patógenos Específicos , Virulencia , beta-Galactosidasa/metabolismoRESUMEN
Three recombinant ß-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze ß-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-ß-D-galactopyranoside and ß-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the ß-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked ß-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.
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Pectinas/metabolismo , Penicillium chrysogenum/enzimología , beta-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Hidrólisis , Penicillium chrysogenum/genética , Filogenia , Pichia/genética , Especificidad por Sustrato , beta-Galactosidasa/genéticaRESUMEN
An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methyl-esterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10-100â¯kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. The data revealed the presence of GalA sequences, having different patterns of methyl-esterification, RG-I composed of unbranched segments and segments heavily substituted with ß-(1â4)-linked galactan chains of varying length. The rheological study showed the shear-thinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did form stable foams at high concentration.
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Galactosa/metabolismo , Cebollas/metabolismo , Polisacáridos/química , Proteínas Bacterianas/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Esterificación , Ácidos Hexurónicos/metabolismo , Peso Molecular , Polisacáridos/metabolismo , Reología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , beta-Galactosidasa/metabolismoRESUMEN
Leishmaniasis, caused by protozoa belonging to the genus Leishmania, is an important public health problem found in >90 countries and with still limited options for treatment. Development of new anti-leishmanial drugs is an urgent need and the identification of new active compounds is a limiting factor that can be accelerated through large scale drug screening. This requires multiple steps and can be expensive and time consuming. Here, we propose an alternative approach for the colorimetric assessment of anti-Leishmania drug activity that can be easily scaled up. L. amazonensis and L. infantum cell lines were generated having the ß-galactosidase (ß-gal) gene integrated into their chromosomal 18S rRNA (ssu) locus. Both cell lines expressed high levels of ß-gal and had their growth easily monitored and quantified colorimetrically. These two cell lines were then evaluated as tools to assess drug susceptibility and their use was validated through in vitro assays with Amphotericin B, which is routinely used against leishmaniasis. ß-gal expression was also confirmed through flow-cytometry, another method of phenotypic detection. With these recombinant parasites, an alternative in vitro model of drug screening against cutaneous and visceral leishmaniasis is now available.