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Biofilms are assemblages of bacteria embedded within a matrix of extracellular polymeric substances (EPS) attached to a substratum. The process of biofilm formation is a complex phenomenon regulated by the intracellular and intercellular signaling systems. Various secondary messenger molecules such as cyclic dimeric guanosine 3',5'-monophosphate (c-di-GMP), cyclic adenosine 3',5'-monophosphate (cAMP), and cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) are involved in complex signaling networks to regulate biofilm development in several bacteria. Moreover, the cell to cell communication system known as Quorum Sensing (QS) also regulates biofilm formation via diverse mechanisms in various bacterial species. Bacteria often switch to the biofilm lifestyle in the presence of toxic pollutants to improve their survivability. Bacteria within a biofilm possess several advantages with regard to the degradation of harmful pollutants, such as increased protection within the biofilm to resist the toxic pollutants, synthesis of extracellular polymeric substances (EPS) that helps in the sequestration of pollutants, elevated catabolic gene expression within the biofilm microenvironment, higher cell density possessing a large pool of genetic resources, adhesion ability to a wide range of substrata, and metabolic heterogeneity. Therefore, a comprehensive account of the various factors regulating biofilm development would provide valuable insights to modulate biofilm formation for improved bioremediation practices. This review summarizes the complex regulatory networks that influence biofilm development in bacteria, with a major focus on the applications of bacterial biofilms for environmental restoration.
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Proteínas Bacterianas , Contaminantes Ambientales , Adenosina/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Biopelículas , Contaminantes Ambientales/metabolismo , Contaminantes Ambientales/toxicidad , Regulación Bacteriana de la Expresión GénicaRESUMEN
Increasing industrialization and urbanization have contributed to a significant rise in wastewater discharge and exerted extensive pressure on the existing natural energy resources. Microbial fuel cell (MFC) is a sustainable technology that utilizes wastewater for electricity generation. MFC comprises a bioelectrochemical system employing electroactive biofilms of several aerobic and anaerobic bacteria, such as Geobacter sulfurreducens, Shewanella oneidensis, Pseudomonas aeruginosa, and Ochrobacterum pseudiintermedium. Since the electroactive biofilms constitute a vital part of the MFC, it is crucial to understand the biofilm-mediated pollutant metabolism and electron transfer mechanisms. Engineering electroactive biofilm communities for improved biofilm formation and extracellular polymeric substances (EPS) secretion can positively impact the bioelectrochemical system and improve fuel cell performance. This review article summarizes the role of electroactive bacterial communities in MFC for wastewater treatment and bioelectricity generation. A significant focus has been laid on understanding the composition, structure, and function of electroactive biofilms in MFC. Various electron transport mechanisms, including direct electron transfer (DET), indirect electron transfer (IET), and long-distance electron transfer (LDET), have been discussed. A detailed summary of the optimization of process parameters and genetic engineering strategies for improving the performance of MFC has been provided. Lastly, the applications of MFC for wastewater treatment, bioelectricity generation, and biosensor development have been reviewed.
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Microfibres (diameter <5 mm) and textile dyes released from textile industries are ubiquitous, cause environmental pollution, and harm aquatic flora, fauna, animals and human life. Therefore, enzymatic abatement of microfibre pollution and textile dye detoxification is essential. Microbial enzymes for such application present major challenges of scale and affordability to clean up large scale pollution. Therefore, enzymes required for the biodegradation of microfibres and indigo dye were expressed in transplastomic tobacco plants through chloroplast genetic engineering. Integration of laccase and lignin peroxidase genes into the tobacco chloroplast genomes and homoplasmy was confirmed by Southern blots. Decolorization (up to 86%) of samples containing indigo dye (100 mg/L) was obtained using cp-laccase (0.5% plant enzyme powder). Significant (8-fold) reduction in commercial microbial cellulase cocktail was achieved in pretreated cotton fibre hydrolysis by supplementing cost effective cellulases (endoglucanases, ß-glucosidases) and accessory enzymes (swollenin, xylanase, lipase) and ligninases (laccase lignin peroxidase) expressed in chloroplasts. Microfibre hydrolysis using cocktail of Cp-cellulases and Cp-accessory enzymes along with minimal dose (0.25% and 0.5%) of commercial cellulase blend (Ctec2) showed 88%-89% of sugar release from pretreated cotton and microfibres. Cp-ligninases, Cp-cellulases and Cp-accessory enzymes were stable in freeze dried leaves up to 15 and 36 months respectively at room temperature, when protected from light. Use of plant powder for decolorization or hydrolysis eliminated the need for preservatives, purification or concentration or cold chain. Evidently, abatement of microfibre pollution and textile dye detoxification using Cp-enzymes is a novel and cost-effective approach to prevent their environmental pollution.
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Biodegradación Ambiental , Celulasa , Carmin de Índigo , Lacasa/metabolismo , Polvos , Textiles , Nicotiana/genéticaRESUMEN
Pseudomonas aeruginosa is a small rod shaped Gram-negative bacterium of Gammaproteobacteria class known for its metabolic versatility. P. aeruginosa PFL-P1 was isolated from Polycyclic Aromatic Hydrocarbons (PAHs) contaminated site of Paradip Port, Odisha Coast, India. The strain showed excellent biofilm formation and could retain its ability to form biofilm grown with different PAHs in monoculture as well as co-cultures. To explore mechanistic insights of PAHs metabolism, the whole genome of the strain was sequenced. Next generation sequencing unfolded a genome size of 6,333,060 bp encoding 5857 CDSs. Gene ontology distribution assigned to a total of 2862 genes, wherein 2235 genes were allocated to biological process, 1549 genes to cellular component and 2339 genes to molecular function. A total of 318 horizontally transferred genes were identified when the genome was compared with the reference genomes of P. aeruginosa PAO1 and P. aeruginosa DSM 50071. Further comparison of P. aeruginosa PFL-P1 genome with P. putida containing TOL plasmids revealed similarities in the meta cleavage pathway employed for degradation of aromatic compounds like xylene and toluene. Gene annotation and pathway analysis unveiled 145 genes involved in xenobiotic biodegradation and metabolism. The biofilm cultures of P. aeruginosa PFL-P1 could degrade ~74% phenanthrene within 120 h while degradation increased up to ~76% in co-culture condition. GC-MS analysis indicated presence of diverse metabolites indicating the involvement of multiple pathways for one of the PAHs (phenanthrene) degradation. The strain also possesses the genetic machinery to utilize diverse toxic aromatic compounds such as naphthalene, benzoate, aminobenzoate, fluorobenzoate, toluene, xylene, styrene, atrazine, caprolactam etc. Common catabolic gene clusters such as benABCD, xylXYZ and catAB were observed within the genome of P. aeruginosa PFL-P1 which play key roles in the degradation of various toxic aromatic compounds.
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Biopelículas/crecimiento & desarrollo , Genoma Bacteriano/genética , Redes y Vías Metabólicas/genética , Fenantrenos/metabolismo , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Biodegradación Ambiental , Biología Marina , Plásmidos/genética , Hidrocarburos Policíclicos Aromáticos/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismoRESUMEN
Chemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf-enzyme products and their validation with 15 commercial microbial-enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH/temperature ranges as crude-leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast-derived enzymes are stable in crude-leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude-extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker-free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site-specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf-production platform offers a novel low-cost approach by the elimination of fermentation, purification, concentration, formulation and cold-chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.
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Detergentes , Lipasa , Hojas de la Planta , Industria Textil , Cloroplastos/enzimología , Cloroplastos/genética , Detergentes/normas , Estabilidad de Enzimas , Lipasa/genética , Lipasa/aislamiento & purificación , Lipasa/metabolismo , Lipasa/normas , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Temperatura , Industria Textil/métodos , beta-Manosidasa/genética , beta-Manosidasa/aislamiento & purificación , beta-Manosidasa/metabolismo , beta-Manosidasa/normasRESUMEN
Bacopa monniera is an important source of metabolites with pharmaceutical value. It has been regarded as a valuable medicinal plant and its entire commercial requirement is met from wild natural population. Recently, metabolic engineering has emerged as an important solution for sustained supply of assured and quality raw material for the production of active principles. Present report describes efficient in vitro multiplication and transformation method for genetic manipulation of this species. MS medium supplemented with 2 mgl(-1) BA and 0.2 mgl(-1) IAA was found optimum for maximum shoot regeneration (98.33 %) from in vitro leaves with 2-3 longitudinal cuts. Agrobacterium tumefaciens-mediated transformation method was used for generating transgenic B. monniera plants. Putative transformants were confirmed by GUS assay and PCR based confirmation of hptII gene. DNA blot analysis showed single copy insertion of transgene cassette. An average of 87.5 % of the regenerated shoots were found PCR positive for hptII gene and GUS activity was detected in leaves of transgenic shoots at a frequency of 82.5 % The efficient multiple shoots regeneration system described herein may help in mass production of B. monniera plant. Also, the high frequency transformation protocol described here can be used for genetic engineering of B. monniera for enhancement of its pharmaceutically important metabolites.
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Triterpenoid saponins are the class of secondary metabolites, synthesized via isoprenoid pathway. Oxidosqualene cyclases (OSCs) catalyzes the cyclization of 2, 3-oxidosqualene to various triterpene skeletons, the first committed step in triterpenoid biosynthesis. A full-length oxidosqualene cyclase cDNA from Bacopa monniera (BmOSC) was isolated and characterized. The open reading frame (ORF) of BmOSC consists of 2,292 bp, encoding 764 amino acid residues with an apparent molecular mass of 87.62 kDa and theoretical pI 6.21. It contained four QxxxxxW motifs, one Asp-Cys-Thr-Ala-Glu (DCTAE) motif which is highly conserved among the triterpene synthases and another MWCYCR motif involved in the formation of triterpenoid skeletons. The deduced amino acid sequence of BmOSC shares 80.5 % & 71.8 % identity and 89.7 % & 83.5 % similarity with Olea europaea mixed amyrin synthase and Panax notoginseng dammarenediol synthase respectively. Phylogenetic analysis revealed that BmOSC is closely related with other plant OSCs. Quantitative real-time PCR (qRT-PCR) data showed that BmOSC is expressed in all tissues examined with higher expression in stem and leaves as compared to roots and floral parts.
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The screening and evaluation of different waste lignocellulosic biomasses to meet the ever-increasing energy demand, from the widely available waste lignocellulosic biomasses evaluated. For the current study, peanut shell biomass is considered for energy procreation. However, the energy content of biomass is still lesser as compared to conventional fossil fuels like coal and petroleum. The dilute acid pretreatment has been proven to improve the energy content of the lignocellulosic biomasses to a significant extent. Various pretreatment process parameters have been reported to have different degrees of impact on the betterment of energy procreation. Among various types of pretreatments, dilute acid pretreatment holds notable cognizance. Accordingly, the current manuscript is to evaluate the impact of various pretreatment process parameters (time, temperature, acid concentration, mass:liquor ratio, and particle size which were defined through an exhaustive literature search) for improving the energy recovery potential. The obtained results indicated notable changes in the devolatilization characteristics of the biomass as a result of pretreatment, thereby resulting in the upgradation of the fuel properties. A sustainability investigation has been carried out to point out the efficacy of the optimized pretreatment of biomass in terms of environmental sustainability and was also compared with the raw variant (untreated form of biomass). The proposed scheme of study will definitely be beneficial toward the mitigation of the energy crisis in the state of Jharkhand.
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INTRODUCTION: Melanocyte progenitors are embryonically derived from the neural crest and subsequently get localized in hair follicles and epidermis to provide hair and skin pigmentation. These progenitor cells in hair follicles repeatedly proliferate and differentiate to maintain pigmentation. Vitiligo, a pigmentary disorder, is associated with loss of melanocytes. Repigmentation of vitiligo lesions mainly depends upon the proliferation, migration and differentiation of melanocyte stem cells (MelSCs) into functional melanocytes. The current study is designed to check the efficacy of lenalidomide, an imide drug in the differentiation of MelSCs into functional melanocytes. OBJECTIVES: The aim of the study is to check the effect of lenalidomide in the proliferation, migration of cultured hair follicle derived melanocyte stem cells and their differentiation into functional melanocytes. METHODS: Primary culture of MelSCs was established from whisker hair of C57BL/6 mice. Proliferation and migration of cultured cells were done by MTT assay and Boyden's chamber migration assay, respectively. Effect of lenalidomide on the MelSCs differentiation was checked at gene level by qPCR and protein expression was checked by immunocytochemistry. RESULTS: A significant increase in the migration of MelSCs in comparison to control was also observed. Lenalidomide treatment significantly increased the expression of melanocyte specific genes in cultured MelSCs as compared to control. CONCLUSIONS: From the results we concluded that lenalidomide induce the proliferation and migration of MelSCs and accelerate the differentiation of MelSCs into functional melanocytes.
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Among the several biofilm-based bioreactors, moving bed biofilm reactors (MBBR) have been extensively used for wastewater treatment due to low operational costs, technical feasibility, and stability. Biofilm forming strains, e.g., Stenotrophomonas maltophila DQ01, achieved 94.21% simultaneous nitrification and denitrification (SND) and 94.43% removal of total nitrogen (TN) at a cycle time of 7 h, and a biofilm consortium consisting of Chryseobacteriumsp. andRhodobactersp. achieved 86.8% removal of total organic carbon (TOC) at hydraulic retention time (HRT) of 24 h using lab-scale MBBR. Modifications in the surface properties of the biocarrier materials achieved 99.5 ± 1.1% chemical oxygen demand (COD) and 93.6 ± 2.3% NH4+-N removal, significantly higher than the conventional commercial carrier. This review article summarizes the application of MBBR technology for wastewater treatment. The importance of bacterial biofilm and extracellular polymeric substances (EPS), anammox-n-DAMO coupled processes, and carrier surface modifications in MBBR technology have also been discussed.
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Reactores Biológicos , Aguas Residuales , Oxidación Anaeróbica del Amoníaco , Biopelículas , Desnitrificación , Matriz Extracelular de Sustancias Poliméricas/química , Nitrificación , Nitrógeno/análisis , Eliminación de Residuos LíquidosRESUMEN
Biofilm formation in bacteria is strongly affected by the nature of substrata. Different substrata such as glass, polystyrene, steel, ceramic, and rubber were used to assess the biofilm forming ability of a marine bacterium Pseudomonas aeruginosa PFL-P1 using a scanning electron microscope (SEM), atomic force microscope (AFM), and confocal laser scanning microscope (CLSM). The bacterium formed dense biofilms with varied aggregation on different substrata. SEM study revealed small rod-shaped cells with diverse arrangements within the biofilms on all the substrata under study. The AFM study revealed the highest roughness of 545 nm on the ceramic substratum. The biofilms formed on ceramic substratum were characterized with maximum roughness (742 nm), maximum peak height (1,480 nm), and maximum arithmetic mean height (611 nm), significantly higher than all the other substrata (p < .05). AFM studies confirmed that P. aeruginosa PFL-P1 exhibited biofilm heterogeneity on all the substrata. The CLSM study indicated a higher fraction of nucleic acids to α-polysaccharides ratio in the biofilms. COMSTAT analysis revealed the highest biofilm biomass of ~18 µm3 /µm2 on the ceramic substratum. The maximum biofilm thickness of ~50 µm in the native state on the ceramic substratum was significantly higher than glass (p = .0015), polystyrene (p = .0001), steel (p = .0035), and rubber substrata (p = .0001). The higher surface roughness of ceramic substratum is accountable for more area for colonization, as evident from higher biomass and thickness of the biofilm. This study provides insight into the substratum properties, which modulate the biofilm forming ability in bacteria.
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Biopelículas , Pseudomonas aeruginosa , Bacterias , Microscopía Confocal , Microscopía Electrónica de RastreoRESUMEN
Dementia linked with cognitive impairments is the most prominent indication of Alzheimer's disease (AD). In the current investigation, we have examined the streptozotocin- (STZ) induced cellular toxicity in mouse neuroblastoma (N2A) cells, and Zn with the high-fat diet- (HFD) induced neurotoxicity in mouse brain. These cells and animals were pretreated with apple cider vinegar (ACV), Chrysin, and Rivastigmine to examine their protection against cellular toxicity and neurotoxicity. Experiments have affirmed that pretreatment of ACV, Chrysin, and Rivastigmine has displayed protective outcomes in MTT reduction, tau phosphorylation, amyloid aggregation, attenuated memory impairment as well as oxidative stress, and protected cholinergic hippocampal neurons from degeneration. ACV showed better antioxidant and neuroprotection potential as compared with Chrysin and Rivastigmine. So the existence of excitatory/inhibitory enzymatic activity and higher antioxidant potential indicate that ACV, as a food beverage in a regular diet, could be promising and effective against neurological complications such as AD. PRACTICAL APPLICATIONS: In the Urban lifestyle, HFD and stress are the critical factors of various chronic and prevalent diseases, including diabetes, obesity, cardiovascular, and neurodegenerative disorders like AD. We are already familiar with the multiple benefits of ACV, such as weight loss, antimicrobial activity, diabetes, skin disorders. So in the current research work, we have gauged the effectiveness of ACV against neurological complications in comparison with a synthetic flavonoid (Chrysin) and an anti-Alzheimer's drug (Rivastigmine). To enhance the pragmatic orientation of our results, we have used the ACV in our study, which is readily available in the market for domestic consumption. All the cellular, biochemical, behavioral, and histopathological data revealed that ACV had high antioxidant potential. Our findings suggest that the addition of ACV as a food additive in the daily diet may reduce the threat of multiple neurodegenerative diseases. Therefore, our study could be the precursor of a new pharmacological therapeutic approach via ACV toward cognitive impairments associated with Alzheimer's disease.
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Malus , Preparaciones Farmacéuticas , Ácido Acético , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Ratones , Estrés OxidativoRESUMEN
BACKGROUND: Paracrine cross-talk exists between the fibroblasts of dermis and epidermal cells through secretions of various growth factors. Melanocytes present at the basement layer of the epidermis and respond to various factors secreted by underlying dermal fibroblasts in the dermis to regulate the function of the skin. OBJECTIVE: Therefore the study was planned to check the expression of fibroblast-derived factor neuregulin1 (NRG1) in vitiligo skin and its effect on melanocytes. METHODS: For this study, relative gene expression at mRNA level of NRG1 in the vitiligo skin was analyzed by qRT-PCR, and protein analysis was done by immunohistochemistry. Effect of different concentrations of NRG1 was checked on the cultured melanocytes by melanin content assay, proliferation assay, and tyrosinase (TYR) assay. The effect of NRG1 was also checked on the level of melanocyte regulatory genes (MITF, c-KIT, TYR, DCT). RESULTS: Expression of NRG1 was significantly less in lesional dermis of vitiligo patients as compared to nonlesional and healthy control dermis both at mRNA as well as protein level. NRG1 treatment showed significant increase in proliferation, melanin content, TYR level, and gene expression level of melanocyte specific genes. CONCLUSION: Treatment of NRG1 to the cultured melanocytes increases proliferation and pigmentation. Lower expression of NRG1 in the lesional dermis of vitiligo patients inhibits the melanocyte growth. Therefore this study hypothesized that low expression of NRG1 in lesional skin of vitiligo patients might have a possible role in the melanocyte loss and vitiligo pathogenesis.
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Neurregulina-1/genética , Neurregulina-1/metabolismo , ARN Mensajero/metabolismo , Piel/metabolismo , Vitíligo/metabolismo , Adulto , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Melaninas/metabolismo , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Neurregulina-1/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Adulto JovenRESUMEN
BACKGROUND: Biosurfactants are amphipathic molecules of microbial origin that reduce surface and interfacial tension at gas-liquid-solid interfaces. Earlier, the biosurfactant was isolated and characterized in our laboratory from Candida parapsilosis. The property of the biosurfactant is further explored in this study by using quantum dots (QDs) as nanocarrier. MATERIALS AND METHODS: Graphene quantum dots (GQDs) were synthesized by bottom-up approach through pyrolysis of citric acid. GQDs were conjugated with both biosurfactant and folic acid (FA) using carbodiimide chemistry. The prepared GQD bioconjugate was studied for diagnostic and therapeutic effects against cancer cells. RESULTS AND DISCUSSION: Photoluminescence quantum yield (QY) of plain GQDs was measured as 12.8%. QY for biosurfactant conjugated GQDs and FA-biosurfactant conjugated GQDs was measured as 10.4% and 9.02%, respectively, and it was sufficient for targeting cancer cells. MTT assay showed that more than 90% of cells remained viable at concentration of 1 mg/mL, hence GQDs seemed to be non-toxic to cells. Biosurfactant conjugated GQDs caused 50% reduction in cellular viability within 24 hours. FA conjugation further increased the specificity of bioconjugated GQDs toward tumor cells, which is clearly evident from the drug internalization studies using confocal laser scanning microscopy. A higher amount of drug uptake was observed when bioconjugated GQDs were decorated with FA. CONCLUSION: The ability of GQD bioconjugate could be used as a theranostic tool for cancer. It is foreseen that in near future cancer can be detected and/or treated at an early stage by utilizing biosurfactant conjugated GQDs. Therefore, the proposed study would provide a stepping stone to improve the life of cancer patients.
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Grafito/química , Neoplasias/diagnóstico , Neoplasias/terapia , Puntos Cuánticos/química , Tensoactivos/química , Nanomedicina Teranóstica/métodos , Supervivencia Celular/efectos de los fármacos , Fluorescencia , Ácido Fólico/farmacología , Humanos , Células MCF-7 , Neoplasias/patología , Puntos Cuánticos/ultraestructura , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) is an important enzyme in the mevalonic acid pathway catalyzing the Mg(2+)-ATP dependant decarboxylation of mevalonate 5-diphosphate (MVAPP) to isopentenyl diphosphate (IPP). Bacopa monniera recombinant MDD (BmMDD) protein was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Km and Vmax for MVAPP were 144 µM and 52 U mg(-1) respectively. The values of turnover (kcat) and kcat/Km for mevalonate 5-diphosphate were determined to be 40s(-1) and 2.77×10(5) M(-1) s(-1) and kcat and kcat/Km values for ATP were found to be 30 s(-1) and 2.20×10(4) M(-1) s(-1), respectively. pH activity profile indicated the involvement of carboxylate ion, lysine and arginine for the activity of enzyme. The apparent activation energy for the BmMDD catalyzed reaction was 12.7 kJ mol(-1). Optimum pH and temperature for the forward reaction was found to be 8.0 and 45 °C. The enzyme was most stable at pH 7 at 20 °C with the deactivation rate constant (Kd(*)) of 1.69×10(-4) and half life (t1/2) of 68 h. The cation studies suggested that BmMDD is a cation dependant enzyme and optimum activity was achieved in the presence of Mg(2+).
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Bacopa/química , Hemiterpenos/química , Magnesio/química , Ácido Mevalónico/análogos & derivados , Compuestos Organofosforados/química , Proteínas de Plantas/química , Bacopa/enzimología , Carboxiliasas , Cationes Bivalentes , Clonación Molecular , Pruebas de Enzimas , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Hemiterpenos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Ácido Mevalónico/química , Ácido Mevalónico/metabolismo , Peso Molecular , Compuestos Organofosforados/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
The present study evaluated the ameliorative potential of ß-carotene (BCT) against experimentally induced arsenic toxicity in Swiss albino mice. BCT (5 and 10 mg/kg) was administered orally to mice 30 min before oral administration of arsenic trioxide (3 mg/kg) for 14 consecutive days. On 15th day, the body weights, organ weights, hematological profiles, serum biochemical profile; hepatic and renal antioxidative parameters viz. lipid peroxidation, reduced and oxidized glutathione, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase; and DNA fragmentation were evaluated. Co-treatment with BCT markedly and significantly normalized body weights, organ weights, hematological profiles, serum biochemical profile and significantly modulated all the hepatic and renal biochemical parameters and DNA fragmentation in arsenic-intoxicated mice. The present findings conclude that ß-carotene possessed remarkable ameliorative effect against arsenic-induced toxicity in albino mice mediated by its antioxidant and antigenotoxic properties.
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Peso Corporal/efectos de los fármacos , Hígado/efectos de los fármacos , Óxidos/toxicidad , beta Caroteno/farmacología , Alanina Transaminasa/sangre , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/toxicidad , Trióxido de Arsénico , Arsenicales/administración & dosificación , Aspartato Aminotransferasas/sangre , Recuento de Células Sanguíneas , Catalasa/metabolismo , Fragmentación del ADN/efectos de los fármacos , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Tamaño de los Órganos/efectos de los fármacos , Óxidos/administración & dosificación , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factores de Tiempo , Vitaminas/administración & dosificación , Vitaminas/farmacología , beta Caroteno/administración & dosificaciónRESUMEN
Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 °C, respectively. The enzyme was most stable at pH 8 at 25 °C with deactivation rate constant (Kd*) 1.398 × 10(-4) and half life (t1/2) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg(2+), having Km and Vmax 331.9 µM and 719.1 pKat µg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2.
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Bacopa/química , Bacopa/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Recombinantes , Secuencia de Aminoácidos , Bacopa/genética , Clonación Molecular , ADN Complementario , Activación Enzimática , Expresión Génica , Concentración de Iones de Hidrógeno , Iones/química , Cinética , Metales/química , Datos de Secuencia Molecular , Peso Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN , TemperaturaRESUMEN
UNLABELLED: Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens. ABBREVIATIONS: IPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.