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1.
Cancer Invest ; 42(4): 319-332, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38695671

RESUMEN

Glioblastoma multiforme (GBM), is a frequent class of malignant brain tumors. Epigenetic therapy, especially with synergistic combinations is highly paid attention for aggressive solid tumors like GBM. Here, RSM optimization has been used to increase the efficient arrest of U87 and U251 cell lines due to synergistic effects. Cell lines were treated with SAHA, 5-Azacytidine, GSK-126, and PTC-209 individually and then RSM was used to find most effective combinations. Results showed that optimized combinations significantly reduce cell survival and induce cell cycle arrest and apoptosis in both cell lines. Expression of cyclin B1 and cyclin D1 were decreased while caspase3 increased expression.


Asunto(s)
Apoptosis , Sinergismo Farmacológico , Epigénesis Genética , Glioblastoma , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/patología , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Azacitidina/farmacología , Azacitidina/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Vorinostat/farmacología , Vorinostat/administración & dosificación , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo
2.
Environ Res ; 238(Pt 1): 116972, 2023 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-37648189

RESUMEN

Meniere's disease (MD) is a severe inner ear condition known by debilitating symptoms, including spontaneous vertigo, fluctuating and progressive hearing loss, tinnitus, and aural fullness or pressure within the affected ear. Prosper Meniere first described the origins of MD in the 1860s, but its underlying mechanisms remain largely elusive today. Nevertheless, researchers have identified a key histopathological feature called Endolymphatic Hydrops (ELH), which refers to the excessive buildup of endolymph fluid in the membranous labyrinth of the inner ear. The exact root of ELH is not fully understood. Still, it is believed to involve several biological and bioenvironmental etiological factors such as genetics, autoimmunity, infection, trauma, allergy, and new theories, such as saccular otoconia blocking the endolymphatic duct and sac. Regarding treatment, there are no reliable and definitive cures for MD. Most therapies focus on managing symptoms and improving the overall quality of patients' life. To make significant advancements in addressing MD, it is crucial to gain a fundamental understanding of the disease process, laying the groundwork for more effective therapeutic approaches. This paper provides a comprehensive review of the pathophysiology of MD with a focus on old and recent theories. Current treatment strategies and future translational approaches (with low-level evidence but promising results) related to MD are also discussed, including patents, drug delivery, and nanotechnology, that may provide future benefits to patients suffering from MD.


Asunto(s)
Hidropesía Endolinfática , Enfermedad de Meniere , Humanos , Enfermedad de Meniere/diagnóstico , Enfermedad de Meniere/terapia , Hidropesía Endolinfática/diagnóstico , Hidropesía Endolinfática/etiología , Membrana Otolítica
3.
Nanomedicine ; 47: 102609, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36228994

RESUMEN

Drug development for multiple sclerosis (MS) clinical management focuses on both neuroprotection and repair strategies, and is challenging due to low permeability of the blood-brain barrier, off-target distribution, and the need for high doses of drugs. The changes in the extracellular matrix have been documented in MS patients. It has been shown that the expression of nidogen-1 increases in MS lesions. Laminin forms a stable complex with nidogen-1 through a heptapeptide which was selected to target the lesion area in this study. Here we showed that the peptide binding was specific to the injured area following lysophosphatidylcholine (LPC) induced demyelination. In vivo data showed enhanced delivery of the peptide-functionalized gold nanoparticles (Pep-GNPs) to the lesion area. In addition, Pep-GNPs administration significantly enhanced myelin content and reduced astrocyte/microglia activation. Results demonstrated the possibility of using this peptide to target and treat lesions in patients suffering from MS.


Asunto(s)
Oro , Nanopartículas del Metal , Humanos , Vaina de Mielina , Péptidos/farmacología
4.
Fish Physiol Biochem ; 49(6): 1409-1419, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37943346

RESUMEN

Approximately 80% of luminous organisms live in the oceans, and considerable diversity of life dependence on bioluminescence has been observed in marine organisms. Among vertebrates, luminous fish species are the only group of vertebrates that have the ability to emit bioluminescent light. Meanwhile, the lantern fish family (Myctophidae), with 33 genera all of which have the ability to emit light, is considered the most prominent family among the luminous fish of the deep oceans and seas. Lantern fish Benthosema pterotum has bioluminescence properties due to the presence of photophores scattered in its ventral-lateral region. However, no research has been performed on its bioluminescence system and light emission mechanism. The present research aimed to assess the type of bioluminescence, pigment, photoprotein, or luciferin-luciferase system in B. pterotum. In order to determine the type of light-emitting system in B. pterotum species, several specific experiments were designed and performed. It was shown that the light emission system in B. pterotum species is categorized into the luciferin-luciferase type. Conducting this research was not only innovative, but it also could be the beginning of further research in the field of marine biochemistry and production of the recombinant active forms of enzymes for industrial, commercial, medical, and pharmaceutical purposes.


Asunto(s)
Peces , Luciferinas , Animales , Luciferasas/genética , Mediciones Luminiscentes
5.
BMC Cancer ; 22(1): 979, 2022 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-36100939

RESUMEN

BACKGROUND: Colorectal cancer is one of the most common cancer and the third leading cause of death worldwide. Increased generation of reactive oxygen species (ROS) is observed in many types of cancer cells. Several studies have reported that an increase in ROS production could affect the expression of proteins involved in ROS-scavenging, detoxification and drug resistance. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a known transcription factor for cellular response to oxidative stress. Several researches exhibited that Nrf2 could exert multiple functions and expected to be a promising therapeutic target in many cancers. Here, Nrf2 was knocked down in colorectal cancer cell line HT29 and changes that occurred in signaling pathways and survival mechanisms were evaluated. METHODS: The influence of chemotherapy drugs (doxorubicin and cisplatin), metastasis and cell viability were investigated. To explore the association between specific pathways and viability in HT29-Nrf2-, proteomic analysis, realtime PCR and western blotting were performed. RESULTS: In the absence of Nrf2 (Nrf2-), ROS scavenging and detoxification potential were dramatically faded and the HT29-Nrf2- cells became more susceptible to drugs. However, a severe decrease in viability was not observed. Bioinformatic analysis of proteomic data revealed that in Nrf2- cells, proteins involved in detoxification processes, respiratory electron transport chain and mitochondrial-related compartment were down regulated. Furthermore, proteins related to MAPKs, JNK and FOXO pathways were up regulated that possibly helped to overcome the detrimental effect of excessive ROS production. CONCLUSIONS: Our results revealed MAPKs, JNK and FOXO pathways connections in reducing the deleterious effect of Nrf2 deficiency, which can be considered in cancer therapy.


Asunto(s)
Neoplasias Colorrectales , Proteómica , Línea Celular , Neoplasias Colorrectales/genética , Humanos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo
6.
Mol Divers ; 26(1): 97-112, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33387185

RESUMEN

Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. In this investigation, we synthesized and characterized ten new N-acetyl phosphoramidate compounds and explored the crystal structure of three others. Furthermore, not only were some kinetic inhibition parameters measured, like IC50, Ki, kp, KD for 7 compounds on human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), but also their hydrophobic parameter was determined by shake-flask technique. All compounds (number 1-10) were investigated for anti-bacterial activity against three Gram-positive and three Gram-negative bacteria, while chloramphenicol was used as a standard antibiotic. In order to find new insecticide, toxicities of 13 acephate (Ace)-derived compounds (number 20-32) were bioassayed on third larval instar of elm leaf beetle and Xanthogaleruca luteola. Additionally, screening in vivo tests revealed that two compounds had had the greatest insecticidal potential in comparison with others. It means these ones inhibited AChE (with mixed mechanisms) and general esterase more than the rest. According to ChE-QSAR models, the inhibitory potency for enzyme and bacteria is directly influenced by the electronic parameters versus structural descriptors. AChE-QSPR model of fluorescence assay indicated that the inhibitory power of AChE is primarily influenced by a set of electronic factors with the priority of: EHB > PL > δ(31P) versus structural descriptor (SA and Mv). Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. Toxicities of 13 acephate (Ace)-derived compounds (number 20-32) were bioassayed on third larval instar of elm leaf beetle and Xanthogaleruca luteola. Insect-QSAR equations of these compounds, based on MLR and PCA, showed that non-descriptor net charge nitrogen atom (which was affected by the polarization of N-H group) had the greatest effect on insecticidal potential.


Asunto(s)
Acetilcolinesterasa , Insecticidas , Acetilcolinesterasa/metabolismo , Butirilcolinesterasa/química , Inhibidores de la Colinesterasa/química , Humanos , Insecticidas/química , Insecticidas/farmacología , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad
7.
Biochemistry (Mosc) ; 86(7): 898-905, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34284714

RESUMEN

One of the main players in the cell-specific replication timing pattern is Rap1 interacting factor-1 (Rif1). Rif1 protein consists of N-terminal and C-terminal domains and an intrinsically disordered region in between. It has been suggested that both N- and C-termini of Rif1 are capable of binding to DNA with particularly high affinity to cruciform DNA structures. In the present study, we expressed, solubilized, and purified the maltose-binding protein-tagged murine Rif1 C-terminal domain (MBP-muRif1-CTD). Biological activity of the purified protein was assessed by the electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). Our results show that the MBP-muRif1-CTD binds G-quadruplex (G4) structure with high affinity (KD 19.0 ± 0.8 nM), as was previously suggested. This study is the first step in investigation of the interaction of MBP-Profinity eXact-muRif1-CTD and G4 by SPR.


Asunto(s)
ADN/metabolismo , G-Cuádruplex , Proteínas de Unión a Telómeros/metabolismo , Animales , Ensayo de Cambio de Movilidad Electroforética , Cinética , Ratones , Unión Proteica , Resonancia por Plasmón de Superficie
8.
Biol Chem ; 401(10): 1143-1151, 2020 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-32673279

RESUMEN

α-Synuclein fibrillation is now regarded as a major pathogenic process in Parkinson's disease and its proteinaceous deposits are also detected in other neurological disorders including Alzheimer's disease. Therefore anti-amyloidegenic compounds may delay or prevent the progression of synucleinopathies disease. Molecular chaperones are group of proteins which mediate correct folding of proteins by preventing unsuitable interactions which may lead to aggregation. The objective of this study was to investigate the anti-amyloidogenic effect of molecular chaperone artemin on α-synuclein. As the concentration of artemin was increased up to 4 µg/ml, a decrease in fibril formation of α-synuclein was observed using thioflavin T (ThT) fluorescence and congo red (CR) assay. Transmission electron microscopy (TEM) images also demonstrated a reduction in fibrils in the presence of artemin. The secondary structure of α-synuclein was similar to its native form prior to fibrillation when incubated with artemin. A cell-based assay has shown that artemin inhibits α-synuclein aggregation and reduce cytotoxicity, apoptosis and reactive oxygen species (ROS) production. Our results revealed that artemin has efficient chaperon activity for preventing α-synuclein fibril formation and toxicity.


Asunto(s)
Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , alfa-Sinucleína/metabolismo , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Humanos , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Especies Reactivas de Oxígeno/análisis , alfa-Sinucleína/química , alfa-Sinucleína/aislamiento & purificación
9.
J Nanobiotechnology ; 18(1): 93, 2020 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-32552818

RESUMEN

BACKGROUND: Since vascular endothelial growth factor (VEGF) is a significant regulator of cancer angiogenesis, it is essential to develop a technology for its sensitive detection. Herein, we sensitized a chemiluminescence (CL) immunoassay through the combination of H2O2-sensitive TGA-CdTe quantum dot (QD) as signal transduction, dextran as a cross-linker to prepare enzyme-labeled antigen and the ultrahigh bioactivity of catalase (CAT) as reporter enzyme. RESULTS: Under the optimized experimental conditions, the chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) method can detect VEGF in the excellent linear range of 2-35,000 pg mL-1, with a detection limit (S/N = 3) of 0.5 pg mL-1 which was approximately ten times lower than the commercial colorimetric immunoassay. This proposed method has been successfully applied to the clinical determination of VEGF in the human serum samples, and the results illustrated an excellent correlation with the conventional ELISA method (R2 = 0.997). The suitable recovery rate of the method in the serum ranged from 97 to 107%, with a relative standard deviation of 1.2% to 13.4%. CONCLUSIONS: The novel immunoassay proposes a highly sensitive, specific, and stable method for very low levels detection of VEGF that can be used in the primary diagnosis of tumors. With the well-designed sensing platform, this approach has a broad potential to be applied for quantitative analysis of numerous disease-related protein biomarkers for which antibodies are available.


Asunto(s)
Catalasa/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Mediciones Luminiscentes/métodos , Puntos Cuánticos , Factor A de Crecimiento Endotelial Vascular/sangre , Compuestos de Cadmio/química , Humanos , Peróxido de Hidrógeno/química , Puntos Cuánticos/química , Puntos Cuánticos/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Telurio/química
10.
Bioconjug Chem ; 30(9): 2349-2357, 2019 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-31429535

RESUMEN

Activated platelets have a high affinity for tumor cells, and consequently, they can protect tumor cells from environmental stress and immune attacks. Therefore, preventing platelet-tumor cell interaction can lead to the elimination of circulating tumor cells via natural killer cells and finally metastasis inhibition. It is also shown that CREKA (Cys-Arg-Glu-Lys-Ala), a tumor-homing pentapeptide, targets fibrin-fibronectin complexes that are found on the tumor stroma and the vessel walls. In this study, we linked CREKA to Ticagrelor, a reversible antagonist of the P2Y12 receptor on platelets. In vitro experiments indicated that CREKA-Ticagrelor could not only inhibit the platelet-induced migration of tumor cells with an invasive phenotype but also prevent tumor-platelet interaction. In vivo antitumor and antimetastasis results of this drug showed that CREKA-Ticagrelor could specifically target the tumor tissues within 24 h post intravenous injection and suppress lung metastasis. Meanwhile, by having this antiplatelet drug targeted, its side effects were minimized, and bleeding risk was decreased. Thus, CREKA-Ticagrelor offers an efficient antimetastatic agent.


Asunto(s)
Péptido Hidrolasas/química , Péptido Hidrolasas/farmacología , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Ticagrelor/química , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia/prevención & control , Péptido Hidrolasas/efectos adversos , Péptido Hidrolasas/farmacocinética , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/farmacocinética , Seguridad , Distribución Tisular , Cicatrización de Heridas/efectos de los fármacos
11.
Mol Biol Rep ; 46(3): 3275-3286, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30993582

RESUMEN

It was confirmed that several enzymes have anti-cancer activity. The enzymes L-asparaginase, L-glutaminase, and L-arginase were chosen according to amino acids starvation in cancer cells and screened in halophilic and halotolerant bacteria, given probably less immunological reactions of halophilic or halotolerant enzymes in patients. Out of 110 halophilic and halotolerant strains, isolated from different saline environments in Iran and screened, some could produce a variety of anticancer enzymes. A total of 29, 4, and 2 strains produced L-asparaginase, L-glutaminase, and L-arginase, respectively. According to the phenotypic characteristics and partial 16S rRNA gene sequence analysis, the positive strains-strains with the ability to produce these anticancer enzymes-were identified as the members of the genera: Bacillus, Dietzia, Halobacillus, Rhodococcus, Paenibacillus and Planococcus as Gram-positive bacteria and Pseudomonas, Marinobacter, Halomonas, Idiomarina, Vibrio and Stappia as Gram-negative bacteria. The production of anticancer enzymes was mostly observed in the rod-shaped Gram-negative isolates, particularly in the members of the genera Halomonas and Marinobacter. Most of the enzymes were produced in the stationary phase of growth and the maximum enzyme activity was experienced in strain GBPx3 (Vibrio sp.) for L-asparaginase at 1.0 IU/ml, strain R2S25 (Rhodococcus sp.) for L-glutaminase at 0.6 IU/ml and strain GAAy3 (Planococcus sp.) for L-arginase at 3.1 IU/ml. The optimum temperature and pH for L-asparaginase and L-glutaminase activities in selected strains were similar to the physiological conditions of human body and the enzymes could tolerate NaCl up to 7.5% concentration.


Asunto(s)
Bacterias/genética , Tolerancia a la Sal/genética , Antineoplásicos , Asparaginasa/metabolismo , ADN Bacteriano/genética , Halobacteriales/genética , Irán , Filogenia , ARN Ribosómico 16S/genética , Solución Salina , Análisis de Secuencia de ADN , Cloruro de Sodio/metabolismo
12.
Int J Mol Sci ; 20(19)2019 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-31569768

RESUMEN

Current cancer therapeutics suffer from a lack of specificity in targeting tumor cells and cause severe side effects. Therefore, the design of highly specialized drugs comprising antibody derivatives inducing apoptosis in targeted cancer cells is considered to be a promising strategy. Drugs acting on death receptor 5 (DR5) such as DR5 agonist antibodies replacing "TNF-related apoptosis-inducing ligand" (TRAIL) offer feasible opportunities in this direction. Although such agonists provided good antitumor activity in preclinical studies, they were less effective in clinical studies, possibly due to a disturbed Fc interaction with Fc-γ receptors. Thus, multimerized antigen binding fragments without Fc have been proposed to increase their efficacy. We generated nanobodies (Nbs), recombinant variable domains of heavy chain-only antibodies of camelids, against the DR5 ectodomain. Nb24 and Nb28 had an affinity in the nM and sub-nM range, but only Nb28 competes with TRAIL for binding to DR5. Bivalent, trivalent, and tetravalent constructs were generated, as well as an innovative pentameric Nb complex, to provoke avidity effects. In our cellular assays, these trimeric, tetrameric, and pentameric Nbs have a higher apoptotic capacity than monomeric Nbs and seem to mimic the activity of the natural TRAIL ligand on various cancer cells.


Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Anticuerpos de Dominio Único/farmacología , Animales , Antineoplásicos Inmunológicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Ratones , Unión Proteica , Receptores de IgG/química , Receptores de IgG/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Proteínas Recombinantes , Anticuerpos de Dominio Único/química , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Biotechnol Appl Biochem ; 65(6): 883-891, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-30132989

RESUMEN

Azoreductases mainly reduce azo dyes, the largest class of colorants, to colorless aromatic amines. AzoH, a new azoreductase from the halophilic bacterium, Halomonas elongata, has been recently cloned and expressed in Escherichia coli. The aim of this study was to improve thermal stability of this enzyme by introducing new disulfide bonds. Since X-ray crystallography was not available, homology modeling and molecular dynamics was used to construct the enzyme three-dimensional structure. Potential disulfide bonds for increasing thermal stability were found using DIScover online software. Appropriate mutations (L49C/D108C) to form a disulfide bond were introduced by the Quik-Change method. Mutant protein expressed in E. coli showed increased thermal stability at 50 °C (increased half-life from 12.6 Min in AzoH to 26.66 Min in a mutated enzyme). The mutated enzyme could also tolerate 5% (w/v) NaCl and retained 30% of original activity after 24 H incubation, whereas the wild-type enzyme was completely inactivated. According to circular dichroism studies, the secondary structure was not altered by this mutation; however, a blue shift in intrinsic florescent graph revealed changes in the tertiary structure. This is the first study to improve thermal stability and salt tolerance of a halophilic azoreductase.


Asunto(s)
Disulfuros/metabolismo , Halomonas/enzimología , Mutagénesis Sitio-Dirigida , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Temperatura , Disulfuros/química , Relación Dosis-Respuesta a Droga , Estabilidad de Enzimas , Halomonas/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Nitrorreductasas , Estructura Terciaria de Proteína , Cloruro de Sodio/farmacología , Programas Informáticos
14.
Mikrochim Acta ; 185(8): 376, 2018 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-30014207

RESUMEN

A method is described for the chemiluminescence based determination of the activity of catalase (CAT) using H2O2-sensitive CdTe quantum dots (QDs). It is based on the finding that the chemiluminescence (CL) of the CdTe/H2O2 system is reduced due to the consumption of H2O2 by the catalytic action of CAT. The Michaelis constant is calculated to be 519 ± 27 mM, showing the potential of the method to accurately measure the Km compared to the standard method. The method does not require QDs to be conjugated to biological/organic molecules and therefore is considered to be a rapid and convenient method for determination of CAT in real samples. At an incubation time of 2 s, the LOD was calculated to be 4.5 unit/mL, with a linear range from 6 to 400 unit/mL. The assay is sensitive, simple, and suitable for practical applications. Graphical abstract Schematic representation of chemiluminescence-based catalase U(CAT) assay using the CdSe QD/H2O2 system. The reduction of H2O2 is reflected by the chemiluminescence of the QDs. A mechanism is put forward based on the changes in chemiluminescence intensity of the QDs by the consumption of H2O2 due to the catalytic action of CAT.

15.
Prep Biochem Biotechnol ; 48(6): 483-489, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29958068

RESUMEN

Escherichia coli is a common host that is widely used for producing recombinant proteins. However, it is a simple approach for production of heterologous proteins; the major drawbacks in using this organism include incorrect protein folding and formation of disordered aggregated proteins as inclusion bodies. Co-expression of target proteins with certain molecular chaperones is a rational approach for this problem. Aequorin is a calcium-activated photoprotein that is often prone to form insoluble inclusion bodies when overexpressed in E. coli cells resulting in low active yields. Therefore, in the present research, our main aim is to increase the soluble yield of aequorin as a model protein and minimize its inclusion body content in the bacterial cells. We have applied the chaperone-assisted protein folding strategy for enhancing the yield of properly folded protein with the assistance of artemin as an efficient molecular chaperone. The results here indicated that the content of the soluble form of aequorin was increased when it was co-expressed with artemin. Moreover, in the co-expressing cells, the bioluminescence activity was higher than the control sample. We presume that this method might be a potential tool to promote the solubility of other aggregation-prone proteins in bacterial cells.


Asunto(s)
Aequorina/genética , Proteínas de Artrópodos/genética , Escherichia coli/genética , Proteínas de Unión a Hierro/genética , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/genética , Aequorina/metabolismo , Animales , Artemia/metabolismo , Proteínas de Artrópodos/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Cuerpos de Inclusión/metabolismo , Proteínas de Unión a Hierro/metabolismo , Luminiscencia , Unión Proteica , Pliegue de Proteína , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad
16.
Water Sci Technol ; 77(11-12): 2867-2875, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-30065139

RESUMEN

The aim of this work was to find a new stable laccase against inhibitors and study the decolorization ability of free and immobilized laccase on different classes of dyes. Spores from a halotolerant bacterium, Bacillus safensis sp. strain S31, isolated from soil samples from a chromite mine in Iran showed laccase activity with maximum activity at 30 °C and pH 5.0 using 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The enzyme retained about 60% of its initial activity in the presence of 10% (v v-1) methanol, ethanol, and acetone. In contrast to many other laccases, NaN3, at 0.1 and 1 mM concentrations, showed a slight inhibitory effect on the enzyme activity. Also, the spore laccase (8 U l-1) decolorized malachite green, toluidine blue, and reactive black 5 at acidic pH values; the highest decolorization percent was 75% against reactive black 5. It was observed that addition of ABTS as a redox mediator enhanced the decolorization activity. Furthermore, immobilized spore laccase encased in calcium alginate beads decolorized 95% of reactive black 5 in the absence of mediators. Overall, this isolated spore laccase might be a potent enzyme to decolorize dyes in polluted wastewaters, especially those containing metals, salts, solvents, and sodium azide.


Asunto(s)
Bacillus/enzimología , Colorantes/química , Lacasa/metabolismo , Azida Sódica/farmacología , Bacillus/aislamiento & purificación , Concentración de Iones de Hidrógeno , Irán , Lacasa/química , Naftalenosulfonatos/química , Colorantes de Rosanilina/química , Microbiología del Suelo , Esporas Bacterianas/enzimología , Cloruro de Tolonio/química , Aguas Residuales/química
17.
Angew Chem Int Ed Engl ; 57(18): 5033-5036, 2018 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-29490117

RESUMEN

Self-assembly of nanoparticles provides unique opportunities as nanoplatforms for controlled delivery. By exploiting the important role of noncovalent hydrophobic interactions in the engineering of stable assemblies, nanoassemblies were formed by the self-assembly of fluorinated quantum dots in aqueous medium through fluorine-fluorine interactions. These nanoassemblies encapsulated different enzymes (laccase and α-galactosidase) with encapsulation efficiencies of ≥74 %. Importantly, the encapsulated enzymes maintained their catalytic activity, following Michaelis-Menten kinetics. Under an acidic environment the nanoassemblies were slowly disassembled, thus allowing the release of encapsulated enzymes. The effective release of the assayed enzymes demonstrated the feasibility of this nanoplatform to be used in pH-mediated enzyme delivery. In addition, the as-synthesized nanoassemblies, having a diameter of about 50 nm, presented high colloidal stability and fluorescence emission, which make them a promising multifunctional nanoplatform.


Asunto(s)
Flúor/química , Lacasa/química , Puntos Cuánticos/química , alfa-Galactosidasa/química , Flúor/metabolismo , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Lacasa/metabolismo , Tamaño de la Partícula , Puntos Cuánticos/metabolismo , Propiedades de Superficie , alfa-Galactosidasa/metabolismo
18.
Biochim Biophys Acta Proteins Proteom ; 1865(6): 652-663, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28315735

RESUMEN

The conversion of proteins from their soluble states into well-organized amyloid fibrils has received abundant attention. This process typically consists of three stages: lag, growth and plateau phases. In this study, the process of amyloid fibril formation by lipase from Pseudomonas sp. after diluting out urea was examined by Thioflavin T (ThT) fluorescence, Congo red (CR) binding, 8-anilinonaphthalene-1-sulfonic acid (ANS) binding, dynamic light scattering (DLS), circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, X-ray diffraction (XRD) and transmission electron microscopy (TEM). To exclude the presence of preformed aggregates in the pure lipase sample, aforementioned assays were also performed for the protein unfolded in urea before dilution. The aggregates formed immediately after dilution were found to bind to ThT and CR and contain a significant amount of ß-sheet structure, as determined by far-UV CD and FTIR spectroscopies, as well as XRD analysis. Moreover, these aggregates present, at least in part, a fibrillar morphology, as deduced with TEM. This examination showed that lipase fibril formation proceeds quickly after dilution, within a few seconds, without a detectable lag phase. We also investigated bacterial inclusion bodies formed after expression of lipase in E. coli, providing evidence for the existence of rapidly formed amyloid-like structural and tinctorial properties in the lipase-containing inclusion bodies.


Asunto(s)
Amiloide/metabolismo , Lipasa/metabolismo , Pseudomonas/enzimología , Dicroismo Circular , Lipasa/química , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Estructura Secundaria de Proteína , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos X
19.
Biopolymers ; 107(10)2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28542806

RESUMEN

Peroxidase-mimicking DNAzyme has a potential to self-assemble into a G-quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase-mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non-labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV-Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G-quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg2+ . Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization.


Asunto(s)
Técnicas Biosensibles/métodos , ADN Catalítico/química , ADN/química , G-Cuádruplex , Magnesio/química , Peroxidasa/química , Espectrofotometría Ultravioleta
20.
Microbiology (Reading) ; 162(2): 246-255, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26747275

RESUMEN

Among nine cyanobacterial strains isolated from oil-contaminated regions in southern Iran, an isolate with maximum cadmium uptake capacity was selected and identified on the basis of analysis of morphological criteria and 16S rRNA gene sequence similarity as Nostoc entophytum (with 99% similarity). The isolate was tentatively designated N. entophytum ISC32. The phylogenetic affiliation of the isolates was determined on the basis of their 16S rRNA gene sequence. The maximum amount of Cd(II) adsorbed by strain ISC32 was 302.91 mg g(-1) from an initial exposure to a solution with a Cd(II) concentration of 150 mg l(-1). The cadmium uptake by metabolically active cells of cyanobacterial strain N. entophytum ISC32, retained in a clinostat for 6 days to simulate microgravity conditions, was examined and compared with that of ground control samples. N. entophytum ISC32 under the influence of microgravity was able to take up cadmium at amounts up to 29% higher than those of controls. The activity of antioxidant enzymes including catalase and peroxidase was increased in strain ISC32 exposed to microgravity conditions in a clinostat for 6 days, as catalase activity of the cells was more than three times higher than that of controls. The activity of the peroxidase enzyme increased by 36% compared with that of the controls. Membrane lipid peroxidation was also increased in the cells retained under microgravity conditions, up to 2.89-fold higher than in non-treated cells. Images obtained using scanning electron microscopy showed that cyanobacterial cells form continuous filaments which are drawn at certain levels, while the cells placed in a clinostat appeared as round-shaped, accumulated together and distorted to some extent.


Asunto(s)
Antioxidantes/metabolismo , Transporte Biológico/fisiología , Cadmio/metabolismo , Contaminantes Ambientales/metabolismo , Nostoc/metabolismo , Biodegradación Ambiental , Biomasa , Catalasa/metabolismo , Citoesqueleto/metabolismo , Peroxidación de Lípido/fisiología , Lípidos de la Membrana/metabolismo , Microscopía Electrónica de Rastreo , Nostoc/genética , Peroxidasa/metabolismo , ARN Ribosómico 16S/genética , Ingravidez
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