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1.
Int J Mol Sci ; 25(2)2024 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-38279319

RESUMEN

Entamoeba histolytica (E. histolytica) exhibits a remarkable capacity to respond to thermal shock stress through a sophisticated genetic regulation mechanism. This process is carried out via Heat Shock Response Elements (HSEs), which are recognized by Heat Shock Transcription Factors (EhHSTFs), enabling fine and precise control of gene expression. Our study focused on screening for HSEs in the promoters of the E. histolytica genome, specifically analyzing six HSEs, including Ehpgp5, EhrabB1, EhrabB4, EhrabB5, Ehmlbp, and Ehhsp100. We discovered 2578 HSEs, with 1412 in promoters of hypothetical genes and 1166 in coding genes. We observed that a single promoter could contain anywhere from one to five HSEs. Gene ontology analysis revealed the presence of HSEs in essential genes for the amoeba, including cysteine proteinases, ribosomal genes, Myb family DNA-binding proteins, and Rab GTPases, among others. Complementarily, our molecular docking analyses indicate that these HSEs are potentially recognized by EhHSTF5, EhHSTF6, and EhHSTF7 factors in their trimeric conformation. These findings suggest that E. histolytica has the capability to regulate a wide range of critical genes via HSE-EhHSTFs, not only for thermal stress response but also for vital functions of the parasite. This is the first comprehensive study of HSEs in the genome of E. histolytica, significantly contributing to the understanding of its genetic regulation and highlighting the complexity and precision of this mechanism in the parasite's survival.


Asunto(s)
Entamoeba histolytica , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Simulación del Acoplamiento Molecular , Regiones Promotoras Genéticas , Respuesta al Choque Térmico/genética , Regulación de la Expresión Génica
2.
Microb Pathog ; 162: 105349, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-34864144

RESUMEN

The heat shock response is a conserved mechanism that allows cells to respond and survive stress damage and is transcriptionally regulated by the heat shock factors and heat shock elements. The P-glycoprotein confer the multidrug resistance phenotype; Entamoeba histolytica has the largest multidrug resistance gene family described so far; one of these genes, the EhPgp5 gene, has an emetine-inducible expression. A functional heat shock element was localized in the EhPgp5 gene promoter, indicating transcriptional regulation by heat shock factors. In this work, we determined the oligomer state of EhHSTF7 and the recognition of the heat shock element of the EhPgp5 gene. The EhHSTF7 recombinant protein was obtained as monomer and oligomer. In silico molecular docking predicts protein-DNA binding between EhHSTF7 and 5'-GAA-3' complementary bases. The rEhHSTF7 protein specifically binds to the heat shock element of the EhPgp5 gene in gel shift assays. The competition assays with heat shock element mutants indicate that 5'-GAA-3' complementary bases are necessary for the rEhHSTF7 binding. Finally, the siRNA-mediated knockdown of Ehhstf7 expression causes downregulation of EhPgp5 expression, suggesting that EhHSTF7 is likely to play a key role in the E. histolytica multidrug resistance. This is the first report of a transcription factor that recognizes a heat shock element from a gene involved in drug resistance in parasites. However, further analysis needs to demonstrate the biological relevance of the EhHSTF7 and the rest of the heat shock factors of E. histolytica, to understand the underlying regulation of transcriptional control in response to stress.


Asunto(s)
Entamoeba histolytica , Parásitos , Animales , Entamoeba histolytica/genética , Respuesta al Choque Térmico , Simulación del Acoplamiento Molecular , Factores de Transcripción
3.
Artículo en Inglés | MEDLINE | ID: mdl-30319995

RESUMEN

Oxygen or nitrogen oxidative species and chemical stress induce the programmed cell death (PCD) of Entamoeba histolytica trophozoites. PCD caused by the aminoglycoside G418 is reduced by incubation with the cysteine protease inhibitor E-64; however, no typical caspases or metacaspases have been detected in this parasite. Calpain, a cysteine protease activated by calcium, has been suggested to be part of a specific PCD pathway in this parasite because the specific calpain inhibitor Z-Leu-Leu-Leu-al diminishes the PCD of trophozoites. Here, we predicted the hypothetical 3D structure of a calpain-like protein of E. histolytica and produced specific antibodies against it. We detected the protein in the cytoplasm and near the nucleus. Its expression gradually increased during incubation with G418, with the highest level after 9 h of treatment. In addition, a specific calpain-like siRNA sequence reduced the cell death rate by 65%. All these results support the hypothesis that the calpain-like protein is one of the proteases involved in the execution phase of PCD in E. histolytica. The hypothetical interactome of the calpain-like protein suggests that it may activate or regulate other proteins that probably participate in PCD, including those with EF-hand domains or other calcium-binding sites.


Asunto(s)
Apoptosis , Calpaína/metabolismo , Entamoeba histolytica/fisiología , Calpaína/antagonistas & inhibidores , Biología Computacional , Entamoeba histolytica/efectos de los fármacos , Activadores de Enzimas/metabolismo , Gentamicinas/metabolismo , Mapas de Interacción de Proteínas
4.
J Med Entomol ; 55(6): 1633-1637, 2018 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-30060188

RESUMEN

Culex quinquefasciatus Say (Diptera: Culicidae) is a mosquito species that has attracted a lot of attention from a medical and veterinary point of view; however, little is known about the frequency of L1014F mutations that have been found in the sodium channel gene, with this being a target for DDT and pyrethroid insecticides. The distribution and frequency of the L1014F mutation in Cx. quinquefasciatus populations was determined in rural and urban areas of Yucatan, Mexico from January 2015 to March 2016. Nine hundred fifty adult females out of 17,727 immature states were collected and analyzed in all sites sampled (n = 10). Susceptible homozygotes were identified (L1014/L1014) in 12% (114/950), heterozygous individuals (F1014/L1014) in 34% (323/950), and mutated homozygotes (F1014/F1014) in 54% (513/950) during the dry and rainy seasons. In this work, study areas with a high frequency of L1014F mutation were identified. These findings may help guarantee a more effective and efficient use of the resources available for the control of this vector.


Asunto(s)
Culex/genética , Mosquitos Vectores/genética , Canales de Sodio/genética , Animales , Femenino , Proteínas de Insectos/genética , México , Mutación
5.
Artículo en Inglés | MEDLINE | ID: mdl-29238701

RESUMEN

Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position -170 to -111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (-151 to -136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Emetina/farmacología , Entamoeba histolytica/efectos de los fármacos , Entamoeba histolytica/metabolismo , Respuesta al Choque Térmico/fisiología , Proteínas Protozoarias/genética , Activación Transcripcional/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Secuencia de Bases , Sitios de Unión , Resistencia a Múltiples Medicamentos/genética , Electroporación , Entamoeba histolytica/genética , Entamoeba histolytica/crecimiento & desarrollo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Protozoarios/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Proteínas Protozoarias/análisis , Proteínas Protozoarias/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Trofozoítos/efectos de los fármacos
6.
J Biomater Sci Polym Ed ; 27(3): 218-34, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26540350

RESUMEN

Silver nanoparticles (AgNPs) induce diverse cell-death mechanisms, similar to those promoted by anticancer chemotherapeutics; however, they have not been tested in vivo because their action is not limited to cancer cells. Therefore, in vivo evaluations of their effectiveness should be developed with targeting systems. Breast cancer shows changes in the sugar expression patterns on cell surfaces, related to cancer progression and metastases; those changes have been identified previously by the specific binding of soybean agglutinin (SBA). Here is proposed the use of SBA to target the AgNP activity in breast cancer. For that, the present work reports the synthesis of AgNPs (3.89 ± 0.90 nm) through the polyol method, the generation of AgNP nanocarriers, and the bioconjugation protocol of the nanocarrier with SBA. The free AgNPs, the AgNP nanocarriers, and the SBA-bioconjugated AgNP nanocarriers were tested for cytotoxicity in breast cancerous (MDA-MB-231and MCF7) and non cancerous (MCF 10A) cells, using the MTT assay. AgNPs demonstrated cytotoxic activity in vitro, the non cancerous cells (MCF 10A) being more sensible than the cancerous cells (MDA-MB-231 and MCF7) showing LD(50) values of 128, 205, and 319 µM Ag, respectively; the nanoencapsulation decreased the cytotoxic effect of AgNPs in non cancerous cells, maintaining or increasing the effect on the cancer-derived cells, whereas the SBA-bioconjugation allowed AgNP cytotoxic activity with a similar behavior to the nanocarriers. Future experiments need to be developed to evaluate the targeting effect of the SBA-bioconjugated AgNP nanocarriers to study their functionality in vivo.


Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Portadores de Fármacos , Nanopartículas del Metal , Lectinas de Plantas/administración & dosificación , Compuestos de Plata , Proteínas de Soja/administración & dosificación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Dispersión Dinámica de Luz , Humanos , Hidrodinámica , Concentración de Iones de Hidrógeno , Nanopartículas del Metal/administración & dosificación , Nanopartículas del Metal/química , Microscopía Electrónica de Transmisión , Paclitaxel/administración & dosificación , Polietilenglicoles/administración & dosificación , Compuestos de Plata/administración & dosificación , Compuestos de Plata/síntesis química , Compuestos de Plata/química , Espectrometría Raman
7.
Exp Parasitol ; 135(3): 532-40, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24012862

RESUMEN

Programmed cell death (PCD) is induced in Entamoeba histolytica by a variety of stimuli in vitro and in vivo. In mammals, intracellular acidification serves as a global switch for inactivating cellular processes and initiates molecular mechanisms implicated in the destruction of the genome. In contrast, intracellular alkalinization produced by P-glycoprotein overexpression in multidrug-resistant cells has been related to apoptosis resistance. Our previous studies showed that overexpression of E. histolytica P-glycoprotein (PGP) altered chloride-dependent currents and triggered trophozoite swelling, the reverse process of cell shrinkage produced during PCD. Here we showed that antisense inhibition of PGP expression produced a synchronous death of trophozoites and the enhancement of biochemical and morphological characteristics of PCD induced by G418. The nucleus was contracted, and the nuclear membrane was disrupted. Moreover, chromatin was extensively fragmented. Ca(2+) concentration was increased, while the intracellular pH (ipH) was acidified. In contrast, PGP overexpression prevented intracellular acidification and circumvented the apoptotic effect of G418.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Amebicidas/farmacología , Apoptosis/fisiología , Entamoeba histolytica/metabolismo , Gentamicinas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Elementos sin Sentido (Genética)/fisiología , Apoptosis/efectos de los fármacos , Entamoeba histolytica/efectos de los fármacos , Entamoeba histolytica/ultraestructura , Expresión Génica , Concentración de Iones de Hidrógeno , Plásmidos , Transfección , Trofozoítos/metabolismo
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