Prevalencia de síndrome metabólico y factores de riesgo asociados en trabajadores de EsSalud Chachapoyas ­ Amazonas / Prevalence of metabolic syndrome and associated risk factors in EsSalud Chachapoyas workers - Amazonas

Los problemas de salud que tienen las personas en estos tiempos es el aumento de peso por malos estilos de vida como inadecuados patrones alimentarios, sedentarismo, altos niveles de estrés laboral, entre otros los que conlleva a aumentar la morbilidad y mortalidad en este marco el estudio fue de tipo descriptivo correlacional, que tuvo como objetivo general determinar la prevalencia de síndrome metabólico y los factores de riesgo asociados en los trabajadores de EsSalud Chachapoyas ­ Amazonas 2015. Para afirmar lo escrito anteriormente se aplicó dos cuestionarios para identificar las variables en estudio a la enfermedad, los cuales tuvieron validez de juicio de expertos y confiabilidad adecuada de alfa de Cronbach, se trabajó con una muestra de 45 trabajadores. Los porcentajes obtenidos nos muestran que la prevalencia de síndrome metabólico según sexo es mayor en las mujeres entre 30 y 59 años y los factores de riesgo son familia/amigos, actividad física/asociatividad, nutrición/alimentación, tabaco, alcohol, sueño y estrés, control de salud. Se concluye que el Síndrome Metabólico está correlacionado significativamente (+0.75) con el factor familia/amigos, (+0.85) actividad Física /asociatividad (+0.93) nutrición y alimentación, (+0.82) alcohol, (+0.75) sueño y estrés, (+0.79) control de Salud/sexualidad.

Anti-Inflammatory Effect of Dialyzable Leukocyte Extract in Autoimmune Prostatitis: Evaluation in Animal Model.

Objective. To evaluate the anti-inflammatory properties of Dialyzable Leukocyte Extract (DLE) in a murine model of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Methods. Histopathological characterization, prostatein Enzyme-Linked Immunosorbent Assay, and immunohistochemical analysis for CD45, TNF-α, IFN-γ, IL-6, IL-17, and IL-4 molecules were done in prostatic Wistar rats treated with DLE, placebo, or Dexamethasone. Results. Histopathological analysis of animals induced to prostatitis showed inflammatory infiltrate, mainly constituted by leucocytes and mast cells as well as Benign Prostatic Hyperplasia. Serum prostatein concentrations were 14 times higher than those displayed by healthy animals. After DLE and Dexamethasone treatments, the inflammatory infiltrate decreased; the tissue morphology was similar to that of a normal prostate, and the prostatein decreased to the basal levels of healthy animals. DLE treatment produced a decreased expression of the cell surface marker CD45 and the proinflammatory cytokines TNF-α, IFN-γ, IL-6, and IL-17. On the other hand, the expression of anti-inflammatory cytokine IL-4 increased in both the Dexamethasone and DLE groups. Conclusion. DLE is able to modulate the inflammatory response in Experimental Autoimmune Prostatitis (EAP).

El efecto de memoria congruente con el estado afectivo reconocimiento diferencial de palabras de tristeza y alegría / The mood congruence memory effect: Differential recognition of sadness and joy words

Los estudios sobre el efecto de congruencia con el estado de ánimo suelen utilizar muestras clínicas de personas depresivas, utilizan programas de inducción de afecto introspectivos, y miden la memoria con tareas de recuerdo libre. Nosotros pretendemos comprobar esta hipótesis induciendo dos estados de ánimo contrapuestos en 100 participantes sin patología. El objetivo fue estudiar la influencia de los estados de ánimo en el reconocimiento de palabras congruentes con dichos estados de ánimo. Para inducir los estados afectivos, utilizamos la asociación de imágenes y música con la dimensión de tristeza y de alegría. Después aprendieron palabras relacionadas con ambos estados emocionales. Encontramos efectos de interacción entre el tipo de estado afectivo inducido y el reconocimiento de las palabras emocionales, tanto en latencias como en índices de discriminación A'. Sin embargo, encontramos diferencias en el reconocimiento dependiendo del tipo de estado de ánimo. El efecto de congruencia con el estado de ánimo se produce en el procesamiento de palabras de la categoría de tristeza, pero no en las palabras de la categoría de alegría. Éstas tienen latencias más rápidas e índices de discriminación más altos, al margen del estado inducido experimentalmente (AU)

Studies on the mood congruence effect commonly used clinical samples of depressed people use affect induction programs introspective, and measure the memory with free recall tasks. We intend to verify this hypothesis induces two conflicting moods in 100 participants without pathology. The aim was to study the influence of mood on the recognition of words congruent with those moods. To induce affective states, we use the association of images and music with the dimension of sadness and joy. After they learned words related to the two emotional states. We found interaction effects between type of induced emotional state and recognition of emotional words in both latencies as indices of discrimination A '. However, we found differences in the recognition depending on the mood. The mood congruence effect occurs in the processing of words in the category of sadness, but not in the words of the category of joy. These latencies are faster and higher rates of discrimination, regardless of the experimentally induced state (AU)

Factores predisponentes al abandono del tratamiento preventivo en una serie de pacientes con migraña / Predisposing factors affecting drop-out rates in preventive treatment in a series of patients with migraine

Introducción. Más de un 30% de pacientes abandona el tratamiento preventivo de la migraña. Esta situación es poco conocida y los factores de riesgo que llevan al abandono del tratamiento tampoco están identificados. Objetivo. Valorar alguno de los factores que pueden predisponer al abandono de un tratamiento preventivo. Pacientes y métodos. Es un estudio prospectivo de pacientes con migraña que precisaron tratamiento preventivo por primera vez con uno de tres fármacos considerados de primera línea: un betabloqueante (nadolol), un neuromodulador (topiramato) o un antagonista del calcio (flunaricina). Se establecieron dos grupos según se produjese abandono o no del tratamiento. Se analizaron y compararon diferentes variables demográficas y clínicas en ambos grupos. Resultados. En un total de 800 pacientes con migraña que precisaron tratamiento preventivo por primera vez, hubo un 19,7% de abandonos. En el grupo que abandonó, las variables ‘edad’, ‘número de crisis previas al tratamiento preventivo’ y ‘efectos adversos’ mostraron diferencias significativas con las del grupo de pacientes que no suspendieron el tratamiento preventivo. Conclusiones. El fármaco utilizado como tratamiento preventivo, los efectos adversos, la edad más joven y el menor número de crisis antes de iniciar el tratamiento preventivo favorecieron su abandono. El tipo de migraña episódica o crónica, la presencia de abuso de fármacos y los fármacos utilizados para el tratamiento de las crisis no guardaron relación con la suspensión del tratamiento preventivo (AU)

Introduction. The drop-out rate among patients receiving preventive treatment for migraine is higher than 30%. This situation is not very widely known and the risk factors that lead patients to drop out from treatment have yet to be identified. Aim. To evaluate some of the factors that can predispose patients to drop out of preventive treatment. Patients and methods. We conducted a prospective study of patients suffering from migraine who required preventive treatment for the first time with one of what are considered the top three first-choice drugs, i.e. a beta-blocker (nadolol a neuromodulator (topiramate) or a calcium antagonist (flunarizine). Two groups were established according to whether patients dropped out of treatment or not. Different demographic and clinical variables were analysed and compared in the two groups. esults. Of 800 patients with migraine who required preventive treatment for the first time, the drop-out rate was 19.7%. In the drop-out group, the variables ‘age’, ‘number of seizures’, ‘number of seizures prior to preventive treatment’ and ‘side effects’ showed significant differences with those from the group of patients who did not drop out of preventive treatment. Conclusions. The drug used as preventive treatment, the side effects, a younger age and a lower number of seizures before starting the preventive treatment favoured higher drop-out rates. Whether the migraine was episodic or chronic, the presence of medication abuse and the drugs used to treat the seizures were not related with dropping out of preventive treatmen (AU)

[Predisposing factors affecting drop-out rates in preventive treatment in a series of patients with migraine]. / Factores predisponentes al abandono del tratamiento preventivo en una serie de pacientes con migraña.

INTRODUCTION: The drop-out rate among patients receiving preventive treatment for migraine is higher than 30%. This situation is not very widely known and the risk factors that lead patients to drop out from treatment have yet to be identified. AIM: To evaluate some of the factors that can predispose patients to drop out of preventive treatment. PATIENTS AND METHODS: We conducted a prospective study of patients suffering from migraine who required preventive treatment for the first time with one of what are considered the top three first-choice drugs, i.e. a beta-blocker (nadolol), a neuromodulator (topiramate) or a calcium antagonist (flunarizine). Two groups were established according to whether patients dropped out of treatment or not. Different demographic and clinical variables were analysed and compared in the two groups. RESULTS: Of 800 patients with migraine who required preventive treatment for the first time, the drop-out rate was 19.7%. In the drop-out group, the variables 'age', 'number of seizures', 'number of seizures prior to preventive treatment' and 'side effects' showed significant differences with those from the group of patients who did not drop out of preventive treatment. CONCLUSIONS: The drug used as preventive treatment, the side effects, a younger age and a lower number of seizures before starting the preventive treatment favoured higher drop-out rates. Whether the migraine was episodic or chronic, the presence of medication abuse and the drugs used to treat the seizures were not related with dropping out of preventive treatment.

[Comparative study of the effectiveness of topiramate and nadolol in the preventive treatment of episodic migraine in independent series of patients]. / Estudio comparativo de la efectividad del topiramato y del nadolol en el tratamiento preventivo de la migraña episodica en series independientes de pacientes.

INTRODUCTION: Topiramate and nadolol with levels A and C of scientific evidence, respectively, would be indicated as preventive treatments of migraine. To date only one study of satisfaction has been carried out to compare the two pharmaceuticals. AIM: To compare the effectiveness parameters in independent groups of patients treated preventively with one of the pharmaceuticals from the study. PATIENTS AND METHODS: From a database of 700 patients with migraine, those with episodic migraine and who had followed a course of preventive treatment, for the first time, with topiramate or nadolol were selected for the study. The effectiveness variables (reduction in the number of crises at four months of preventive treatment and responder rates) were analysed. RESULTS: Altogether 208 patients with were included for treatment: 140 with topiramate (77.8% females; mean age, 37.9) and 68 with nadolol (69% females; mean age, 36.9). The mean number of crises in the month prior to treatment was: topiramate group, 6.3 +/- 2.6; nadolol group 5.3 +/- 2.0 (p = 0.0066). At four months after starting treatment: topiramate group, 2.69 +/- 2.6; nadolol group 2.6 +/- 2.2 (NS). The percentage of reduction in the number of migraines was 56.6% with topiramate and 51.6% with nadolol (NS). The responder rate (reduction in the frequency of crises by at least 50%) was 71.3% with topiramate versus 69% with nadolol (NS). The excellent response rate (reduction in crises by at least 75%) was 53.3% with topiramate versus 32.2% with nadolol (p = 0.0077). Adverse side effects were reported by 54% of patients treated with topiramate versus 30.8% of those treated with nadolol (p = 0.0015). The rate of satisfaction was 61% for the topiramate group and 71% for the group with nadolol (NS). CONCLUSIONS: Both topiramate and nadolol proved to be effective in the preventive treatment of episodic migraine. Topiramate was found to be more effective than nadolol, although it was used in patients with a higher frequency of crises, and was not tolerated so well.

Estudio comparativo de la efectividad del topiramato y del nadolol en el tratamiento preventivo de la migraña episódica en series independientes de pacientes / Comparative study of the effectiveness of topiramate and nadolol in the preventive treatment of episodic migraine in independent series of patients

Introducción. El topiramato con nivel A y el nadolol con nivel C de evidencia científica estarían indicados como tratamientos preventivos de la migraña. Sólo existe un estudio de satisfacción que compare ambos fármacos. Objetivo. Comparar los parámetros de efectividad en grupos independientes de pacientes tratados preventivamente con uno de los fármacos del estudio. Pacientes y métodos. De una base de datos de 700 pacientes con migraña, se seleccionaron aquéllos con migraña episódica y que habían llevado tratamiento preventivo, por primera vez, con topiramato o nadolol. Se analizaron las variables de efectividad (reducción del número de crisis al cuarto mes de tratamiento preventivo y tasa de respondedores). Resultados. Fueron incluidos 208 pacientes con intención de tratar; 140 con topiramato (77,8% mujeres; edad media: 37,9 años) y 68 con nadolol (69% mujeres; edad media: 36,9 años). La media de crisis en el mes previo al tratamiento fue: grupo con topiramato, 6,3 ± 2,6; grupo con nadolol, 5,3 ± 2,0 (p = 0,0066). Al cuarto mes de tratamiento: grupo con topiramato, 2,69 ± 2,6; grupo con nadolol, 2,6 ± 2,2 (NS). El porcentaje de reducción de migrañas fue del 56,6% con topiramato y del 51,6% con nadolol (NS). La tasa de respondedores (reducción en la frecuencia de crisis al menos del 50%) fue del 71,3% con topiramato y del 69% con nadolol (NS). La tasa de respuesta excelente (reducción de las crisis al menos un 75%) fue del 53,3% con topiramato y del 32,2% con nadolol (p = 0,0077). El 54% de los pacientes tratados con topiramato y el 30,8% de los pacientes tratados con nadolol presentaron efectos adversos (p = 0,0015). La tasa de satisfacción fue del 61% en el grupo de topiramato y del 71% en el grupo de nadolol (NS). Conclusión. El topiramato y el nadolol mostraron ser efectivos en el tratamiento preventivo de la migraña episódica. El topiramato mostró mayor efectividad y se utilizó en pacientes con mayor frecuencia de crisis, pero se toleró peor que el nadolol (AU)

Introduction. Topiramate and nadolol with levels A and C of scientific evidence, respectively, would be indicated as preventive treatments of migraine. To date only one study of satisfaction has been carried out to compare the two pharmaceuticals. Aim. To compare the effectiveness parameters in independent groups of patients treated preventively with one of the pharmaceuticals from the study. Patients and methods. From a database of 700 patients with migraine, those with episodic migraine and who had followed a course of preventive treatment, for the first time, with topiramate or nadolol were selected for the study. The effectiveness variables (reduction in the number of crises at four months of preventive treatment and responder rates) were analysed. Results. Altogether 208 patients with were included for treatment: 140 with topiramate (77.8% females; mean age, 37.9) and 68 with nadolol (69% females; mean age, 36.9). The mean number of crises in the month prior to treatment was: topiramate group, 6.3 ± 2.6; nadolol group 5.3 ± 2.0 (p = 0.0066). At four months after starting treatment: topiramate group, 2.69 ± 2.6; nadolol group 2.6 ± 2.2 (NS). The percentage of reduction in the number of migraines was 56.6% with topiramate and 51.6% with nadolol (NS). The responder rate (reduction in the frequency of crises by at least 50%) was 71.3% with topiramate versus 69% with nadolol (NS). The excellent response rate (reduction in crises by at least 75%) was 53.3% with topiramate versus 32.2% with nadolol (p = 0.0077). Adverse side effects were reported by 54% of patients treated with topiramate versus 30.8% of those treated with nadolol (p = 0.0015). The rate of satisfaction was 61% for the topiramate group and 71% for the group with nadolol (NS). Conclusions. Both topiramate and nadolol proved to be effective in the preventive treatment of episodic migraine. Topiramate was found to be more effective than nadolol, although it was used in patients with a higher frequency of crises, and was not tolerated so well (AU)

Regulation of gene expression in protozoa parasites.

Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

Ultrastructural changes on Entamoeba histolytica HM1-IMSS caused by the flavan-3-ol, (-)-epicatechin.

The flavan-3-ol, (-)-epicatechin has been previously identified as the most important antiamoebic compound among the extracts from two medicinal plants: Rubus coriifolius and Geranium mexicanum. Here we report the effects of epicatechin on Entamoeba histolytica morphology, analyzed by electronic microscopy. E. histolytica trophozoites were incubated for 48 h at 37 degrees C in the presence of 1.9 microg/mL epicatechin and processed for electronic microscopy analysis. Epicatechin induced nuclear and cytoplasmic changes in the treated trophozoites. These morphological alterations are identical to the cellular changes experienced by E. histolytica trophozoites undergoing programmed cell death (PCD), suggesting that epicatechin could be an alternative compound to treat amoebiasis.

Programmed cell death in Entamoeba histolytica induced by the aminoglycoside G418.

This study presents morphological and biochemical evidence of programmed cell death (PCD) in Entamoeba histolytica induced by exposure of trophozoites to the aminoglycoside antibiotic G418. Morphological characteristics of PCD, including cell shrinkage, reduced cellular volume, nuclear condensation, DNA fragmentation and vacuolization were observed, with preservation of trophozoite membrane integrity. PCD is orchestrated biochemically by alterations in intracellular ion fluxes. In G418-treated trophozoites, overproduction of reactive oxygen species (ROS), decreased intracellular K+, increased cytosolic calcium, and decreased intracellular pH levels were observed. However, externalization of phosphatidylserine was not detected. These results suggest that amoebae can undergo PCD under stress conditions, and that this PCD shares several properties with PCD reported in mammals and in a variety of unicellular organisms.

Structural and functional analysis of the Entamoeba histolytica EhrabB gene promoter.

BACKGROUND: The Entamoeba histolytica EhrabB gene encodes for a Rab GTPase involved in phagocytosis. It is located at a virulence locus where the Ehcp112 gene is in the complementary strand at 332 bp of EhrabB start codon, suggesting a finely regulated transcription of both genes. However, the transcription regulation in this parasite is poorly understood. RESULTS: To initiate the knowledge of EhrabB gene expression regulation, here we studied the structural characteristics of its gene promoter and its control transcription elements. In silico searches of the EhrabB 5'-flanking region revealed that it contains a motif similar to the upstream regulatory element 1 (URE1) of the E. histolytica hgl5 gene. It also has sequences with homology to C/EBP and GATA1 binding sites, and heat shock elements (HSE). Primer extension experiments revealed that EhrabB has at least four transcription initiation sites. The elements at the 5'-flanking region that drive EhrabB gene expression were detected and characterized using transitory transfected trophozoites with a plasmid carrying the CAT reporter gene. EhrabB transcription is negatively regulated by a sequence located between positions -491 to -428 with respect to the first transcription initiation site. We also showed that the URE1-like motif activates EhrabB transcription. In addition, heat shock activated the EhrabB promoter in episomal constructs and lead to an increase in de novo EhrabB transcription. CONCLUSION: The data suggest that EhrabB transcription is controlled negatively by an unidentified sequence, but it is activated by an URE1-like motif. Our analyses also revealed the presence of activator HSE that function under stress.

Entamoeba histolytica: functional characterization of the -234 to -196 bp promoter region of the multidrug resistance EhPgp1 gene.

The multidrug resistance EhPgp1 gene is constitutively expressed in drug resistant trophozoites from Entamoeba histolytica. It has been demonstrated that two CCAAT/enhancer binding sites located in the EhPgp1 gene promoter control its transcriptional activation. However, functional assays of the 5' end of its promoter showed that region from -234 to -196 bp (38 bp) is also important for the EhPgp1 gene transcription. Here, we demonstrated that in the 38 bp region putative cis-activator sequences are located. In silico analysis showed the presence of GATA1, Gal4, Nit-2, and C/EBP consensus sequences. Additionally, we identified three specific DNA-protein complexes, which were competed by a C/EBP, GATA1, and HOX oligonucleotides. Finally, we partially purified three proteins of 64.4, 56.7, and 27.4 kDa. Further investigations are currently in progress to determine the identity of these nuclear factors and how they are interacting with the EhPgp1 gene promoter.

Entamoeba histolytica EhPgp5 transcriptional activation depends on putative emetine response elements.

The multidrug resistance EhPgp5 gene promoter is active in drug resistant clone C2 trophozoites and its activity increases when trophozoites are cultured in the presence of emetine, suggesting that the EhPgp5 gene shows an inducible drug dependent mechanism. We analyzed different promoter fragments to detect those regions that activate transcription in the presence of emetine. Trophozoites were transfected with p375Pgp5, p259Pgp5, p187Pgp5, and p76Pgp5 plasmids and incubated with different emetine concentrations. p375Pgp5 and p259Pgp5 plasmids were able to drive CAT expression in A and C2 trophozoites only in the presence of emetine. CAT activity was turned off in the absence of drug. Interestingly, no CAT activity was detected in the presence or in the absence of emetine with p187Pgp5 plasmid in which 59 bp were deleted at the 5' end of the EhPgp5 minimal promoter (p259Pgp5). These results suggest that the overexpression of the EhPgp5 gene is a consequence of transcriptional activation of the gene promoter by putative drug responsive elements, located within the -111 to -170 bp of the transcription initiation site.

Two CCAAT/enhancer binding protein sites are cis-activator elements of the Entamoeba histolytica EhPgp1 (mdr-like) gene expression.

Here, we show the relevance of promoter regions (-74 to +24, -167 to -75 and -259 to -168 bp) in the transcriptional activation of the multidrug resistance gene EhPgp1 in Entamoeba histolytica, using mutated plasmids and transfection assays. We also demonstrate that both CCAAT/enhancer binding protein sites (-54 to -43 bp and -198 to -186 bp) are cis-activating elements of gene expression in the drug-resistant (clone C2) and -sensitive (clone A) trophozoites. Nuclear proteins from trophozoites of both clones and C/EBP sequences of the core promoter formed specific complexes, which were abolished by anti-human C/EBPbeta antibodies. UV cross-linking and Western blot assays revealed 25 and 65 kDa bands in urea treated and untreated proteins respectively. The nuclear factors that bind to C/EBP sites were semi-purified by affinity chromatography. They were immunodetected by anti-human C/EBPbeta antibodies and formed a specific complex with the C/EBP probe. The antibodies recognized proteins in the cytoplasm, nucleus and EhkO organelles in immunofluorescence and confocal microscopy experiments. Based on our results, we propose that the C/EBP site at -54 bp stabilizes the transcription pre-initiation complex, whereas the other site at -198 bp may be involved in the formation of a multiprotein complex, which provokes DNA folding and promotes the EhPgp1 gene transcription.

La resistencia a múltiples drogas (MDR) en Entamoeba histolytica / Multiple drug resistance in Entamoeba histolytica

El desarrollo de mecanismos para evadir la acción de los fármacos y desarrollar resistencia a los mismos es un evento universal en los organismos vivos. Se presenta en virus, bacterias, hongos, plantas y animales. En una población de seres vivos algunos individuos son capaces de encender genes apagados, de mutar la secuencia de otros genes, de sintetizar nuevas moléculas para desarrollar los mecanismos que les permiten sobrevivir y perpetuar la especie en circunstancias adversas. La acumulación de eventos de adaptación y mutagénesis pueden dar lugar a la aparición de organismos con características diferentes a la mayoría de los individuos de su especie, las cuales al acumularse podrían constituir nuevas especies. De manera que los cambios para la adaptación y la sobrevivencia de los individuos constituyen en última instancia las bases de la evolución. Uno de los mecanismos que las células han desarrollado para sobrevivir en presencia de sustancias tóxicas es el llamado fenotipo de resistencia a múltiples drogas o MDR (por sus siglas en inglés, multidrug resistance). Este evento se caracteriza porque las células presentan resistencia a drogas con estructuras químicas y blancos de acción distintos. El fenotipo de MDR se descubrió primeramente en células transformadas de mamífero, las cuales expresan una glicoproteína de aproximadamente 170 kDa en su superficie, denominada Pgp. Posteriormente este mecanismo se ha descubierto en muchas especies, incluyendo a los protozoarios parásitos...

The Entamoeba histolytica EhPgp5 (MDR-like) protein induces swelling of the trophozoites and alters chloride-dependent currents in Xenopus laevis oocytes.

Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents the multidrug resistant phenotype due to the expression of the E. histolytica P-glycoproteins EhPgpl and EhPgp5. Here, we studied the protein EhPgp5 encoded by the EhPgp5 gene in emetine-sensitive trophozoites transfected with the pEhNEOPgp5 plasmid carrying the EhPgp5 gene. The transfected trophozoites increased their drug resistance slightly, but became bigger and globular. To investigate other EhPgp5 functions further, we microinjected the EhPgp5 mRNA in Xenopus laevis oocytes. Microinjected oocytes expressed EhPgp5 protein in their membranes and exhibited an ion current not present in the control oocytes. The antisense EhPgp5AS transcript, co-injected with the EhPgp5 mRNA, abolished the exogenous current, showing its specificity. Exogenous current was outward during depolarizing pulses. Reduction of the extracellular Cl- concentration displayed a reversible decrease of the current amplitude. Niflumic acid, 4,4-diisothiocyanatostilbene-2, 2'-disulfonic acid, and other Cl- channel blockers abolished the exogenous current, which was poorly modified by verapamil and changes in osmolarity of the medium. Our results suggest that the EhPgp5 protein could function as a Cl- current inductor and as a coadjuvant factor to avoid drug accumulation in the cell.

Cellular location and function of the P-glycoproteins (EhPgps) in Entamoeba histolytica multidrug-resistant trophozoites.

We have studied the cellular location and the efflux pump function of the Entamoeba histolytica P-glycoproteins (EhPgps) in drug-sensitive and -resistant trophozoites. Polyclonal antibodies against the EhPgp384 polypeptide (375-759 amino acids) revealed a 147-kDa protein by Western blot. The band intensity correlated with the emetine-resistance of the trophozoites. Through the confocal microscope, using the anti-EhPgp384 and fluorescein secondary antibodies, the EhPgps were found in a complex vesicular network, in the plasma membrane and outside of the cells. Transmission electron microscopy assays confirmed that drug-resistant trophozoites presented four to five times more EhPgps than sensitive cells. Fluorescence co-localization experiments using rhodamine-123 (R123) and the anti-EhPgp384 antibodies suggested the interaction between EhPgps and the drug. R123 efflux kinetics evidenced that the emetine-resistant trophozoites displayed a drug efflux kinetic four times higher than the drug-sensitive trophozoites, which was reduced by verapamil in both cases. EhPgps may participate in avoiding drug accumulation in the trophozoites by two putative mechanisms: (1) the direct extrusion of the drug from the plasma membrane, and (2) an indirect transport mechanism in which the drug is trapped by EhPgps and concentrated within vesicles that drive the drug to the plasma membrane.

Physiology and molecular genetics of multidrug resistance in Entamoeba histolytica.

Entamoeba histolytica presents the evolutionarily conserved multidrug-resistance (MDR) phenotype, discovered in mammalian cells. MDR cells overexpress the membrane P-glycoprotein, which excludes unrelated drugs from the cytoplasm. E. histolytica mutants exhibit cross-resistance to unrelated drugs, which are pumped out from the cytoplasm. In drug-resistant trophozoites, the constitutively expressed EhPg1 gene appears to be up-regulated by a C/EBP-like factor and a multiprotein complex that were not found in drug-sensitive trophozoites. The drug-induced EhPgp5 gene, on the other hand, appears to be up-regulated by AP-1 and HOX factors. Here we review the main physiological and molecular facts of the MDR phenotype in E. histolytica. Copyright 1999 Harcourt Publishers Ltd.