Identification and functional characterization of EhClC-A, an Entamoeba histolytica ClC chloride channel located at plasma membrane.

ClC chloride channels perform a wide variety of physiological functions and they had been characterized in animals, yeast, plants and bacteria but not in protozoa. By blast search we found in Entamoeba histolytica, the protozoan responsible for human amoebiasis, two genes (Ehclc-A and Ehclc-B) encoding for putative polypeptides with 25-30% identity to ClC chloride channels of several organisms. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that both genes are transcribed in trophozoites. Phylogenetic analysis revealed that EhClC-A and EhClC-B polypeptides belong to the eukaryotic branch of plasma membrane ClCs. Specific antibodies against EhClC-A confirmed that it is located at the trophozoite plasma membrane. Xenopus laevis oocytes microinjected with Ehclc-A cRNA elicited anion currents not detected in oocytes microinjected with water. Induced currents were inwardly rectifying and had a permeability sequence of Cl->Br->I->F->>NO3-. The chloride channel blocker 4-acetamido-4'isothiocyanostilbene-2, 2'-disulphonic acid (SITS) strongly inhibited the oocytes anion currents and trophozoites growth. Experiments at diverse pHs suggested that EhClC-A is not a Cl-/H+ exchanger, but it is an ion channel that could be involved in pH regulation. EhClC-A may also participate in cell volume regulation. As far as we know, EhClC-A is the first chloride channel characterized in protozoa.

Entamoeba histolytica: alterations in EhRabB protein in a phagocytosis deficient mutant correlate with the Entamoeba dispar RabB sequence.

We analyzed the expression and location of EhRabB in clone L-6, a phagocytosis-deficient mutant of Entamoeba histolytica, in comparison with the wild-type clone A. Intriguingly, trophozoites of clone L-6 express more EhRabB than those of clone A. However, the majority of EhRabB-containing vesicles remained in the cytoplasm of clone L-6 during phagocytosis. To investigate molecular alterations in EhRabB of clone L-6 we compared the EhrabB gene sequences from clones L-6 and A. We also isolated, sequenced and compared the RabB protein of Entamoeba dispar. Results showed that EhrabB gene of clone L-6 is 98.2 and 94.1% identical to rabB genes of E. dispar and clone A, respectively. The rabB genes from clone A and E. dispar have 92.2% identity. Four out of five amino acids changes in RabB proteins of clone L-6 and E. dispar are shared. These changes may alter the binding of effector proteins and the specific subcellular location of EhRabB.

EhCP112 is an Entamoeba histolytica secreted cysteine protease that may be involved in the parasite-virulence.

EhCP112 is an Entamoeba histolytica protease that together with the EhADH112 protein forms the EhCPADH complex involved in trophozoite virulence. Here, we produced the recombinant EhCP112 and studied its relationships with extracellular matrix components and with target cells. A DNA fragment containing the pro-peptide and the mature enzyme was expressed in bacteria as an active enzyme (rEhCP112), whereas the full gene containing the signal peptide, the pro-peptide and the mature enzyme expressed a non-active protein. The fragment only with the mature enzyme was not expressed. rEhCP112 purified by affinity columns digested azocasein and had a strong autoproteolytic activity. Four hours after purification the protein appeared degraded. Anti-tag antibodies, monoclonal antibodies against the EhCP112 and sera from human patients with amoebiasis recognized rEhCP112. rEhCP112 digested gelatin, collagen type I, fibronectin and haemoglobin; it destroyed MDCK cell monolayers and bound to red blood cells. The native EhCP112 was poorly expressed in a virulence-deficient mutant, and in the wild-type clone it was located in secreted vesicles, forming the EhCPADH complex. Altogether these results show that EhCP112 is a molecule able to disrupt cell monolayers and digest proteins of the extracellular matrix and haemoglobin, and it is secreted by the trophozoites.

Usos alternativos del dinero dedicado a fumar y valoración del riesgo por exposición al humo de tabaco ambiental / Alternative uses of the money spent in smoking and assessment of the risk for exposure to the environmental cigarette smoke

En la lucha antitabáquica, lo referente al costo que el tabaquismo representa para el fumador, su familia, las instituciones de salud y la sociedad no ha sido incluido con frecuencia. Los objetivos de esta investigación fueron identificar en qué emplearían los fumadores y los no fumadores el dinero que los primeros dedican a su adicción, así como describir en ambos grupos la valoración del daño producido por el humo de tabaco ambiental. Se emplearon 3 cohortes que incluyeron 1 104 personas, pareadas por sexo y edad (± 5 años). Entre los resultados más significativos se observó que la alimentación fue la alternativa de uso del dinero más recurrida por ambos grupos. Aunque 9 de cada 10 encuestados valoraron como dañina la exposición al humo de tabaco ambiental, la mayor proporción de ellos fueron no fumadores. Puede concluirse que los fumadores asumen sus adicciones conscientes de que renuncian a otras actividades de la vida cotidiana, así como que en el desarrollo de las estrategias actuales en el país contra el tabaquismo es aún insuficiente el énfasis sobre la dimensión social que tiene la conducta de fumar(AU)

Usos alternativos del dinero dedicado a fumar y valoración del riesgo por exposición al humo de tabaco ambiental / Alternative uses of the money spent in smoking and assessment of the risk for exposure to the environmental cigarette smoke

En la lucha antitabáquica, lo referente al costo que el tabaquismo representa para el fumador, su familia, las instituciones de salud y la sociedad no ha sido incluido con frecuencia. Los objetivos de esta investigación fueron identificar en qué emplearían los fumadores y los no fumadores el dinero que los primeros dedican a su adicción, así como describir en ambos grupos la valoración del daño producido por el humo de tabaco ambiental. Se emplearon 3 cohortes que incluyeron 1 104 personas, pareadas por sexo y edad (± 5 años). Entre los resultados más significativos se observó que la alimentación fue la alternativa de uso del dinero más recurrida por ambos grupos. Aunque 9 de cada 10 encuestados valoraron como dañina la exposición al humo de tabaco ambiental, la mayor proporción de ellos fueron no fumadores. Puede concluirse que los fumadores asumen sus adicciones conscientes de que renuncian a otras actividades de la vida cotidiana, así como que en el desarrollo de las estrategias actuales en el país contra el tabaquismo es aún insuficiente el énfasis sobre la dimensión social que tiene la conducta de fumar

The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites.

The Entamoeba histolytica EhCPADH complex, formed by a cysteine proteinase (EhCP112) and an adhesin (EhADH112), is involved in adherence, phagocytosis and cytolysis. This makes this complex an attractive candidate as a vaccine against amoebiasis. Here, we produced the recombinant polypeptide EhADH243, which includes the adherence epitope detected by a monoclonal antibody against the EhCPADH complex. EhADH243 was purified, and the effect of the polypeptide on in vitro and in vivo virulence was studied. Antibodies against EhADH243 reacted with the EhCPADH complex and with the recombinant polypeptide. EhADH243 and antibodies against this polypeptide inhibited adherence, phagocytosis and destruction of cell monolayers by live trophozoites, but had little effect on cell monolayer destruction by trophozoite extracts. EhADH243 recognized a 97 kDa protein in the MDCK membrane fraction that could be a putative receptor for E. histolytica trophozoites. Hamsters immunized with EhADH243 developed humoral response against EhCPADH, and animals were partially protected from amoebic liver abscess.