Role of the NO-cGMP-K+ channels pathway in the peripheral antinociception induced by α-bisabolol.

The aim of this study was to examine if the peripheral antinociception of α-bisabolol involves the participation of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) synthesis followed by K+ channel opening in the formalin test. Wistar rats were injected in the dorsal surface of the right hind paw with formalin (1%). Rats received a subcutaneous injection into the dorsal surface of the paw of vehicles or increasing doses of α-bisabolol (100-300 µg/paw). To determine whether the peripheral antinociception induced by α-bisabolol was mediated by either the opioid receptors or the NO-cGMP-K+ channels pathway, the effect of pretreatment (10 min before formalin injection) with the appropriate vehicles, naloxone, naltrexone, NG-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]-oxadiazolo[4,2-a]quinoxalin-1-one (ODQ), glibenclamide, glipizide, apamin, charybdotoxin, tetraethylammonium, or 4-aminopyridine on the antinociceptive effects induced by local peripheral α-bisabolol (300 µg/paw) were assessed. α-Bisabolol produced antinociception during both phases of the formalin test. α-Bisabolol antinociception was blocked by L-NAME, ODQ, and all the K+ channels blockers. The peripheral antinociceptive effect produced by α-bisabolol was not blocked by the opioid receptor inhibitors. α-Bisabolol was able to active the NO-cGMP-K+ channels pathway to produce its antinoceptive effect. The participation of opioid receptors in the peripheral local antinociception induced by α-bisabolol is excluded.

Insoluble Vascular Amyloid Deposits Trigger Disruption of the Neurovascular Unit in Alzheimer's Disease Brains.

Alzheimer's disease (AD) is a neurodegenerative disease, characterized histopathologically by intra-neuronal tau-related lesions and by the accumulation of amyloid ß-peptide (Aß) in the brain parenchyma and around cerebral blood vessels. According to the vascular hypothesis of AD, an alteration in the neurovascular unit (NVU) could lead to Aß vascular accumulation and promote neuronal dysfunction, accelerating neurodegeneration and dementia. To date, the effects of insoluble vascular Aß deposits on the NVU and the blood-brain barrier (BBB) are unknown. In this study, we analyze different Aß species and their association with the cells that make up the NVU. We evaluated post-mortem AD brain tissue. Multiple immunofluorescence assays were performed against different species of Aß and the main elements that constitute the NVU. Our results showed that there are insoluble vascular deposits of both full-length and truncated Aß species. Besides, insoluble aggregates are associated with a decrease in the phenotype of the cellular components that constitute the NVU and with BBB disruption. This approach could help identify new therapeutic targets against key molecules and receptors in the NVU that can prevent the accumulation of vascular fibrillar Aß in AD.

A Calcium/Cation Exchanger Participates in the Programmed Cell Death and in vitro Virulence of Entamoeba histolytica.

Entamoeba histolytica is the etiologic agent of human amoebiasis, disease that causes 40,000 to 100,000 deaths annually worldwide. The cytopathic activity as well as the growth and differentiation of this microorganism is dependent on both, extracellular and free cytoplasmic calcium. However, few is known about the proteins that regulate the calcium flux in this parasite. In many cells, the calcium extrusion from the cytosol is performed by plasma membrane Ca2+-ATPases and calcium/cation exchangers. The aim of this work was to identify a calcium/cation exchanger of E. histolytica and to analyze its possible role in some cellular processes triggered by calcium flux, such as the programmed cell death and in vitro virulence. By searching putative calcium/cation exchangers in the genome database of E. histolyica we identified a protein belonging to the CCX family (EhCCX). We generated a specific antibody against EhCCX, which showed that this protein was expressed in higher levels in E. histolytica than its orthologous in the non-pathogenic amoeba E. dispar. In addition, the expression of EhCCX was increased in trophozoites incubated with hydrogen peroxide. This E. histolytica exchanger was localized in the plasma membrane and in the membrane of some cytoplasmic vesicles. However, after 10 min of erythrophagocytosis, EhCCX was found predominantly in the plasma membrane of the trophozoites. On the other hand, the parasites that overexpress this exchanger contained higher cytosolic calcium levels than control, but the extrusion of calcium after the addition of hydrogen peroxide was more efficient in EhCCX-overexpressing trophozoites; consequently, the programmed cell death was retarded in these parasites. Interestingly, the overexpression of EhCCX increased the in vitro virulence of trophozoites. These results suggest that EhCCX plays important roles in the programmed cell death and in the in vitro virulence of E. histolytica.

A putative calcium-ATPase of the secretory pathway family may regulate calcium/manganese levels in the Golgi apparatus of Entamoeba histolytica.

Calcium regulates many cellular processes in protozoa, including growth, differentiation, programmed cell death, exocytosis, endocytosis, phagocytosis, fusion of the endosomes of distinct stages with phagosomes, fusion of phagosomes with lysosomes, and recycling the membrane. In Entamoeba histolytica, the protozoa responsible for human amoebiasis, calcium ions are essential for signaling pathways that lead to growth and development. In addition, calcium is crucial in the modulation of gene expression in this microorganism. However, there is scant information about the proteins responsible for regulating calcium levels in this parasite. In this work, we characterized a protein of E. histolytica that shows a close phylogenetic relationship with Ca2+ pumps that belong to the family of secretory pathway calcium ATPases (SPCA), which for several organisms are located in the Golgi apparatus. The amoeba protein analyzed herein has several amino acid residues that are characteristic of SPCA members. By an immunofluorescent technique using specific antibodies and immunoelectron microscopy, the protein was detected on the membrane of some cytoplasmic vacuoles. Moreover, this putative calcium-ATPase was located in vacuoles stained with NBD C6-ceramide, a Golgi marker. Overall, the current findings support the hypothesis that the presently analyzed protein corresponds to the SPCA of E. histolytica.

Identification of calcium-transporting ATPases of Entamoeba histolytica and cellular localization of the putative SERCA.

Calcium has an important role on signaling of different cellular processes in the protozoa parasite Entamoeba histolytica, including development and pathogenesis. However, the systems that control calcium responses in this parasite are incompletely understood. Calcium-ATPases (Ca(2+)-ATPases) are proteins that play an important role in calcium homeostasis by catalyzing the active efflux of this ion from cytoplasm and are essential to the correct functioning of the cell machinery. Here, we reported the identification of five E. histolytica genes encoding putative Ca(2+)-ATPases, three related to PMCA, and two related to organellar ATPases. RT-PCR assays showed that all those genes are expressed in trophozoites and specific antibodies against the SERCA-like member located this protein in a continuous cytoplasmic network, supporting the hypothesis that it corresponds to the Ca(2+)-ATPase responsible to sequester calcium in the endoplasmic reticulum of this parasite.

The small GTPase EhRabB of Entamoeba histolytica is differentially expressed during phagocytosis.

It has been described that the pathogenicity of Entamoeba histolytica is influenced by environmental conditions and that transcription profile changes occur during invasion, suggesting that gene expression may be involved in the virulence of this parasite. However, the molecular mechanisms that are implicated in the control of gene expression in this microorganism are poorly understood. Here, we showed that the expression of the EhRabB protein, a small GTPase involved in phagocytosis, is modified through the interaction with red blood cells. By ELISA, Western blot, and immunofluorescence assays, we observed that the expression of EhRabB diminished after 5 min of the interaction of trophozoites with red blood cells, but protein level was recovered at subsequent times. In the EhRabB amino acid sequence, we found two lysine residues that could be target for ubiquitin modification and trigger the degradation of this GTPase at early times of phagocytosis. The analysis of the expression of the EhrabB mRNA showed that the interaction of trophozoites with red blood cells produces a drastic diminishing in its half-life. In addition, promoter assays using the chloramphenicol acetyltransferase reporter gene and electrophoretic mobility shift assays experiments showed that the URE1 motif located in the promoter region of EhrabB is involved in the expression regulation of this gene during phagocytosis. Moreover, the immunolocalization of the URE1-binding protein during phagocytosis indicated that the transcription of the EhrabB gene is determined, at least in part, by the translocation of this transcription factor to nuclei. These results suggested that the expression of particular genes of this parasite is controlled at several stages.

Identification of a polypeptide containing Tudor and staphyloccocal nuclease-like domains as the sequence-specific binding protein to the upstream regulatory element 1 of Entamoeba histolytica.

Transcription initiation is the most regulated stage for the control of gene expression. This event requires that a complex of proteins called transcription factors bind to DNA through cis-regulatory elements located in the gene promoters. However, little is known about transcription regulation in Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis. Some genes encoding for proteins involved in the parasite pathogenicity contain specific upstream regulatory elements (URE1-URE5) in their promoters. Here, we identified the protein that specifically binds to the URE1 sequence (EhURE1BP). This protein contains five SNase domains and one Tudor motif, and has 21% identity and 36% similarity to the multifunctional eukaryotic protein known as the protein containing Tudor and staphyloccocal nuclease-like domains (TSN). To obtain antibodies against EhURE1BP, the recombinant protein was expressed and immunised in rabbits. Western blot and immunofluorescence assays showed that EhURE1BP is located in both nuclei and cytoplasm. Electrophoretic mobility shift assays and supershift assays demonstrated that EhURE1PB specifically binds to URE1 and that the C-terminus that includes the Tudor motif contains the DNA-binding domain of this protein. Results suggest that this TSN-like protein is the transcription factor that activates the transcription of some pathogenicity-related genes of E. histolytica.

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.