Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity.

BACKGROUND: It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES: To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS: CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20: showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS: The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Characterisation of the in vitro activity of a Nitazoxanide-N-methyl-1H-benzimidazole hybrid molecule against albendazole and nitazoxanide susceptible and resistant strains of Giardia intestinalis and its in vivo giardicidal activity

BACKGROUND It was previously demonstrated that CMC-20, a nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, had higher in vitro activity against Giardia intestinalis WB strain than metronidazole and albendazole and similar to nitazoxanide. OBJETIVES To evaluate the in vitro activity of CMC-20 against G. intestinalis strains with different susceptibility/resistance to albendazole and nitazoxanide and evaluate its effect on the distribution of parasite cytoskeletal proteins and its in vivo giardicidal activity. METHODS CMC-20 activity was tested against two isolates from patients with chronic and acute giardiasis, an experimentally induced albendazole resistant strain and a nitazoxanide resistant clinical isolate. CMC-20 effect on the distribution of parasite cytoskeletal proteins was analysed by indirect immunofluorescence and its activity was evaluated in a murine model of giardiasis. FINDINGS CMC-20 showed broad activity against susceptible and resistant strains to albendazole and nitaxozanide. It affected the parasite microtubule reservoir and triggered the parasite encystation. In this process, alpha-7.2 giardin co-localised with CWP-1 protein. CMC-20 reduced the infection time and cyst load in feces of G. muris infected mice similar to albendazole. MAIN CONCLUSIONS The in vitro and in vivo giardicidal activity of CMC-20 suggests its potential use in the treatment of giardiasis.

Morphological and oxidative alterations on Sertoli cells cytoskeleton due to retinol-induced reactive oxygen species.

Retinol (vitamin A) is involved in several cellular processes, like cell division, differentiation, transformation and apoptosis. Although it has been shown that retinol is a limitant factor for all these processes, the precise mechanisms by which retinol acts are still unknown. In the present study we hypothesised that alterations in the cytoskeleton of Sertoli cells induced by retinol supplementation could indicate an adaptive maintenance of its functions, since it plays an important role in the transformation process that we observed. Previous results demonstrated that Sertoli cells treated with retinol showed an oxidative imbalance, that leads the cell to two phenotypes: apoptosis or transformation. Our group has identified characteristics of Sertoli cells transformed by retinol which results in normal cell functions modification. In the present study the actin filament fluorescence assay and the deformation coefficient showed a modification in the morphology induced by retinol. We also observed an oxidative alteration in isolated cytoskeleton proteins and did not show alterations when these proteins are analyzed by electrophoreses. Our results showed an increase in mitochondria superoxide production and a decrease in nitric oxide levels. All results were partially or completely reverted by co-treatment of the antioxidant Trolox. These findings suggest that the cytoskeleton components suffer individual alterations in different levels and that these alterations generate a global phenotype modification and that these processes are probably ROS dependent. We believe that the results from this study indicate an adaptation of the cytoskeleton to oxidative imbalance since there was not a loss of its function.

The anti-inflammatory and cholinesterase inhibitor bifunctional compound IBU-PO protects from beta-amyloid neurotoxicity by acting on Wnt signaling components.

Changes in signal transduction are implicated in neuronal responses to the Alzheimer's amyloid-beta-peptide (Abeta), which include neurotransmitter systems and pathways involved in the maintenance of the nervous system. We report here that a new bifunctional compound IBU-PO, which combines a non-steroidal anti-inflammatory drug (NSAID) (Ibuprofen) and a cholinesterase (ChE) inhibitor (Octyl-Pyridostigmine), is neuroprotective against Abeta-neurotoxicity, and its activity is associated to Wnt signaling components in rat hippocampal and mouse cortical neurons. IBU-PO (0.01-1 microM) inhibits glycogen-synthase-kinase-3beta (GSK-3beta) and stabilizes cytoplasmic beta-catenin reverting the silencing of the Wnt pathway caused by Abeta-toxicity and GSK-3beta overexpression. In addition, IBU-PO enhances, dose-dependently, the non-amyloidogenic amyloid precursor protein (APP) cleavage by increasing secreted APP and decreasing endogenous Abeta1-40 in rat hippocampal neurons.

Wnt-3a overcomes beta-amyloid toxicity in rat hippocampal neurons.

The aim of this study was to evaluate whether the direct activation of the Wnt signaling pathway by its endogenous Wnt-3a ligand prevents the toxic effects induced by amyloid-beta-peptide (Abeta) in rat hippocampal neurons. We report herein that the Wnt-3a ligand was indeed able to overcome toxic effects induced by Abeta in hippocampal neurons, including a neuronal impairment on cell survival, an increase in glycogen synthase kinase-3beta (GSK-3beta) and tau phosphorylation, a decrease in cytoplasmic beta-catenin and a decrease in the expression of the Wnt target gene engrailed-1. We further demonstrate that Wnt-3a protects hippocampal neurons from apoptosis induced by Abeta. Our results support the hypothesis that a loss of function of Wnt signaling may play a role in the progression of neurodegenerative diseases such as Alzheimer's disease.

S100B protein stimulates calcineurin activity.

S100B is a calcium binding protein from astrocytes that regulates protein phosphorylation by binding to substrates and protein kinases. S100B might also regulate protein phosphatases and this was investigated for protein phosphatase 2B (calcineurin). The results indicate that S100B (5-10 microM) increased the activity of both purified and cytoskeletal calcineurin in a Ca-dependent manner. This effect was blocked by a specific inhibitor of calcineurin activity, but not by TRTK-12 (an inhibitor of S100B binding to other protein targets). The present results and the known co-localization of S100B and calcineurin in the astrocyte cytoskeleton suggest that S100B may play a role in the phosphorylation state of cytoskeletal proteins.

Changes in the expression of cytoskeletal proteins in the CNS of transferrin transgenic mice.

Apotransferrin injected intracranially into young rats has been shown in our laboratories to induce an early differentiation of oligodendroglial cells and an increased deposition of myelin. The expression of some myelin-specific proteins such as myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and of their mRNAs were significantly increased in these animals. Also, in the cytoskeleton obtained from isolated myelin, it was found that several microtubule associated proteins (MAPs), particularly the stable tubule only peptide (STOP) and MAP 1B, as well as actin and tubulin were markedly increased. In the present paper, we compare the changes in expression of brain and myelin cytoskeletal proteins in a newly generated transferrin transgenic mouse (Tg), overexpressing the human transferrin gene, with the results obtained in aTf-injected rats. In the myelin cytoskeletal fraction of Tg mice there was a significant increase in the expression of MBP, tubulin, tau and STOP, similarly to what was previously found in the aTf-injected rats. Immunohistochemical studies showed that a variance with what occurs in the aTf-injected model, in which the above mentioned changes were limited to the corpus callosum, in the Tg mice the changes in expression of cytoskeletal proteins were observed in the various anatomical areas studied such as cerebral cortex, brain stem and cerebellum. There was also an increased expression of neurofilaments in the Tg animals, in contrast with results obtained in aTf-injected rats, suggesting that in the Tg mice, the continuous overexpression of Tf might also induce some neuronal changes. Changes in tau, total and acetylated tubulin and MAP 1B were observed in both neurons and OLGc. The increase in STOP was more significant in OLGc while the changes in MAP2 were exclusively found in neurons.

Ghost protein damage by peroxynitrite and its protection by melatonin.

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 microM ONOO-) disappearance at pH 7. 4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ss-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 microM ONOO-). However, a lack of effect of ss-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that (ONOO-)-induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 microM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Ghost protein damage by peroxynitrite and its protection by melatonin

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 µM ONOO-) disappearance at pH 7.4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ß-mercaptoethanol (2 percent) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50 percent at 250 µM ONOO-). However, a lack of effect of ß-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that ONOO--induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox® (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90 percent) at 500 µM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes

Cytoskeletal organization and incorporation of beta 3 integrin in thrombin-stimulated platelets: effect of acetylsalicylic acid.

Platelet stimulation by agonists is followed by changes in cytoskeletal organization that includes actin polymerization and association of the membrane skeleton (which is connected with the integrin alpha IIb beta 3) with the underlying cytoplasmic actin filaments. The effect of orally administered acetylsalicylic acid to healthy volunteers on incorporation of contractile protein and beta 3 integrin into the cytoskeletal core of thrombin-stimulated platelets was studied. Stimulation was followed by increased contractile protein and beta 3 incorporation into the cytoskeleton. Acetylsalicylic acid intake resulted in decreased incorporation of myosin and actin (32% and 20%, respectively), and a decrease (36%) in the association of beta 3 integrin with the cytoskeletal elements was evident. In conclusion, we have shown that acetylsalicylic acid, besides the known inhibitory effect on thromboxane synthesis, promotes changes in the cytoskeletal organization of thrombin-stimulated platelets that could limit thrombus formation.

Human sperm beta-glucuronidase is poorly extractable by Triton X-100.

A part of sperm glycosidase activities was detected as detergent-insoluble after sequential extractions with Triton X-100. Sixty per cent of total beta-glucuronidase activity was found in the detergent-insoluble fraction. This portion of beta-glucuronidase was resistant to extractions in the presence of 1 M KCl, chaotropic agents, colchicine or cytochalasine B, being only partially solubilized by 3 M KCl or DNAse I treatment. Results demonstrate that beta-glucuronidase is tightly associated to the Triton X-100 resistant fraction.