Irreversible inactivation of trypanothione reductase by unsaturated Mannich bases: a divinyl ketone as key intermediate

Trypanothione reductase is a flavoenzyme unique to trypanosomatid parasites. Here we show that unsaturated Mannich bases irreversibly inactivate trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The inhibitory potency of the compounds strongly increased upon storage of the DMSO stock solutions. HPLC, NMR, and mass spectrometry data of potential intermediates revealed a divinyl ketone as the active compound inactivating the enzyme. ESI- and MALDI-TOF mass spectrometry of trypanothione reductase modified by the Mannich base or the divinyl ketone showed specific alkylation of the active site Cys52 by a 5-(2'chlorophenyl)-3-oxo-4-pentenyl substituent. The reaction mechanism and the site of alkylation differ from those in Plasmodium falciparum thioredoxin reductase where the C-terminal redox active dithiol is modified. After deamination, unsaturated Mannich bases are highly reactive in polycondensation with trypanothione. Interaction of these compounds with both trypanothione and trypanothione reductase could account for their potent trypanocidal effect against Trypanosoma brucei.     

Design and synthesis of a series of melamine-based nitroheterocycles with activity against Trypanosomatid parasites

The parasites that give rise to human African trypanosomiasis (HAT) are auxotrophs for various nutrients from the human host, including purines. They have specialist nucleoside transporters to import these metabolites. In addition to uptake of purine nucleobases and purine nucleosides, one of these transporters, the P2 transporter, can carry melamine derivatives; these derivatives are not substrates for the corresponding mammalian transporters. In this paper, we report the coupling of the melamine moiety to selected nitro heterocycles with the aim of selectively delivering these compounds to the parasites. Some compounds prepared have similar in vitro trypanocidal activities as melarsoprol, the principal drug used against late-stage HAT, with 50% growth inhibitory concentrations in the submicromolar range. Selected compounds were also evaluated in vivo in rodent models infected with Trypanosoma brucei brucei and T. brucei rhodesiense and showed pronounced activity and in two cases were curative without overt signs of toxicity. Compounds were also tested against other trypanosomatid pathogens, Leishmania donovani and Trypanosoma cruzi, and significant activity in vitro was noted for T. cruzi against which various nitro heterocycles are already registered for use.     

Beneficial effects of vitamin C and vitamin E on reserpine-induced oral dyskinesia in rats: critical role of striatal catalase activity

Oral dyskinesias are implicated in a series of neuropathologies and have been associated to an increase in oxidative stress. Several antioxidants, including vitamin E, decrease reserpine-induced oral dyskinesia (OD) in rodents and we have described a protective role of striatal catalase against the development of OD. The aim of this study was to verify the effects of vitamin C alone or in combination with vitamin E on reserpine-induced OD as well as to determine a possible role of catalase in the antidyskinetic property of these vitamins. Different doses of vitamin C attenuated reserpine-induced increase in OD. A similar treatment with an effective dose of vitamin C concomitant to an effective dose of vitamin E potentiated the antidyskinetic effect of both vitamins when administered alone. The administration of these vitamins alone produced an increase in striatal catalase activity that likewise was potentiated by their combined administration. In addition, the antidyskinetic property of vitamin E and vitamin C was abolished by a concomitant treatment with the catalase inhibitor aminotriazole. These results indicate a beneficial effect of these vitamins and reinforce the critical role of striatal catalase against the development of oral dyskinesias.     

Panicolytic-like effect induced by the stimulation of GABAA and GABAB receptors in the dorsal periaqueductal grey of rats

Activation of GABA(A) and benzodiazepine receptors within the dorsal periaqueductal grey inhibits the escape behaviour evoked by the electrical stimulation of this midbrain area, a defensive reaction that has been related to panic. Nevertheless, there is no evidence indicating whether the same antiaversive effect is also observed in escape responses evoked by species-specific threatening stimuli. In the present study, male Wistar rats were injected intra-dorsal periaqueductal grey with the benzodiazepine receptor agonist midazolam (10, 20 and 40 nmol), the GABA(A) receptor agonist muscimol (2, 4 and 8 nmol), the GABA(B) receptor agonist baclofen (2, 4 and 8 nmol), or with the benzodiazepine inverse agonist FG 7142 (20, 40 and 80 pmol) and tested in an ethologically-based animal model of anxiety, the elevated T-maze. Besides escape, this test also allows the measurement of inhibitory avoidance which has been related to generalised anxiety disorder. Midazolam, muscimol and baclofen impaired escape, a panicolytic-like effect, without altering inhibitory avoidance. FG 7142, on the other hand, facilitated both avoidance and escape reactions, suggesting an anxiogenic and panicogenic-like effect, respectively. The data suggest that GABA(A)/benzodiazepine and GABA(B) receptors within the dorsal periaqueductal grey are involved in the control of escape behaviour and that a failure in this regulatory mechanism may be of importance in panic disorder.     

Chronic corticosterone treatment increases the endocannabinoid 2-arachidonylglycerol in the rat amygdala

This research was designed to examine the effect of three weeks of administration of corticosterone (20 mg/kg) on endocannabinoid content and cannabinoid CB1 receptor binding in the amygdala. It was found that the endocannabinoid 2-arachidonylglycerol was significantly increased in the amygdala following chronic corticosterone treatment. However, there was no change in either the maximal binding (Bmax) or binding affinity (KD) of [3H]-CP 55,940 to the CB1 receptor in the amygdala. Given the role of amygdalar endocannabinoids in the regulation of emotionality, this suggests that the ability of glucocorticoids to influence affective behavior may involve interactions with regulation of endocannabinoid content.     

Diffuse lamellar keratitis complicating laser in situ keratomileusis: post-marketing surveillance of an emerging disease in British Columbia, Canada, 2000-2002

PURPOSE: To describe a surveillance system and summarize data between January 2000 and December 2002 regarding diffuse lamellar keratitis (DLK), a complication of laser in situ keratomileusis (LASIK) surgery. SETTING: Community-based clinics in British Columbia, Canada, in which LASIK surgery is performed. METHODS: Monthly, all clinics in which LASIK is performed reported the number of LASIK procedures and nonnominal cases of DLK (by grade and onset date) to the British Columbia Centre for Disease Control. Diffuse lamellar keratitis outbreaks were investigated, and prevention and control measures were recommended. RESULTS: From 2000 to 2002, approximately 72,000 LASIK procedures were performed, with a mean DLK incidence rate of 0.67% (95% confidence interval, 0.61-0.73). The overall proportion of DLK cases attributed to outbreaks was 64%, decreasing from 72% in 2000 to 40% in 2003. CONCLUSIONS: An effective DLK surveillance program was implemented at all laser refractive clinics in British Columbia. Reported DLK incidence was 0.67 cases per 100 procedures, with 64% occurring in outbreaks.     

Transport deficient (TR-) hyperbilirubinemic rats are resistant to acetaminophen hepatotoxicity

The biliary excretion of acetaminophen (APAP) is reduced in transport deficient (TR⁻) hyperbilirubinemic rats lacking the multidrug resistance-associated protein 2 (Mrp2). This mutant strain of Wistar rats has impaired biliary excretion of organic anions and increased hepatic glutathione. The rational for this study was to determine if there is an altered risk for liver damage by APAP in the absence of Mrp2. Therefore, the susceptibility of TR⁻ rats to APAP hepatotoxicity was investigated. Male Wistar and TR⁻ rats were fasted overnight before APAP treatment (1 g/kg). Hepatotoxicity was assessed 24 h later by plasma sorbitol dehydrogenase activity and histopathology. In other studies, TR⁻ rats received buthionine sulfoximine before APAP to reduce hepatic glutathione to values similar to those in Wistar rats. mRNA expression of APAP metabolizing enzymes was also measured in naïve animals. Wistar rats treated with APAP showed significant elevations in plasma sorbitol dehydrogenase activity, while no increases in enzyme activity were observed in TR⁻ rats. Histopathology was in agreement. Hepatic non-protein sulfhydryls were significantly lower in Wistar rats receiving APAP than in TR⁻ rats. TR⁻ rats treated with buthionine sulfoximine and APAP showed dramatic increases in hepatotoxicity. TR⁻ rats had increased mRNA expression of several APAP metabolizing enzymes. Mrp2 expression not only is important in biliary excretion, but also influences the toxic potential of reactive intermediates by controlling intrahepatic GSH and possibly drug metabolism.     

Cell cycle arrest and proapoptotic effects of the anticancer cyclodepsipeptide serratamolide (AT514) are independent of p53 status in breast cancer cells

In a search for new anticancer agents, we have identified serratamolide (AT514), a cyclodepsipeptide from Serratia marcescens 2170 that induces cell cycle arrest and apoptosis in various cancer cell lines. A cell viability assay showed that the concentrations that cause 50% inhibition (IC50) in human cancer cell lines range from 5.6 to 11.5 microM depending on the cell line. Flow cytometry analysis revealed that AT514 caused cell cycle arrest in G0/G1 or cell death, depending on the cell type and the length of time for which the cells were exposed to the drug. Subsequent studies revealed that AT514-induced cell death is caused by apoptosis, as indicated by caspases activation (8, 9, 2 and 3) and cleavage of poly (ADP-ribose) polymerase (PARP), release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, and the appearance of apoptotic bodies and DNA laddering. Alterations in protein levels of Bcl-2 family members might be involved in the mitochondrial disruption observed. AT514 induced p53 accumulation in wild-type p53 cells but cell death was observed in both deficient and wild-type p53 cells. Our results indicate that AT514 induces cell cycle arrest and apoptosis in breast cancer cells irrespectively of p53 status, suggesting that it might represent a potential new chemotherapeutic agent.     

Aging of porous silicon in physiological conditions: cell adhesion modes on scaled 1D micropatterns

The surface properties of porous silicon (PSi) evolve rapidly in phosphate-buffered saline. X-ray photoelectron spectra indicate the formation of a Si-OH and C-O enriched surface, which becomes increasingly hydrophilic with aging time. Multiscale stripe micropatterns of Si and PSi have been fabricated by means of a high-energy ion-beam irradiation process. These micropatterns have been aged in physiological conditions and used to analyze human mesenchymal stem cell (hMSC) adhesion. The actin cytoskeleton of hMSCs orients following the uniaxial micropatterns. In the wider Si stripes, hMSCs are dominantly located on Si areas. However, for reduced Si widths, adhesion is avoided on PSi by a split assembly of the actin cytoskeleton on two parallel Si areas. These results confirm that nanostructured Si-OH/C-O-rich surfaces with hydrophilic character are specially adapted for the creation of cell adhesion surface contrasts.     

Multiple exposures to familiar conspecific withdrawal is a novel robust stress paradigm in ewes

Paradigms used so far to study the effects of social isolation in sheep confound the effects of social isolation with those of other stressors (e.g. new environment) and showed contradictory effects after multiple social isolations. We propose here to characterize and examine the repeatable effects of social isolation induced by the familiar conspecific withdrawal (FCW). This latter test consists of socially isolating the ewe by the removal of group mates from the room test for 3 h. Behavioral and endocrine responses of adult ovariectomized-estradiol implanted ewes were compared 90 min before and 90 min after FCW, which was applied three times every fourteen days. We observed that each FCW induced significant increases in plasma cortisol level, in the number of vocalizations, foot pawing, circling attempts and a significant decrease in time spent lying down. An increase in plasma cortisol levels and decrease in duration of maintenance behaviors were significantly lower after the third FCW than after the first one. These differences could be explained by higher plasma cortisol levels and lower duration of maintenance behaviors before the third FCW than before the first FCW suggesting an anticipation of the social isolation period. These data indicate that social isolation is sufficient to induce distress with stable stressful responses after multiple exposures to familiar conspecific withdrawal.