Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2.

Peroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 ± 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 ± 0.08 s-1 when the alternate active site was present as a sulfenic acid and 2.29 ± 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions.

A chalcone derivative binds a putative allosteric site of YopH: Inhibition of a virulence factor of Yersinia.

Identification of allosteric inhibitors of PTPs has attracted great interest as a new strategy to overcome the challenge of discover potent and selective molecules for therapeutic intervention. YopH is a virulence factor of the genus Yersinia, validated as an antimicrobial target. The finding of a second substrate binding site in YopH has revealed a putative allosteric site that could be further exploited. Novel chalcone compounds that inhibit PTPs activity were designed and synthesized. Compound 3j was the most potent inhibitor, interestingly, with different mechanisms of inhibition for the panel of enzymes evaluated. Further, our results showed that compound 3j is an irreversible non-competitive inhibitor of YopH that binds to a site different than the catalytic site, but close to the well-known second binding site of YopH.

Antioxidant and Antibacterial Activity of Sulfonamides Derived from Carvacrol: A Structure-Activity Relationship Study.

BACKGROUND: Bacterial resistance to antibiotics is a growing problem in all countries and has been discussed worldwide. In this sense, the development of new drugs with antibiotic properties is highly desirable in the context of medicinal chemistry. METHODOLOGY: In this paper we investigate the antioxidant and antibacterial potential of sulfonamides derived from carvacrol, a small molecule with drug-like properties. Most sulfonamides had antioxidant and antibacterial potential, especially compound S-6, derived from beta-naphthylamine. RESULTS: To understand the possible mechanisms of action involved in biological activity, the experimental results were compared with molecular docking data. CONCLUSION: This research allows appropriate discussion on the identified structure activity relationships.

Peroxiredoxin expression and redox status in neutrophils and HL-60 cells.

Peroxiredoxins (Prxs) are thiol peroxidases with a key role in antioxidant defense and redox signaling. They could be important in neutrophils for handling the large amount of oxidants that these cells produce. We investigated the redox state of Prx1 and Prx2 in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and in human neutrophils. HL-60 cell differentiation with dimethyl sulfoxide caused a large decrease in expression of both Prxs, and all-trans retinoic acid also decreased Prx1 expression. Prx1 was mostly reduced in dHL-60 cells. NADPH oxidase activation by phorbol myristate acetate (PMA) or ingestion of Staphylococcus aureus induced rapid oxidation to disulfide-linked dimers, and eventually hyperoxidation. The NADPH oxidase inhibitor, diphenyleneiodonium, prevented Prx1 dimerization in stimulated dHL-60 cells, and decreased the extent of oxidation under resting conditions. In contrast, Prx1 and Prx2 were present in neutrophils from human blood as disulfides, and PMA or S. aureus caused no further oxidation. They remained oxidized on incubation with diphenyleneiodonium in media. Although this suggests that Prx redox cycling could be deficient in neutrophils, thioredoxin expression and thioredoxin reductase activity were similar in neutrophils and dHL-60 cells. Additionally, neutrophil thioredoxin was initially reduced and underwent oxidation after PMA activation. Thus, although the Prxs respond to oxidant generation in dHL-60 cells, in neutrophils they appear "locked" as disulfides. On this basis we propose that neutrophil Prxs are inefficient antioxidants and contribute little to peroxide removal during the oxidative burst, and speculate that they might be involved in other cell processes.

Phaseolin ingestion affects vesicular traffic causing oxidative stress in the midgut of Callosobruchus maculatus larvae.

It has been reported that phaseolin, the major storage globulin of the common bean (Phaseolus vulgaris), is toxic to Callosobruchus maculatus larvae, an Old World bruchid beetle that is not capable of infesting this New World edible bean. It has also been demonstrated that vicilin, the major storage globulin found in cowpea (Vigna unguiculata) seeds, is absorbed through receptor-mediated endocytosis in the insect midgut. A putative vicilin receptor has been purified and showed high homology to α-tocopherol transfer protein. However, the ingestion of a variant vicilin purified from C. maculatus resistant seeds inhibits transcytosis, resulting in the accumulation of vicilins in the midgut cells and ultimately antibiosis. In the present work, we studied the cellular up-take of phaseolin in C. maculatus larvae with the aim of discovering if this protein is also capable of inhibiting endocytic traffic in the enterocytes. FITC-labelled vicilin and FITC-labelled phaseolin were incorporated into the diet of the larvae at a physiological concentration of 0.5% w/w. The fate of labelled and non-labelled globulins was monitored by confocal microscopy. Here we demonstrated that phaseolin is also endocytosed by enterocytes causing an accumulation of endocytic vesicles in the midgut when compared to the ingestion of vicilin obtained from a susceptible V. unguiculata cultivar. From the results obtained for HNE, MDA and TBARS, a pro-oxidative scenario was established in the intestinal epithelial cells of the larvae, which may explain the deleterious effect observed in larvae developing inside P. vulgaris seeds.

RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

Pramipexole, a Dopamine D2/D3 Receptor-Preferring Agonist, Prevents Experimental Autoimmune Encephalomyelitis Development in Mice.

Experimental autoimmune encephalomyelitis (EAE) is the most used animal model of multiple sclerosis (MS) for the development of new therapies. Dopamine receptors can modulate EAE and MS development, thus highlighting the potential use of dopaminergic agonists in the treatment of MS, which has been poorly explored. Herein, we hypothesized that pramipexole (PPX), a dopamine D2/D3 receptor-preferring agonist commonly used to treat Parkinson's disease (PD), would be a suitable therapeutic drug for EAE. Thus, we report the effects and the underlying mechanisms of action of PPX in the prevention of EAE. PPX (0.1 and 1 mg/kg) was administered intraperitoneally (i.p.) from day 0 to 40 post-immunization (p.i.). Our results showed that PPX 1 mg/kg prevented EAE development, abolishing EAE signs by blocking neuroinflammatory response, demyelination, and astroglial activation in spinal cord. Moreover, PPX inhibited the production of inflammatory cytokines, such as IL-17, IL-1ß, and TNF-α in peripheral lymphoid tissue. PPX was also able to restore basal levels of a number of EAE-induced effects in spinal cord and striatum, such as reactive oxygen species, glutathione peroxidase, parkin, and α-synuclein (α-syn). Thus, our findings highlight the usefulness of PPX in preventing EAE-induced motor symptoms, possibly by modulating immune cell responses, such as those found in MS and other T helper cell-mediated inflammatory diseases.

A porcine circovirus-2 mutant isolated in Brazil contains low-frequency substitutions in regions of immunoprotective epitopes in the capsid protein.

Porcine circovirus-2 (PCV2) is the etiologic agent of several diseases in pigs, including multi-systemic wasting syndrome (PMWS). In this work, a new mutant PCV2b was isolated from PMWS-affected pigs on a Brazilian farm. Its genome showed high sequence similarity (>99% identity) to those from a group of emerging mutants isolated from cases of PMWS outbreaks in vaccinated pigs in China, the USA and South Korea. Here, we show that these isolates share a combination of low-frequency substitutions (single amino acid polymorphisms with a frequency of ≤25%) in the viral capsid protein, mainly in regions of immunoprotective epitopes, and an additional lysine residue at position 234. These isolates were phylogenetically grouped in the PCV2b clade, reinforcing the idea of the emergence of a new group of mutants PCV2b associated with outbreaks worldwide. The identification of these polymorphisms in the viral capsid highlights the importance of considering these isolates for the development of more-effective vaccines.

Interaction of curcumin with manganese may compromise metal and neurotransmitter homeostasis in the hippocampus of young mice.

Manganese (Mn) exposure is related to industrial activities, where absorption by inhalation has high relevance. Manganism, a syndrome caused as a result of excessive accumulation of Mn in the central nervous system, has numerous symptoms similar to those seen in idiopathic Parkinson disease (IPD). Some of these symptoms, such as learning, memory, sensorial, and neurochemical changes, appear before the onset of motor deficits in both manganism and IPD. The aim of this study was to evaluate the possible neuroprotective effects of curcumin against behavioral deficits induced by Mn toxicity in young (2 months old) Swiss mice. We evaluated the effect of chronic inhalation of a Mn mixture [Mn(OAc)3 and MnCl2 (20:40 mM)], 1 h/session, three times a week, over a 14-week period on behavioral and neurochemical parameters. Curcumin was supplemented in the diet (500 or 1,500 ppm in food pellets). The Mn disrupted the motor performance evaluated in the single-pellet reach task, as well as the short- and long-term spatial memory evaluated in the step-down inhibitory avoidance task. Surprisingly, curcumin also produced similar deleterious effects in such behavioral tests. Moreover, the association of Mn plus curcumin significantly increased the levels of Mn and iron, and decreased the levels of dopamine and serotonin in the hippocampus. These alterations were not observed in the striatum. In conclusion, the current Mn treatment protocol resulted in mild deficits in motor and memory functions, resembling the early phases of IPD. Additionally, curcumin showed no beneficial effects against Mn-induced disruption of hippocampal metal and neurotransmitter homeostasis.

Identification of an Emergent Porcine Circovirus-2 in Vaccinated Pigs from a Brazilian Farm during a Postweaning Multisystemic Wasting Syndrome Outbreak.

Three porcine circovirus-2 strains were isolated from pigs on a Brazilian farm during an outbreak, indicating a vaccine failure. They present identical genomic sequences, with high identities to other isolates that were also related to vaccination failures, supporting the recent theory about an antigen drift being associated with vaccine failures throughout the world.

Antidepressant-like action of the bark ethanolic extract from Tabebuia avellanedae in the olfactory bulbectomized mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Tabebuia avellanedae Lorentz ex Griseb is a plant employed in tropical America folk medicine for the treatment of several diseases, including depressive disorders. AIM OF THE STUDY: To investigate the ability of Tabebuia avellanedae ethanolic extract (EET) administered chronically to cause an antidepressant-like effect in the tail suspension test (TST), a predictive test of antidepressant activity, and to reverse behavioral (hyperactivity, anhedonic-like behavior and increased immobility time in the TST) and biochemical changes induced by olfactory bulbectomy (OB), a model of depression, in mice. MATERIALS AND METHODS: Mice were submitted to OB to induce depressive-related behaviors, which were evaluated in the open-field test (hyperactivity), splash test (loss of motivational and self-care behavior indicative of an anhedonic-like behavior) and TST (increased immobility time). Phosphorylation levels of Akt, GSK-3ß, ERK1/2 and CREB, as well as BDNF immunocontent, were evaluated in the hippocampus of bulbectomized mice or sham-operated mice treated for 14 days by p.o. route with EET or vehicle. RESULTS: EET (10 and 30mg/kg) given 14 days by p.o route to mice reduced the immobility time in the TST without altering locomotor activity, an indicative of an antidepressant-like effect. EET per se increased both CREB (Ser(133)) and GSK-3ß (Ser(9)) phosphorylation (at doses of 10-30 and 30mg/kg, respectively) in sham-operated mice. OB caused hyperactivity, loss of motivational and self-care behavior, increased immobility time in the TST and an increase in CREB and ERK1 phosphorylation, as well as BDNF immunocontent. EET abolished all these OB-induced alterations except the increment of CREB phosphorylation. Akt (Ser(473)) and ERK2 phosphorylation levels were not altered in any group. CONCLUSIONS: EET ability to abolish the behavioral changes induced by OB was accompanied by modulation of ERK1 and BDNF signaling pathways, being a promising target of EET. Results indicate that this plant could constitute an attractive strategy for the management of depressive disorders, once more validating the traditional use of this plant.

Detection of avian paramyxoviruses in migratory and resident birds in the state of Rio de Janeiro, Brazil.

Paramyxoviruses and avian influenza viruses are present worldwide, and wild birds are known natural reservoirs of these viruses. This study monitored the circulation of these viruses in migratory and resident coastal birds captured in the state of Rio de Janeiro, Brazil. In total, 494 birds were trapped, and their fecal samples were collected and inoculated into embryonated chicken eggs. The allantoic fluids were evaluated using a hemagglutination test and PCR amplification of the genes of the M and L proteins of influenza A virus and paramyxovirus, respectively. Avian paramyxovirus was detected in 5 (1.01%) of the birds. The majority of these viruses were isolated from migratory birds classified into the order Charadriiformes (families Scolopacidae and Charadriidae). Four samples were characterized as avian paramyxovirus serotype-2 (APMV-2) by a hemagglutination inhibition test. These results reinforce the importance of continuous surveillance of wild species in Brazil.

Retinol and retinoic acid modulate catalase activity in Sertoli cells by distinct and gene expression-independent mechanisms.

Vitamin A (retinol) exerts a major role in several biological functions. However, it was observed that retinol induces oxidative stress on different cellular types. Catalase (EC 1.11.1.6; CAT) is a hydrogen peroxide metabolizing enzyme, and its activity and expression is widely used as an index to measure oxidative stress and perturbations in the cellular redox state. The aim of this study was to investigate the effects of retinol and its major biologically active metabolite, all-trans retinoic acid (RA), on CAT regulation. For this purpose, cultured Sertoli cells (a physiological target of vitamin A) were treated with retinol or RA. Retinol (7 microM, 14 microM) and RA (100 nM, 1 microM) enhanced intracellular reactive species production and increased CAT activity after 24 h of treatment. Retinol increased CAT immunocontent but did not alter CAT mRNA expression, while the increase in CAT activity by RA was not related to alterations in immunocontent or mRNA expression. In vitro incubation of purified CAT with retinol or RA did not alter enzyme activity.