Soyuretox, an Intrinsically Disordered Polypeptide Derived from Soybean (Glycine Max) Ubiquitous Urease with Potential Use as a Biopesticide.

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.

The side-by-side exploratory test: a simple automated protocol for the evaluation of adult zebrafish behavior simultaneously with social interaction.

The assessment of shoaling in adult zebrafish is technically difficult, but important, given their social nature. The present study aimed to characterize a new protocol using simple automated tracking software to evaluate general behavior and social interaction simultaneously. To this end, we used a single tank with a central transparent glass division and placed one zebrafish on each side for 5 min. This strategy allows fish to interact visually at the same time that individual automated evaluation of behavior can be easily performed. Our results showed that, when two fish are placed side-by-side, there is an increase in their height in the tank compared with isolated fish and they remain close to each other. The pharmacological treatments with benzodiazepines (bromazepam and clonazepam) and the serotonergic drugs buspirone, fluoxetine, and escitalopram did not affect locomotion at the concentrations tested, except for the highest concentration of buspirone. Nevertheless, benzodiazepines increased interfish distance (i.e. reduced shoaling behavior) and serotonergic drugs elevated height in the tank. These results support the use of the side-by-side exploratory test for behavioral studies with the zebrafish, including high-throughput behavioral screening for antidepressants and anxiolytics.

Mild hyperhomocysteinemia reduces the activity and immunocontent, but does not alter the gene expression, of catalytic α subunits of cerebral Na+,K+-ATPase.

Na(+),K(+)-ATPase is a membrane protein which plays a key role in the maintenance of ion homeostasis that is necessary to neuronal excitability, secondary transport and neurotransmitter uptake. Mild hyperhomocysteinemia leads to several clinical manifestations and particularly cerebral diseases; however, little is known about the mechanisms of homocysteine on cerebral Na(+),K(+)-ATPase. In the present study, we investigated the effect of mild hyperhomocysteinemia on the activity, the immunocontent of catalytic subunits (α1, α2, and α3) and the gene expression of this enzyme. We used the experimental model of mild hyperhomocysteinemia that was induced by homocysteine administration (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th postpartum day. Controls received saline in the same volumes. Results showed that mild hyperhomocysteinemia significantly decreased the activity and the immunocontent of the α 1 and α 2 subunits of the Na(+),K(+)-ATPase in cerebral cortex and hippocampus of adult rats. On the other hand, we did not observe any change in levels of Na(+),K(+)-ATPase mRNA transcripts in such cerebral structures of rats after chronic exposure to homocysteine. The present findings support that the homocysteine modulates the Na(+),K(+)-ATPase and this could be associated, at least in part, with the risk to the development of cerebral diseases in individuals with mild hyperhomocysteinemia.

Y-Maze memory task in zebrafish (Danio rerio): the role of glutamatergic and cholinergic systems on the acquisition and consolidation periods.

The interest in the behavioral features of zebrafish has significantly increased over the past two decades. However, most available protocols have used longer training periods and have been based on reinforcement/reward or avoidance. The Y-Maze memory task has the advantage of using a simple and rapid training session, but it has not been established in zebrafish. Here, we have characterized this task for zebrafish, with the addition of pharmacological interventions in the acquisition and consolidation memory phases. The results show that zebrafish spend more time in the novel arm than in the other arms of the Y-Maze, both in response to novelty and spatial memory training-test intervals (TTIs). We have also studied the involvement of the glutamatergic and cholinergic systems with pre- and post-training treatments with the NMDA receptor antagonist MK-801 (20 µM) and the cholinergic blocker scopolamine (200 µM). After 1h of TTI, pre-training MK-801 and scopolamine-treated fish reduced their exploration of the novel arm when compared to the control group, with no changes in their locomotor activity. Post-training of MK-801 treatment also impaired their Y-Maze performance, while post-training of any scopolamine treatment failed to affect novel arm exploration. In conclusion, the Y-Maze memory task can be reliably used for zebrafish, providing a new, rapid, and preference/avoidance independent task for the study of memory in this teleost. In addition, our results highlight the implication of the glutamatergic and cholinergic systems in the memory of zebrafish.

Long-term methionine exposure induces memory impairment on inhibitory avoidance task and alters acetylcholinesterase activity and expression in zebrafish (Danio rerio).

Hypermethioninemic patients exhibit a variable degree of neurological dysfunction. However, the mechanisms involved in these alterations have not been completely clarified. Cholinergic system has been implicated in many physiological processes, including cognitive performances, as learning, and memory. Parameters of cholinergic signaling have already been characterized in zebrafish brain. Since zebrafish is a small freshwater teleost which is a vertebrate model for modeling behavioral and functional parameters related to human pathogenesis and for clinical treatment screenings, in the present study we investigated the effects of short- and long-term methionine exposure on cognitive impairment, AChE activity and gene expression in zebrafish. For the studies, animals were exposed at two methionine concentrations (1.5 and 3.0 mM) during 1 h or 7 days (short- or long-term treatments, respectively). We observed a significant increase in AChE activity of zebrafish brain membranes after long-term methionine exposure at 3.0 mM. However, AChE gene expression decreased significantly in both concentrations tested after 7 days of treatment, suggesting that post-translational events are involved in the enhancement of AChE activity. Methionine treatment induces memory deficit in zebrafish after long-term exposure to this amino acid, which could be related, at least in part, with cognitive impairment observed in hypermethioninemia. Therefore, the results here presented raise a new perspective to use the zebrafish as a complementary vertebrate model for studying inborn errors of metabolism, which may help to better understand the pathophysiology of this disease.

Chronic mild hyperhomocysteinemia alters ectonucleotidase activities and gene expression of ecto-5'-nucleotidase/CD73 in rat lymphocytes.

Since mild hyperhomocysteinemia is a risk factor for cardiovascular and cerebral diseases and extracellular nucleotides/nucleosides, which are controlled by the enzymatic action of ectonucleotidases, can induce an immune response, in the present study, we investigated the effect of chronic mild hyperhomocysteinemia on ectonucleotidase activities and expression in lymphocytes from mesenteric lymph nodes and serum of adult rats. For the chronic chemically induced mild hyperhomocysteinemia, Hcy (0.03 µmol/g of body weight) or saline (control) were administered subcutaneously from the 30th to the 60th day of life. Results showed that homocysteine significantly decreased ATP, ADP, and AMP hydrolysis in lymphocytes of adult rats. E-NTPDases transcriptions were not affected, while the ecto-5'-nucleotidase transcription was significantly decreased in mesenteric lymph nodes of hyperhomocysteinemic rats. ATP, ADP, and AMP hydrolysis were not affected by homocysteine in rat serum. Our findings suggest that Hcy in levels similar to considered risk factor to development of vascular diseases modulates the ectonucleotidases, which could lead to a pro-inflammatory status.

Atrazine and Diuron Effects on Survival, Embryo Development, and Behavior in Larvae and Adult Zebrafish.

Atrazine and Diuron are widely used herbicides. The use of pesticides contaminates the aquatic environment, threatening biodiversity and non-target organisms such as fish. In this study, we investigated the effects of acute exposure for 96 h hours to atrazine and diuron commercial formulations in zebrafish (Danio rerio, wild-type AB) embryos and larvae and adult stages. We observed a significant concentration-dependent survival decrease and hatching delays in animals exposed to both herbicides and in the frequency of malformations compared to the control groups. Morphological defects included cardiac edema, tail reduction, and head malformation. At 7 days post-fertilization (dpf), atrazine exposure resulted in a reduction in the head length at 2, 2.5, and 5 mg/L and increased the ocular distance at 1, 2, 2.5, and 5 mg/L atrazine when compared to controls. At the same age, diuron increased the ocular distance in animals exposed to diuron (1.0 and 1.5 mg/L) and no effects were observed on the head length. We also evaluated a behavioral repertoire in larvae at 7 dpf, and there were no significant differences in distance traveled, mean speed, time in movement, and thigmotaxis for atrazine and diuron when animals were individually placed in a new environment. The cognitive ability of the larvae was tested at 7 dpf for avoidance and optomotor responses, and neither atrazine nor diuron had significant impacts when treated groups were compared to their corresponding controls. Adults' behavior was evaluated 7 and 8 days after the end of the acute herbicide exposure. Exploration of a new environment and associated anxiety-like parameters, social interaction, and aggressiveness were not altered. Our results highlight the need for further studies on the sublethal effects of both herbicides and the consideration of the effects of commercial formulas vs. isolated active ingredients. It also emphasizes the need to take sublethal effects into consideration when establishing the environmental limits of residues.

Acute restraint stress in zebrafish: behavioral parameters and purinergic signaling.

Despite the extensive knowledge about the effects of acute restraint stress (ARS) in rodents, zebrafish research is still elementary in this field, and the consequences of stress on purinergic system are unclear. Therefore, we evaluated the effects of ARS on behavior, biochemical, and molecular parameters in zebrafish brain. Animals were submitted to a 90 min ARS protocol and tested for anxiety levels, exploratory behavior, and memory performance. Furthermore, we analyzed ectonucleotidase and adenosine deaminase activities and their gene expression profile, as well as transcription of adenosine receptors. ARS increased anxiety, but did not impair locomotion or cognition. ARS significantly increased ATP hydrolysis, decreased cytosolic ADA activity, and changed the entpd and adora gene expression. In conclusion, ARS disturbed zebrafish behavior, and we hypothesize that the augmentation in adenosine-mediated signaling may be a strategy to reestablish homeostasis and normal behavior after a stressful event.

Role of antioxidants on Na(+),K (+)-ATPase activity and gene expression in cerebral cortex of hyperprolinemic rats.

Considering that Na(+),K(+)-ATPase is an embedded-membrane enzyme and that experimental chronic hyperprolinemia decreases the activity of this enzyme in brain synaptic plasma membranes, the present study investigated the effect of chronic proline administration on thiobarbituric acid-reactive substances, as well as the influence of antioxidant vitamins E plus C on the effects mediated by proline on Na(+),K(+)-ATPase activity in cerebral cortex of rats. The expression of Na(+),K(+)-ATPase catalytic subunits was also evaluated. Results showed that proline increased thiobarbituric acid-reactive substances, suggesting an increase of lipid peroxidation. Furthermore, concomitant administration of vitamins E plus C significantly prevented the increase of lipid peroxidation, as well as the inhibition of Na(+),K(+)-ATPase activity caused by proline. We did not observe any change in levels of Na(+),K(+)-ATPase mRNA transcripts after chronic exposure to proline and vitamins E plus C. These findings provide insights into the mechanisms through which proline exerts its effects on brain function and suggest that treatment with antioxidants may be beneficial to treat neurological dysfunctions present in hyperprolinemic patients.

Decreased convulsive threshold and memory loss after anti-NMDAR positive CSF injection in zebrafish.

Anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis initially promotes memory deficits, behavioral changes, and epileptic seizures. We developed a new animal model of anti-NMDAR encephalitis using a single cerebroventricular injection of CSF from patients in adult zebrafish. We observed a reduction of the seizure threshold and recent memory deficits in those animals injected with CSF from patients with anti-NMDAR encephalitis. The locomotor activity was similar in the CSF and control groups. This zebrafish model consistently recapitulates symptoms seen in patients with anti-NMDAR encephalitis. It may provide a reliable, fast and cost-effective platform to investigate new therapeutic strategies to anti-NMDAR encephalitis.

Zebrafish as a model for inflammation and drug discovery.

Zebrafish is a small teleost (bony) fish used in many areas of pharmacology and toxicology. This animal model has advantages for the discovery of anti-inflammatory drugs, such as the potential for real-time assessment of cell migration mechanisms. Additionally, zebrafish display a repertoire of inflammatory cells, mediators, and receptors that are similar to those in mammals, including humans. Inflammatory disease modeling in either larvae or adult zebrafish represents a promising tool for the screening of new anti-inflammatory compounds, contributing to our understanding of the mechanisms involved in chronic inflammatory conditions. In this review, we provide an overview of the characterization of inflammatory responses in zebrafish, emphasizing its relevance for drug discovery in this research area.

Tryptophan alleviates neuroendocrine and behavioral responses to stress in zebrafish.

Stressful experiences are related to the triggering of anxiety and mood disorders. Tryptophan (amino acid precursor of serotonin synthesis) emerges as important treatment of these disorders. Here, we evaluate the effects of pre-treatment with tryptophan (300 mg/L) and fluoxetine (50 µg/L) in response to acute stress in zebrafish. Overall, acute stress decreased the distance traveled, entries and time in top of tank, as well as increased the cortisol levels, demonstrating an anxiogenic behavior. Tryptophan and fluoxetine prevented anxiogenic effects. This study showed the importance of tryptophan and fluoxetine in the regulation of stress and anxiety-like behavior in adult zebrafish. Collectively, our data support tryptophan effects on stress responses in zebrafish and reinforce the growing utility of this aquatic model to screen CNS therapies.

Targeting Adenosine Signaling in Parkinson's Disease: From Pharmacological to Non-pharmacological Approaches.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disease displaying negative impacts on both the health and social ability of patients and considerable economical costs. The classical anti-parkinsonian drugs based in dopaminergic replacement are the standard treatment, but several motor side effects emerge during long-term use. This mini-review presents the rationale to several efforts from pre-clinical and clinical studies using adenosine receptor antagonists as a non-dopaminergic therapy. As several studies have indicated that the monotherapy with adenosine receptor antagonists reaches limited efficacy, the usage as a co-adjuvant appeared to be a promising strategy. The formulation of multi-targeted drugs, using adenosine receptor antagonists and other neurotransmitter systems than the dopaminergic one as targets, have been receiving attention since Parkinson's disease presents a complex biological impact. While pharmacological approaches to cure or ameliorate the conditions of PD are the leading strategy in this area, emerging positive aspects have arisen from non-pharmacological approaches and adenosine function inhibition appears to improve both strategies.

Gender differences in aggression and cortisol levels in zebrafish subjected to unpredictable chronic stress.

Chronic stress may cause physical, behavioral and neuropsychiatric changes, affecting the health condition of an individual. Aggression is a universal behavior with great relevance on human and animal social systems. Despite studies showing the influence of chronic stress on aggression, the effects of unpredictable chronic stress (UCS) on aggressive behavior in male and female zebrafish remain unknown. Thus, the aim of this study was to evaluate the effects of UCS on the aggressive behavior and cortisol levels in adult zebrafish of both sexes. Our results showed that UCS increased aggression in males, but not in females, which displayed more aggressive behavior at baseline than control males. Increased whole-body cortisol levels were observed in stressed males; however, no differences were found between female groups. In conclusion, we reported for the first time gender differences on behavioral parameters and cortisol levels in response to UCS in zebrafish. These results highlight the relevance of studying behavioral and physiological parameters in both sexes separately.

Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme.

Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van't Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

Fluoxetine and diazepam acutely modulate stress induced-behavior.

Drug residue contamination in aquatic ecosystems has been studied extensively, but the behavioral effects exerted by the presence of these drugs are not well known. Here, we investigated the effects of acute stress on anxiety, memory, social interaction, and aggressiveness in zebrafish exposed to fluoxetine and diazepam at concentrations that disrupt the hypothalamic-pituitary-interrenal (HPI) axis. Stress increased the locomotor activity and time spent in the bottom area of the tank (novel tank). Fluoxetine and diazepam prevented these behaviors. We also observed that stress and fluoxetine and diazepam exposures decreased social interaction. Stress also increased aggressive behavior, which was not reversed by fluoxetine or diazepam. These data suggest that the presence of these drugs in aquatic ecosystems causes significant behavioral alterations in fish.

Acute exposure to waterborne psychoactive drugs attract zebrafish.

Psychotropic medications are widely used, and their prescription has increased worldwide, consequently increasing their presence in aquatic environments. Therefore, aquatic organisms can be exposed to psychotropic drugs that may be potentially dangerous, raising the question of whether these drugs are attractive or aversive to fish. To answer this question, adult zebrafish were tested in a chamber that allows the fish to escape or seek a lane of contaminated water. These attraction and aversion paradigms were evaluated by exposing the zebrafish to the presence of acute contamination with these compounds. The zebrafish were attracted by certain concentrations of diazepam, fluoxetine, risperidone and buspirone, which were most likely detected by olfaction, because this behavior was absent in anosmic fish. These findings suggest that despite their deleterious effects, certain psychoactive drugs attract fish.

Behavioral and cognitive profile of mice with high and low exploratory phenotypes.

Temperament is the heritable and relatively stable pattern of basic emotions, such as fear and anger. We explored behavioral features in mice to select distinct phenotypes with extremes of temperament. In a new environment (open-field) with a central object, two groups of 15 mice from 79 screened were separated according to high or low exploration of the object to compose the high and low exploratory groups, respectively. Their performance was mostly identical in the same task 1 week later and still distinguishable 8 months later, suggesting the presence of trait or temperamental features. These mice were further tested in other behavioral tasks. Compared to low exploratory mice, high exploratory mice were less anxious in the light/dark task and the elevated plus maze, showed increased locomotion in an open-field, improved their performance along trials in the Lashley maze (with appetitive stimulus) and had higher latency to step-down in the inhibitory avoidance task (with aversive stimulus). High exploratory mice were aggressive in the intruder test, whereas low exploratory mice were non-aggressive or submissive. These results show that individual differences in temperament influence a range of behaviors in mice. The behavioral profile of low and high exploratory mice resembled the depressive and hyperthymic temperaments of patients with unipolar depression and bipolar disorders, respectively, which may be relevant for modeling mood disorders.

Aqueous extract of Ilex paraguariensis decreases nucleotide hydrolysis in rat blood serum.

Mate is a xanthine-containing beverage, which is prepared as an infusion of the dried and ground leaves of Ilex paraguariensis St. Hil. (Aquifoliacea). Previous reports have shown that Ilex paraguariensis has the highest levels of caffeine and theobromine when compared to other Ilex species. Furthermore, mate is able to interfere in the circulatory system, acting as a diuretic and hypotensive agent. Many processes of vascular injury result in the release of adenine nucleotides, which exert a variety of effects. Nucleoside 5' tri- and diphosphates may be hydrolyzed by members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. The synchronic action of a NTPDase and a 5'-nucleotidase promotes the catabolism of ATP to adenosine, which is able to control the extracellular nucleotides/nucleosides ratio. The chronic ingestion of aqueous extract of Ilex paraguariensis by rats during 15 days significantly decreased ATP (55%), ADP (50%) and AMP (40%) hydrolysis in blood serum. These results suggest changes in the balance of purine levels induced by Ilex paraguariensis ingestion. Considering the potential effects of Ilex paraguariensis in the circulatory system, these results may be relevant since NTPDases are a novel drug target for the treatment of cardiovascular diseases.

Maternal caffeine exposure alters neuromotor development and hippocampus acetylcholinesterase activity in rat offspring.

The objective of this study was to evaluate the effects of maternal caffeine intake on the neuromotor development of rat offspring and on acetylcholine degradation and acetylcholinesterase (AChE) expression in the hippocampus of 14-day-old infant rats. Rat dams were treated with caffeine (0.3g/L) throughout gestation and lactation until the pups were 14 days old. The pups were divided into three groups: (1) control, (2) caffeine, and (3) washout caffeine. The washout group received a caffeine solution until the seventh postnatal day (P7). Righting reflex (RR) and negative geotaxis (NG) were assessed to evaluate postural parameters as an index of neuromotor reflexes. An open-field (OF) test was conducted to assess locomotor and exploratory activities as well as anxiety-like behaviors. Caffeine treatment increased both RR and NG latency times. In the OF test, the caffeine group had fewer outer crossings and reduced locomotion compared to control, while the washout group showed increased inner crossings in relation to the other groups and fewer rearings only in comparison to the control group. We found decreased AChE activity in the caffeine group compared to the other groups, with no alteration in AChE transcriptional regulation. Chronic maternal exposure to caffeine promotes important alterations in neuromotor development. These results highlight the ability of maternal caffeine intake to interfere with cholinergic neurotransmission during brain development.