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Guanosine is a purine nucleoside that has been shown to exhibit antidepressant effects, but the mechanisms underlying its effect are not well established. We investigated if the antidepressant-like effect induced by guanosine in the tail suspension test (TST) in mice involves the modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-dependent calcium channel (VDCC), and brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) pathway. We also evaluated if the antidepressant-like effect of guanosine is accompanied by an acute increase in hippocampal and prefrontocortical BDNF levels. Additionally, we investigated if the ability of guanosine to elicit a fast behavioral response in the novelty suppressed feeding (NSF) test is associated with morphological changes related to hippocampal synaptogenesis. The antidepressant-like effect of guanosine (0.05 mg/kg, p.o.) in the TST was prevented by DNQX (AMPA receptor antagonist), verapamil (VDCC blocker), K-252a (TrkBantagonist), or BDNF antibody. Increased P70S6K phosphorylation and higher synapsin I immunocontent in the hippocampus, but not in the prefrontal cortex, were observed 1 h after guanosine administration. Guanosine exerted an antidepressant-like effect 1, 6, and 24 h after its administration, an effect accompanied by increased hippocampal BDNF level. In the prefrontal cortex, BDNF level was increased only 1 h after guanosine treatment. Finally, guanosine was effective in the NSF test (after 1 h) but caused no alterations in dendritic spine density and remodeling in the ventral dentate gyrus (DG). Altogether, the results indicate that guanosine modulates targets known to be implicated in fast antidepressant behavioral responses (AMPA receptor, VDCC, and TrkB/BDNF pathway). [Image: see text]

Fluoxetine is the foremost prescribed antidepressant. Drugs acting on monoaminergic system may also regulate glutamatergic system. Indeed, the investigation of proteins associated with this system, such as Narp (neuronal activity-dependent pentraxin) and GluA4 subunit of AMPA receptor may reveal poorly explored modulations triggered by conventional antidepressants. This study aimed to uncover neurochemical mechanisms underlying the chronic fluoxetine treatment, mainly by evaluating these protein targets in the prefrontal cortex and in the hippocampus. Mice received a daily administration of fluoxetine (0.1, 1 or 10 mg/kg, p.o.) or potable water (vehicle group) for 21 days. These animals were submitted to the forced swim test (FST) to verify antidepressant-like responses and the open-field test (OFT) to assess locomotor activity. Modulation of signaling proteins was analyzed by western blot. Chronic treatment with fluoxetine (1 and 10 mg/kg) was effective, since it reduced the immobility time in the FST, without altering locomotor activity. Fluoxetine 10 mg/kg increased CREB phosphorylation and BDNF expression in the prefrontal cortex and hippocampus. Noteworthy, in the hippocampus fluoxetine also promoted Akt activation and augmented Narp expression. In the prefrontal cortex, a significant decrease in the expression of the GluA4 subunit and Narp were observed following fluoxetine administration (10 mg/kg). The results provide evidence of novel molecular targets potentially involved in the antidepressant effects of fluoxetine, since in mature rodents Narp and GluA4 are mainly expressed in the GABAergic parvalbumin-positive (PV+) interneurons. This may bring new insights into the molecular elements involved in the mechanisms underlying the antidepressant effects of fluoxetine.

The central autonomic network, which is connected to the limbic system structures including the amygdala (AMY) and anterior hippocampus (aHIP), regulates the sympathetic and parasympathetic modulation of visceromotor, neuroendocrine, pain, and behavior manifestations during stress responses. Heart rate variability (HRV) is useful to estimate the cardiac autonomic tone. The levels of phosphorylation on the Ser831 and Ser845 sites of the GluA1 subunit of the AMPAr (P-GluA1-Ser845 and P-GluA1-Ser831) are useful markers of synaptic plasticity. The relation between synaptic plasticity in the human limbic system structures and autonomic regulation in humans is unknown. This study investigated the association between HRV and neurochemistry biomarkers of synaptic plasticity in AMY and aHIP. HRV indices were obtained from the resting state electrocardiogram of patients with drug-resistant mesial temporal lobe epilepsy (MTLE, n = 18) and the levels of P-GluA1-Ser845 and P-GluA1-Ser831 in the AMY and aHIP resected during the epilepsy surgery. A backward stepwise multiple linear regression models were used to analyze the association between HRV and synaptic plasticity biomarkers controlling for imbalances in the distribution of sociodemographic, clinical, neuroimaging, and neurosurgical variables. P-GluA1-Ser845 levels in AMY show a negative association (p

Methylglyoxal (MGO) is an endogenous toxin, mainly produced as a by-product of glycolysis that has been associated to aging, Alzheimer’s disease, and inflammation. Cell culture studies reported that MGO could impair the glyoxalase, thioredoxin, and glutathione systems. Thus, we investigated the effect of in vivo MGO administration on these systems, but no major changes were observed in the glyoxalase, thioredoxin, and glutathione systems, as evaluated in the prefrontal cortex and the hippocampus of mice. A previous study from our group indicated that MGO administration produced learning/memory deficits and depression-like behavior. Confirming these findings, the tail suspension test indicated that MGO treatment for 7 days leads to depression-like behavior in three different mice strains. MGO treatment for 12 days induced working memory impairment, as evaluated in the Y maze spontaneous alternation test, which was paralleled by low dopamine and serotonin levels in the cerebral cortex. Increased DARPP32 Thr75/Thr34 phosphorylation ratio was observed, suggesting a suppression of phosphatase 1 inhibition, which may be involved in behavioral responses to MGO. Co-treatment with a dopamine/noradrenaline reuptake inhibitor (bupropion, 10 mg/kg, p.o.) reversed the depression-like behavior and working memory impairment and restored the serotonin and dopamine levels in the cerebral cortex. Overall, the cerebral cortex monoaminergic system appears to be a preferential target of MGO toxicity, a new potential therapeutic target that remains to be addressed.

Lectins from Diocleinae subtribe species (family Leguminosae) are of special interest since they present a wide spectrum of biological activities, despite their high structural similarity. During their synthesis in plant cells, these proteins undergo post-translational processing resulting in the formation of three chains (α, β, γ), which constitute the lectins' subunits. Furthermore, such wild-type proteins are presented as isolectins or with different combinations of these chains, which undermine their biotechnological potential. Thus, the present study aimed to produce a recombinant form of the lectin from Dioclea sclerocarpa seeds (DSL), exclusively constituted by α-chain. The recombinant DSL (rDSL) was successfully expressed in E. coli BL21 (DE3) and purified by affinity chromatography (Sephadex G-50), showing a final yield of 74 mg of protein per liter of culture medium and specificity for D-mannose, α-methyl-mannoside and melibiose, unlike the wild-type protein. rDSL presented an effective vasorelaxant effect in rat aortas up to 100% and also interacted with glioma cells C6 and U87. Our results demonstrated an efficient recombinant production of rDSL in a bacterial system that retained some biochemical properties of the wild-type protein, showing wider versatility in sugar specificities and better efficacy in its activity in the biological models evaluated in this work.

The molecular mechanisms underlying the capability of pituitary tumours to avoid unregulated cell proliferation are still not well understood. However, the NF-κB transcription factor, which is able to modulate not only cellular senescence but also tumour progression, has emerged as a targeted candidate. This work was focused on the NF-κB role in cellular senescence during the progression of experimental pituitary tumours. Also, the contribution of the signalling pathways in senescence-associated NF-κB activation and the senescence-associated secretory phenotype (SASP) and pro-survival-NF-κB target genes transcription were analysed. A robust NF-κB activation was seen at E20-E40 of tumour development accompanied by a marked SA-β-Gal co-reactivity in the tumour pituitary parenchyma. The induction of TNFα and IL1-β as specific SASP-related NF-κB target genes as well as Bcl-2 and Bcl-xl pro-survival genes was shown to be accompanied by increases in the p-p38 MAPK protein levels, starting at the E20 stage and strengthening from 40 to 60 days of tumour growth. It is noteworthy that p-JNK displayed a similar pattern of activation during pituitary tumour development, while p-AKT and p-ERK1/2 were downregulated. By employing a pharmacological strategy to abrogate NF-κB activity, we demonstrated a marked reduction in SA-β-Gal activity and a slight decrease in Ki67 immunopositive cells after NF-κB blockade. These results suggest a central role for NF-κB in the regulation of the cellular senescence programme, leading to the strikingly benign intrinsic nature of pituitary adenomas. Fil: Mongi Bragato, Bethania del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Grondona, Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Sosa, Liliana del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Zlocowski, Natacha. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Venier, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Torres, Alicia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Latini, Alexandra. Universidade Federal de Santa Catarina; Brasil Fil: Bainy Leal, Rodrigo. Universidade Federal de Santa Catarina; Brasil Fil: Gutiérrez, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: de Paul, Ana Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina

Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant whose mechanisms of action involve oxidation of endogenous nucleophilic groups (mainly thiols and selenols), depletion of antioxidant defenses, and disruption of neurotransmitter homeostasis. Diphenyl diselenide-(PhSe)

We demonstrate that the rate of extracellular signal-related kinase phosphorylation (P-ERK1,2/Total-ERK1,2) in the amygdala is negatively and independently associated with anxiety symptoms in 23 consecutive patients with drug-resistant mesial temporal lobe epilepsy that was surgically treated. In naive Wistar rats, the P-ERK1,2/Total-ERK1,2 ratio in the amygdala correlates negatively with innate anxiety-related behavior on the elevated plus maze (n = 20) but positively with expression of defensive-learned behavior (i.e., freezing) on Pavlovian aversive (fear) conditioning (n = 29). The microinfusion of ERK1/2 inhibitor (FR180204, n = 8-13/group) or MEK inhibitor (U0126, n = 8-9/group) into the basolateral amygdala did not affect anxiety-related behavior but impaired the evocation (anticipation) of conditioned-defensive behavior (n = 9-11/group). In conclusion, the P-ERK1,2/Total-ERK1,2 ratio in the amygdala predicts anxiety in humans and the innate anxiety- and conditioned freezing behaviors in rats. However, the ERK1/2 in the basolateral AMY is only required for the expression of defensive-learned behavior. These results support a dissociate ERK-dependent mechanism in the amygdala between innate anxiety-like responses and the anticipation of learned-defensive behavior. These findings have implications for understanding highly prevalent psychiatric disorders related to the defensive circuit manifested by anxiety and fear. HIGHLIGHTS: The P-ERK1,2/Total-ERK1,2 ratio in the amygdala (AMY) correlates negatively with anxiety symptoms in patients with mesial temporal lobe epilepsy. The P-ERK1,2/Total-ERK1,2 in the amygdala correlates negatively with the anxiety-like behavior and positively with freezing-learned behavior in naive rats. ERK1,2 in the basolateral amygdala is required for learned-defensive but not for the anxiety-like behavior expression in rats.

Although guanosine is an endogenous nucleoside that displays antidepressant-like properties in several animal models, the mechanism underlying its antidepressant-like effects is not well characterized. The present study aimed at investigating the involvement of ERK/GSK-3β and Nrf2/HO-1 signaling pathways in the antidepressant-like effect of guanosine in the mouse tail suspension test (TST). The immobility time in the TST was taken as an indicative of antidepressant-like responses and the locomotor activity was assessed in the open-field test. Biochemical analyses were performed by Western blotting in the hippocampus and prefrontal cortex (PFC). The combined treatment with sub-effective doses of guanosine (0.01 mg/kg, p.o.) and lithium chloride (a non-selective GSK-3β inhibitor, 10 mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, 0.01 μg/site, i.c.v.) produced a synergistic antidepressant-like effect in the TST. The antidepressant-like effect of guanosine (0.05 mg/kg, p.o.) was completely prevented by the treatment with MEK1/2 inhibitors U0126 (5 μg/site, i.c.v.), PD98059 (5 μg/site, i.c.v.), or zinc protoporphyrin IX (ZnPP) (HO-1 inhibitor, 10 μg/site, i.c.v). Guanosine administration (0.05 mg/kg, p.o.) increased the immunocontent of β-catenin in the nuclear fraction and Nrf2 in the cytosolic fraction in the hippocampus and PFC. The immunocontent of HO-1 was also increased in the hippocampus and PFC. Altogether, the results provide evidence that the antidepressant-like effect of guanosine in the TST involves the inhibition of GSK-3β, as well as activation of MAPK/ERK and Nrf2/HO-1 signaling pathways, highlighting the relevance of these molecular targets for antidepressant responses.

Lectins are proteins that can bind specifically and reversibly to carbohydrates. This capacity gives lectins multiple biological roles and biotechnological applications. Although lectins can be found in all organisms, plant lectins, especially legume lectins, are undoubtedly the most thoroughly studied. Among legume lectins, the lectin from Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr), both from Diocleinae subtribe, are two of the most well-known lectins. It has been 100 years since the first report of ConA and 40 years since the first report of ConBr, making 2019 an important year for lectinology. Structural data of these lectins in combination with biological activity tests clearly indicate that even a small shift in amino acid sequence can affect the tertiary and quaternary structures, consequently affecting the biological activity of these proteins. It is in this context that the present paper aims to review the structural data of ConA and ConBr, focusing on the primary structure, crystallography, tertiary and quaternary structures of these lectins, as well as their binding sites. This paper also expands the structural data by employing molecular dynamics to evaluate carbohydrate-binding properties and structural stability. It is anticipated that these data will increase knowledge about the structure-function relationships of these proteins.

Fluoxetine is the foremost prescribed antidepressant. Drugs acting on monoaminergic system may also regulate glutamatergic system. Indeed, the investigation of proteins associated with this system, such as Narp (neuronal activity-dependent pentraxin) and GluA4 subunit of AMPA receptor may reveal poorly explored modulations triggered by conventional antidepressants. This study aimed to uncover neurochemical mechanisms underlying the chronic fluoxetine treatment, mainly by evaluating these protein targets in the prefrontal cortex and in the hippocampus. Mice received a daily administration of fluoxetine (0.1, 1 or 10 mg/kg, p.o.) or potable water (vehicle group) for 21 days. These animals were submitted to the forced swim test (FST) to verify antidepressant-like responses and the open-field test (OFT) to assess locomotor activity. Modulation of signaling proteins was analyzed by western blot. Chronic treatment with fluoxetine (1 and 10 mg/kg) was effective, since it reduced the immobility time in the FST, without altering locomotor activity. Fluoxetine 10 mg/kg increased CREB phosphorylation and BDNF expression in the prefrontal cortex and hippocampus. Noteworthy, in the hippocampus fluoxetine also promoted Akt activation and augmented Narp expression. In the prefrontal cortex, a significant decrease in the expression of the GluA4 subunit and Narp were observed following fluoxetine administration (10 mg/kg). The results provide evidence of novel molecular targets potentially involved in the antidepressant effects of fluoxetine, since in mature rodents Narp and GluA4 are mainly expressed in the GABAergic parvalbumin-positive (PV+) interneurons. This may bring new insights into the molecular elements involved in the mechanisms underlying the antidepressant effects of fluoxetine.

Glioblastoma multiforme is the most aggressive type of glioma, with limited treatment and poor prognosis. Despite some advances over the last decade, validation of novel and selective antiglioma agents remains a challenge in clinical pharmacology. Prior studies have shown that leguminous lectins may exert various biological effects, including antitumor properties. Accordingly, this study aimed to evaluate the mechanisms underlying the antiglioma activity of ConBr, a lectin extracted from the Canavalia brasiliensis seeds. ConBr at lower concentrations inhibited C6 glioma cell migration while higher levels promoted cell death dependent upon carbohydrate recognition domain (CRD) structure. ConBr increased p38MAPK and JNK and decreased ERK1/2 and Akt phosphorylation. Moreover, ConBr inhibited mTORC1 phosphorylation associated with accumulation of autophagic markers, such as acidic vacuoles and LC3 cleavage. Inhibition of early steps of autophagy with 3-methyl-adenine (3-MA) partially protected whereas the later autophagy inhibitor Chloroquine (CQ) had no protective effect upon ConBr cytotoxicity. ConBr also augmented caspase-3 activation without affecting mitochondrial function. Noteworthy, the caspase-8 inhibitor IETF-fmk attenuated ConBr induced autophagy and C6 glioma cell death. Finally, ConBr did not show cytotoxicity against primary astrocytes, suggesting a selective antiglioma activity. In summary, our results indicate that ConBr requires functional CRD lectin domain to exert antiglioma activity, and its cytotoxicity is associated with MAPKs and Akt pathways modulation and autophagy- and caspase-8- dependent cell death. Fil: Wolin, Ingrid A. V.. Universidade Federal de Santa Catarina; Brasil Fil: Heinrich, Isabella A.. Universidade Federal de Santa Catarina; Brasil Fil: Nascimento, Ana Paula M.. Universidade Federal de Santa Catarina; Brasil Fil: Welter, Priscilla G.. Universidade Federal de Santa Catarina; Brasil Fil: Sosa, Liliana del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: de Paul, Ana Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Zanotto Filho, Alfeu. Universidade Federal de Santa Catarina; Brasil Fil: Nedel, Cláudia Beatriz. Universidade Federal de Santa Catarina; Brasil Fil: Lima, Lara Dias. Universidade Estadual do Ceará; Brasil Fil: Osterne, Vinicius Jose Silva. Universidade Estadual do Ceará; Brasil Fil: Pinto Junior, Vanir Reis. Universidade Estadual do Ceará; Brasil Fil: Nascimento, Kyria S.. Universidade Estadual do Ceará; Brasil Fil: Cavada, Benildo S.. Universidade Estadual do Ceará; Brasil Fil: Leal, Rodrigo B.. Universidade Federal de Santa Catarina; Brasil

Lectins are a group of proteins of non-immune origin recognized for their ability to bind reversibly to carbohydrates. Researchers have been intrigued by oligosaccharides and glycoconjugates for their involvement as mediators of complex cellular events and then many biotechnological applications of lectins are based on glycocode decoding and their activities. Here, we report a structural and biological study of a ConA-like mannose/glucose-specific lectin from Canavalia bonariensis seeds, CaBo. More specifically, we evaluate the binding of CaBo with α-methyl-D-mannoside (MMA) and mannose-1,3-α-D-mannose (M13) and the resultant in vivo effects on a rat model of acute inflammation. A virtual screening was also carried out to cover a larger number of possible bindings of CaBo. In silico analysis demonstrated the stability of CaBo interaction with mannose-type ligands, and the lectin was able to induce acute inflammation in rats with the participation of the carbohydrate recognition domain (CRD) and histamine release. These results confirm the ability of CaBo to interact with hybrid and high-mannose N-glycans, supporting the hypothesis that CaBo's biological activity occurs primarily through its interaction with cell surface glycosylated receptors.

A type II lectin, designated as DlyL2, was purified from Dioclea lasiophylla Mart. ex Benth seeds and some of its physicochemical properties determined. The lectin demonstrated specificity for α-lactose and N-acetyl- d -galactosamine and was able to interact with porcine stomach mucin. DlyL2 has 0.78 % carbohydrates in its composition, therefore can be considered a glycoprotein. In addition, its hemagglutinating activity remained stable at a temperature of 90 °C and in a pH range from 5 to 10. Metal chelation treatment did not affect DlyL2 activity suggesting it's not a metalloprotein. Dlyl2 showed an apparent mass of 31 kDa and average molecular mass of 26.371 kDa. Primary structure data could be generated from the partial amino acid sequence of DlyL2, with about 192 residues sequenced by a combination of Edman degradation and tandem mass spectrometry. The lectin is similar to other type II lectins from Diocleinae subtribe, as well as lectins derived from species of more ancient tribes of the Fabaceae family. In addition, DlyL2 exhibited no toxicity to Artemia sp. nauplii. The present study expands the knowledge about the specificity, structural and physicochemical properties of Diocleinae type II lectins.

Plant lectins have been studied owing to their structural properties and biological effects that include agglutinating activity, antidepressant-like effect and antitumor property. The results from this work showed the effects of the lectin extracted from the Dioclea violacea plant (DVL) on the C6 rat glioma cell line. DVL treatment was able to induce caspase-3 activation, apoptotic cell death and cellular membrane damage. Furthermore, DVL decreased mitochondrial membrane potential and increased the number of acidic vesicles and cleavage of LC3, indicating activation of autophagic processes. DVL also significantly inhibited cell migration. Compared to ConA, a well-studied lectin extracted from Canavalia ensiformes seeds, some effects of DVL were more potent, including decreasing C6 glioma cell viability and migration ability. Taken together, the results suggest that DVL can induce glioma cell death, autophagy and inhibition of cell migration, displaying potential anti-glioma activity. Fil: Nascimento, Ana Paula M.. Universidade Federal de Santa Catarina; Brasil Fil: Knaut, Jhônatas L.. Universidade Federal de Santa Catarina; Brasil Fil: Rieger, Débora K.. Universidade Federal de Santa Catarina; Brasil Fil: Wolin, Ingrid A.V.. Universidade Federal de Santa Catarina; Brasil Fil: Heinrich, Isabella A.. Universidade Federal de Santa Catarina; Brasil Fil: Mann, Josiane. Universidade Federal de Santa Catarina; Brasil Fil: Juarez, Andrea Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Universidad Nacional de Córdoba. Facultad de Medicina. Centro de Microscopía Electrónica; Argentina Fil: Sosa, Liliana del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Universidad Nacional de Córdoba. Facultad de Medicina. Centro de Microscopía Electrónica; Argentina Fil: de Paul, Ana Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Universidad Nacional de Córdoba. Facultad de Medicina. Centro de Microscopía Electrónica; Argentina Fil: Moreira, Cleane G.. Universidade Estadual do Ceará; Brasil Fil: Silva, Ivanice B.. Universidade Estadual do Ceará; Brasil Fil: Nobre, Clareane S.. Universidade Estadual do Ceará; Brasil Fil: Osterne, Vinicius J.S.. Universidade Estadual do Ceará; Brasil Fil: Nascimento, Kyria S.. Universidade Estadual do Ceará; Brasil Fil: Cavada, Benildo S.. Universidade Estadual do Ceará; Brasil Fil: Leal, Rodrigo B.. Universidade Federal de Santa Catarina; Brasil

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is one of the most robust neurobiological findings in the pathophysiology of major depressive disorder (MDD) over the last 40 years. The persistent increase in glucocorticoids levels induces morphological and anatomical changes in the brain, especially in the hippocampus. Ketamine represents a major advance for the treatment of MDD, however the psychotomimetic effects of this compound limit its widespread use. Agmatine is a neuromodulator that has been shown to be a putative novel and well-tolerated antidepressant/augmenter drug. In this study, the exposure of HT22 hippocampal neuronal cell line to corticosterone (50 μM) induced a significant neuronal cell death. Interestingly, the incubation of HT22 cells with the fast-acting antidepressant drug ketamine (1 μM) prevented the corticosterone-induced toxicity. Similarly, agmatine caused a significant cytoprotection at the concentration of 0.1 μM against corticosterone (50 μM) cell damage. Notably, the incubation with a subthreshold concentration of ketamine (0.01 μM) in combination with a subthreshold concentration of agmatine (0.001 μM) prevented the neuronal damage elicited by corticosterone (50 μM). A 24 h co-incubation with subthreshold concentrations of ketamine (0.01 μM) and agmatine (0.001 μM) was able to cause a significant increase in the phosphorylation levels of Akt (Ser473) and p70S6 kinase (Thr389) as well as PSD95 immunocontent. Neither glycogen synthase kinase-3β (Ser9) phosphorylation nor β catenin immunocontent were altered by a 24 h co-incubation period. Finally, the co-incubation of cells for 30 min did not produce any effect in the phosphorylation or immunocontent of any protein investigated. Taken together, our results support the notion that the combination of subthreshold concentrations of ketamine and agmatine has cytoprotective effects against corticosterone-induced cell death. This effect is accompanied by its ability to activate Akt and mTOR/S6 kinase signaling pathway, and increase the expression of synaptic proteins.

Folic acid has been reported to exert antidepressant effects, but its ability to abrogate the depressive-like behavior and signaling pathways alterations elicited by an inflammatory model of depression remains to be established. This study examined: a) the efficacy of folic acid in a mouse model of depression induced by tumor necrosis factor (TNF-α); b) whether the administration of subthreshold doses of folic acid and antidepressants (fluoxetine, imipramine, and bupropion), MK-801, or 7-nitroindazole cause antidepressant-like effects; c) the effects of TNF-α and/or folic acid on hippocampal p38MAPK, Akt, ERK, and JNK phosphorylation. Folic acid reduced the immobility time in the tail suspension test (TST) in control mice (10–50 mg/kg, p.o) and abolished the depressive-like behavior elicited by TNF-α (0.001 fg/site, i.c.v.) in this test (1–50 mg/kg, p.o). Coadministration of subthreshold doses of folic acid (1 mg/kg, p.o.) and fluoxetine, imipramine, bupropion, MK-801, or 7-nitroindazole produced an antidepressant-like effect in mice exposed or not to TNF-α. TNF-α-treated mice presented increased p38MAPK phosphorylation and decreased Akt phosphorylation, and the later effect was prevented by folic acid (10 mg/kg, p.o.). Additionally, ERK1 phosphorylation was increased in mice treated with TNF-α + folic acid (1 mg/kg), but no effects on ERK2 or JNK1/2/3 phosphorylation were found in any group. The results indicate the efficacy of folic acid to counteract the depressive-like behavior induced by a pro-inflammatory cytokine, an effect that might be associated with the activation of monoaminergic systems, inhibition of N-methyl- d -aspartate (NMDA) receptors and nitric oxide (NO) synthesis, as well as Akt modulation.

Lectins are multidomain proteins that specifically recognize various carbohydrates. The structural characterization of these molecules is crucial in understanding their function and activity in systems and organisms. Most cancer cells exhibit changes in glycosylation patterns, and lectins may be able to recognize these changes. In this work, Dioclea lasiocarpa seed lectin (DLL) was structurally characterized. The lectin presented a high degree of similarity with other lectins isolated from legumes, presenting a jelly roll motif and a metal-binding site stabilizing the carbohydrate-recognition domain. DLL demonstrated differential interactions with carbohydrates, depending on type of glycosidic linkage present in ligands. As observed by the reduction of cell viability in C6 cells, DLL showed strong antiglioma activity by mechanisms involving activation of caspase 3.

The lectin from Platypodium elegans seeds (PELa) was purified by affinity chromatography in a mannose-agarose column. The lectin agglutinated rabbit erythrocytes and the agglutinating effect was inhibited by previous incubation with the glycoprotein fetuin, along with N -acetyl- d -glucosamine, D-mannose and its derivatives. The lectin maintained complete activity in temperatures ranging from 40 to 60 °C and pH values ranging from 9 to 10. As a glycoprotein, PELa has a carbohydrate content of 2.2%, and its activity requires divalent cations such as Ca 2+ and Mn 2+ . Based on SDS-PAGE, PELa displays a profile similar to that of other Dalbergieae lectins with the main chain of molecular mass around 30 kDa and two subunits of 19 kDa and 10 kDa each. Two-dimensional (2D) electrophoresis revealed the presence of isoforms with different isoelectric points, and high-performance size exclusion chromatography (HPSEC) was performed to confirm the purity of the sample. The lectin was immobilized in CNBr-activated Sepharose 4 B and successfully captured fetuin in solution, demonstrating that this lectin remains active and capable of binding carbohydrates. PELa showed effects different from those of its recombinant form in both pro- and anti-inflammatory tests.

Glutamatergic system and mTOR signaling pathway have been proposed to be important targets for pharmacological treatment of major depressive disorder. Previous studies have shown that inosine, an endogenous purine, is able to exert a remarkable antidepressant-like effect in mice. Nevertheless, the role of glutamatergic system and mTOR in this effect was not previously determined. This study was designed to investigate the possible modulation of NMDA receptors (NMDAR), AMPA receptors (AMPAR) and mTOR complex 1 (mTORC1) signaling pathway in the inosine anti-immobility effect in the tail suspension test (TST) in mice. Pre-treatment of mice with NMDA (0.1 pmol/mouse, NMDAR agonist, i.c.v.) and D-serine (30 μg/mouse, NMDAR co-agonist, i.c.v.) prevented inosine (10 mg/kg, i.p.) anti-immobility effect in the TST. In addition, a synergistic antidepressant-like effect was observed when a sub-effective dose of inosine (0.1 mg/kg, i.p.) was combined with sub-effective doses of NMDAR antagonists MK-801 (0.001 mg/kg, p.o.) or ketamine (0.1 mg/kg, i.p.). Conversely, the antidepressant-like effect elicited by inosine was not altered by pre-treatment with AMPAR antagonist, DNQX (2.5 μg/mouse, i.c.v.). The mTORC1 inhibitor rapamycin (0.2 nmol/mouse, i.c.v.) prevented the inosine anti-immobility effect in the TST. Noteworthy, inosine treatment did not change the immunocontent of the synaptic proteins PSD95, GluA1 and synapsin I. Mice locomotor activity assessed by open-field test, was not altered by treatments. Taken together, this study shows a pivotal role of NMDAR inhibition and mTORC1 activation for inosine antidepressant-like effect and extends the knowledge concerning the molecular mechanism and potential of inosine for antidepressant strategies.

The three-dimensional structure of Dioclea reflexa seed lectin (DrfL) was studied in detail by a combination of X-ray crystallography, molecular docking and molecular dynamics. DrfL was purified by affinity chromatography using Sephadex G-50 matrix. Its primary structure was obtained by mass spectrometry, and crystals belonging to orthorhombic space group P2(1)2(1)2(1) were grown by the vapor diffusion method at 293 K. The crystal structure was solved at 1.765 A and was very similar to that of other lectins from the same subtribe. The structure presented R-factor and R-free of 21.69% and 24.89%, respectively, with no residues in nonallowed regions of Ramachandran plot. Similar to other Diocleinae lectins, DrfL was capable of relaxing aortic rings via NO induction, with CRD participation, albeit with low intensity (32%). In silica analysis results demonstrated that DrfL could strongly interact with complex N-glycans, components of blood vessel glycoconjugates. Despite the high similarity among Diocleinae lectins, it was also reported that each lectin has unique CRD properties that influence carbohydrate binding, resulting in different biological effects presented by these molecules. (C) 2017 Elsevier B.V. All rights reserved.

Neuronal hippocampal death can be induced by exacerbated levels of cortisol, a condition usually observed in patients with Major depressive disorder (MDD). Previous in vitro and in vivo studies showed that ursolic acid (UA) elicits antidepressant and neuroprotective properties. However, the protective effects of UA against glucocorticoid-induced cytotoxicity have never been addressed. Using an in vitro model of hippocampal cellular death induced by elevated levels of corticosterone, we investigated if UA prevents corticosterone-induced cytotoxicity in HT22 mouse hippocampal derived cells. Concentrations lower than 25 µM UA did not alter cell viability. Co-incubation with UA for 48 h was able to protect HT22 cells from the reduction on cell viability and from the increase in apoptotic cells induced by corticosterone. Inhibition of protein kinase A (PKA), protein kinase C (PKC) and, Ca2+/calmodulin-dependent protein kinase II (CaMKII), but not phosphoinositide 3-kinase(PI3K), by using the pharmacological the inhibitors: H-89, chelerythrine, KN-62, and LY294002, respectively totally abolished the cytoprotective effects of UA. Finally, UA abrogated the reduction in phospho-extracellular signal–regulated kinases 1 and 2 (ERK1/2) but not in phospho-c-Jun kinases induced by corticosterone. These results indicate that the protective effect of UA against the cytotoxicity induced by corticosterone in HT22 cells may involve PKA, PKC, CaMKII, and ERK1/2 activation. The cytoprotective potential of UA against corticosterone-induced cytotoxicity and its ability to modulate intracellular signaling pathways involved in cell proliferation and survival suggest that UA may be a relevant strategy to manage stress-related disorders such as MDD.

The antitumor activity of DVL, a lectin purified from Dioclea violacea seeds, on the U87 human glioma cell line was evaluated and compared with Canavalia ensiformis lectin (ConA). Treatment with DVL (10–100 μg/mL; 24–96 h) induced alterations in cell morphology, decreased cell numbers and clonogenic survival in a time- and concentration-dependent manner. DVL caused significant decreases in cell viability and impaired cell migration. Mechanistically, DVL treatment (12 h) disrupted mitochondrial electrochemical gradient, without ROS accumulation or caspase activation. In the absence of apoptosis, DVL (30–100 μg/mL), instead, induced autophagy, as detected by acridine orange staining and cleavage of LC3I. Inhibition of autophagy with 3-Methyladenine (3-MA) and Chloroquine partially abrogated DVL, but not ConA, cytotoxicity. The modulation of signaling pathways that orchestrate autophagic and cell survival processes were analyzed. DVL (30–100 μg/mL) decreased Akt, mTORC1 and ERK1/2 phosphorylation and augmented JNK(p54) and p38MAPK phosphorylation. DVL was more potent than ConA for most parameters analyzed. Even though both lectins showed cytotoxicity to glioma cells, they spared primary astrocyte cultures. The results suggest a selective antiglioma activity of DVL by inhibiting U87 glioma cell migration and proliferation and inducing cell death, partially associated with autophagy, and likely involving Akt and mTORC1 dephosphorylation.

α-Tocopheryl phosphate (αTP) is a phosphorylated form of α-tocopherol. Since it is phosphorylated in the hydroxyl group that is essential for the antioxidant property of α-tocopherol, we hypothesized that αTP would modulate the antioxidant system, rather than being an antioxidant agent per se. α-TP demonstrated antioxidant activity in vitro against iron-induced oxidative stress in a mitochondria-enriched fraction preparation treated with 30 or 100 µM α-TP. However, this effect was not observed ex vivo with mitochondrial-enriched fraction from mice treated with an intracerebroventricular injection of 0.1 or 1 nmol/site of αTP. Two days after treatment (1 nmol/site αTP), peroxiredoxin 2 (Prx2) and glutathione reductase (GR) expression and GR activity were decreased in cerebral cortex and hippocampus. Glutathione content, glutathione peroxidase, and thioredoxin reductase activities were not affected by αTP. In conclusion, the persistent decrease in GR and Prx2 protein content is the first report of an in vivo effect of αTP on protein expression in the mouse brain, potentially associated to a novel and biologically relevant function of this naturally occurring compound.

Selenium (Se) is an essential trace element for humans; its intake is needed to allow the proper synthesis of 25 different selenoproteins that are necessary to the normal functioning of several organs, including the brain. Accordingly, decreased Se levels have been associated with neurological disorders. In the present study, we investigated the potential beneficial effects of Se, as sodium selenite, against 3-nitropropionic acid (3-NP)-induced oxidative stress in primary cultures of mouse cortical neurons. 3-NP treatment caused a significant decrease in cellular viability, which was accompanied by decreases in mitochondrial complex II activity and reduced glutathione (GSH) content, as well as increases in reactive oxygen species (ROS) generation and oxidized glutathione (GSSG) levels. Sodium selenite pretreatment (6 days) attenuated 3-NP-induced decrease in cell viability. In addition, sodium selenite pretreatment significantly protected against 3-NP-induced increase in ROS generation and decrease in GSH/GSSG ratio. Of note, sodium selenite pretreatment did not change 3-NP-induced decrease of mitochondrial complex II activity, suggesting that Se modulates secondary events resultant from 3-NP-induced mitochondrial dyshomeostasis. In addition, sodium selenite pretreatment significantly increased glutathione peroxidase (GPx) activity. Our data provide insights into the mechanism of protection by sodium selenite, which is related, at least in part, to GPx induction.

Excessive levels of the essential metal manganese (Mn) may cause a syndrome similar to Parkinson’s disease. The model organism Caenorhabditis elegans mimics some of Mn effects in mammals, including dopaminergic neurodegeneration, oxidative stress, and increased levels of AKT. The evolutionarily conserved insulin/insulin-like growth factor-1 signaling pathway (IIS) modulates worm longevity, metabolism, and antioxidant responses by antagonizing the transcription factors DAF-16/FOXO and SKN-1/Nrf-2. AKT-1, AKT-2, and SGK-1 act upstream of these transcription factors. To study the role of these proteins in C. elegans response to Mn intoxication, wild-type N2 and loss-of-function mutants were exposed to Mn (2.5 to 100 mM) for 1 h at the L1 larval stage. Strains with loss-of-function in akt-1, akt-2, and sgk-1 had higher resistance to Mn compared to N2 in the survival test. All strains tested accumulated Mn similarly, as shown by ICP-MS. DAF-16 nuclear translocation was observed by fluorescence microscopy in WT and loss-of-function strains exposed to Mn. qRT-PCR data indicate increased expression of γ-glutamyl cysteine synthetase (GCS-1) antioxidant enzyme in akt-1 mutants. The expression of sod-3 (superoxide dismutase homologue) was increased in the akt-1 mutant worms, independent of Mn treatment. However, dopaminergic neurons degenerated even in the more resistant strains. Dopaminergic function was evaluated with the basal slowing response behavioral test and dopaminergic neuron integrity was evaluated using worms expressing green fluorescent protein (GFP) under the dopamine transporter (DAT-1) promoter. These results suggest that AKT-1/2 and SGK-1 play a role in C. elegans response to Mn intoxication. However, tissue-specific responses may occur in dopaminergic neurons, contributing to degeneration.

Agmatine is a neuromodulator that has been proposed as a therapeutic strategy for the treatment of major depressive disorder (MDD). A previous study reported that agmatine caused a fast-acting effect in mice subjected to chronic mild stress without causing changes in the levels of synaptic proteins in the prefrontal cortex. We examined whether a single administration of agmatine is able to counteract the depressive-like behavior induced by chronic administration of corticosterone, a pharmacological model of stress, paralleled with the modulation of synaptic protein levels in the prefrontal cortex and hippocampus. Female mice received corticosterone (20 mg/kg, p.o.) for 21 days and, in the last day of treatment, were administered with a single dose of agmatine (0.1 mg/kg, p.o.), fluoxetine (10 mg/kg, p.o.; control for a conventional antidepressant) or ketamine (1 mg/kg, i.p.; control for a fast-acting antidepressant). Agmatine, similar to ketamine, reversed the depressive-like behavior induced by corticosterone in the tail suspension test (TST), an effect that was not observed in mice treated with fluoxetine. The immunocontent of GluA1 was increased by all the treatments in the hippocampus of control mice, whereas PSD95 was not significantly altered by treatments in any brain structure. Although the levels of synaptic proteins do not seem to account for the behavioral findings reported here, the present study provides clear evidence for the fast-acting antidepressant profile of agmatine in the TST, similar to ketamine.

Several research efforts have been focused on finding newer and more efficient photosensitizers for photodynamic therapy (PDT). Although, it was demonstrated that riboflavin is an efficient photosensitizer for PDT, the effect of its ester derivate, riboflavin 2′,3′,4′,5′-tetraacetate (RFTA), which has higher cellular uptake, has not been well defined. To evaluate the cell death generated by applying RFTA as the photosensitizer in PDT in a human cancer cell line of squamous carcinoma (SCC-13), these cells were incubated with riboflavin and its ester derivate, RFTA followed by irradiation with different blue light doses. Cell viability was evaluated using neutral red uptake assay and cell death was evaluated using transmission electron microscopy, TUNEL assay and annexin V-PE/7AAD double staining. The expression of caspase-3, Bax, Bcl-2, ERK 1/2 and p38MAPK was evaluated by Western blotting and generation of intracellular ROS and changes in anion superoxide levels were analyzed using 2′,7′-dichlorofluorescein-diacetate and dihydroethidium dye, respectively. RFTA-PDT generated a decrease in cancer cell viability in a light dose–response. Treated SCC-13 cells exhibited chromatin condensation, formation of apoptotic bodies, increases in TUNEL-positive cells, phosphatidylserine externalization and decreased procaspase-3 and Bcl-2 protein expression and increment of ERK 1/2 phosphorylation. Moreover, trolox abolished the effect of PDT on cell viability linking the increase in intracellular ROS levels with the cell death observed, whereas that the pre-treatment with MEK inhibitor did not induce changes in SCC-13 cell survival. These findings demonstrate the effects of RFTA in triggering apoptosis induced by ROS (\O2−) production after visible light irradiation of squamous carcinoma cells. Fil: Juarez, Andrea Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Sosa, Liliana del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: de Paul, Ana Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Costa, Ana Paula. Universidade Federal de Santa Catarina; Brasil Fil: Farina, Marcelo. Universidade Federal de Santa Catarina; Brasil Fil: Leal, Rodrigo B.. Universidade Federal de Santa Catarina; Brasil Fil: Torres, Alicia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Pons, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina

Exposure to high manganese (Mn) levels may damage the basal ganglia, leading to a syndrome analogous to Parkinson's disease, with motor and cognitive impairments. The molecular mechanisms underlying Mn neurotoxicity, particularly during development, still deserve further investigation. Herein, we addressed whether early-life Mn exposure affects motor coordination and cognitive function in adulthood and potential underlying mechanisms. Male Wistar rats were exposed intraperitoneally to saline (control) or MnCl2 (5, 10 or 20 mg/kg/day) from post-natal day (PND) 8-12. Behavioral tests were performed on PND 60-65 and biochemical analysis in the striatum and hippocampus were performed on PND14 or PND70. Rats exposed to Mn (10 and 20 mg/kg) performed significantly worse on the rotarod test than controls indicating motor coordination and balance impairments. The object and social recognition tasks were used to evaluate short-term memory. Rats exposed to the highest Mn dose failed to recognize a familiar object when replaced by a novel object as well as to recognize a familiar juvenile rat after a short period of time. However, Mn did not alter olfactory discrimination ability. In addition, Mn-treated rats displayed decreased levels of non-protein thiols (e.g. glutathione) and increased levels of glial fibrillary acidic protein (GFAP) in the striatum. Moreover, Mn significantly increased hippocampal glutathione peroxidase (GPx) activity. These findings demonstrate that acute low-level exposure to Mn during a critical neurodevelopmental period causes cognitive and motor dysfunctions that last into adulthood, that are accompanied by alterations in antioxidant defense system in both the hippocampus and striatum. (C) 2015 Elsevier Inc. All rights reserved.

Creatine has been proposed to exert beneficial effects in the management of depression, but the cell signaling pathways implicated in its antidepressant effects are not well established. This study investigated the involvement of PI3K/Akt signaling pathway and its downstream intracellular targets in the antidepressant-like effect of creatine. The acute treatment of mice with creatine (1 mg/kg, po) increased the Akt and P70S6K phosphorylation, and HO-1, GPx and PSD95 immunocontents. The pretreatment of mice with LY294002 (10 nmol/mouse, icv, PI3K inhibitor), wortmannin (0.1 μg/mouse, icv, PI3K inhibitor), ZnPP (10 μg/mouse, icv, HO-1 inhibitor), or rapamycin (0.2 nmol/mouse, icv, mTOR inhibitor) prevented the antidepressant-like effect of creatine (1 mg/kg, po) in the TST. In addition, the administration of subeffective dose of either the selective GSK3 inhibitor AR-A014418 (0.01 μg/mouse, icv), the nonselective GSK3 inhibitor lithium chloride (10 mg/kg, po), or the HO-1 inductor CoPP (0.01 μg/mouse, icv), in combination with a subeffective dose of creatine (0.01 mg/kg, po) reduced the immobility time in the TST as compared with either drug alone. No treatment caused significant changes in the locomotor activity of mice. These results indicate that the antidepressant-like effect of creatine in the TST depends on the activation of Akt, Nrf2/HO-1, GPx, and mTOR, and GSK3 inhibition.

Here we show that a systemic injection of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaB) immediately after training in a step-down inhibitory avoidance task produced an enhancement of memory consolidation that persisted across consecutive retention tests during 14 days in aged rats, while it did not significantly affect memory in young adults. Control aged and young adult rats showed comparable basal levels of memory retention. Our results suggest that HDACis can display memory-enhancing effects specific for aged animals, even in the absence of age-related memory impairment.

Lectins are a class of carbohydrate-binding proteins or glycoproteins with diverse specificities and functions. The determination and characterization of the three-dimensional structures of these proteins are keys to understanding their biological effects. Recent studies have explored the anticancer potential of Diocleinae lectins (from Leguminoseae family), evaluating their antiproliferative effect and their ability to induce glioma cell death via apoptosis and autophagy. In this work, the three-dimensional structure of Dioclea lasiophylla seed lectin (DlyL) complexed with Xman (5-bromo-6-chloro-3-indolyl-α- d -mannopyranoside) was determined by X-ray crystallography. Moreover, interactions with relevant N-glycans were evaluated by molecular docking. DlyL presented the jellyroll motif, and both metal binding site (MBS) and carbohydrate-recognition domain (CRD) were determined and characterized. Molecular docking simulations indicated that DlyL interacts favorably with N-glycans, especially those of the complex and hybrid types, unlike previously studied Diocleinae lectins. DlyL also showed antitumor potential against rat C6 glioma cells impairing cell migration, inducing autophagy and cell death via activation of caspase 3. These results indicate that small structural differences among Diocleinae lectins can, in turn, result in differential modulation of autophagy and cell apoptosis processes.

Fear is a conscious state caused by exposure to real or imagined threats that trigger stress responses that affect the body and brain, particularly limbic structures. A sub-group of patients with mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) have seizures with fear, which is called ictal fear (IF), due to epileptic activity within the brain defensive survival circuit structures. Synaptic transmission efficacy can be bi-directionally modified through potentiation (long-term potentiation (LTP)) or depression (long-term depression (LTD)) as well as the phosphorylation state of Ser831 and Ser845 sites at the GluA1 subunit of the glutamate AMPA receptors, which has been characterized as a critical event for this synaptic plasticity. In this study, GluA1 levels and the phosphorylation at Ser845 and Ser831 in the amygdala (AMY), anterior hippocampus (aHIP) and middle gyrus of temporal neocortex (CX) were determined with western blots and compared between MTLE-HS patients who were showing (n = 06) or not showing (n = 25) IF. Patients with IF had an 11% decrease of AMY levels of the GluA1 subunit (p = 0.05) and a 21.5% decrease of aHIP levels of P-GluA1-Ser845 (p = 0.009) compared to patients not showing IF. The observed associations were not related to imbalances in the distribution of other concomitant types of aura, demographic, clinical or neurosurgical variables. The lower levels of P-GluA1-Ser845 in the aHIP of patients with IF were not related to changes in the levels of the serine/threonine-protein phosphatase PP1-alpha catalytic subunit or protein kinase A activation. Taken together, the GluA1 subunit levels in AMY and P-GluA1-Ser845 levels in the aHIP show an overall accuracy of 89.3% (specificity 95.5% and sensitivity 66.7%) to predict the presence of IF. AMY levels of the GluA1 subunit and aHIP levels of P-GluA1-Ser845 were not associated with the psychiatric diagnosis and symptoms of patients. Taken together with previous findings in MTLE-HS patients with IF who were evaluated by stereotactic implanted depth electrodes, we speculate our findings are consistent with the hypothesis that AMY is not a centre of fear but together with other sub-cortical and cortical structures integrates the defensive circuit that detect and respond to threats. This is the first report to address neuroplasticity features in human limbic structures connected to the defensive survival circuits, which has implications for the comprehension of highly prevalent psychiatric disorders and symptoms.

(1) Objectives: Epilepsy disorder is likely to increase with aging, leading to an increased incidence of comorbidities and mortality. In spite of that, there is a lack of information regarding this issue and little knowledge of cognitive and emotional responses in aging subjects following epileptogenesis. We investigated whether and how aging distress epilepsy-related behavioral and biochemical outcomes are associated with cognition and emotion. (2) Methods: Young and middle-aged Wistar rats (3 or 12 months old) were treated with pentylenetetrazol (PTZ, 35 mg/kg) and injected on alternated days for 20 (young rats) and 32 days (middle-aged rats). Kindling was reached after two consecutive stages 4 plus one stage 5 or 6 in Racine scale. Control and kindled rats were evaluated in the elevated plus-maze (EPM) and object-recognition tests and their hippocampus was collected 24 h later for mitogen-activated protein kinases (MAPK) dosage. (3) Results: Middle-aged rats presented a higher resistance to develop kindling, with a decrease in the seizure severity index observed following the 4th and 9th PTZ injections. Middle-aged rats displayed an increased duration of the first myoclonic seizure and an increased latency to the first generalized seizure when compared to younger rats. The induction of kindling did not impair the animals’ performance (regardless of age) in the object-recognition task and the EPM test as well as it did not alter the hippocampal levels of MAPKs. (4) Significance: Our findings reveal that, despite age-related differences during epileptogenesis, middle-aged rats evaluated after kindling performed similarly during discriminative learning and emotional tasks in comparison to young animals, with no alteration of hippocampal MAPKs. Additional investigation must be carried out to explore the electrophysiological mechanisms underlying these responses, as well as the long-term effects displayed after kindling.

CaBo is a mannose/glucose-specific lectin purified from seeds of Canavalia bonariensis . In the present work, we report the CaBo crystal structure determined to atomic resolution in the presence of X-man, a specific ligand. Similar to the structural characteristics of other legume lectins, CaBo presented the jellyroll motif, a metal binding site occupied by calcium and manganese ions close to the carbohydrate-recognition domain (CRD). In vitro test of CaBo cytotoxicity against glioma cells demonstrated its ability to decrease the cellular viability and migration by induction of autophagy and cell death. Molecular docking simulations corroborate previous data indicating that the lectin’s biological activities occur mostly through interactions with glycoproteins since the lectin interacted favorably with several N -glycans, especially those of the high-mannose type. Together, these results suggest that CaBo interacts with glycosylated cell targets and elicits a remarkable antiglioma activity.

The Pisum arvense lectin (PAL), a legume protein belonging to the Vicieae tribe, is capable of specific recognition of mannose, glucose and its derivatives without altering its structure. In this work, the three-dimensional structure of PAL was determined by X-ray crystallography and studied in detail by a combination of molecular docking and molecular dynamics (MD). Crystals belonging to monoclinic space group P21 were grown by the vapor diffusion method at 293 K. The structure was solved at 2.16 A and was similar to that of other Vicieae lectins. The structure presented Rfactor and Rfree of 17.04% and 22.08%, respectively, with all acceptable geometric parameters. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and high-mannose N-glycans. PAL demonstrated different affinities on carbohydrates, depending on bond orientation and glycosidic linkage present in ligands. Furthermore, the lectin interacted with representative N-glycans in a manner consistent with the biological effects described for Vicieae lectins. Carbohydrate-recognition domain (CRD) in-depth analysis was performed by MD, describing the behavior of CRD residues in complex with ligand, stability, flexibility of the protein over time, CRD volume and topology. This is a first report of its kind for a lectin of the Vicieae tribe.

Lectins are proteins, or glycoproteins, capable of reversibly binding to specific mono- or oligosaccharides via a noncatalytic domain. The Diocleinae subtribe presents lectins with high structural similarity, but different effects based on biological activity assays. This variability results from small structural differences. Therefore, in this context, the present study aimed to perform a structural analysis of the lectin from Dioclea lasiophylla Mart. ex Benth seeds (DlyL) and evaluate its inflammatory effect. To accomplish this, DlyL was purified in a single step by affinity chromatography on Sephadex® G-50 matrix. DlyL primary structure was determined through a combination of tandem mass spectrometry and DNA sequencing. DlyL showed high similarity with other species from the same genus. Its theoretical three-dimensional structure was predicted by homology modelling, and the protein was subjected to ligand screening with monosaccharides, oligosaccharides and complex N-glycans by molecular docking. Stability and binding of the lectin with α-methyl-d-mannoside were assessed by molecular dynamics. DlyL showed acute inflammatory response with hypernociceptive effect in the paw edema model, possibly by interaction with glycans present at the cell surface.

Agmatine is an endogenous neuromodulator which, based on animal and human studies, is a putative novel antidepressant drug. In this study, we investigated the ability of sub-chronic (21 days) p.o. agmatine administration to produce an antidepressant-like effect in the tail suspension test and examined the hippocampal cell signaling pathways implicated in such an effect. Agmatine at doses of 0.01 and 0.1 mg/kg (p.o.) produced a significant antidepressant-like effect in the tail suspension test and no effect in the open-field test. Additionally, agmatine (0.001-0.1 mg/kg, p.o.) increased the phosphorylation of protein kinase A substrates (237-258% of control), protein kinase B/Akt (Ser(473)) (116-127% of control), glycogen synthase kinase-3β (Ser(9)) (110-113% of control), extracellular signal-regulated kinases 1/2 (119-137% and 121-138% of control, respectively) and cAMP response elements (Ser(133)) (127-152% of control), and brain-derived-neurotrophic factor (137-175% of control) immunocontent in a dose-dependent manner in the hippocampus. Agmatine (0.001-0.1 mg/kg, p.o.) also reduced the c-jun N-terminal kinase 1/2 phosphorylation (77-71% and 65-51% of control, respectively). Neither protein kinase C nor p38(MAPK) phosphorylation was altered under any experimental conditions. Taken together, the present study extends the available data on the mechanisms that underlie the antidepressant action of agmatine by showing an antidepressant-like effect following sub-chronic administration. In addition, our results are the first to demonstrate the ability of agmatine to elicit the activation of cellular signaling pathways associated with neuroplasticity/cell survival and the inhibition of signaling pathways associated with cell death in the hippocampus.

In the central nervous system, many receptors, ion channels and neurotransmitter transporters are glycoproteins, where the glycan chains are modulator elements. Lectins are proteins, which recognize and bind carbohydrate complexes. We have previously shown that ConBr, a lectin purified from Canavalia brasiliensis seeds, produced antidepressant-like effect and blocked hippocampal neurotoxicity induced by quinolinic acid and glutamate. Noteworthy, all these effects occurred in a dependence of its carbohydrate recognition domain. Therefore, the present study was undertaken in order to elucidate intracellular signaling pathways regulated by ConBr that may be potentially associated with the antidepressant and neuroprotective effects previously reported to be dependent on carbohydrate interaction. ConBr (10 µg/site) was injected into the ventricle (i.c.v.) of mice, and the hippocampi were removed 0.5, 1, 3, 6, 8, 12, 18, and 24 h after treatment. Our results showed that in the period of 0.5–3 h, ConBr induced activation of the protein kinases Akt, ERK1, and PKA. Furthermore, the phosphorylation of CREB-Ser133 was stimulated by ConBr (1–6 h), while brain-derived neurotrophic factor (BDNF) mRNA was increased at 12 h and BDNF protein at 18–24 h. Our data suggest that an early activation of protein kinases may trigger CREB-dependent BDNF transcription, resulting in a subsequent increase of BDNF protein in response to ConBr. Later, increment of Akt phosphorylation was observed 24 h after ConBr administration, possibly due to BDNF/TrkB-dependent activation of Akt. Our findings indicate that ConBr is a multifunctional molecule capable to activate signaling pathways involved in neuroplasticity and neuroprotection. Copyright © 2014 John Wiley & Sons, Ltd.

Epidermal neural crest stem cells (EPI-NCSCs), which reside in the bulge of hair follicles, are attractive candidates for several applications in cell therapy, drug screening and tissue engineering. As suggested remnants of the embryonic neural crest (NC) in an adult location, EPI-NCSCs are able to generate a wide variety of cell types and are readily accessible by a minimally invasive procedure. Since the combination of epidermal growth factor (EGF) and fibroblast growth factor type 2 (FGF 2 ) is mitogenic and promotes the neuronal commitment of various stem cell populations, we examined its effects in the proliferation and neuronal potential of mouse EPI-NCSCs. By using a recognized culture protocol of bulge whiskers follicles, we were able to isolate a population of EPI-NCSCs, characterized by the migratory potential, cell morphology and expression of phenotypic markers of NC cells. EPI-NCSCs expressed neuronal, glial and smooth muscle markers and exhibited the NC-like fibroblastic morphology. The treatment with the combination EGF and FGF 2 , however, increased their proliferation rate and promoted the acquisition of a neuronal-like morphology accompanied by reorganization of neural cytoskeletal proteins βIII-tubulin and nestin, as well as upregulation of the pan neuronal marker βIII-tubulin and down regulation of the undifferentiated NC, glial and smooth muscle cell markers. Moreover, the treatment enhanced the response of EPI-NCSCs to neurogenic stimulation, as evidenced by induction of GAP43, and increased expression of Mash-1 in neuron-like cell, both neuronal-specific proteins. Together, the results suggest that the combination of EGF–FGF2 stimulates the proliferation and improves the neuronal potential of EPI-NCSCs similarly to embryonic NC cells, ES cells and neural progenitor/stem cells of the central nervous system and highlights the advantage of using EGF–FGF 2 in neuronal differentiation protocols.

Lectins recognize and reversibly bind to carbohydrates attached to proteins and lipids modulating a variety of signaling pathways. We previously showed that ConBr, a lectin from Canavalia brasiliensis seeds, produced an antidepressant-like effect in mice by modulating the monoaminergic neurotransmitter systems. Moreover, ConBr blocked hippocampal neurotoxicity induced by quinolinic acid in vivo and by glutamate in vitro, suggesting a neuroprotective activity of ConBr via glutamatergic system modulation. Therefore, the present study was undertaken to investigate the involvement of the N-methyl-D-aspartate (NMDA) receptor and the L-arginine-nitric oxide (NO) pathway in the antidepressant-like action displayed by ConBr in the forced swimming test (FST). With the aim of verifying the involvement of NMDA receptors in the antidepressant-like effect of ConBr (10 μg/site, i.c.v.), an intracerebroventricular (i.c.v.) pretreatment with either NMDA (0.1 pmol/site) or D-serine (30 μg/site) was carried out. The results show that both treatments blocked the effect of ConBr. Furthermore, the coadministration of subeffective doses of the NMDA receptor antagonist MK-801 (0.001 mg/kg, i.p.) or ketamine (0.1 mg/kg, i.p.; NMDA receptor antagonist) and ConBr (0.1 μg/site, i.c.v.) caused a synergistic reduction in immobility time. In order to verify the dependence of the L-arginine-NO-cGMP pathway, on the effect of ConBr in the FST, a pretreatment with the NO precursor, L-arginine (750 mg/kg, i.p.), or the PDE5 inhibitor, sildenafil (5 mg/kg, i.p.), was performed. Both drugs abolished the antidepressant-like action of ConBr. Finally, the administration of subeffective doses of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 30 pmol/site, i.c.v.) and ConBr (0.1 μg/site, i.c.v.) produced a synergistic antidepressant-like effect in the FST. Taken together, the results suggest that the antidepressant-like effect of ConBr in the FST involves NMDA receptor inhibition and reduction in NO and cGMP synthesis.

While manganese (Mn) is essential for proper central nervous system (CNS) development, excessive Mn exposure may lead to neurotoxicity. Mn preferentially accumulates in the basal ganglia, and in adults it may cause Parkinson’s disease–like disorder. Compared to adults, younger individuals accumulate greater Mn levels in the CNS and are more vulnerable to its toxicity. Moreover, the mechanisms mediating developmental Mn-induced neurotoxicity are not completely understood. The present study investigated the developmental neurotoxicity elicited by Mn exposure (5, 10 and 20 mg/kg; i.p.) from postnatal day 8 to PN27 in rats. Neurochemical analyses were carried out on PN29, with a particular focus on striatal alterations in intracellular signaling pathways (MAPKs, Akt and DARPP-32), oxidative stress generation and cell death. Motor alterations were evaluated later in life at 3, 4 or 5 weeks of age. Mn exposure (20 mg/kg) increased p38MAPK and Akt phosphorylation, but decreased DARPP-32-Thr-34 phosphorylation. Mn (10 and 20 mg/kg) increased caspase activity and F2-isoprostane production (a biological marker of lipid peroxidation). Paralleling the changes in striatal biochemical parameters, Mn (20 mg/kg) also caused motor impairment, evidenced by increased falling latency in the rotarod test, decreased distance traveled and motor speed in the open-field test. Notably, the antioxidant Trolox™ reversed the Mn (20 mg/kg)-dependent augmentation in p38MAPK phosphorylation and reduced the Mn (20 mg/kg)-induced caspase activity and F2-isoprostane production. Trolox™ also reversed the Mn-induced motor coordination deficits. These findings are the first to show that long-term exposure to Mn during a critical period of neurodevelopment causes motor coordination dysfunction with parallel increment in oxidative stress markers, p38MAPK phosphorylation and caspase activity in the striatum. Moreover, we establish Trolox™ as a potential neuroprotective agent given its efficacy in reversing the Mn-induced neurodevelopmental effects.

The molecular mechanisms mediating manganese (Mn)-induced neurotoxicity, particularly in the immature central nervous system, have yet to be completely understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs) and tyrosine hydroxylase (TH) could represent potential targets of Mn in striatal and hippocampal slices obtained from immature rats (14 days old). The aim of this study was to evaluate if the MAPK pathways are modulated after subtoxic Mn exposure, which do not significantly affect cell viability. The concentrations of manganese chloride (MnCl2; 10–1,000 μM) caused no change in cell viability in slices exposed for 3 or 6 hours. However, Mn exposure significantly increased extracellular signal-regulated kinase (ERK) 1/2, as well as c-Jun N-terminal kinase (JNK) 1/2/3 phosphorylation at both 3 and 6 hours incubations, in both brain structures. Furthermore, Mn exposure did not change the total content or phosphorylation of TH at the serine 40 site in striatal slices. Thus, Mn at concentrations that do not disrupt cell viability causes activation of MAPKs (ERK1/2 and JNK1/2/3) in immature hippocampal and striatal slices. These findings suggest that altered intracellular MAPKs signaling pathways may represent an early event concerning the effects of Mn in the immature brain.

The olfactory bulbectomy (OB) animal model of depression is a well-established model that is capable of detecting antidepressant activity following chronic drug therapy, and the surgery results in behavioral and biochemical changes that are reminiscent of various symptoms of depression. In the present study, we investigated the degree to which 14 days of p.o. administration of the classic antidepressant fluoxetine (10 mg/kg) were able to reverse OB-induced changes in behavior (namely, hyperactivity in the open-field test and reduced motivational and self-care behaviors in the splash test) and in the activation of hippocampal cell signaling pathways that are thought to be involved in synaptic plasticity. OB caused significant increases in ERK1 and CREB (Ser 133 ) phosphorylation and in the expression of BDNF immunocontent, all of which were prevented by fluoxetine administration. Moreover, fluoxetine administration also caused a significant decrease in ERK2 phosphorylation in mice that had undergone OB. Neither Akt nor GSK-3β phosphorylation was altered in any experimental condition. In conclusion, the present study shows that OB can induce significant behavioral changes that are accompanied by the activation of hippocampal signaling pathways, namely the ERK1/CREB/BDNF pathway, which is involved in the synaptic plasticity. Conversely, fluoxetine prevented these OB-induced behavioral changes and avoided the activation of ERK1/CREB/BDNF in the hippocampus. Taken together, our results extend the data from the existing literature regarding OB-induced behavioral and neurochemical changes, and suggest a possible underlying mechanism that can account for the antidepressant effect of fluoxetine in this model.

Agmatine is an endogenous neuromodulator that has been shown to have antidepressant-like properties. We have previously demonstrated that it can induce a rapid increase in BDNF levels after acute administration, suggesting that agmatine may be a fast-acting antidepressant. To investigate this hypothesis, the present study evaluated the effects of a single administration of agmatine in mice subjected to chronic unpredictable stress (CUS), a model of depression responsive only to chronic treatment with conventional antidepressants. The ability of agmatine to reverse CUS-induced behavioral and biochemical alterations was evaluated and compared with those elicited by the fast-acting antidepressant (ketamine) and the conventional antidepressant (fluoxetine). After exposed to CUS for 14 days, mice received a single oral dose of agmatine (0.1 mg/kg), ketamine (1 mg/kg) or fluoxetine (10 mg/kg), and were submitted to behavioral evaluation after 24 h. The exposure to CUS caused an increased immobility time in the tail suspension test (TST) but did not change anhedonic-related parameters in the splash test. Our findings provided evidence that, similarly to ketamine, agmatine is able to reverse CUS-induced depressive-like behavior in the TST. Western blot analyses of prefrontal cortex (PFC) demonstrated that mice exposed to CUS and/or treated with agmatine, fluoxetine or ketamine did not present alterations in the immunocontent of synaptic proteins [i.e. GluA1, postsynaptic density protein 95 (PSD-95) and synapsin]. Altogether, our findings indicate that a single administration of agmatine is able to reverse behavioral alterations induced by CUS in the TST, suggesting that this compound may have fast-acting antidepressant-like properties. However, there was no alteration in the levels of synaptic proteins in the PFC, a result that need to be further investigated in other time points.

Inosine is a purine nucleoside formed by the breakdown of adenosine that elicits an antidepressant-like effect in mice through activation of adenosine A1 and A2A receptors. However, the signaling pathways underlying this effect are largely unknown. To address this issue, the present study investigated the influence of extracellular-regulated protein kinase (ERK)1/2, Ca2+/calmoduline-dependent protein kinase (CaMKII), protein kinase A (PKA), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase 3beta (GSK-3β) modulation in the antiimmobility effect of inosine in the tail suspension test (TST) in mice. In addition, we attempted to verify if inosine treatment was capable of altering the immunocontent and phosphorylation of the transcription factor cyclic adenosine monophosphatate (cAMP) response-binding element protein (CREB) in mouse prefrontal cortex and hippocampus. Intracerebroventricular administration of U0126 (5 μg/mouse, MEK1/2 inhibitor), KN-62 (1 μg/mouse, CaMKII inhibitor), H-89 (1 μg/mouse, PKA inhibitor), and wortmannin (0.1 μg/mouse, PI3K inhibitor) prevented the antiimmobility effect of inosine (10 mg/kg, intraperitoneal (i.p.)) in the TST. Also, administration of a sub-effective dose of inosine (0.1 mg/kg, i.p.) in combination with a sub-effective dose of AR-A014418 (0.001 μg/mouse, GSK-3β inhibitor) induced a synergic antidepressant-like effect. None of the treatments altered locomotor activity of mice. Moreover, 24 h after a single administration of inosine (10 mg/kg, i.p.), CREB phosphorylation was increased in the hippocampus. Our findings provided new evidence that the antidepressant-like effect of inosine in the TST involves the activation of PKA, PI3K/Akt, ERK1/2, and CaMKII and the inhibition of GSK-3β. These results contribute to the comprehension of the mechanisms underlying the purinergic system modulation and indicate the intracellular signaling pathways involved in the antidepressant-like effect of inosine in a preclinical test of depression.

A lectin from Canavalia virosa, Diocleinae subtribe, was purified by affinity chromatography with Sephadex G-50 matrix and named ConV. The primary structure of ConV was obtained by mass spectrometry and crystals were obtained by the vapor diffusion method at 293K and belonged to orthorhombic space group P21221 with two molecules in its asymmetric unit. The structure obtained presented Rfactor and Rfree of 18.91% and 24.92% respectively, with no residues in nonallowed regions of Ramachandran plot. The crystal structure was solved at 2.53A and was demonstrated to be very similar to other lectins from the same subtribe. In inflammatory tests, ConV elicited paw edema, but incubation of lectin with glucose beforehand was able to reduce the edematogenic effect, indicating the involvement of the carbohydrate recognition domain in this process. The lectin also showed toxicity to rat C6 glioma cells, disrupting the mitochondrial membrane potential (ΔYm) and decreasing cell viability, indicating an anticancer potential for ConV. In silico studies confirmed that ConV interacts strongly with carbohydrates that comprise the N-glycans of glycoproteins. This finding corroborates the hypothesis which holds that the lectin domain interacts with glycans in molecular targets and that this contributes to the effects observed in biological activities.