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Introduction: Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer, often referred to as the Brazilian wandering spider, banana spider or "armed" spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. Methods: In this study, we explored the neuroactivity and molecular diversity of P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Results: Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Discussion: Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggest that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.

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Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.

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The phylogenetic affiliations of organisms responsible for aerobic CO oxidation in hypersaline soils and sediments were assessed using media containing 3.8 M NaCl. CO-oxidizing strains of the euryarchaeotes, Haloarcula, Halorubrum, Haloterrigena and Natronorubrum, were isolated from the Bonneville Salt Flats (UT) and Atacama Desert salterns (Chile). A halophilic euryarchaeote, Haloferax strain Mke2.3(T), was isolated from Hawai'i Island saline cinders. Haloferax strain Mke2.3(T) was most closely related to Haloferax larsenii JCM 13917(T) (97.0% 16S rRNA sequence identity). It grew with a limited range of substrates, and oxidized CO at a headspace concentration of 0.1%. However, it did not grow with CO as a sole carbon and energy source. Its ability to oxidize CO, its polar lipid composition, substrate utilization and numerous other traits distinguished it from H. larsenii JCM 13917(T), and supported designation of the novel isolate as Haloferax namakaokahaiae Mke2.3(T), sp. nov (= DSM 29988, = LMG 29162). CO oxidation was also documented for 'Natronorubrum thiooxidans' HG1 (Sorokin, Tourova and Muyzer 2005), N. bangense (Xu, Zhou and Tian 1999) and N. sulfidifaciens AD2(T) (Cui et al. 2007). Collectively, these results established a previously unsuspected capacity for extremely halophilic aerobic CO oxidation, and indicated that the trait might be widespread among the Halobacteriaceae, and occur in a wide range of hypersaline habitats.

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Olfactory neuroepithelial cells in culture have been proposed as a model to study the physiopathology of psychiatric disorders and biomarker characterization for diagnosis. In patients with schizophrenia (SZ) and bipolar disorder (BD) diminished microtubule-associated proteins expression occurs, which might lead to aberrant microtubular organization and which in turn may affect Ca(2+) voltage-activated currents. The aim of this work was to characterize of microtubule organization as well as of the L-type Ca(2+) current in neuronal precursors obtained from nasal exfoliates of patients with SZ and BD. Microtubule organization was studied by immunofluorescence with a specific anti-III ß-tubulin antibody and by quantification of globular and assembled tubulin by Western blot. L-type current recording was performed by whole-cell patch-clamp technique and nifedipine superfusion. The results showed differential altered microtubular organization in neuronal precursors of SZ and BD. Short microtubules were observed in BD neurons, while extensive, unstained subcellular areas and disorganized microtubules were evident in SZ neuronal precursors. Patients with BD showed a decrease in amounts of tubulin in total homogenates and 40% decrease in the globular fraction. However, L-type current in BD was similar to that in healthy subjects (HS). In contrast, this current in SZ was 50% lower. These reduction in L-type current in SZ together with differential microtubule alterations are potential biomarkers that may differentiates SZ and BD.

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Brain imaging and histopathological studies suggest that neurodevelopmental anomalies play a key role in the etiology of schizophrenia (SZ) and bipolar disorder (BD). New neuron formation and maturation occur in human olfactory epithelium throughout life. Therefore, the olfactory epithelium has been proposed as a model to study alterations in neurodevelopment, particularly in some psychiatric diseases. However, former studies were done with olfactory epithelium biopsies taken post mortem or under anesthesia from patients with SZ and BD. In this work we have developed a new method to obtain viable neural precursors by exfoliation of the anterior region of the medial lateral turbinate of the nasal cavity from healthy controls, and ambulatory patients. Cells were propagated to establish neural precursor banks. Thawed cells showed cytoskeletal phenotypes typical of developing neurons. They also conserved the ability to differentiate in presence of 2mM dibutyril-cyclic adenosine monophosphate, and maintained voltage-operated Ca(2+) currents in culture. Moreover, proportions of neuronal maturation stages were maintained in cultured exfoliates obtained from SZ and BD patients. Data support that neural precursors obtained from a nasal exfoliate are an excellent experimental model to later approach studies on biomarkers, neural development and cellular alterations in the pathophysiology of SZ and BD.

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AIMS: Nitric oxide (NO) is a free radical which reportedly causes damage to living cells. This study evaluated the damaging effect of NO and the protection of melatonin on the retina in vivo. METHODS: Female Wistar rats (230-250 g) received two intraperitoneal injections of either melatonin (5 mg/kg) or vehicle alone. After general anaesthesia, the animals received 1 microl intravitreal injections of 0.9% saline and 1 mM sodium nitroprusside (SNP) into the right eye and the left eye, respectively. The animals were divided into two groups and then sacrificed after 24 hours (day 1) and 96 hours (day 4). The mean inner retinal layer thickness (mIRLT), the number of retinas expressing hyperchromatic (HC) nuclei in the inner nuclear layer (INL) and the apoptotic ganglion cell detection were compared. RESULTS: After 1 day, SNP significantly increased the mIRLT by 45% (p = 0.004), initiated more INL nuclear HC expression (p = 0.01) and apoptotic nuclei (p<0.05) compared with the control eyes. Injection of melatonin ameliorated these changes. On day 4, SNP demonstrated similar effects in all parameters on the retina. After the injection of melatonin, both INL HC expression and apoptotic ganglion nuclei in the SNP treated eyes were similar to the controls but the mIRLT was significantly greater than in controls (p = 0.006). CONCLUSION: Uncontrolled NO elevation caused morphological and nuclear changes in the retina. Melatonin significantly suppressed the NO induced increase in mIRLT, INL HC expression, and apoptotic ganglion cells on day 1, but not after day 4. Melatonin may have a protective role in the NO elevated retina.

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Progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, both in neural aging and in neurodegenerative diseases. Cytoskeleton in neurodegenerative diseases is characterized by hyperphosphorylated tau assembled in neurofibrillary tangles. Tau protein promotes microtubule enlargement and its hyperphosphorylation inhibits tubulin assembly. Okadaic acid (OA) causes oxidative stress, tau hyperphosphorylation, and altered cytoskeletal organization similar to those observed in neurons of patients with dementia. Since melatonin acts by both enlarging microtubules and as a free-radical scavenger, in this work we studied the effects of melatonin on altered cytoskeletal organization induced by OA in N1E-115 neuroblastoma cells. Optic microscopy, morphometric analysis, and tubulin immunofluorescence staining of neuroblastoma cells incubated with 50 nM OA showed an intact microtubule network following the neurite profile similar to that observed in the vehicle-incubated cells when melatonin was added to the incubation media 2 h before OA. The melatonin effects on altered cytoskeletal organization induced by OA were dose-dependent and were not abolished by luzindole, the mt(1) melatonin antagonist receptor. Also, increased lipid peroxidation and augmented apoptosis in N1E-115 cells incubated with 50 nM OA were prevented by melatonin. The results support the hypothesis that melatonin can be useful in the treatment of neurodegenerative diseases.

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Melatonin, vitamin E and estrogen have been shown to exert neuroprotective effects against kainic acid (KA)-induced damage in the hippocampus. The aim of the present study was to examine the changes in potassium-evoked gamma-aminobutyric acid (GABA) release in the hippocampus of KA-treated rats and to test the possible protective effects of melatonin, vitamin E or estrogen. Following the treatment of mice with KA, a marked reduction in potassium-evoked [3H]GABA release was observed. Melatonin or estrogen prevented the reduction in potassium-evoked GABA release due to kainate administration. Vitamin E also exhibited some protective effect, but it was less than that provided by melatonin or estrogen. Melatonin, estrogen and, to a lesser extent, vitamin E reduce the physiological toxicity of KA. Since KA is believed to cause neuronal alterations via oxidative processes, it is assumed that the free radical scavenging and oxidative properties of melatonin, estrogen and vitamin E account for the protective effects of these agents.

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Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.

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