Holistic profiling of the venom from the Brazilian wandering spider Phoneutria nigriventer by combining high-throughput ion channel screens with venomics.

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.

Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus.

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.

Isolation and characterization of extremely halophilic CO-oxidizing Euryarchaeota from hypersaline cinders, sediments and soils and description of a novel CO oxidizer, Haloferax namakaokahaiae Mke2.3T, sp. nov.

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.

Microtubule organization and L-type voltage-activated calcium current in olfactory neuronal cells obtained from patients with schizophrenia and bipolar disorder.

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.

A non-invasive method to isolate the neuronal linage from the nasal epithelium from schizophrenic and bipolar diseases.

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.

Effects of melatonin on the nitric oxide treated retina.

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.

Melatonin prevents cytoskeletal alterations and oxidative stress induced by okadaic acid in N1E-115 cells.

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.

Melatonin, vitamin E, and estrogen reduce damage induced by kainic acid in the hippocampus: potassium-stimulated GABA release.

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.

Melatonin activates PKC-alpha but not PKC-epsilon in N1E-115 cells.

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.

O sistema imune inato intestinal em pacientes com estrongiloidíase / The innate intestinal immune system in patients with strongyloidiasis

Foram estudados fragmentos da mucosa duodenal obtidos de nove pacientes apresentando sintomas leves de estrogiloidiase, nove com sintomas moderados, sete com sintomas graves e sete indivíduos aparentemente normais. A muramidase(lisozima) foi imunocitoquimicamente demonstrada em cortes contracorados pela técnica do PAS. Havia aparente aumento progressivo na secreçao de muramidase pela célula de Paneth aaacompanhado o agravamento dos sintomas, näo obstante o fato de que a sua populaçao permanecesse constante. Decréscimo progressivo no número de células caliciformes foi observado enquanto, concomitantemente, haavia aumento na populaçao de células intermediarias. Esses resultados foram interpretados como a indicaçao da participaçao do sistema imune inato intestinal no estabelecimento da interaçao Strongyloides stercoralis/hospedeiro, através do aumento da secreçao da enzima mucolítica muramidase

Absence of lysozyme (muramidase) in the intestinal Paneth cells of newborn infants with necrotising enterocolitis.

AIM: To determine immunocytochemically whether preterm and newborn infants with necrotising enterocolitis (NEC) show differences in numbers of lysozyme positive Paneth cells compared with normal controls, and to relate the findings to the possibility that lysozyme deficiency may facilitate the bacterial infections thought to be associated with this condition. METHODS: Tissues from 10 infants with NEC and from 11 matched controls were sectioned and stained immunocytochemically for lysozyme. Differences in the numbers of Paneth cells and degree of lysozyme positivity in the tissues were assessed. RESULTS: Tissues from NEC patients showed no, or very few, lysozyme positive Paneth cells, whereas controls showed strong positive staining. CONCLUSIONS: A deficiency or developmental defect in Paneth cells, resulting in an absence of lysozyme, may render the intestine more susceptible to bacterial infection, allowing organisms to adhere and translocate across the mucosa. Such enhancement of infection may contribute to the pathogenesis of NEC.

In vitro stimulation of protein kinase C by melatonin.

It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca2+ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca2+ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [3H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 microM) and protein kinase C inhibitors (100 microM) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [3H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca2+. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca2+ dependent proteins.

Intra-abdominal sepsis: an immunocytochemical study of the small intestine mucosa.

AIM: To investigate immunocytochemical changes in intestinal tissues from patients with intra-abdominal sepsis, and to relate the changes to the possibility of enhanced bacterial adhesion and translocation. METHODS: Tissues from 17 patients suffering from intra-abdominal sepsis and from controls were sectioned and stained immunocytochemically for IgA, IgM, secretory component, J chain, and HLA-DR. Differences in the distribution and characteristics of positively staining cells between the patient groups were assessed. RESULTS: Patients with intra-abdominal sepsis had noticeable reductions in numbers of IgA and IgM plasma cells, reduced J chain staining, and had little immunoglobulin on the surfaces of enterocytes. In contrast, HLA-DR positive cells were increased in the sepsis compared with the control group. The plasma cells present showed cytological changes suggestive of apoptosis. CONCLUSIONS: Stress associated with sepsis and its immediate causes might result in increased plasma glucocorticoid levels that bring about apoptosis of mucosal plasma cells (or their precursors). The consequent reduction in expression of IgA and IgM may favour bacterial adhesion to the enterocytes and facilitate bacterial translocation into the tissues.

Immunocytochemistry of mucosal changes in patients infected with the intestinal nematode Strongyloides stercoralis.

AIM: To investigate the immunopathological changes in duodenal tissues induced by strongyloidiasis and to relate these to degrees of clinical severity. METHODS: Tissues taken from 21 patients showing mild, moderate or severe symptoms of strongyloidiasis, and from non-infected controls, were sectioned and stained immunocytochemically for IgA, secretory component (SC) and HLA-DR. Immunopathology was assessed by changes in numbers, intensity and distribution of stained cells. RESULTS: Parasitised individuals showed villous atrophy and crypt hyperplasia. There was notable infiltration of the lamina propria by IgA positive plasma cells and of the epithelium by intraepithelial lymphocytes. Infection was also associated with increased expression of SC and decreased expression of HLA-DR in epithelial cells. Changes in all parameters correlated with degree of clinical severity. CONCLUSIONS: Profound mucosal changes are induced by strongyloidiasis. Some are analogous to those seen in coeliac disease, but others seem quite unusual. It is likely that these changes are functionally related to the immunopathophysiological consequences of infection seen in patients with severe disease.

Immunocytochemical demonstration that human duodenal Brunner's glands may participate in intestinal defence.

The immunocytochemical demonstration of IgA and IgM in some secretory units of human Brunner's glands, associated with the presence of secretory component in all secretory cells, indicates the possibility that these glands assist the function of the intestinal crypts in transporting immunoglobulins into the gut lumen. In addition, the presence of muramidase (lysozyme) in the cells of the secretory units suggests that Brunner's glands continuously secrete bactericidal enzyme, thus reinforcing the function of the Paneth cells as contributors to nonspecific defence (innate immunity) in the intestinal tract.

In vitro inhibition of Ca2+/calmodulin-dependent kinase II activity by melatonin.

Recent evidence suggests that a melatonin (MEL) mechanism of action may be through modulation of Ca2+-activated calmodulin (CaM). MEL binds to CaM with a high affinity, and has been shown to act as a CaM antagonist. Among the CaM-dependent enzymes, Ca2+/Calmodulin-dependent protein kinase II (CaM-kinase II) is a particularly abundant enzyme in the nervous system. In the brain it phosphorylates a broad spectrum of substrates, thus modulating important neuronal functions. We describe the MEL effect on CaM-kinase II activity in vitro. CaM-kinase II was purified from rat brain by column chromatography, and identified by Western immunoblotting. CaM-kinase II activity was assessed in the presence of Ca2+/CaM by the kinase's ability to phosphorylate the synthetic substrate syntide-2 and by enzyme autophosphorylation. MEL inhibited CaM-kinase II activity, and enzyme autophosphorylation. Inhibition of the enzyme by 10(-9) M MEL was nearly of 30%. Trifluoperazine (10 microM), W7 (10 microM), and compound 48/80 (30 micrograms/ml), inhibited CaM-kinase II activity by 40%, 42%, and 93%, respectively. Both EGTA (5 mM) and MEL (10(-5) M) abolished autophosphorylation. The effect of MEL on CaM-kinase II activity was specific, since neither serotonin, N-acetylserotonin, nor 6-hydroxymelatonin inhibited its activity. Our results support the hypothesis that MEL acts as a CaM antagonist and cellular functions may be rhythmically regulated by MEL modulation of CaM-dependent protein phosphorylation.

Immunocytochemical study of the ontogeny of the marsupial Didelphis albiventris immune system.

Marsupials have considerable merits as models for studying the developmental dynamics of the mammalian immune system, but until recently there has been a conspicuous lack of specific immune probes to facilitate such studies. To begin a precise study of the ontogeny of the marsupial Didelphis albiventris we have used cross-reactive polyclonal antibodies raised against evolutionarily highly conserved peptides which form part of the antigen specific receptor complexes of human differentiated lymphocytes. Moreover, because of antigen receptor conservation, the antibodies also recognise specifically the immunocompetent T and B lymphocytes of other species including those in the organs of the opossum. Use of the antipeptide antibodies together with other cross-reacting antibodies has allowed us to study the cellular immunology of T and B cells and antigen presenting cells (APC) during the development of thymus, skin, lymph nodes and spleen in the Brazilian white-belly opossum. The molecular nature and identity of the T cell antigens detected in opossum tissues were confirmed by immunoblotting. These findings indicate that it is now possible to exploit these antibody probes for comparative mammalian studies, and indeed to investigate interesting features of the opossum, such as reaction of the immature immune system of the pouch young to antigenic stimulation.

Entamoeba histolytica: PKC transduction pathway activation in the trophozoite-fibronectin interaction.

Interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces reorganization of the actin cytoskeleton and an increase in proteolytic activities that results in the degradation of the bound protein. The binding is mediated by a 37-kDa FN "receptor" localized in the trophozoite surface and associated to the cytoskeleton. The intracellular signals triggered by the ligand-receptor interaction are not well understood but it is plausible that they drive the observed responses. To address this issue, the activation of protein kinase C (PKC) pathways by FN binding was explored. Stimulation with phorbol myristate acetate (PMA) or FN produced a rapid increase in the amebas adhesion to the substrate and local release of proteases. Two PKC inhibitors, H7 and staurosporine, reverted the PMA stimulus and inhibited the response induced by FN. Interaction with FN as well as treatment with PMA produced transient changes of F-actin levels susceptible to inhibition by H7. Furthermore, phosphorylation of amebic proteins was enhanced in response to FN binding and PMA, while the presence of the PKC inhibitor diminished their phosphorylation. Inositol triphosphate production was stimulated by the FN binding, and PKC activation and translation was registered in cell extracts obtained from the stimulated amebas. Our results suggest that PKC pathways are activated in amebas by information transduced as a result of trophozoite binding to FN.