Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids

This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography-mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18-23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential. (C) 2003 Elsevier B.V All rights reserved.

In situ characterization of stickiness of sugar-rich foods using a linear actuator driven stickiness testing device

A stickiness testing device based on the probe tack test has been designed and tested. It was used to perform in situ characterization of drying hemispherical drops with an initial radius 3.5 mm. Tests were carried out in two drying temperatures, 63 and 95 degreesC. Moisture and temperature histories of the drying drops of fructose, honey, sucrose, maltodextrin and sucrose-maltodextrin mixtures were determined. The rates of moisture evaporation of the fructose solution was the fastest while those of the maltodextrin solution was the lowest. A profile reversal was observed when the temperature profiles of these materials were compared. Different modes of failure were observed during the stickiness tests. Pure fructose and honey solutions remained completely sticky and failed cohesively until the end of drying. Pure sucrose solution remained sticky and failed cohesively until complete crystallization occurred. The surface of the maltodextrin drops formed a skin shortly after the start of drying. It exhibited adhesive failure and reached a state of non-adhesion. Addition of maltodextrin significantly altered the stickiness of sucrose solution. (C) 2002 Elsevier Science Ltd. All rights reserved.

Characterization of the surface stickiness of fructose-maltodextrin solutions during drying

A probe tack test has been used for the in situ characterization of the surface stickiness of hemispherical drops with an initial radius of 3.5 mm while drying. Surface stickiness of drops of fructose and maltodextrin solutions dried at 63degreesC and 95degreesC was determined. The effect of addition of maltodextrin on fructose solution-was studied with fructose/maltodextrin solid mass ratios of 4: 1, 1: 1, and 1:4. Pure fructose solutions remained completely sticky and failed cohesively even when their moisture approached zero. Shortly after the start of drying, the surface of the maltodextrin drops formed a skin, which rapidly grew in thickness. Subsequently the drop surface became completely nonsticky probably due to transformation of outer layers into a glassy material. Addition of malto,dextrin significantly altered the surface stickiness of drops of fructose solutions, demonstrating its use as an effective drying aid.

Biased EPR entanglement and its application to teleportation

We consider pure continuous variable entanglement with non-equal correlations between orthogonal quadratures. We introduce a simple protocol which equates these correlations and in the process transforms the entanglement onto a state with the minimum allowed number of photons. As an example we show that our protocol transforms, through unitary local operations, a single squeezed beam split on a beam splitter into the same entanglement that is produced when two squeezed beams are mixed orthogonally. We demonstrate that this technique can in principle facilitate perfect teleportation utilizing only one squeezed beam.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

The synthesis and X-ray crystal structure of 6-bromo-2,4,4-trimethyl-cyclohex-2-enone

Regiospecific bromination of 2,4,4-trimethyl-cyclohex-2-enone was achieved and the X-ray crystal structure of 6-bromo-2,4,4-trimethyl-cyclohex-2-enone is presented.

Preparation and characterization of Nickel-and cobalt-doped magnetites

Trabalho apresentado no I Simpósio Mineiro de Ciências dos Materiais, Ouro Preto, Novembro de 2001.

Effects of small doses of ouabain on the arterial blood pressure of anesthetized hypertensive and normotensive rats

Ouabain increases vascular resistance and may induce hypertension by inhibiting the Na+ pump. The effects of 0.18 and 18 µg/kg, and 1.8 mg/kg ouabain pretreatment on the phenylephrine (PHE; 0.1, 0.25 and 0.5 µg, in bolus)-evoked pressor responses were investigated using anesthetized normotensive (control and uninephrectomized) and hypertensive (1K1C and DOCA-salt treated) rats. Treatment with 18 µg/kg ouabain increased systolic and diastolic blood pressure in all groups studied. However, the magnitude of this increase was larger for the hypertensive 1K1C and DOCA-salt rats than for normotensive animals, while the pressor effect of 0.18 µg/kg ouabain was greater only in DOCA-salt rats. A very large dose (1.8 mg/kg) produced toxic effects on the normotensive control but not on uninephrectomized or 1K1C rats. Rat tail vascular beds were perfused to analyze the effects of 10 nM ouabain on the pressor response to PHE. In all animals, 10 nM ouabain increased the PHE pressor response, but this increase was larger in hypertensive DOCA-salt rats than in normotensive and 1K1C rats. Results suggested that a) increases in diastolic blood pressure induced by 18 µg/kg ouabain were larger in hypertensive than normotensive rats; b) in DOCA-salt rats, smaller ouabain doses had a stronger effect than in other groups; c) hypertensive and uninephrectomized rats were less sensitive to toxic doses of ouabain, and d) after treatment with 10 nM ouabain isolated tail vascular beds from DOCA-salt rats were more sensitive to the pressor effect of PHE than those from normotensive and 1K1C hypertensive rats. These data suggest that very small doses of ouabain, which might produce nanomolar plasma concentrations, enhance pressor reactivity in DOCA-salt hypertensive rats, supporting the idea that endogenous ouabain may contribute to the increase and maintenance of vascular tone in hypertension.

Reactivity of the isolated perfused rat tail vascular bed

Isolated segments of the perfused rat tail artery display a high basal tone when compared to other isolated arteries such as the mesenteric and are suitable for the assay of vasopressor agents. However, the perfusion of this artery in the entire tail has not yet been used for functional studies. The main purpose of the present study was to identify some aspects of the vascular reactivity of the rat tail vascular bed and validate this method to measure vascular reactivity. The tail severed from the body was perfused with Krebs solution containing different Ca2+ concentrations at different flow rates. Rats were anesthetized with sodium pentobarbital (65 mg/kg) and heparinized (500 U). The tail artery was dissected near the tail insertion, cannulated and perfused with Krebs solution plus 30 µM EDTA at 36oC and 2.5 ml/min and the procedures were started after equilibration of the perfusion pressure. In the first group a dose-response curve to phenylephrine (PE) (0.5, 1, 2 and 5 µg, bolus injection) was obtained at different flow rates (1.5, 2.5 and 3.5 ml/min). The mean perfusion pressure increased with flow as well as PE vasopressor responses. In a second group the flow was changed (1.5, 2, 2.5, 3 and 3.5 ml/min) at different Ca2+ concentrations (0.62, 1.25, 2.5 and 3.75 mM) in the Krebs solution. Increasing Ca2+ concentrations did not alter the flow-pressure relationship. In the third group a similar protocol was performed but the rat tail vascular bed was perfused with Krebs solution containing PE (0.1 µg/ml). There was an enhancement of the effect of PE with increasing external Ca2+ and flow. PE vasopressor responses increased after endothelial damage with air and CHAPS, suggesting an endothelial modulation of the tone of the rat tail vascular bed. These experiments validate the perfusion of the rat tail vascular bed as a method to investigate vascular reactivity.

Effects of ouabain on vascular reactivity

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the a-subunit of Na+,K+-ATPase inhibiting the Na+-pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension.

Formas de aplicação de inoculante e seus efeitos sobre a nodulação da soja

Atualmente, tem-se difundido a aplicação de inoculante no sulco de semeadura na cultura da soja, mas há poucas informações que dão suporte a essa prática e comprovam sua eficiência em diferentes ambientes manejados sob plantio direto. Este trabalho teve como objetivo avaliar a viabilidade da aplicação de inoculantes na cultura da soja, via semente e sulco de semeadura, em solo já cultivado ou não com soja. Foram realizados dois experimentos em campo a partir de dezembro de 2004 em Latossolo Vermelho-Amarelo, seguindo o mesmo método e tratamentos, porém em dois locais distintos, com ou sem cultivo anterior de soja. Foram testados oito tratamentos: (1) inoculação via semente (inoculante + fungicida + micronutriente); (2) sem inoculação (fungicida + micronutriente); (3) testemunha (semente pura, sem tratamento); (4) aplicação no sulco-dose 1 (dose do inoculante recomendada no sulco); (5) aplicação no sulco-dose 2 (duas vezes a dose recomendada no sulco); (6) aplicação no sulco-dose 3 (três vezes a dose recomendada no sulco); (7) sulco-dose 1 + inoculação via semente; e (8) adubação com N (200 kg ha-1 N). Foram avaliados massa de matéria seca de nódulos e número de nódulos totais e nódulos viáveis e não-viáveis aos 30 e 75 dias após emergência. A melhor nodulação foi obtida com aplicação de inoculante + fungicida + micronutriente via semente no solo ainda não cultivado. No solo previamente cultivado com soja, destacaram-se os tratamentos uma e duas vezes a dose do inoculante no sulco. Menores valores de massa seca de nódulos na soja foram obtidos no tratamento com adubação mineral. A aplicação via sulco do inoculante mostrou-se uma prática viável, em razão da semelhança dos resultados obtidos com a aplicação tradicional via semente.

Eletrococleografia extratimpânica na neuropatia/dessincronia auditiva

O potencial evocado auditivo de tronco encefálico (PEATE) vem sendo amplamente utilizado como método para avaliação da função coclear em indivíduos com diagnóstico de neuropatia/dessincronia auditiva (NA/DA). Na ausência das emissões otoacústicas, muitos casos de NA/DA foram diagnosticados pela presença do microfonismo coclear (MC) identificado no PEATE. OBJETIVO: Demonstrar a aplicabilidade clínica da eletrococleografia extratimpânica (Ecog-ET) no diagnóstico diferencial da NA/DA quando comparada ao PEATE. MATERIAL E MÉTODO: Uma criança com 4 anos de idade, com diagnóstico de NA/DA atendida no Centro de Pesquisas Audiológicas realizou a Ecog-ET com tone burst de 2000Hz nas polaridades de rarefação e condensação. RESULTADOS: Ilustrou-se o registro da Ecog-ET. Com a utilização de protocolo apropriado, o MC pode ser evidenciado e confirmado na Ecog, com qualidade de registro superior ao obtido no PEATE. CONCLUSÃO: A Ecog-ET permitiu uma análise mais detalhada do MC quando comparada ao PEATE tendo, portanto aplicabilidade clínica na investigação da função coclear na NA/DA.

O Mismatch Negativity em pacientes com distúrbios do processamento auditivo (central)

O Mismatch Negativity (MMN) é um potencial evocado auditivo de longa latência que fornece uma medida objetiva das habilidades de discriminação e memória sensorial auditiva. Assim, pode ser utilizado como uma avaliação eletrofisiológica do processamento auditivo (central). OBJETIVO: Estudar o MMN em pacientes com Distúrbios do Processamento Auditivo (Central) - DPA(C). FORMA DO ESTUDO: Clínico prospectivo. MATERIAL E MÉTODO: Foram avaliados oito sujeitos com DPA(C), na faixa etária de nove a 14 anos, bem como um grupo controle. O MMN foi eliciado para estímulos tonais (tone bursts) diferindo quanto à freqüência (MMNf - estímulo padrão: 750Hz e estímulo diferente: 1000Hz ), bem como quanto à duração (MMNd - estímulo padrão: 100 ms e estímulo diferente: 50 ms; na freqüência de 1000Hz). RESULTADOS: A presença do MMNf e do MMNd foi comprovada estatisticamente em ambos os grupos estudados. Não foram constatadas diferenças significantes estatisticamente entre os valores de latência e de amplitude do MMNf e do MMNd obtidos nos dois grupos. Também não foram verificadas diferenças significantes estatisticamente entre o MMNf e o MMNd em nenhum dos grupos estudados. CONCLUSÃO: Os sujeitos com DPA(C) avaliados não apresentaram alterações no MMNf, nem no MMNd.

Potenciais miogênicos evocados vestibulares: metodologias de registro em homens e cobaias

O potencial miogênico evocado vestibular (VEMP) é um teste clínico que avalia a função vestibular através de um reflexo vestíbulo-cervical inibitório captado nos músculos do corpo em resposta à estimulação acústica de alta intensidade. OBJETIVO: Verificar e analisar os diversos métodos de registro dos potenciais miogênicos evocados vestibulares no homem e em cobaias. MATERIAL E MÉTODO: Realizou-se busca eletrônica nas bases de dados MEDLINE, LILACS, SCIELO e COCHRANE. RESULTADOS: Foram verificadas divergências quanto às formas de registro dos potenciais miogênicos evocados vestibulares, relacionadas com os seguintes fatores: posição do paciente no momento do registro, tipo de estímulo sonoro utilizado (clicks ou tone bursts), parâmetros para a promediação dos estímulos (intensidade, freqüência, tempo de apresentação, filtros, ganho de amplificação das respostas e janelas para captação dos estímulos), tipo de fone utilizado e forma de apresentação dos estímulos (monoaural ou binaural, ipsi ou contralateral). CONCLUSÃO: Não existe consenso na literatura quanto ao melhor método de registro dos potenciais evocados miogênicos vestibulares, havendo necessidade de pesquisas mais específicas para comparação entre estes registros e a definição de um modelo padrão para a utilização na prática clínica.