Chemical profile, anti 5-lipoxygenase and cyclooxegenase inhibitory effects of ginger (Zingiber officinale) rhizome, callus and callus treated with elicitors / Perfil químico, anti-5-lipoxigenase e efeitos inibitórios da ciclooxegenase do rizoma de gengibre (Zingiber officinale), do calo e calos tratados com elicitores

The present study investigated the chemical profiles and evaluated the inhibitory effect against 5-Lipoxygenase (5-Lox) activity for extracts of ginger rhizome, callus, and callus treated with the elicitors; yeast extract (100, 300 and 500 mg/L), glycine (100, 200 and 300 mg/L) and salicylic acid (100 and 200 mg/L). Oils and chloroform: methanol (CM) extracts were prepared by maceration in petroleum ether and CM (1:1, v/v), respectively. Chemical profiles were determined by gas chromatography/mass spectrometry (GC/MS) analysis. Oil of the callus recorded higher 5-Lox inhibitory effect (IC50 58.33±4.66 µg/mL) than the oil of rhizome (IC50168.34±15.64 µg/mL) and comparable to that of the positive control; Nordihydroguaiaretic acid (IC50 61.25±1.02 µg/mL). The chemical profile of the callus oil contained large amounts of fatty acids, mainly the unsaturated fatty acid oleic acid (31.11%) and saturated fatty acid palmitic acid (28.56%). Elicitors modified the chemical profile of the callus and ameliorated the anti-5-Lox activity of CM extract of the callus. CM extracts of callus treated with 100 and 300 mg/L yeast extract and 50 mg/L salicylic acid significantly suppressed (P ≤ 0.05) the 5-Lox activity by 33.16%, 25.46% and 16%, respectively as compared to the CM extract of untreated callus. In conclusion, ginger callus could be considered as a valuable dietary supplement in the treatment of various inflammatory disorders.

O presente estudo teve como objetivo investigar os perfis químicos e avaliar o efeito inibitório da atividade da 5-Lipoxigenase (5-Lox) em extratos de rizoma, calo e calo de gengibre tratados com os eliciadores; extrato de levedura (100, 300 e 500 mg / L), glicina (100, 200 e 300 mg / L) e ácido salicílico (100 e 200 mg / L). Extratos de óleos e clorofórmio: metanol (CM) foram preparados por maceração em éter e CM (1: 1, v / v), respectivamente. Os perfis químicos foram determinados por análise de cromatografia gasosa / espectrometria de massa (GC / MS). O óleo do calo registrou maior efeito inibitório de 5-Lox (IC50 58,33 ± 4,66 µg / mL) do que o óleo de rizoma (IC50168,34 ± 15,64 µg / mL) e comparável ao do controle positivo; Ácido nordi-hidroguaiarético (IC50 61,25 ± 1,02 µg / mL). O perfil químico do óleo de calo continha grandes quantidades de ácidos graxos, principalmente o ácido graxo insaturado ácido oleico (31,11%) e ácido graxo saturado palmítico (28,56%). Os elicitores modificaram o perfil químico do calo e melhoraram a atividade anti-5-Lox do extrato de CM do calo. Extratos de CM de calos tratados com 100 e 300 mg / L de extrato de levedura e 50 mg / L de ácido salicílico suprimiram significativamente (P ≤ 0,05) a atividade de 5-Lox em 33,16%, 25,46% e 16%, respectivamente, em comparação com o extrato de CM de calo não tratado. Em conclusão, o calo de gengibre pode ser considerado um suplemento dietético valioso no tratamento de vários distúrbios inflamatórios.

Beneficial effects of methanolic extract of Indigofera linnaei Ali. on the inflammatory and nociceptive responses in rodent models

ABSTRACT Indigofera linnaei Ali. (Tamil Name: Cheppu Nerinjil) belongs to the family Fabaceae, used for the treatment of various ailments in the traditional system of medicine. In the present study, the beneficial effects of methanol extract of whole plant of I. linnaei (MEIL) were evaluated on inflammation and nociception responses in rodent models. In vitro nitric oxide (NO), lipoxygenase (LOX) and cyclooxygense (COX) inhibitory activities were also performed to understand the mode of action. MEIL at the dose of 200 & 400 mg/kg, p.o. significantly inhibited carrageenan induced rat paw volume and reduced the weight of granuloma in cotton pellet granuloma model. The results obtained were comparable with the standard drug aceclofenac. The anti-nociceptive effect of MEIL in mice was evaluated in hot plate and acetic acid induced writhing model. The plant extract significantly reduced the number of writhes and the analgesic effect was higher than that of the standard drug aspirin. However, the extract fails to increase the latency period in hot plate method suggesting that the extract produce nociception by peripheral activity. The extract produced inhibitory effect on NO, LOX and COX in concentration dependent manner. The extract exhibited pronounced and selective COX-2 inhibition. Altogether, these results suggested that the methanol extract of Indigofera linnaei could be considered as a potential anti-inflammatory and analgesic agent.

RESUMO Indigofera linnaei Ali pertence à família Leguminosae e é utilizada para o tratamento de várias doenças na medicina tradicional. No presente estudo, os efeitos benéficos do extrato metanólico da planta inteira de I. linnaei (MEIL) foram avaliados em respostas inflamatórias e nocicepção em modelos de roedores. Testes in vitro de atividade inibitória do óxido nítrico (NO), lipoxigenase (LOX) e ciclooxigenase (COX) também foram realizados para compreender o modo de ação. MEIL nas doses de 200 e 400 mg/kg, p.o. inibiu significativamente o volume da pata de rato induzido por carragenana e reduziu o peso do granuloma no modelo de pélete de algodão. Os resultados obtidos foram comparáveis ao do fármaco padrão, aceclofenaco. O efeito anti-nociceptivo de MEIL foi avaliado em camundongos no modelo de placa quente e de contorção induzida por ácido acético. O extrato da planta reduziu significativamente o número de contorções e o efeito analgésico foi maior do que o do fármaco padrão, ácido acetilsalicílico. Porém, o extrato não conseguiu aumentar o período de latência no método da placa quente, sugerindo que este produz nocicepção por atividade periférica. O extrato produziu efeito inibitório sobre o NO, LOX e COX dependente da concentração. O extrato exibiu inibição acentuada e seletiva da COX-2. No seu conjunto, estes resultados sugerem que o extrato metanólico de Indigofera linnaei poderia ser considerado como agente anti-inflamatório e analgésico potencial.

Apontamentos acerca da Cadeia do Ser e o lugar dos negros na filosofia natural na Europa setecentista / Notes on the Chain of Being and the place of blacks in eighteenth-century European natural philosophy

Por meio da análise de obras acadêmicas produzidas por filósofos naturais no século XVIII, pretendemos discutir algumas ideias recorrentes acerca da Grande Cadeia do Ser. Para tal, analisamos as relações entre filosofia e teologia natural no período. Reavaliamos ainda alguns elementos da Cadeia do Ser, investigando autores que discorreram sobre o tema em seus escritos. Por fim, elencamos um ponto específico das discussões setecentistas sobre a scala naturae, qual seja, as diversas e nem sempre convergentes ideias de que, a partir de características específicas, haveria diferenças entre os homens, bem como seu consequente lugar na Cadeia do Ser.

This examination of academic works produced by eighteenth-century natural philosophers discusses some recurring ideas about the Chain of Being. To this end, the article analyzes the relations between natural philosophy and theology during the period. It also re-evaluates some elements of the Chain of Being through an exploration of authors who addressed the topic in their writings. Lastly, it identifies a specific element within eighteenth-century discussions of scala naturae, to wit, the various and not always convergent ideas about whether there are differences between humans based on specific characteristics and, consequently, about the places they occupy in the chain of being.

Avaliação da atividade da lipoxigenase na castanha do Brasil (Bertholletia excelsa) / Evaluation of activity in Brazil nuts lipoxigenase (Bertholletia excelsa)

Para determinar as condições ótimas de atividade da lipoxigenase (LOX) presente na castanha-do-brasil (Bertholletia excelsa) verificou-se sua atividade em função do pH em soluções tampão (pH 4,00; 5,00; 6,00; 6,50; 7,00; 8,00 e 9,00), variando-se o tipo de substrato (ácido linoleico e linolênico) e suas concentrações (1,00; 2,50; 5,00; 10,00; 15,00; 20,00 e 40,00 mM), bem como a concentração de extrato enzimático (0,50; 1,00; 1,50; 2,00; 2,50 e 3,00%). Monitorou-se a atividade da LOX pelo acompanhamento do aumento da absorbância a 234 nm durante 240 segundos, expressos em milimolar por segundo por grama (mM/s.g). Ambos os ácidos graxos estudados mostraram picos de atividade de LOX em pH 6,5 e concentração de substrato a 1,5 mM. Aplicando-se os dados experimentais à equação de Michaelis-Menten e da linearização de Lineawer-Burk, os valores obtidos para a constante de Michaelis-Menten (KM) e de velocidade enzimática máxima (VMAX) variaram entre: 0,164 a 0,169 mM e 0,083 a 0,093 mM/s.g, respectivamente. O ácido linoleico revelou-se o melhor substrato para a enzima e foi utilizado para determinação da atividade da LOX em função da concentração de enzima, que apresentou maior atividade a 3,00%.

Panificación con harina de arvejas (Pisum sativum) previamente sometidas a inactivación enzimática / Inactivated pea flour (Pisum sativum) in bread making

La harina de arveja (Pisum sativum) es una fuente proteica de relativo bajo costo y escasamente utilizada en la elaboración de productos de consumo masivo. Su incorporación a la harina de trigo, en la elaboración de productos panificados, ofrece una buena alternativa para complementar un perfil de aminoácidos. En este trabajo se evalúa el efecto de la inactivación enzimática de la arveja sobre las características de los panes de molde formulados con niveles de sustitución de 5 por ciento, 10 por ciento y 15 por ciento. Se efectuaron determinaciones de proteínas y de lisina y por cálculo se obtuvieron los valores de score químico, siendo la lisina el aminoácido limitante. La evaluación sensorial se realizó con un panel de seis evaluadores entrenados y se aplicó el Análisis Descriptivo Cuantitativo. Los puntajes asignados por el panel fueron procesados estadísticamente mediante Análisis de Varianza al nivel de significación p = 0,05. La actividad residual de lipoxigenasa para el tratamiento térmico de un minuto fue de 48.6 por ciento mientras que para un minuto y medio resultó prácticamente nula (2.1 por ciento). En cuanto al efecto del tratamiento térmico de la arveja sobre el volumen específico, se aprecia que el mejor resultado se obtiene cuando el tiempo de tratamiento térmico es de un minuto. A su vez la valoración sensorial del panel asignó los mayores puntajes a los panes con nivel de sustitución de 5 por ciento de harina de arveja, para ambos tiempos de tratamiento térmico (1.0 y 1.5 min). Mayores porcentajes de harina de arveja producen un efecto negativo sobre el volumen y sobre los atributos sensoriales del pan.

Pea flour (Pisum sativum) is a relatively cheap protein source and it is scarcely utilized in making widely consumed products. It provides a good opportunity to improve the amino acidic profile. The purpose of this study was to determine the effect of the enzymatic inactivation of pea on bread characteristics, made with levels of 5, 10 and 15 percent of pea flour. Protein and lysine contents were determined and then chemical score obtained considering lysine as limiting amino acid. Sensory evaluation was carry out by six trained panelists using quantitative descriptive analysis (QDA) and analysis of variance (ANOVA) at p = 0.05. Residual lipoxygenase activity was 48.6 percent when heat treatment was made during 1 minute, and only 2.1 percent when the heat treatment was carry out during 1.5 minutes. Highest specific volumes of bread were obtained with pea flour treated during 1 minute. The sensory evaluation by panel determined that pea flour at a level of 5 percent could be successfully substituted for both heat treatments. But pea flour substitution at levels of 10 and 15 percent had adverse effects on specific volume and sensory characteristics.

Asma induzida por aspirina / Aspirin-induced asthma

Asma induzida por aspirina (AIA) é doença comum e freqüentemente subdiagnosticada. Aproximadamente 20% da população asmática é sensível à aspirina e a outros antiinflamatórios não hormonais (AINEs) e apresentam a tríade de rinossinusite com pólipos nasais, asma e hipersensibilidade à aspirina. A patogênese da AIA está associada às vias do metabolismo do ácido araquidânico: a via da lipo-oxigenase (LO) e a via da ciclo-oxigenase (CCX). Ao inibir a via da ciclo-oxigenase (CCX), a aspirina desvia os metabóhtos do ácido araquidânico para a via da LO. Isso leva à queda nos níveis de prostaglandina E2, associado ao aumento da síntese de leucotrienos cisteínicos (Cys-LT5). Os anti-leucotrienos são eficazes em bloquear a broncoconstrição provocada pela aspirina e são utilizados no tratamento desta condição. A dessensibilização tem papel importante no manejo da AIA, principalmente em pacientes que necessitam de profilaxia para doenças tromboembólicas, infarto agudo do miocárdio, e AVE. Esta revisão tem por objetivo dissertar sobre alguns aspectos atuais desta doença.

Determinantes microambientais da progressão do melanoma murino: contribuição da resposta inflamatória / Microenvironment factors in murine melanoma progression: contributions from the imflammatory response

Doses subtumorigênicas de células (103) de melanoma murino Tm1 ou Tm5, (derivados da linhagem não tumorigênica, melan-a) apresentam progressão rápida após inoculação em conjunto com células apoptóticas, mas não com células necróticas. A coinjeção de células tumorais viáveis e células apoptóticas induz o aparecimento de um infiltrado inflamatório transitório, composto principalmente de neutrófilos e macrófagos. Neutrófilos e linfócitos 131 são importantes para a indução do crescimento tumoral neste modelo, ao contrário de linfócitos T. A utilização de animais geneticamente depletados do receptor 1 de bradicinina (BradBl R KO) mostrou que esta via de sinalização inflamatória é importante para a indução da resposta dependente de neutrófilos, e nestes animais há um atraso significativo na indução do crescimento tumoral. Além disso, LPS um potente agente inflamatório bacteriano, também induz o desenvolvimento tumoral quando associado a doses subtumorigênicas de melanoma. Enzimas da via do ácido araquidônico participam da indução da resposta inflamatória neste modelo através da ação da 5-lipooxigenase, já que seus inibidores (ácido cafeico e MK886) inibem a progressão tumoral induzida por células apoptóticas ou LPS. As ciclooxigenases COM e 2 não têm participação na promoção do crescimento tumoral, o que foi demonstrado com a utilização de inibidores específicos das ciclooxigenases, que não foram capazes de inibir o crescimento tumoral associado à resposta inflamatória...

Qualidade protéica de linhagens de soja com ausência do Inibidor de Tripsina Kunitz e das isoenzimas Lipoxigenases / Protein quality of soy lines lacking Kunitz's Trypsin Inhibitor and Lipoxygenase isozymes

OBJETIVO: Avaliar a qualidade protéica de farinhas de soja, que diferem entre si com relação à presença de lipoxigenases e/ou Inibidor de Tripsina Kunitz. MÉTODOS: Procedeu-se aos ensaios biológicos com ratos, em que foram avaliados a Razão da Eficiência Protéica, Razão Protéica Líquida, Utilização Protéica Líquida e a digestibilidade. Determinou-se a composição aminoacídica das farinhas e cálculo do Escore Químico Corrigido pela Digestibilidade. RESULTADOS: Os valores de Razão da Eficiência Protéica, Razão Protéica Líquida, Utilização Protéica Líquida para as variedades de soja foram inferiores aos valores obtidos para caseína. Para as farinhas sem Inibidor de Tripsina Kunitz foram obtidos valores de digestibilidade maiores que para as farinhas com Inibidor de Tripsina Kunitz, e esses foram bem próximos aos da caseína. Com relação ao teor aminoacídico, constatou-se que o aminoácido limitante dessa soja é lisina e não metionina, ao contrário do que aponta a literatura. CONCLUSAO: A eliminação genética do Inibidor de Tripsina Kunitz melhora consideravelmente a digestibilidade da proteína de soja. Os resultados do Escore Químico Corrigido pela Digestibilidade não indicaram diferença entre as farinhas sem Inibidor de Tripsina Kunitz das farinhas derivadas de linhagens com Inibidor de Tripsina Kunitz, como foi observado pelos resultados da digestibilidade in vivo.

Stimulatory effects of adenosine on prolactin secretion in the pituitary gland of the rat

We investigated the effects of adenosine on prolactin (PRL) secretion from rat anterior pituitaries incubated in vitro. The administration of 5-N-methylcarboxamidoadenosine (MECA), an analog agonist that preferentially activates A2 receptors, induced a dose-dependent (1 nM to 1 æM) increase in the levels of PRL released, an effect abolished by 1,3-dipropyl-7-methylxanthine, an antagonist of A2 adenosine receptors. In addition, the basal levels of PRL secretion were decreased by the blockade of cyclooxygenase or lipoxygenase pathways, with indomethacin and nordihydroguaiaretic acid (NDGA), respectively. The stimulatory effects of MECA on PRL secretion persisted even after the addition of indomethacin, but not of NDGA, to the medium. MECA was unable to stimulate PRL secretion in the presence of dopamine, the strongest inhibitor of PRL release that works by inducing a decrease in adenylyl cyclase activity. Furthermore, the addition of adenosine (10 nM) mimicked the effects of MECA on PRL secretion, an effect that persisted regardless of the presence of LiCl (5 mM). The basal secretion of PRL was significatively reduced by LiCl, and restored by the concomitant addition of both LiCl and myo-inositol. These results indicate that PRL secretion is under a multifactorial regulatory mechanism, with the participation of different enzymes, including adenylyl cyclase, inositol-1-phosphatase, cyclooxygenase, and lipoxygenase. However, the increase in PRL secretion observed in the lactotroph in response to A2 adenosine receptor activation probably was mediated by mechanisms involving regulation of adenylyl cyclase, independent of membrane phosphoinositide synthesis or cyclooxygenase activity and partially dependent on lipoxygenase arachidonic acid-derived substances

Mechanisms in anti-inflammation and resolution: the role of lipoxins and aspirin-triggered lipoxins

Multicellular host responses to infection, injury or inflammatory stimuli lead to the formation of a broad range of chemical mediators by the host. The integrated response of the host is essential to health and disease; thus it is important to achieve a more complete understanding of the molecular and cellular events governing the formation and actions of endogenous mediators of resolution that appear to control the duration of inflammation. Lipoxins are trihydroxytetraene-containing lipid mediators that can be formed during cell-cell interactions and are predominantly counterregulators of some well-known mediators of inflammation. Since this circuit of lipoxin formation and action appears to be of physiological relevance for the resolution of inflammation, therapeutic modalities targeted at this system are likely to have fewer unwanted side effects than other candidates and current anti-inflammatory therapies. Here, we present an overview of the recent knowledge about the biosynthesis and bioactions of these anti-inflammatory lipid mediators

Inactivación de lipoxigenasas e inhibidoeres de tripsina en un proceso de obtención de leche de soja por molienda directa y ultra alta temperatura (MD-UAT) / Lipoxygenases and trypsin inhibitors inactivation in a soymilk processing by direct milling and ultra high temperature (DM-UHT)

La elaboración de leche de soja mediante un nuevo proceso de Molienda Directa de sémola de soja y Ultra Alta Temperatura (MD-UAT), ha sido objeto de estudios de laboatorio en sus etapas de molienda y tratamientos térmicos, a los efectos de evaluar la extracción de sólidos y proteínas, como así también la inactivación de lipoxigenasas e inhibidores de tripsina (IT). Para la determinación de IT en extractos y leche de soja se modificó el método clásico de Kakade (17). Los mayores rendimientos de extracción en la molienda se obtienen a 70 grados Celsius durante 2 minutos. La inactivación de lipoxigenasas es notablemente más eficiente durante la molienda si se utiliza solución de carbonato de sodio 0,01 M en lugar de agua como medio dispersante (actividad residual de 14 por ciento en carbonato frente a 46 por ciento en agua), reduciéndose así la generación de sustancias indeseables. Se completa la destrucción de lipoxigenasas con un breve calentamiento de 30 segundos con vapor desde 70 grados Celsius hasta la temperatura de ebullición a 96 grados Celsius. Por otra parte, los inhibidores de tripsina no son adecuadamente inactivados en la molienda ni tampoco en el tratamiento con vapor a 96 degrees Celsius durante varios minutos. Posteriormente, en la etapa de UAT a 139 grados Celsius durante dos minutos se logra la inactivación de los IT hasta los valores considerados aceptables, posibilitando la destrucción simultánea de microorganismos. Estas condiciones se logran sin dificuldad en el proceso MD-UAT, cuya flexibilidad permite adecuar temperaturas y tiempos a requerimentos específicos.

Effect of germination on the protein content and on the level of specific activity of lipoxygenase-1 in seedlings of three soybean cultivars / Efecto de la germinación sobre el contenido de proteína y sobre los niveles de actividad específica de lipoxigenasa-1 en plantines de tres cultivares de soya

The effect of germination on the protein content and in the level of specific activity of lipoxygenase-1 on seedlings of Santa Rosa, FT-2 and Davis soybean cultivars were studied. Soybean seeds of the three cultivars were germinated from ) to 72 hours in a seed germinator at 30ºC temperature, 100 por ciento relative humidity, and absence of light. The seeds were then frozen in liquid nitrogen, freeze-dryed and milled. The lipoxygenase-1 was extracted with 0,02M thris-HCI buffer at pH=8,2 and 13 por ciento saccharose for 30 minutes at 4ºC without shaking. The highst levels of specific activity of lipoxygenase-1 per miligram of flour (dry basis) in the Santa Rosa, FT 2 and Davis cultivars were observed between 18 and 36, 18 and 24, and 0 and 12 hours, respectively. The polinomial quadratic model best fits the regression analysis of the variable levels of specific lipoxygenase activity for cultivas Davis (R²=0,09197). Although for cultivars Santa Rosa (R²=0,9041), and FT-2 (R²=0,7486) the 4th degree polinomial has shown the best fit. the protein content in the three cultivars studied the maximum values at 48h germination. The germination induced a relative increase of the protein contein but caused a reduction of the level of specifit lipoxigenase-1 activity

Use of the random amplified polymorphic DNA technique to characterize soybean (Glycine max (L.) Merrill)genotypes

We have started a breeding program to genetically eliminate the lipoxygenase isozymes (LOX) from soybean seeds. These enzymes are believed to be the main cause of off-flavors in soybean products. LOX are present in the seed as three isozymes encoded by three different genes, which are inherited in a simple mendelian fashion. Mutants lacking each one of these isozymes have been identified in the world germplasm. To introduce these null alleles into the Brazilian variety Cristalina, three mutant progenitors were chosen: PI 408.251 (LOX1 minus), PI 86.023 (LOX2 minus), and Ichigowase (LOX3 minus). The random amplified polymorphic DNA (RAPD) technique was used to charactetize these progenitors, as well as lines lacking LOX1 (CR1), LOX3 (CR3), LOX1 and 3 (CR1,3), and LOX2 and 3 (UFV 91-263, UFV 91-401 and UFV 91-717). The results enabled us to establish the fingerprint of each genotype and the genetic distances among them.

Intracerebroventricular administration of canatoxin to rats increases the circulating levels of luteinizing hormone

Recent evidence has implicated the central nervous system as a target organ for canatoxin, a toxic protein present in Canavalia ensiformis seeds. This toxin activates the lipoxygenase pathway of arachidonic acid metabolism and can thus induce the release of substances mediated by lipoxygenase products. In the present study, the circulating levels of luteinizing hormone (LH) were measured by RIA in male Wistar rats (200-240 g) after the administration of canatoxin into the lateral cerebral ventricle. Canatoxin (0.5-2 micrograms in 2 microliters daily for 3 days) caused a dose- and time-dependent increase in the plasma levels of LH. The total dose of canatoxin used is subconvulsive. At 2, 4 and 24 h after 2 micrograms of canatoxin LH levels were increased by 10 per cent , 43 per cent and 61 per cent , respectively, compared to vehicle-injected animals (0.18 +/- 0.03 ng/ml). This response to 2 micrograms of canatoxin was not attenuated by pretreatment with two different lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 125 mg/kg) or esculetin (ECLT, 125 mg/kg), ip, 1 h before each canatoxin (CNTX) injection; per cent increase in LH with CNTX alone: 61 per cent ; CNTX+NDGA: 54 per cent ; CNTX+ECLT:76 per cent ; N = 5/group. These data show that intracerebral injection of CNTX in rats increases circulating levels of LH via a mechanism that is independent of the lipoxygenase pathway

Further studies on the hypoxia produced by canatoxin in rats

Canatoxin, a convulsant neurotoxin from the seeds of Canavalia ensiformis, induces lipoxygenase-dependent hypoxia in rats which is blocked by hexamethonium. The purpose of the present study was to examine the relationship between canatoxin-induced hypoxia and bronchoconstriction. Since several effects of the toxin are very similar to those described for morphine and opioid-like peptides, the effects of opioid antagonists were also investigated. Pretreatment of male, adult Wistar rats (200-250 g) with cyproheptadine (80 ug/kg, ip, N=6) and isoproterenol (100 ug/kg, ip, N+6) partially blocked (% variation of pO2:CNTX alone: -26.67 ñ 2.56, N=6; with cyproheptadine: 16.15 ñ 2.97*, N=6; with isoproterenol: 17.73 ñ 1.93*, N=6; *P<0.05 as compared to CNTX alone) the hypoxia but no effect was observed with diphenhydramine (2 mg/kg, ip, N=6) or atrophine (2 mg/kg, ip, N=6). The hypoxemic effect of canatoxin (100 ug/kg iv 20 min, N=6) was also almost completely blocked with either naloxone (1 mg/kg, sc, N=6) or naltrexone (5 mg/kg, sc, N=6). The results presented here provide evidence suggesting that both opioid peptides and bronchoconstriction seem to play a role in the hypoxia caused by canatoxin

ELISA na determinaçäo quantitativa e análise genética nas lipoxigenases L1 e L2 em soja / ELISA in the quantification and genetic analysis of lipoxygenases L1 and L2 from soybeans

A soybean breeding program has been undertaken at the Federal University of Viçosa aiming to reduce lipoxygenase lelvels ib the seeds of commercial varieties. A direct ELISA (Enzyme Linked Immunosorbent Assay) procedure has been developed to quantify lipoxygenases L1 and L2 in soybean seeds. Polyclonal antibodies were reised in rabbits against pure L1 and L2 and purified further by affinity chromatography. The best conditions to perform the analaysis were determined as foloow: coating antibody dilution 1:100, crude soybean protein extract 1:2 x 105 and conjugate 1:100 for both L1 and L2 isozymes. The method proved to be sensible enough to identify homozygous and heterozygous genotypes in the breeding program performing single non destructive analysis of seeds or even cotileldones right after germination. Besides, L1 was quantified in about 120 soybean Brazilian cultivars an L2 in some of them. The results showed a significant variability in the level of lipoxygenase-1 among the cultivars analysed. With respect to L2, it was found that the level of this isozyme was significantly greater in the seeds than observed for L1, in spite of its lower "in vitro" specific activity

Dupla difusäo radial na análise genética das lipoxigenases L1 e L2 em soja / Double gel immunodiffusion in the genetic analysis of lipoxygenases L1 and L2 from soybeans

In the effort to genetically manipulate soybean seeds aiming to improve flavor quality toward human consuption, lipoxygenase null alleles are being transferred to a brazilian commercial variety. Immunochemical approach has been taken to perform analysus of hundred of seeds derived from the breeding program. For that reason lipoxygenases L1 and L2 were puridied to hemogeneity by gel filtration and affinity chromatography. Policlonal antibodies were prepared in rabbits and purified further by affinity chromatogrphy. By using the purified anti-L1 and anti-L2 an Ouchterlony double immunodifusion method has been developed in our lab to detect the presence/absence of the isozymes simultaneously in soybean seeds. The method proved to be highly specific and efficient enought to perform single non destructive seed analysis

Convulsions induced by canatoxin in rats are probably a consequence of hypoxia

Canatoxin, a convulsant neurotoxin from the seeds of Canavalia ensiformis, induces lipoxygenase-dependent hypoxia and sex-related alterations of carbohydrate metabolism in rats which are blocked by glucose, diazepam and hexamethonium. The present study analyzes the possible causal relationship between the convulsant action of canatoxin and its effects on carbohydrate metabolism. The incidence of canatoxin-induced convulsions was greater in male than in female rats. Pretreatment of male rats with drugs that block hypoxia, such as glucose (2.5 g/kg,iv,15 min), diazepam (5 mg/kg,ip, at 48 h, 24 animals against convulsions, respiratory distress and death. These results suggest that canatoxin/induced convulsions are probably the consequence of hypoxia and both effects are mediated by lipoxygenase activation