Resumen Rhinella schneideri (o Bufo paracnemis), conocido popularmente en Brasil como sapo cururu, también se encuentra en otros países de América del Sur. Los efectos cardiovasculares de este veneno son ampliamente conocidos y recientemente se demostró que es capaz de afectar la unión neuromuscular en la preparación aislada de aves y ratones. En este trabajo, utilizamos microscopía electrónica de transmisión para investigar la ultraestructura de la terminación nerviosa motora y pliegues de unión postsináptica de preparaciones de nervio frénico-hemidiafragma incubadas durante 5 o 60 min con el extracto metanólico de R. schneideri (50 μg/mL). Además, se examinó el estado de los receptores de acetilcolina (AChR) mediante la ubicación de inmunofluorescencia de TRITC-α-bungarotoxina en la membrana de la placa terminal. Los resultados muestran que 5 min de incubación con el extracto de secreción de glándula disminuyeron significativamente (32 %) el número de vesículas sinápticas en el terminal del nervio motor, pero no disminuyeron la densidad electrónica en la parte superior de los pliegues de unión donde se concentran los receptores nicotínicos. Sin embargo, 60 min de incubación condujeron a una recarga significativa de los terminales nerviosos en las vesículas sinápticas, mientras que la inmunorreactividad del AChR no fue tan marcada como en el control y después de 5 min de incubación. Las fibras musculares estaban bien conservadas, pero los axones motores intramusculares no. Los hallazgos corroboraron los datos farmacológicos ya que la disminución en el número de vesículas sinápticas (5 min) seguida de recuperación (60 min) está de acuerdo con el aumento transitorio de la frecuencia de MEPPs, lo que significa una mayor liberación de neurotransmisores. Estos datos apoyan el modo de acción presináptico predominante de R. schneideri, pero no excluyen la posibilidad de una acción postsináptica secundaria dependiendo del tiempo en que la preparación esté expuesta al veneno.

Abstract Rhinella schneideri (or Bufo paracnemis), popularly known in Brazil as cururu toad, is also found in other countries in South America. The cardiovascular effects of this poison are largely known and recently was shown that it is capable to affect the neuromuscular junction on avian and mice isolated preparation. In this work, we used transmission electron microscopy to investigate the ultrastructure of the motor nerve terminal and postsynaptic junctional folds of phrenic nerve-hemidiaphragm preparations incubated for either 5 or 60 min with the methanolic extract of R. schneideri (50 µg/mL). In addition, the status of the acetylcholine receptors (AChR) was examined by TRITC-α-bungarotoxin immunofluorescence location at the endplate membrane. The results show that 5 min of incubation with the gland secretion extract significantly decreased (32 %) the number of synaptic vesicles into the motor nerve terminal, but did not decrease the electron density on the top of the junctional folds where nicotinic receptors are concentrated; however, 60 min of incubation led to significant nerve terminal reloading in synaptic vesicles whereas the AChR immunoreactivity was not as marked as in control and after 5 min incubation. Muscle fibers were well-preserved but intramuscular motor axons were not. The findings corroborated pharmacological data since the decrease in the number of synaptic vesicles (5 min) followed by recovery (60 min) is in accordance with the transient increase of MEPPs frequency meaning increased neurotransmitter release. These data support the predominant presynaptic mode of action of the R. schneideri, but do not exclude the possibility of a secondary postsynaptic action depending on the time the preparation is exposed to poison. Rev. Biol. Trop. 66(3): 1290-1297. Epub 2018 September 01.

Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (µBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10µg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0% (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71% homology with bothropstoxin-II and 54% homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92% homology with Basp-III and 62% homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2.

In the present study, manganese (Mn2+), a neuromuscular blocker with pre and postsynaptic actions, was used to verify the neurotoxicity and myotoxicity induced by Crotalus durissus terrificus (Cdt) and Bothrops jararacussu (Bjssu) venoms in biventer cervicis preparations (BCp). Preparations pretreated with 0.66 and 1.6mM Mn2+ did not affect Cdt venom-induced blockage nor change KCl-induced contracture but partially reduced ACh-induced contracture. However, both Mn2+ concentrations partially hindered Bjssu venom-induced blockage after washing the preparations with Krebs solution, and only 1.6mM Mn2+ preparations significantly recovered ACh-induced contracture. The effect of Cdt venom myotoxicity on contractile responses was different from that of Bjssu venom myotoxicity. Pretreatment with 1.6mM Mn2+ partially reduced muscle damage percentage and creatine kinase (CK) activity (U/l) induced by both venoms. In conclusion, Mn2+ interfered in ACh-induced contracture of the nicotinic receptor; did not prevent Cdt venom neurotoxicity but partially reduced its myotoxicity in vitro due to the stabilizing action of this venom on the sarcolemmal membrane; and partially attenuated myotoxicity and neuromuscular blockage induced by Bjssu venom. The Mn2+ dual action (pre and postsynaptic) is useful to study snake venoms since most of them present one or both of these actions; besides, Mn2+ allowed recovering coherent interpretation of experimental versus clinical results.

O objetivo deste trabalho foi avaliar linhas quase isogênicas de trigo com o caráter permanência verde e de maturação sincronizada quanto à tolerância ao alumínio e à sensibilidade ao ácido giberélico (AG3). O experimento foi conduzido no Laboratório de Duplo-haplóides e Hidroponia da FAEM/UFPel, em delineamento de blocos ao acaso, com parcelas subdivididas com três repetições, através do teste simultâneo utilizando solução nutritiva com concentrações de 10mg L-1 de Al3+ e 100mg L-1 de AG3. Foram determinados: a inserção da 1ª folha, o comprimento da 1ª folha, a inserção da 2ª folha, comprimento da 2ª folha, diferença da inserção entre a 1ª e 2ª folha, estatura de plântula, estatura de planta, área foliar, área radicular e o crescimento de raiz. Os caracteres estatura da plântula, inserção da 1ª folha e comprimento da 2ª folha foram os mais expressivos para a seleção de indivíduos com estatura reduzida através da reação do AG3. O grupo do caráter permanência verde apresentou maior insensibilidade à ação do AG3, enquanto o grupo de plantas sincronizadas proporcionou maior tolerância ao Al3+. Contudo, existe possibilidade de selecionar linhas quase isogênicas dentro do grupo permanência verde com tolerância ao Al3+ e insensibilidade ao AG3 através da técnica de cultivo hidropônico com solução nutritiva em ambiente controlado.

The objective was to evaluate wheat near-isogenic lines differing from the trait stay-green and synchronized maturation regarding their alluminum tolerance and sensibility to the giberellic acid (GA3). The experiment was conducted in the Double-haploids and Hydroponics Laboratory of FAEM/UFPel, in random block with split plot design with three replications, using the simultaneous test in nutrient solutions with 10mg L-1 of Al3+ and 100mg L-1 of GA3. The following characters were evaluated: insertion of the 1st leaf, length of the 1st leaf, insertion of the 2nd leaf, length of the 2nd leaf, difference between the 1st and 2nd leaf insertion, seedling stature, plant stature, leaf area, root area and root growth. The characters seedling stature, insertion of the 1st leaf and length of the 2nd leaf were the most expressive for the selection of individuals with reduced stature caused by the GA3. The group stay-green presented higher insensibility to GA3 compared to the group of synchronized plants showed larger tolerance to Al3+. However, it is possible to select near-isogenic lines within the stay-green group with Al3+ tolerance using hydroponic culture under controlled environmental conditions.

Numerous plants are used as snakebite antidotes in Brazilian folk medicine, including Casearia sylvestris Swartz, popularly known as guaçatonga. In this study, we examined the action of a hydroalcoholic extract from C. sylvestris on the neuromuscular blockade caused by bothropstoxin-I (BthTX-I), a myotoxin from Bothrops jararacussu venom, in mouse isolated phrenic nerve-diaphragm (PND) preparations. Aqueous (8 and 12 mg/ml, n=4 and 5, respectively) and hydroalcoholic (12 mg/ml, n=12) extracts of the leaves of C. sylvestris caused facilitation in PND preparations followed by partial neuromuscular blockade. BthTX-I (20 µg/ml, n=4) caused 50% paralysis after 65±15 min (mean ± S.E.M). Preincubation (30 min at 37° C) of BthTX-I (20 µg/ml, n=4) with a concentration of the hydroalcoholic extract (4 mg/ml) that had no neuromuscular activity, such as the control (n=5), prevented the neuromuscular blockade caused by the toxin. This protection may be mediated by compounds such as flavonoids and phenols identified by thin-layer chromatography and colorimetric assays.

The pharmacological effects of Bothrops neuwiedi pauloensis venom on mouse phrenic nerve-diaphragm (PND) preparations were studied. Venom (20 mug/ml) irreversibly inhibited indirectly evoked twitches in PND preparations (60 ± 10% inhibition, mean ± SEM; p<0.05; n=6). At 50 mug/ml, the venom blocked indirectly and directly (curarized preparations) evoked twitches in mouse hemidiaphragms. In the absence of Ca2+, venom (50 mug/ml), produced partial blockade only after an 80 min incubation, which reached 40.3 ± 7.8% (p<0.05; n=3) after 120 min. Venom (20 mug/ml) increased (25 ± 2%, p< 0.05) the frequency of giant miniature end-plate potentials in 9 of 10 end-plates after 30 min and the number of miniature end-plate potentials which was maximum (562 ± 3%, p<0.05) after 120 min. During the same period, the resting membrane potential decreased from - 81 ± 1.4 mV to - 41.3 ± 3.6 mV 24 fibers; p<0.01; n=4) in the end-plate region and from - 77.4 ± 1.4 to -44.6 ± 3.9 mV (24 fibers; p<0.01; n=4) in regions distant from the end-plate. These results indicate that B. n. pauloensis venom acts primarily at presynaptic sites. They also suggest that enzymatic activity may be involved in this pharmacological action.

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 µg/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 µg/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 µM acetylcholine alone and cumulative concentrations of 1 µM to 10 mM were unaffected. At venom concentrations higher than 50 µg/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24ºC, the venom (50 µg/ml) produced only partial neuromuscular blockade (30.7 ± 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.

Snake venoms frequently vary in composition. In this work, we compared the neurotoxic and myotoxic activities of 16 lots of Bothrops neuwiedii venoms from different regions of Brazil, using chick biventer cervicis preparations. The neuromuscular blockade varied from 2% to 100% after 120 min incubation with venoms (50 mug/ml). In all cases, this blockade was irreversible and concentration-dependent; at low concentrations (10-20 mug/ml), 15 of the 16 venom lots failed to abolish responses to acetylcholine (110 muM), but blocked responses to KCl (13.4 muM), and induced contracture. At 5-20 mug/ml, the most active venom totally blocked twitch-tension without affecting responses to acetylcholine and KCl. Polyacrylamide gel electrophoresis for basic proteins showed that the most active samples contained a band that was absent in the less active venoms. These results indicate that there may be considerable intraspecific variation in the neurotoxic activity of B. neuwiedii venoms, whereas myotoxic activity is less variable.

Bothrops jararacussu venom and its major toxin bothropstoxin-I (BthTX-I) possess myotoxic and neurotoxic properties. The efficacy of a rabbit antivenom raised against B. jararacussu venom in the neutralization of physiological, biochemical, and morphological changes induced by the venom and its major toxin BthTX-I was studied in mouse isolated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations. The times required for 50% neuromuscular blockade in PND and EDL preparations for venom were 70+11.5 (S.E.M., n=5) min and 58+8 (n=16) (50 mu g/mL), and for BthTX-I 31+6 (n=3) min and 30+3 (n=5) min (20 mu g/mL), respectively. After 120 min incubation, creatine kinase (CK) concentrations in solution containing the EDL preparations were 3464+346 U/L after exposure to venom (50 mu g/mL, n=5) and 3422+135 U/L to BthTX-I (20mu g/mL, n=4), respectively. Rabbit antivenom dose-dependently neutralized venom and toxin-induced neuromuscular blockade in both preparations and effectively prevented venom and toxin-induced CK release from EDL. Histological analysis showed that rabbit antivenom neutralized morphological damage caused by B. jararacussu venom and BthTX-I in EDL preparations. These results indicate that rabbit antivenom effectively neutralized the biological activities of B. jararacussu venom and BthTX-I.

O objetivo deste experimento foi identificar material promissor para ser utilizado como uma forrageira alternativa nas condições do sul do Brasil. Uma população de híbridos, em gerações avançadas, entre Leucaena leucocephala e L. diversifolia (ambas espécies tetraplóides, com 2n=104 cromossomos) foi avaliada quanto à taxa de crescimento em altura, época de florescimento e frutificação, número de flores e de legumes por inflorescência e de sementes por legume, tolerância ao frio (expressa em retenção de folhas durante o inverno) e capacidade de rebrote após o inverno. Foi detectada grande variabilidade intrapopulacional para todas as características avaliadas. Foram identificados indivíduos, reunindo tolerância a frio com características desejáveis, que podem ser utilizados na continuidade dos trabalho de seleção e melhoramento e comprovam a potencialidade de Leucaena como espécie forrageira para o sul do Brasil.

The objective of this experiment was to identify material to be used as an alternative forage in the conditions of Southern Brazil. A population of hybrids between L. leucocephala and L. diversifolia (both tetraploid species, with 2n=104 chromosomes) in advanced generations was evaluated. The rate of growth in height, duration of flowering and frutification, number of flowers and legumes per inflorescence, seeds per legume, cold tolerance (expressed as foliage retention during winter) and regrowth ability in early spring were evaluated. A great intrapopulational variability was detected for all evaluated characteristics. Some individuals allying cold tolerance and desirable characteristics were identified to be used for further selection and breeding, which confirms the potentiality of Leucaena as a forage for Southern Brazil.

Uma população de 513 híbridos entre as espécies tetraplóides (2n = 104) L. leucocephala e L. diversifolia ssp. diversifolia, que estão sendo analisados quanto a diversas características agronômicas e morfológicas, com o objetivo de selecionar plantas tolerantes ao frio, foi também analisada considerando a cor das partes florais. É proposto um modelo de herança dominante e aditiva (rosa dominante sobre branco, ou presença versus ausência de pigmento e o número de alelos dominantes determinando a intensidade da cor rosa). Também sugere-se a ação de dois conjuntos independentes de locos, um conjunto para os órgãos masculinos (com dois genes estruturais principais) e o outro conjunto para os órgãos femininos (um gene estrutural principal), e que a expressão da cor é afetada por genes regulatórios.

A population of 513 hybrids of Leucaena leucocephala (4x) and L. diversifolia ssp. diversifolia (4x), that is being analyzed for several morphological and agronomical characteristics, aiming at selecting cold tolerant plants, was also analyzed for floral color. A model of additive dominant inheritance (pink color dominant over white color, or presence versus absence of pigment, and the number of dominant alleles present determining the intensity of the pink color) is proposed. The action of two independent locus sets is also suggested, one for the male (with two main structural genes) and another for the female floral organs (one main structural gene). It is suggested that an undetermined number of regulatory genes affect color expression.