Abstract Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

Um Tymovirus isolado de sementes de tomateiro (Solanum lycopersicum) 'Paronset' com sintomas de necrose, denominado TyToRS08, foi caracterizado quanto ao círculo de hospedeiras, propriedades físico-químicas, sorológicas e moleculares. O vírus foi transmitido mecanicamente a partir de folhas, frutos e sementes infectadas. A gama de hospedeiras incluiu espécies de Amaranthaceae, Chenopodiaceae e Solanaceae, induzindo sintomas local e sistêmicos na maior parte das espécies, exceto para Nicotiana sylvestris, N. tabacum 'Samsun', 'White Burley' e 'Xanthi' que não manifestaram sintomas. Em tomateiros 'Alambra', 'Thomas', 'Diva', 'Romana' e 'Graziela', o vírus induziu sintomas de mosaico e necrose branca. A presença do vírus foi detectada, nas sementes infectadas de todas as cultivares avaliadas, porém não se notou em plântulas após a germinação destas sementes. Foi obtida uma preparação purificada com concentração de 1,04 mg mL-1, empregada na produção de um antissoro policlonal que reagiu em PTA-ELISA com um título de 1/32768. Utilizou-se um par de oligonucleotídeos degenerados, desenhados para anelar na ORF 1 de espécies de Tymovirus, que permitiu a amplificação por RT-PCR de fragmentos com cerca de 700 pb. Após o alinhamento e a análise das sequências, verificou-se que o isolado TyToRS08 possuía valores abaixo de 70% de identidade com as espécies de Tymovirus, indicando que pode se tratar de uma espécie distinta das descritas no gênero, com potencial epidemiológico, pela sua estabilidade e disseminação por sementes de tomate.

A Tymovirus isolated from seeds of tomato (Solanum lycopersicum) cv. 'Paronset' with necrosis, named TyToRS08 isolate, was characterized by its host range, physical-chemical, serological and molecular properties. Firstly, it was successfully transmitted by mechanical inoculation by infected seeds, and the host range was restricted to species of Amaranthaceae, Chenopo diaceae and Solanaceae. The symptoms induced by the virus in tomatoes 'Alambra', 'Thomas', 'Diva', 'Romana' and 'Graziela' were mainly mosaic and white necrosis. Nicotiana sylvestris, N. tabacum 'Samsun', 'White Burley' and 'Xanthi' were symptomless. The virus was detected in seeds from five tomato cultivars, but it was not detected in seedlings from these infected seeds. The purified preparation had a high concentration of 1.04 mg mL-1, and the title of the polyclonal antiserum produced, evaluated in PTA-ELISA, was 1/32.768. RT-PCR was performed with degenerated primers directed to the ORF 1 of the tymoviruses, resulting in a DNA fragment of 700 bp. The sequence analysis revealed that TyToRS08 isolate presents higher values of percentage of identity with other species of Tymovirus. The highest identity values were under 70%, indicating that TyToRS08 should be a distinct species of the genus Tymovirus, with great epidemiological importance due to its high stability and potential to be spread by tomato seeds.

RESUMO Introdução: O fator tissular ativador do plasminogênio, um componente do sistema fibrinolítico, ativa o plasminogênio em plasmina, que degrada a rede de fibrina. O t-PA recombinante (rt-PA) tem sido utilizado em oftalmologia para diminuir as complicações fibrino-proliferativas pós-operatórias. A preparação comercial excede a dose preconizada para uso oftalmológico e o custo por injeção poderia ser reduzido se o produto fosse dividido em doses. Objetivo: Avaliar se o rt-PA, para uso cardiológico, pode ser diluido e utilizado em oftalmologia, se a atividade é mantida após estocagem e, eventualmente determinar quais os melhores métodos de estocagem e de utilização. Materiais e métodos: Este trabalho estudou a atividade inicial do rt-PA em placa de fibrina, a atividade residual, após 6 meses de conservação em freezer à -20ºC e à -70ºC; e ainda a esterilidade do material. Resultados: A atividade residual após 6 meses de conservação em freezer à -70ºC foi 40,7% da atividade inicial e em congelador de geladeira à -20ºC foi 34,9%. Se descongelado e deixado à temperatura ambiente 24 horas antes de ser testado a atividade residual cai para 27,9%. Conclusão: O rt-PA manteve-se estéril após 6 meses, independente da forma de estocagem. A melhor forma de conservação foi o freezer à -70ºC. A utilização do rt-PA deve ser portanto imediata após o descongelamento.

SUMMARY Introduction: The tissue plasminogen activator (t-PA), a fibrinolitic system component, activates the plasminogen into plasmin, which degrades the fibrin net. The recombinant tissue plasminogen activator has been used in ophthalmology to control the post-operative fibrin formation complications. The comercial t-PA available has a higher dose than necessary in ophthalmology. If it can be shared in doses the coasts will reduced. Purpose: To check if the comercial rt-PA, for cardiologic use, can be, diluted and used in ophthalmology, and if the activity remains after storage, and eventualy determine the best storage and utilization methods. Material and methods: The rt-PA initial lytic activity was studied on fibrin net substract, and residual activity after long term storage at -20ºC and -70ºC. The material sterility was tested in culture plates. Results: The rt-PA activity after 6 months storage at -70ºC was 40,7% of non storage rt-PA activity, and at -20ºC it was 34,9%. Samples kept at -20ºC for 6 months and then left at roam temperature 24 hours before testing, showed an activity of 27,9%. No contamination was found in any sample. Conclusion: The best way tested of storage rt-PA is at -70ºC. And in arder to keep the total potential activity, it should be used immediately after defrost.