Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix

© 2016 Elsevier Ltd Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD50 of 1687 pmol/g

Chemical synthesis, 3D structure and ASIC binding site of mambalgin-2

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify -helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide

Inhibition or stimulation of ochratoxin a synthesis on inoculated barley triggered by diffuse coplanar surface barrier discharge plasma

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins. Besides their high toxicity, mycotoxins are highly stable to physical, chemical or biological detoxification. Therefore, the treatment with cold atmospheric plasma could be one approach to reduce the amount of mycotoxins in different products. The aim of this study was to determine the influence of cold atmospheric plasma on the inactivation of Aspergillus niger and Penicillium verrucosum inoculated on barley and their production of OTA. Inoculated barley was treated with plasma generated by dry air, CO2 or CO2 + O2 for 1 or 3 min and stored for up to two weeks at 9, 25, or 37°C. Three minutes of air plasma treatment effectively significantly reduced the total mold count of both microorganisms by 2.5–3 log cycles. The production of OTA from A. niger was only low, therefore the treatment effect was indistinguishable. The treatment of P. verrucosum on barley after an incubation of five days using a CO2 + O2 plasma resulted in a reduction of the OTA content from 49.0 (untreated) to 27.5 (1 min) and 23.8 ng/g (3 min), respectively. In contrast, CO2 plasma caused an increase of the OTA amount from 49.0 (untreated) to 55.8 (1 min) and 72.9 ng/g (3 min). Finally, the use of air plasma resulted likewise in a decrease of the OTA concentration from 56.9 (untreated) to 25.7 (1 min) and 20.2 ng/g (3 min), respectively. Reducing the incubation time before the treatment to 24 h caused in contrast an increase of the OTA content from 3.1 (untreated) to 29.1 (1 min) and 20.7 ng/g (3 min). Due to the high standard deviation, these changes were not significant, but the tendencies were clearly visible, showing the strong impact of the plasma gas on the OTA production. The results show, that even if the total mold count was reduced, under certain conditions the OTA amount was yet enhanced, probably due to a stress reaction of the mold. Concluding, the plasma gas and incubation conditions have to be considered to allow a successful inactivation of molds and in particular their toxic metabolites

NMR structure of μ-conotoxin GIIIC : leucine 18 induces local repacking of the N-terminus resulting in reduced NaV channel potency

mu-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of mu-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of mu-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical mu-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (Na-V), albeit with similar to 2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of Na(V)1.4 channels, but the same Na-V selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications

Nicotiana alata defensin chimeras reveal differences in the mechanism of fungal and tumor cell killing and an enhanced antifungal variant

The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized alpha-beta motif which consists of an alpha helix and a triple-stranded beta-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P-2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P-2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P-2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants

Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain

Loss-of-function mutations of NaV1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of NaV1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of NaV1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of NaV1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with NaV1.7 inhibitors and significantly reduced in NaV1.7−/− mice. To validate the use of the model for profiling NaV1.7 inhibitors, we determined the NaV selectivity and tested the efficacy of the reported NaV1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited NaV1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited NaV1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited NaV channels and was only effective in the OD1 model when delivered systemically. Our novel model of NaV1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of NaV1.7 inhibitors

Efficient chemical synthesis of human complement protein C3a

We report the total chemical synthesis of human C3a by one-pot native chemical ligation of three unprotected peptide segments, followed by efficient in vitro folding that yielded the anaphylatoxin C3a in high yield and excellent purity. Synthetic C3a was fully active and its crystal structure at 2.1 Å resolution showed 3 helices and a C-terminal turn motif

"Świat idealny" - systemowa koncepcja pracy z dzieckiem niepłnosprawnym intelektualnie

The first part of text is concentrated on intellectual disability in the theoretical aspect. In the second part the author is concentrated on description of “Ideal World” proj­ect. This project is being carried out through the staff of Special School No. 4 in Sosnowiec. An equalization of practical disproportions between fully abled and disabled persons and a holistic approach are their purpose for the keeping of the disabled person from the birth for the rest of their life

Verificaçăo das boas práticas de fabricaçăo em indústrias de leite e derivados, registradas no Serviço de Inspeçăo Federal - SIF /

Orientadora: Lys Mary B. CandidoDissertaçăo (mestrado) - Universidade Federal do Paraná, Setor de Cięncias Agrárias, Programa de Pós-Graduaçăo em Ciencias Veterinárias. Defesa: Curitiba, 2005Inclui bibliografi

Reflexão sobre as fraudes fiscais nas contribuições previdenciárias

Orientador: Blenio Cesar Severo PeixeMonografia (Especialização) - Universidade Federal do Paraná, Setor de Ciências Sociais Aplicadas, Curso de Especialização em Auditoria IntegralResumo: Reflexão sobre as Fraudes fiscais nas Contribuições Previdenciárias. Esta reflexão pretende contribuir para o aperfeiçoamento das ações desenvolvidas e no estudo bem como na busca de estratégias para o combate à sonegação e às fraudes previdenciárias, cujos resultados serão envolvidos no tema da matéria tratada para que seja possível encontrar uma fórmula capaz de minorar os efeitos de um mal tido como incurável. Observa-se que poucos estão pagando muito porque muitos sonegam e fraudam os tributos. Em decorrência deste trabalho, espera-se colaborar com as áreas fiscais, com os dirigentes públicos no sentido de dar informações para combater os sonegadores/fraudadores e demonstrar que uma boa legislação e um bom planejamento de atuação pode possibilitar a recuperação de valores fraudados, e conseqüente ação punitiva aos aplicadores desta "arte" de enganar o fisco. Dados preliminares divulgados indicam que a sonegação de contribuições previdenciárias ultrapassa a cifra de bilhões, sendo um fator de deslealdade na concorrência entre as empresas. Muitos contribuintes não pagam contribuições previdenciárias porque os seus concorrentes não pagam. A sonegação e a fraude fiscal são problemas culturais. Pelo menos, é isto o que se propaga nas campanhas de combate à sonegação e à fraude pelas entidades fiscalizadoras, federal, estadual e municipal, no entanto, é necessário enfrentar o problema e envidar esforços no sentido de mudar esta cultura através de esclarecimentos, orientações e demonstrações dos procedimentos obrigatórios nas áreas administrativas, contábeis e judiciais na tentativa de evitar práticas erradas ou fraudulenta