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1.
Rev. Bras. Parasitol. Vet. (Online) ; 32(1): e013522, 2023. tab, graf
Artigo em Inglês | VETINDEX | ID: biblio-1416496

Resumo

Around the world, the main problems of livestock are caused by ectoparasites, however, commercial acaracide are toxic to the environment and detrimental to One Health. Therefore, research has increasingly focused on development of natural products as alternatives for tick control. The purpose of this study was to evaluate the larvicidal effect on Rhipicephalus (Boophilus) microplus, through use of essential oils (EOs) extracted from the leaves, flower buds and stems of Tetradenia riparia. The chemical composition of these EOs was determined through gas chromatography coupled to mass spectrometry (GC-MS). They were tested on larvae at concentrations of 100.000 to 40 µg/mL, using the larval packet test and under semi-natural conditions. The main class of compounds in the chemical composition was sesquiterpenes (both oxygenates and hydrocarbons), whereas the predominant compounds in the leaves, flower buds and stems were 14-hydroxy-9-epi-caryophyllene, T-cadinol and 6-7-dehydroroyleanone, respectively. The leaves proved to be the most effective, with highest larvicidal activity (LC99.9 = 83.53 µg/mL). When tested under semi-natural conditions, the oils obtained efficiency above 98% in all compound tests. The results indicated that these EOs were effective against R. (B.) microplus larvae in vitro and ex-situ, proving that this plant has bioactive molecules with significant larvicidal activity.(AU)


Os principais problemas para a pecuária estão relacionados às ectoparasitoses, e ao fato dos carrapaticidas apresentarem elevada toxicidade ao meio ambiente e à saúde única. Surgem, então, demandas na busca por inovações e desenvolvimento de produtos naturais, como alternativas para o controle dos carrapatos. O objetivo deste trabalho foi avaliar o potencial da atividade larvicida sobre Rhipicephalus (Boophilus) microplus a partir dos óleos essenciais de Tetradenia riparia (TrOEs) extraídos das folhas, botões florais e caules. A composição química foi determinada por cromatografia gasosa acoplada à espectrometria de massa (GC/MS). Os TrOEs foram testados sobre larvas nas concentrações de 100.000 a 40 µg/mL pelo teste de pacote de larvas e em condições seminaturais. Na composição química, a classe majoritária foi os sesquiterpenos (oxigenados e hidrocarbonetos); já os compostos em destaques foram 14-hidroxy-9-epi-caryophyllene, T-cadinol e 6-7-dehidroroyleanone para folhas, botões florais e caules, respectivamente. As folhas demonstraram ser mais eficientes e com maior poder larvicida (CL99.9 = 83.53 µg/mL). Quando testado em condições seminaturais os óleos obtiveram eficiência acima de 98% em todos os compostos testados. Os resultados indicaram que os TrOEs, foram eficazes sobre as larvas de R. (B.) microplus in vitro e ex-situ, evidenciando que esta planta possui moléculas bioativas com ação larvicidas significativas.(AU)


Assuntos
Óleos Voláteis/efeitos adversos , Rhipicephalus/imunologia , Larvicidas , Acaricidas/análise , Espectrometria de Massas/métodos , Cromatografia Gasosa/métodos , Lamiaceae/química
2.
J. venom. anim. toxins incl. trop. dis ; 28: e20210116, 2022. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1375812

Resumo

Background: Conopeptides from cone snail venom have aroused great interest related to the discovery of novel bioactive candidates, due to their excellent prospects for the treatment of various health problems such as pain, addiction, psychosis and epilepsy. In order to explore novel biopeptides, we investigated the structure and function of five novel conopeptides isolated from the venom of Conus marmoreus from South China Sea. Methods: C. marmoreus crude venom was prepared, fractionated and purified by HPLC system. The primary sequences of the five novel disulfide-poor conopeptides Mr-1 to Mr-5 were identified by comprehensive analysis of de novo MALDI-TOF tandem mass spectrometry and Edman degradation data. In order to investigate their function, these five conopeptides were synthesized by Fmoc-SPPS chemistry, and their biological effects at several heterologous rat nicotinic acetylcholine receptor (nAChR) subtypes (α1β1δε, α3β2, α3β4, α4β2) were determined by electrophysiological technique. Results: Five novel disulfide-poor conopeptides were identified and named as follows: Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPP NTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. None of them contains a disulfide bond. The sequences of conopeptides Mr-2 to Mr-5 do not belong to any category of the known disulfide-poor conopeptides. No significant activity against the above nAChR subtypes were observed for the five conopeptides at 100 µM. Conclusion: We purified and structurally characterized five novel disulfide-poor conopeptides from C. marmoreus crude venom and first investigated their nAChR inhibitory effects. This work expanded our knowledge on the structure and function of disulfide-poor conopeptides from this cone snail venom.(AU)


Assuntos
Animais , Conotoxinas/isolamento & purificação , Dissulfetos/efeitos adversos , Venenos de Moluscos , Espectrometria de Massas
3.
Ciênc. rural (Online) ; 52(2): e20200894, 2022. ilus, tab, graf
Artigo em Inglês | VETINDEX, LILACS | ID: biblio-1339655

Resumo

Seed germination is a complex process controlled by many factors, in which physical and biochemical mechanisms are involved and the mobilization of reserves is crucial for this process to occur. Although, seed reserve mobilization is usually thought to be a post-germination process, seed reserve proteins mobilization occurs during germination. This study quantified seed proteins of bean genotypes during different hydration times, in order to understand the process of protein mobilization and whether there is relationship of this biochemical component with seed vigor. This study was conducted using seeds with different levels of vigor, genotypes with highest (13, 42, 55 and 81) and lowest (07, 23, 44, 50, IPR-88-Uirapurú and Iapar 81) physiological quality. High vigor genotypes showed greater efficiency in hydrolysis and mobilization of protein component, because they presented low globulins content in cotyledons at radicle protrusion in relation to low vigor genotypes (07, 23 and 50). The protein alpha-amylase inhibitor, observed in all genotypes, is involved with the longer time needed for radicle protrusion, according to the band intensity difference in genotypes 07, 44 and Iapar 81.


A germinação de sementes é um processo complexo controlado por muitos fatores, nos quais mecanismos físicos e bioquímicos estão envolvidos e a mobilização de reservas é decisiva para que esse processo ocorra. Embora a mobilização de reservas de sementes seja considerada um processo pós-germinativo, a mobilização das proteínas de reserva de sementes ocorre durante a germinação. Este estudo teve como objetivo quantificar as proteínas de sementes de genótipos de feijão durante os diferentes tempos de hidratação, a fim de compreender o processo de mobilização proteica e se há relação desse componente bioquímico com o vigor das sementes. Este estudo foi realizado utilizando sementes com diferentes níveis de vigor, genótipos com maior (13, 42, 55 e 81) e menor (07, 23, 44, 50, IPR-88-Uirapurú e Iapar 81) qualidade fisiológica. Os genótipos de alto vigor apresentaram maior eficiência na hidrólise e mobilização do componente proteico, pois apresentaram baixo teor de globulinas nos cotilédones na protrusão radicular em relação aos genótipos de baixo vigor (07, 23 e 50). A proteína inibidora da alfa-amilase, observada em todos os genótipos, está envolvida com o maior tempo necessário para a protrusão da radícula, de acordo com a diferença de intensidade da banda nos genótipos 07, 44 e Iapar 81.


Assuntos
Sementes/química , Variação Genética/genética , Proteínas/análise , Phaseolus/embriologia , Espectrometria de Massas , Eletroforese em Gel de Poliacrilamida
4.
J. venom. anim. toxins incl. trop. dis ; 28: e20210042, 2022. graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1360568

Resumo

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)


Assuntos
Animais , Espectrometria de Massas/instrumentação , Venenos de Aranha/análise , Aranhas , Isoformas de Proteínas/biossíntese , Hialuronoglucosaminidase , Preparações Farmacêuticas
5.
J. venom. anim. toxins incl. trop. dis ; 28: e20210047, 2022. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1375811

Resumo

Accidents with venomous animals are a public health issue worldwide. Among the species involved in these accidents are scorpions, spiders, bees, wasps, and other members of the phylum Arthropoda. The knowledge of the function of proteins present in these venoms is important to guide diagnosis, therapeutics, besides being a source of a large variety of biotechnological active molecules. Although our understanding about the characteristics and function of arthropod venoms has been evolving in the last decades, a major aspect crucial for the function of these proteins remains poorly studied, the posttranslational modifications (PTMs). Comprehension of such modifications can contribute to better understanding the basis of envenomation, leading to improvements in the specificities of potential therapeutic toxins. Therefore, in this review, we bring to light protein/toxin PTMs in arthropod venoms by accessing the information present in the UniProtKB/Swiss-Prot database, including experimental and putative inferences. Then, we concentrate our discussion on the current knowledge on protein phosphorylation and glycosylation, highlighting the potential functionality of these modifications in arthropod venom. We also briefly describe general approaches to study "PTM-functional-venomics", herein referred to the integration of PTM-venomics with a functional investigation of PTM impact on venom biology. Furthermore, we discuss the bottlenecks in toxinology studies covering PTM investigation. In conclusion, through the mining of PTMs in arthropod venoms, we observed a large gap in this field that limits our understanding on the biology of these venoms, affecting the diagnosis and therapeutics development. Hence, we encourage community efforts to draw attention to a better understanding of PTM in arthropod venom toxins.(AU)


Assuntos
Animais , Venenos de Artrópodes/toxicidade , Processamento de Proteína Pós-Traducional , Fosforilação , Escorpiões , Espectrometria de Massas/métodos , Aranhas , Vespas , Abelhas , Glicosilação
6.
Acta sci., Biol. sci ; 43: e53534, 2021. map, ilus, graf, tab
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1460982

Resumo

This research assessed the fumigant activity of the essential oil from Piper sancti-felicis Trel and five of its components on the Tribolium castaneum (Herbst) biological model. Hydrodistillation was used for extraction of the essential oil, with separation and identification of the compounds through gas chromatography coupled to mass spectrometry (GC-MS). The fumigant was evaluated through gas dispersion on the T. castaneum. The majority compounds found in the EO were b-nerolidol (15.4%), 3-carene (14.9%), p-cymene (9.1%), spathulenol (8.2%), a-cubebene (6.2%) and calamenene (5.2%). Piper sancti-felicis displayed fumigant activity with a LC50 = 108.5 & 956;g L-1 air, and other individual monoterpenes tested such as & 945;-terpinolene (LC50 = 110.1 & 956;g L-1 air), p-cymene (LC50 = 120.3 & 956;g L-1 air), 3-carene (LC50 = 130.6 & 956;g L-1 air), (R) -limonene (CL50 = 189.6 & 956;g L-1 air), and a-pinene (LC50 = 213.1 & 956;g L-1 air), were significantly less toxic than methyl pyrimiphos used as a positive control, CL50 = 87.4 & 956;g L-1 air. The essential oil of P. sancti-felicis can be considered as a natural source of biocides.


Assuntos
Espectrometria de Massas , Piper/química , Tribolium/química , Óleos Voláteis/química
7.
Acta Sci. Biol. Sci. ; 43: e53534, 2021. mapas, ilus, graf, tab
Artigo em Inglês | VETINDEX | ID: vti-764591

Resumo

This research assessed the fumigant activity of the essential oil from Piper sancti-felicis Trel and five of its components on the Tribolium castaneum (Herbst) biological model. Hydrodistillation was used for extraction of the essential oil, with separation and identification of the compounds through gas chromatography coupled to mass spectrometry (GC-MS). The fumigant was evaluated through gas dispersion on the T. castaneum. The majority compounds found in the EO were b-nerolidol (15.4%), 3-carene (14.9%), p-cymene (9.1%), spathulenol (8.2%), a-cubebene (6.2%) and calamenene (5.2%). Piper sancti-felicis displayed fumigant activity with a LC50 = 108.5 & 956;g L-1 air, and other individual monoterpenes tested such as & 945;-terpinolene (LC50 = 110.1 & 956;g L-1 air), p-cymene (LC50 = 120.3 & 956;g L-1 air), 3-carene (LC50 = 130.6 & 956;g L-1 air), (R) -limonene (CL50 = 189.6 & 956;g L-1 air), and a-pinene (LC50 = 213.1 & 956;g L-1 air), were significantly less toxic than methyl pyrimiphos used as a positive control, CL50 = 87.4 & 956;g L-1 air. The essential oil of P. sancti-felicis can be considered as a natural source of biocides.(AU)


Assuntos
Óleos Voláteis/química , Piper/química , Tribolium/química , Espectrometria de Massas
8.
J. venom. anim. toxins incl. trop. dis ; 26: e20200025, 2020. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135152

Resumo

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)


Assuntos
Espectrometria de Massas , Antivenenos , Cromatografia , Corrente Jusante , Plasma , Imunoterapia
9.
Arq. Inst. Biol ; 87: e0602019, 2020. ilus, tab
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1130104

Resumo

Substances with cytotoxic activity present in vaccines against the foot-and-mouth disease may interfere with methods used to detect residual live virus in the product or cause undesirable postvaccination reactions. This study describes a rapid in vitro test to detect cytotoxic activity in water-in-oil vaccines against foot-and-mouth disease using a commercial saponin as a cytotoxic agent and a solution of sheep's red blood cells as substrate. Hemolytic and cytotoxic activity was analyzed using experimental and commercial vaccines prepared with and without saponin. The hemolytic and cytotoxic potential of preparations containing saponin was evident. In contrast, hemolytic and cytotoxic activities were not observed in vaccines without saponin in their composition. The method described here allows to easily detect if the vaccine under study has cytotoxic activity, making it possible to select the most appropriate method to process the sample to be used for the innocuity test. Additionally, due to undesirable effects that may be observed in animals receiving vaccines containing saponin in their formulation, the use of the rapid test described here allows to identify those vaccines with cytotoxic activity and to verify the presence of saponin on them, through the mass spectrometry method.(AU)


Substâncias com atividade citotóxica presentes em vacinas contra febre aftosa podem interferir com o método utilizado para a detecção de vírus ativo residual no produto ou causar reações pós-vacinais indesejáveis. O presente trabalho descreve uma prova rápida in vitro para detectar atividade citotóxica em vacinas oleosas contra febre aftosa utilizando uma saponina comercial como agente citotóxico e uma solução de hemácias de carneiro como substrato. Analisaram-se as atividades hemolítica e citotóxica utilizando-se vacinas experimentais e comerciais preparadas com e sem saponina. O potencial hemolítico e citotóxico dos preparados que continham saponina na sua formulação foi evidente. Em contraste, não se observou atividade hemolítica e citotóxica nas vacinas sem saponina. O método descrito permite conhecer rapidamente se a vacina em estudo apresenta atividade citotóxica e, dessa maneira, selecionar o método mais adequado para processar a amostra que será utilizada para investigar a presença de vírus ativo residual. Adicionalmente, devido aos efeitos indesejáveis que podem ser observados em animais que recebem vacinas que contêm saponina na sua formulação, o uso da prova rápida descrita permite selecionar aquelas vacinas com atividade citotóxica para posteriormente verificar a presença de saponina através de técnicas analíticas como a espectrometria de massas.(AU)


Assuntos
Bovinos , Vacinas , Vírus da Febre Aftosa , Febre Aftosa , Saponinas , Espectrometria de Massas , Técnicas In Vitro , Eritrócitos
10.
J. venom. anim. toxins incl. trop. dis ; 26: e20200037, 2020. graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135157

Resumo

Proteases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated with several pathologies. Therefore, proteases have become potential therapeutic targets regarding the search for inhibitors. Snake venoms are complex mixtures of molecules that can feature a variety of functions, including peptidase inhibition. Considering this, the present study reports the purification and characterization of a Kunitz-type peptide present in the Dendroaspis polylepis venom as a simultaneous inhibitor of elastase-1 and cathepsin L. Methods: The low molecular weight pool from D. polylepis venom was fractionated in reverse phase HPLC and all peaks were tested in fluorimetric assays. The selected fraction that presented inhibitory activity over both proteases was submitted to mass spectrometry analysis, and the obtained sequence was determined as a Kunitz-type serine protease inhibitor homolog dendrotoxin I. The molecular docking of the Kunitz peptide on the elastase was carried out in the program Z-DOCK, and the program RosettaDock was used to add hydrogens to the models, which were re-ranked using ZRANK program. Results: The fraction containing the Kunitz molecule presented similar inhibition of both elastase-1 and cathepsin L. This Kunitz-type peptide was characterized as an uncompetitive inhibitor for elastase-1, presenting an inhibition constant (Ki) of 8 μM. The docking analysis led us to synthesize two peptides: PEP1, which was substrate for both elastase-1 and cathepsin L, and PEP2, a 30-mer cyclic peptide, which showed to be a cathepsin L competitive inhibitor, with a Ki of 1.96 µM, and an elastase-1 substrate. Conclusion: This work describes a Kunitz-type peptide toxin presenting inhibitory potential over serine and cysteine proteases, and this could contribute to further understand the envenomation process by D. polylepis. In addition, the PEP2 inhibits the cathepsin L activity with a low inhibition constant.(AU)


Assuntos
Animais , Peptídeos , Serina , Venenos de Serpentes , Cisteína Proteases , Elapidae , Peptídeo Hidrolases/isolamento & purificação , Espectrometria de Massas
11.
J. venom. anim. toxins incl. trop. dis ; 26: e20200018, 2020. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135146

Resumo

Variability in snake venoms is a well-studied phenomenon. However, sex-based variation of Bothrops atrox snake venom using siblings is poorly investigated. Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Differences in the venom composition of Bothrops genus have been linked to several factors such as ontogeny, geographical distribution, prey preferences and sex. Thus, in the current study, venom samples of Bothrops atrox male and female siblings were analyzed in order to compare their biochemical and biological characteristics. Methods: Venoms were collected from five females and four males born from a snake captured from the wild in São Bento (Maranhão, Brazil), and kept in the Laboratory of Herpetology of Butantan Intitute. The venoms were analyzed individually and as a pool of each gender. The assays consisted in protein quantification, 1-DE, mass spectrometry, proteolytic, phospholipase A2, L-amino acid oxidase activities, minimum coagulant dose upon plasma, minimum hemorrhagic dose and lethal dose 50%. Results: Electrophoretic profiles of male's and female's venom pools were quite similar, with minor sex-based variation. Male venom showed higher LAAO, PLA2 and hemorrhagic activities, while female venom showed higher coagulant activity. On the other hand, the proteolytic activities did not show statistical differences between pools, although some individual variations were observed. Meanwhile, proteomic profile revealed 112 different protein compounds; of which 105 were common proteins of female's and male's venom pools and seven were unique to females. Despite individual variations, lethality of both pools showed similar values. Conclusion: Although differences between female and male venoms were observed, our results show that individual variations are significant even between siblings, highlighting that biological activities of venoms and its composition are influenced by other factors beyond gender.(AU)


Assuntos
Animais , Mordeduras de Serpentes , Venenos de Serpentes , Espectrometria de Massas , Bothrops , L-Aminoácido Oxidase , Fosfolipases A2 , Produtos Biológicos
12.
Arq. Inst. Biol. ; 87: e0602019, 2020. ilus, tab
Artigo em Inglês | VETINDEX | ID: vti-29344

Resumo

Substances with cytotoxic activity present in vaccines against the foot-and-mouth disease may interfere with methods used to detect residual live virus in the product or cause undesirable postvaccination reactions. This study describes a rapid in vitro test to detect cytotoxic activity in water-in-oil vaccines against foot-and-mouth disease using a commercial saponin as a cytotoxic agent and a solution of sheep's red blood cells as substrate. Hemolytic and cytotoxic activity was analyzed using experimental and commercial vaccines prepared with and without saponin. The hemolytic and cytotoxic potential of preparations containing saponin was evident. In contrast, hemolytic and cytotoxic activities were not observed in vaccines without saponin in their composition. The method described here allows to easily detect if the vaccine under study has cytotoxic activity, making it possible to select the most appropriate method to process the sample to be used for the innocuity test. Additionally, due to undesirable effects that may be observed in animals receiving vaccines containing saponin in their formulation, the use of the rapid test described here allows to identify those vaccines with cytotoxic activity and to verify the presence of saponin on them, through the mass spectrometry method.(AU)


Substâncias com atividade citotóxica presentes em vacinas contra febre aftosa podem interferir com o método utilizado para a detecção de vírus ativo residual no produto ou causar reações pós-vacinais indesejáveis. O presente trabalho descreve uma prova rápida in vitro para detectar atividade citotóxica em vacinas oleosas contra febre aftosa utilizando uma saponina comercial como agente citotóxico e uma solução de hemácias de carneiro como substrato. Analisaram-se as atividades hemolítica e citotóxica utilizando-se vacinas experimentais e comerciais preparadas com e sem saponina. O potencial hemolítico e citotóxico dos preparados que continham saponina na sua formulação foi evidente. Em contraste, não se observou atividade hemolítica e citotóxica nas vacinas sem saponina. O método descrito permite conhecer rapidamente se a vacina em estudo apresenta atividade citotóxica e, dessa maneira, selecionar o método mais adequado para processar a amostra que será utilizada para investigar a presença de vírus ativo residual. Adicionalmente, devido aos efeitos indesejáveis que podem ser observados em animais que recebem vacinas que contêm saponina na sua formulação, o uso da prova rápida descrita permite selecionar aquelas vacinas com atividade citotóxica para posteriormente verificar a presença de saponina através de técnicas analíticas como a espectrometria de massas.(AU)


Assuntos
Animais , Bovinos , Vacinas , Vírus da Febre Aftosa , Febre Aftosa , Saponinas , Espectrometria de Massas , Técnicas In Vitro , Eritrócitos
13.
J. venom. anim. toxins incl. trop. dis ; 26: e20190078, 2020. graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1091025

Resumo

Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.(AU)


Assuntos
Animais , Anuros , Peptídeos , Espectrometria de Massas , Produtos Biológicos , Eletroforese em Gel de Poliacrilamida , Fosfolipases A2 , Reações Bioquímicas/classificação , Citotoxinas
14.
J. Venom. Anim. Toxins incl. Trop. Dis. ; 26: e20200025, 2020. tab, graf
Artigo em Inglês | VETINDEX | ID: vti-32211

Resumo

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)


Assuntos
Antivenenos , Corrente Jusante , Imunoterapia , Cromatografia por Troca Iônica , Espectrometria de Massas
15.
Anim. Reprod. (Online) ; 17(4): e20200533, 2020. ilus, graf, tab
Artigo em Inglês | VETINDEX | ID: biblio-1461537

Resumo

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P 0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.


Assuntos
Feminino , Animais , Espectrometria de Massas , Oócitos/classificação , Partenogênese , Suínos/embriologia , Suínos/fisiologia
16.
Anim. Reprod. (Online) ; 17(4): e20200552, 2020. tab, graf, ilus
Artigo em Inglês | VETINDEX | ID: biblio-1461538

Resumo

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.


Assuntos
Feminino , Animais , Cavalos/fisiologia , Cavalos/metabolismo , Eletroforese em Gel Diferencial Bidimensional/veterinária , Espectrometria de Massas
17.
Anim. Reprod. ; 17(4): e20200552, 2020. tab, graf, ilus
Artigo em Inglês | VETINDEX | ID: vti-29835

Resumo

This experiment aimed to compare at day seven after ovulation, the protein profile of uterine fluid in cyclic mares with mares infused two days before with Day 13 conceptus fragments. Experimental animals were ten healthy cyclic mares, examined daily to detect ovulation (Day 0) as soon as estrus was confirmed. On day seven, after ovulation, uterine fluid was collected, constituting the Cyclic group (n = 10). The same mares were examined in the second cycle until ovulation was detected. On day five, after ovulation, fragments from a previously collected concepti were infused into each mare's uterus. Two days after infusion, uterine fluid was collected, constituting the Fragment group (n = 10). Two-dimensional electrophoresis technique processed uterine fluid samples. A total of 373 spots were detected. MALDI-TOF/TOF and NanoUHPLC-QTOF mass spectrometry identified twenty spots with differences in abundance between the Cyclic and Fragment group. Thirteen proteins were identified, with different abundance between groups. Identified proteins may be related to embryo-maternal communication, which involves adhesion, nutrition, endothelial cell proliferation, transport, and immunological tolerance. In conclusion, conceptus fragments signalized changes in the protein profile of uterine fluid seven days after ovulation in comparison to the observed at Day 7 in the same cyclic mares.(AU)


Assuntos
Animais , Feminino , Cavalos/metabolismo , Cavalos/fisiologia , Eletroforese em Gel Diferencial Bidimensional/veterinária , Espectrometria de Massas
18.
Anim. Reprod. ; 17(4): e20200533, 2020. ilus, graf, tab
Artigo em Inglês | VETINDEX | ID: vti-29797

Resumo

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P 0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.(AU)


Assuntos
Animais , Feminino , Suínos/embriologia , Suínos/fisiologia , Oócitos/classificação , Espectrometria de Massas , Partenogênese
19.
Sci. agric ; 76(4): 311-320, July-Aug. 2019. tab, graf
Artigo em Inglês | VETINDEX | ID: biblio-1497793

Resumo

This research investigated the potential of beef products with acid whey to release bioactive peptides and thereby emphasize their health-promoting potential. Peptide sequences were isolated and identified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Firstly, the antihealth properties (toxicity, allergenicity) of the peptides were estimated based on the peptide sequences. Next, their health-promoting potential was demonstrated based on an in silico analysis by determining their bioactivity scores (PeptideRanker). Their various biological actions were also determined using BIOPEP-UWM tools. We presented peptide sequences with properties relevant to ensuring good health and well-being, including cardiovascular system, nervous and immune systems, or their support for the maintenance of general homeostasis. We obtained information on generation of biologically active peptides in uncured beef with acid whey and it can be considered as a new knowledge as it contributes to science development of functional and nutraceutical foods. In the long term, this information can be used in designing products with desired nutritional and health-promoting properties that are important for the well-being and for preventing the occurrence of noncommunicable diseases.


Assuntos
Alimento Funcional , Alérgenos , Medidas de Toxicidade , Peptídeos , Produtos da Carne/análise , Bovinos , Cromatografia Líquida , Espectrometria de Massas
20.
R. bras. Saúde Prod. Anim. ; 20: e04102019, Nov. 11, 2019. tab, graf
Artigo em Inglês | VETINDEX | ID: vti-24676

Resumo

Anthelmintics are used to combat nematodes. The misuse of anthelmintics can raise the cost of milk production. The objective of this research was to determine the presence of anthelmintics in goat milk. Twenty goats were used, divided into four groups of five animals: I- animals treated with an ivermectin-based anthelmintic; II- animals treated with moxidectin; III- animals treated with levamisole hydrochloride; and IV: animals treated with albendazole. Milk samples were collected individually: before, and 1, 2, 3, 15 and 21 days after administration of the anthelmintics. Determination of anthelmintic residues was performed by liquid chromatography-mass spectrometry (LC-MS). According to the results, there was an exponential effect (P<0.05) for ivermectin and moxidectin. Moxidectin was the anthelmintic that left a residue in the milk for the longest time, up to 21 days. However, with all the anthelmintics researched, residues were below the maximum limit recommended by the inspecting agencies.(AU)


Anthelmintics são usados ​​para combater nematodes. O mau uso de anti-helmínticos pode aumentar o custo da produção de leite. O objetivo desta pesquisa foi determinar a presença de anti-helmínticos no leite de cabra. Foram utilizados vinte cabras, divididos em quatro grupos de cinco animais: I - animais tratados com um antihelmíntico à base de ivermectina; II - animais tratados com moxidectina; III- animais tratados com cloridrato de levamisol; E IV: animais tratados com albendazole. Amostras de leite foram coletadas individualmente: antes, e 1, 2, 3, 15 e 21 dias após a administração dos anti-helmínticos. A determinação dos resíduos antihelmínticos foi realizada por cromatografia líquida-espectrometria de massa (LC-MS). De acordo com os resultados, houve um efeito exponencial (P <0.05) para ivermectina e moxidectina. A moxidectina foi o anti-helmíntico que deixou um resíduo no leite por mais tempo, até 21 dias. Contudo, com todos os anthelmintics pesquisados, os resíduos estavam abaixo do limite máximo recomendado pelas agências de inspeção.(AU)


Assuntos
Animais , Ruminantes , Leite/toxicidade , Contaminantes Químicos em Alimentos , Anti-Helmínticos/administração & dosagem , Ivermectina , Espectrometria de Massas/veterinária
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