Molecular transduction in receptor-ligand systems by planar electromagnetic fields

Abstract The coupling of a ligand with a molecular receptor induces a signal that travels through the receptor, reaching the internal domain and triggering a response cascade. In previous work on T-cell receptors and their coupling with foreign antigens, we observed the presence of planar molecular patterns able to generate electromagnetic fields within the proteins. These planes showed a coherent (synchronized) behavior, replicating immediately in the intracellular domain that which occurred in the extracellular domain as the ligand was coupled. In the present study, we examined this molecular transduction - the capacity of the coupling signal to penetrate deep inside the receptor molecule and induce a response. We verified the presence of synchronized behavior in diverse receptor-ligand systems. To appreciate this diversity, we present four biochemically different systems - TCR-peptide, calcium pump-ADP, haemoglobin-oxygen, and gp120-CD4 viral coupling. The confirmation of synchronized molecular transduction in each of these systems suggests that the proposed mechanism would occur in all biochemical receptor-ligand systems.

Resumo A ligação de um ligante com um receptor molecular induz um sinal que viaja através do receptor, chegando ao domínio interno e disparando uma cascata de resposta. Em trabalhos anteriores em receptores de células T e sua ligação com antígenos estranhos, observamos a presença de padrões moleculares planares capazes de gerar campos eletromagnéticos dentro das proteínas. Esses planos mostraram um comportamento coerente (sincronizado), replicando, instantaneamente, no domínio intracelular o que ocorreu no domínio extracelular, enquanto o ligante era acoplado. No presente estudo, examinamos essa transdução a capacidade de um sinal de acoplamento de penetrar profundamente a molécula receptora e induzir uma resposta. Verificamos a presença de um comportamento coerente em sistemas diversos de receptor-ligante. Para apreciar essa diversidade, apresentamos quatro sistemas bioquímicos diferentes: TCR-peptídeo, ADP-bomba de cálcio, hemoglobina-oxigênio e gp120-CD4 acoplamento viral. A confirmação de transdução molecular sincronizada em cada um desses sistemas sugere que o mecanismo proposto ocorreria em todos os sistemas bioquímicos receptor-ligante.

Molecular transduction in receptor-ligand systems by planar electromagnetic fields / Transdução molecular em sistemas receptor-ligante por campos electromagnéticos planares

The coupling of a ligand with a molecular receptor induces a signal that travels through the receptor, reaching the internal domain and triggering a response cascade. In previous work on T-cell receptors and their coupling with foreign antigens, we observed the presence of planar molecular patterns able to generate electromagnetic fields within the proteins. These planes showed a coherent (synchronized) behavior, replicating immediately in the intracellular domain that which occurred in the extracellular domain as the ligand was coupled. In the present study, we examined this molecular transduction - the capacity of the coupling signal to penetrate deep inside the receptor molecule and induce a response. We verified the presence of synchronized behavior in diverse receptor ligand systems. To appreciate this diversity, we present four biochemically different systems - TCR-peptide, calcium pump-ADP, haemoglobin-oxygen, and gp120-CD4 viral coupling. The confirmation of synchronized molecular transduction in each of these systems suggests that the proposed mechanism would occur in all biochemical receptor-ligand systems.

A ligação de um ligante com um receptor molecular induz um sinal que viaja através do receptor, chegando ao domínio interno e disparando uma cascata de resposta. Em trabalhos anteriores em receptores de células T e sua ligação com antígenos estranhos, observamos a presença de padrões moleculares planares capazes de gerar campos eletromagnéticos dentro das proteínas. Esses planos mostraram um comportamento coerente (sincronizado), replicando, instantaneamente, no domínio intracelular o que ocorreu no domínio extracelular, enquanto o ligante era acoplado. No presente estudo, examinamos essa transdução – a capacidade de um sinal de acoplamento de penetrar profundamente a molécula receptora e induzir uma resposta. Verificamos a presença de um comportamento coerente em sistemas diversos de receptor-ligante. Para apreciar essa diversidade, apresentamos quatro sistemas bioquímicos diferentes: TCR-peptídeo, ADP-bomba de cálcio, hemoglobina-oxigênio e gp120-CD4 acoplamento viral. A confirmação de transdução molecular sincronizada em cada um desses sistemas sugere que o mecanismo proposto ocorreria em todos os sistemas bioquímicos receptor-ligante.

Molecular transduction in receptor-ligand systems by planar electromagnetic fields / Transdução molecular em sistemas receptor-ligante por campos electromagnéticos planares

The coupling of a ligand with a molecular receptor induces a signal that travels through the receptor, reaching the internal domain and triggering a response cascade. In previous work on T-cell receptors and their coupling with foreign antigens, we observed the presence of planar molecular patterns able to generate electromagnetic fields within the proteins. These planes showed a coherent (synchronized) behavior, replicating immediately in the intracellular domain that which occurred in the extracellular domain as the ligand was coupled. In the present study, we examined this molecular transduction - the capacity of the coupling signal to penetrate deep inside the receptor molecule and induce a response. We verified the presence of synchronized behavior in diverse receptor ligand systems. To appreciate this diversity, we present four biochemically different systems - TCR-peptide, calcium pump-ADP, haemoglobin-oxygen, and gp120-CD4 viral coupling. The confirmation of synchronized molecular transduction in each of these systems suggests that the proposed mechanism would occur in all biochemical receptor-ligand systems.(AU)

A ligação de um ligante com um receptor molecular induz um sinal que viaja através do receptor, chegando ao domínio interno e disparando uma cascata de resposta. Em trabalhos anteriores em receptores de células T e sua ligação com antígenos estranhos, observamos a presença de padrões moleculares planares capazes de gerar campos eletromagnéticos dentro das proteínas. Esses planos mostraram um comportamento coerente (sincronizado), replicando, instantaneamente, no domínio intracelular o que ocorreu no domínio extracelular, enquanto o ligante era acoplado. No presente estudo, examinamos essa transdução a capacidade de um sinal de acoplamento de penetrar profundamente a molécula receptora e induzir uma resposta. Verificamos a presença de um comportamento coerente em sistemas diversos de receptor-ligante. Para apreciar essa diversidade, apresentamos quatro sistemas bioquímicos diferentes: TCR-peptídeo, ADP-bomba de cálcio, hemoglobina-oxigênio e gp120-CD4 acoplamento viral. A confirmação de transdução molecular sincronizada em cada um desses sistemas sugere que o mecanismo proposto ocorreria em todos os sistemas bioquímicos receptor-ligante.(AU)

Molecular transduction in receptor-ligand systems by planar electromagnetic fields / Transdução molecular em sistemas receptor-ligante por campos electromagnéticos planares

The coupling of a ligand with a molecular receptor induces a signal that travels through the receptor, reaching the internal domain and triggering a response cascade. In previous work on T-cell receptors and their coupling with foreign antigens, we observed the presence of planar molecular patterns able to generate electromagnetic fields within the proteins. These planes showed a coherent (synchronized) behavior, replicating immediately in the intracellular domain that which occurred in the extracellular domain as the ligand was coupled. In the present study, we examined this molecular transduction - the capacity of the coupling signal to penetrate deep inside the receptor molecule and induce a response. We verified the presence of synchronized behavior in diverse receptorligand systems. To appreciate this diversity, we present four biochemically different systems - TCR-peptide, calcium pump-ADP, haemoglobin-oxygen, and gp120-CD4 viral coupling. The confirmation of synchronized molecular transduction in each of these systems suggests that the proposed mechanism would occur in all biochemical receptor-ligand systems.

A ligação de um ligante com um receptor molecular induz um sinal que viaja através do receptor, chegando ao domínio interno e disparando uma cascata de resposta. Em trabalhos anteriores em receptores de células T e sua ligação com antígenos estranhos, observamos a presença de padrões moleculares planares capazes de gerar campos eletromagnéticos dentro das proteínas. Esses planos mostraram um comportamento coerente (sincronizado), replicando, instantaneamente, no domínio intracelular o que ocorreu no domínio extracelular, enquanto o ligante era acoplado. No presente estudo, examinamos essa transdução ­ a capacidade de um sinal de acoplamento de penetrar profundamente a molécula receptora e induzir uma resposta. Verificamos a presença de um comportamento coerente em sistemas diversos de receptor-ligante. Para apreciar essa diversidade, apresentamos quatro sistemas bioquímicos diferentes: TCR-peptídeo, ADP-bomba de cálcio, hemoglobina-oxigênio e gp120-CD4 acoplamento viral. A confirmação de transdução molecular sincronizada em cada um desses sistemas sugere que o mecanismo proposto ocorreria em todos os sistemas bioquímicos receptor-ligante.

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)

Suppression of cardiomyocyte functions by ß-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. Methods: ß-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of ß-CTX concentration. Results: Purified ß-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. ß-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in ß-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. Conclusion: We present an alternative purification method for ß-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from ß-CTX.(AU)

Neurobehavioral and neuroprotector effects of caffeine in animal models

This review aims to analyze and contrast the neurological effects associated with the use of caffeine on neurobehavior and neuroprotection in animal models. Caffeine belongs to the group of methylxanthines that exert a direct effect on adenosine receptors associated with inhibitory or excitatory G proteins, generating modification of cyclic AMP activity and intracellular calcium flow which produces alterations in the modulation system of the neurotransmitters dopamine and glutamate. The regulation of the neurotransmission systems generates protection against the inflammation of the central nervous system, by activation of the microglia and reinforcement of the blood-brain barrier. This drug will also restore cognition or prevent memory loss in Parkinson's or Alzheimer's diseases. It is important to establish new study models in other species to assess whether the behavior of the molecule is similar and to obtain other clinical applications in its behavioral and neuroprotective effects.(AU)

Neurobehavioral and neuroprotector effects of caffeine in animal models

This review aims to analyze and contrast the neurological effects associated with the use of caffeine on neurobehavior and neuroprotection in animal models. Caffeine belongs to the group of methylxanthines that exert a direct effect on adenosine receptors associated with inhibitory or excitatory G proteins, generating modification of cyclic AMP activity and intracellular calcium flow which produces alterations in the modulation system of the neurotransmitters dopamine and glutamate. The regulation of the neurotransmission systems generates protection against the inflammation of the central nervous system, by activation of the microglia and reinforcement of the blood-brain barrier. This drug will also restore cognition or prevent memory loss in Parkinson's or Alzheimer's diseases. It is important to establish new study models in other species to assess whether the behavior of the molecule is similar and to obtain other clinical applications in its behavioral and neuroprotective effects.

Eventos fisiológicos e bioquímicos relacionados a capacitação espermática de garanhões em condições in vitro

Apenas dois potros foram nascidos de FIV (fertilização in vitro) convencional em equinos, ambos no início dos anos 90. O principal motivo do insucesso da técnica é direcionado ao espermatozoide do garanhão, que não é capaz de penetrar a zona pelúcida e fertilizar o oócito provavelmente devido a sua incompleta ativação (capacitação) em condições in vitro. Capacitação pode ser definida como o conjunto de mudanças físicas e biológicas que acontecem no espermatozoide o tornando apto a fertilizar o oócito em condições in vivo ou in vitro. Para que esse processo ocorra, o espermatozoide deve estar submetido a um ambiente que contenha bicarbonato (HCO3 - ), cálcio (Ca2+) e albumina. Essas substâncias causarão mudanças nas concentrações de adenosina monofosfato cíclico (AMPc), pH e Ca2+ intracelular, além alteração no potencial de membrana causando reorganização da membrana plasmática e depleção do colesterol, levando ao espermatozoide à reação acrossomal e fertilização do oócito. Infelizmente, nos equinos várias etapas da capacitação ainda não estão bem elucidadas. O objetivo desse trabalho foi demonstrar, por meio do uso de citometria de fluxo, microscopia de fluorescência, crio-eletromicroscopia e imunohistoquímica, algumas etapas da capacitação espermática de garanhões na presença de moléculas que podem estimular esse processo, além de compreender qual adenilato ciclase (solúvel ou transmembrana) está envolvida em algumas das etapas desse evento. O uso da probe Annexina-V em condições capacitantes demonstrou a ocorrência da exposição da fosfatidilserina (PS) em espermatozoides de garanhão durante o processo de capacitação, porém em uma pequena porcentagem da população (p0,05). Além disso, o uso de cafeína e dibutiril-AMPc indicam a relevância da via sAC/pkA para esse processo (p0,05). A observação de três diferentes padrões de fluorescência da Annexina-V no espermatozoide viável de garanhão sugere etapas sequenciais na remodelação da membrana plasmática. Em relação aos efeitos na fluidez de membrana e reação acrossomal induzida, diferentes concentrações de Ca2+ não interferiram na população espermática viva, merocianina e FITC-PNA 647 positiva (p0,05). O cálcio deve estar presente no meio capacitante uma vez que, na presença de seu quelante EGTA, essas etapas da capacitação foram bloqueadas. Porém o cálcio não deve estar obrigatoriamente presente em elevadas concentrações para iniciar a capacitação em espermatozoides de garanhão como é necessário em outras espécies. Diferentes concentrações de progesterona (P4) e albumina sérica bovina (BSA) também não foram capazes de aumentar a fluidez de membrana ou induzir reação acrossomal nos espermatozoides de garanhão (p0,05). Em relação as adenilato ciclases, a adenilato ciclase solúvel (sAC) no espermatozoide de garanhão demonstrou ser responsável pelo aumento da fluidez de membrana na presença de bicarbonato. Esse resultado foi demonstrado pelo uso do LRE1, um inibidor específico da sAC que demonstrou não causar efeitos colaterais em outros eventos fisiológicos espermáticos como alteração do potencial mitocondrial (p0,05). A imunohistoquímica da sAC em espermatozoide de garanhões demonstrou diferente localização comparada à sAC de suínos. No espermatozoide de garanhão essa enzima está presente formando uma linha pontilhada como um cordão na parte distal do acrosoma enquanto no suíno essa enzima está presente na parte apical da região acrossomal e na porção inicial da peça intermediária. A ação da forskolina indicou que a adenilato ciclase transmembrana (tmAC) possivelmente está presente no espermatozoide de garanhão e pode desempenhar um papel na reação acrossomal. Contudo, o fato da necessidade de uma alta concentração (500 µM) necessária para demonstrar seu efeito comparada a outras espécies indica que mais estudos como o uso de inibidores específicos (como o ddAdo) ou imunohistoquímica da tmAC (AC1-9) devem ser realizados no intuito de corroborar e validar os resultados encontrados. Em conclusão os resultados obtidos no presente trabalho elucidam diversos questionamentos na capacitação espermática de garanhões como exposição da PS, papel de diferentes concentrações Ca2+, P4 e BSA nesse evento além do papel de diferentes adenilato ciclases em alguns estágios da capacitação demonstrando que a capacitação é um processo complexo que varia entre as espécies.

Only two foals have ever been born from conventional equine IVF (in vitro fertilization), both in the early 1990s. The main reason for the conventional IVF failure is thought to be the stallion spermatozoa, which are not able to penetrate the zona pellucida, most likely due to incomplete activation (capacitation) under in vitro conditions. Capacitation is considered as a consecutive activation of different signaling pathways inducing physiological and biochemical modifications which primes the sperm for fertilization in vitro. To have the capacitation occur, the sperm must be under an environment (in vivo or in vitro) that contains bicarbonate (HCO3 - ), calcium (Ca2+) and albumin. These elements will besides changes in membrane potential, provide changes in cyclic adenosine monophosphate (cAMP) levels, intracellular pH and intracellular Ca2+ causing plasma membrane reorganization and cholesterol depletion leading the sperm to undergo the acrosome reaction and fertilize the oocyte. The aim of this work was to show by flow cytometry, fluorescence microscopy, cryo-electron microscopy and immunohistochemistry some capacitation steps in stallion spermatozoa in the presence of molecules that can trigger this process and also understand which molecules may be involve in some capacitation steps. The use of the probe Annexin-V in capacitating conditions showed a significant increase in the live, Annexin-V positive sperm population indicating a phosphatidylserine (PS) exposure in stallion sperm during this process (p0.05), but it was only in a small percentage of viable sperm. Stimulation of the sAC/cAMP/PKA pathway by caffeine and dibutyryl-cAMP indicated the relevance of this pathway for PS exposure. The observation of three different staining patterns for Annexin-V in live stallion sperm may warrant further investigation with respect to whether these represent sequential steps in membrane remodeling. Regarding the effects in membrane fluidity and induced acrosome reaction different concentrations of calcium did not interfere in the population live merocyanine and PNA-positive spermatozoa (p>0.05). Also, different concentrations of progesterone (P4) and bovine serum albumin (BSA) were not able to increase membrane fluidity or induced acrosome reaction (p0.05). However, calcium should be present in the medium since EGTA (ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid; 2 mM) was able to block all this capacitation steps, but there is no need for higher concentrations (2 mM) to start capacitation in stallion sperm as is needed in other species. Regarding the adenilato cyclases (ACs), the presence of sAC (soluble) in the stallion sperm was shown to be responsible for the increase in membrane fluidity in the presence of bicarbonate. This finding was possible to demonstrate with the use of LRE1, a sAC specific inhibitor that showed no effect in other sperm physiological events (p>0.05). The immunostaining of sAC in stallion sperm indicated different localization compared to boar. In stallions, the enzyme was present as a dotted line (diadem-like pattern) distal from the acrosomal area, while in boars, the sAC was present in the acrosomal area and in the neck. The action of forskolin indicated that tmAC (transmembrane) may be present in the stallion sperm and may play a role in the acrosome reaction. However, the fact that a relatively high concentration (500 µM) was needed to show this effect compared to other reports, indicates that more studies like use of specific inhibitors of tmAC (as ddAdo) or immunostaining of the AC1 to 9 must be performed in stallion sperm to confirm the findings. In conclusion, the findings in this work elucidate several questions in stallion capacitation as PS exposure, role of different concentrations of calcium, progesterone and BSA in this process and the role of adenylyl cyclases in some stallion capacitation events showing that capacitation is a complex process that differs among species.

Exposure to 50 Hz electromagnetic field changes the efficiency of the scorpion alpha toxin

Background Extremely low-frequency (50 Hz) electromagnetic field (ELF-EMF) is produced by electric power transmission lines and electronic devices of everyday use. Some phenomena are proposed as "first effects" of ELF-EMF: the discrete changes in the membrane potential and the increase of the calcium channel activity as well as the intracellular concentration of Ca 2+ . Interaction of the scorpion alpha toxin with the sodium channel depends on the orientation of the charges and may be perturbed by changes in the membrane polarization. The toxin induces overexcitability in the nervous system and an increase in the neurotransmitters released with different consequences, mainly the paralysis of muscles. We assumed that the exposure to ELF-EMF 0.7 mT will change the effects of the insect selective scorpion alpha toxin (recombinant LqhαIT from Leiurus quinquestriatus hebraeus) at the level of the cercal nerve function, the synaptic transmission and on the level of entire insect organism. Taking into account the compensatory mechanisms in organisms, we tested in addition ten times higher ELF-EMF on whole insects.Methods Experiments were performed in vivo on cockroaches (Periplaneta americana) and in vitro - on isolated cockroach abdominal nerve cord with cerci. In biotests, the effects of LqhαIT (10 −8 M) were estimated on the basis of the insect ability to turn back from dorsal to ventral side. Three groups were compared: the control one and the two exposed to ELF-EMF - 0.7 and 7 mT. Bioelectrical activity of the cercal nerve and of the connective nerve that leaves the terminal abdominal ganglion was recorded using extracellular electrodes. LqhαIT (5 × 10 −8 M) induced modifications of neuronal activity that were observed in the control cockroach preparations and in the ones exposed to ELF-EMF (0.7 mT). The exposure to ELF-EMF was carried out using coils with a size appropriate to the examined objects.Results The exposure to ELF-EMF (0.7 mT) modified the effects of LqhαIT (5 × 10−8 M) on activity of the cercal nerve and of the connective nerve. We observed a decrease of the toxin effect on the cercal nerve activity, but the toxic effect of LqhαIT on the connective nerve was increased. Biotests showed that toxicity of LqhαIT (10 −8 M) on cockroaches was reduced by the exposure to ELF-EMF (0.7 and 7 mT).Conclusions The exposure to 50 Hz ELF-EMF modified the mode of action of the anti-insect scorpion alpha toxin LqhαIT at cellular level of the cockroach nervous system and in biotests. Toxin appeared as a usefull tool in distinguishing between the primary and the secondary effects of ELF-EMF.(AU)

SUPLEMENTAÇÃO DE BOVINOS A PASTO COM FONTES DE LIPÍDIOS PROTEGIDOS SOBRE AS CARACTERÍSTICAS SEMINAIS, PARÊNQUIMA TESTICULAR E PRODUÇÃO IN VITRO DE EMBRIÕES.

A hipótese do presente estudo é de que o uso de gordura de palma e gordura de soja, protegidos com sais de cálcio e fornecidos via dieta afetam a histologia testicular e melhoram a qualidade do sêmen fresco e congelado de touros Nelores jovens, e pode interferir diretamente no perfil lipídico das células espermáticas e na fertilidade das doses de sêmen crio preservadas a ponto de melhorar a produção in vitro de embriões. No primeiro capítulo o objetivo foi avaliar a qualidade do sêmen fresco e a histologia testicular de 48 touros jovens da raça Nelore distribuídos aleatoriamente em quatro grupos consumindo os seguintes suplementos: Controle (CO, suplemento proteico-energético sem adição de gordura protegida); gordura de palma (OP, suplemento controle + adição de 285 g de gordura de palma protegida); gordura de soja (OS, suplemento controle + 285g de gordura de soja protegida) ou associação (OP+OS, suplemento controle + 145 g de gordura de soja protegida + 145 g de palma protegida). O período de suplementação durou 84 dias sendo então realizada a coleta e as avaliações do sêmen e a histologia dos testículos. As variáveis testiculares analisadas foram: circunferência escrotal (CE), consistência do testículo esquerdo (CONSTE), volume testicular do lado esquerdo (VOLTE), consistência do testículo direito (CONSTD) e volume testicular do testículo direito (VOLTD). Foram avaliados também os parâmetros seminais do sêmen fresco: Turbilhonamento (TURB), Motilidade (MOT), Vigor (VIG), Concentração (CONC) e o Volume seminal (VOL). Além disso, avaliou-se a Atividade Citoquímica mitocondrial (DAB) e a integridade funcional da membrana plasmática (HIPO). Com o auxílio da análise histológica avaliou-se o número de túbulos seminíferos (NTF), a densidade de superfície tubular (DST) e as características morfológicas das células germinativas: Célula (área (ARC), perímetro (PTC), circularidade (CIR), diâmetro mínimo (DMiC) e diâmetro máximo (DMaC) e Núcleo (área (ARN), perímetro, circularidade (PTN), diâmetro mínimo (DMiN), diâmetro máximo (DMaN), e relação núcleo/célula (NC). No segundo capítulo o objetivo foi avaliar o sêmen pós descongelamento na produção in vitro de embriões, associando com possíveis mudanças no perfil lipídico dos espermatozóides, após os 84 dias de suplementação foi realizada a criopreservação seminal dos ejaculados dos touros experimentais. No sêmen pós descongelamento as variáveis de movimentação foram avaliadas com técnica assistida por computador (CASA). Analisou-se também a atividade mitocondrial (DAB) e o estresse oxidativo induzido (TBARS induzido). Com o uso de sondas fluorescentes e análise em citometro de fluxo avaliou-se: a integridade de membrana plasmática e acrossomal (FITC-PI), o potencial de membrana mitocondrial sonda (JC-1), a quantidade de radicais livres intracelular (CellROX) e a desnaturação do DNA espermático através estabilidade da cromatina (LA). Foi realizada também a análise do perfil lipidico (LIP) dos espermatozóides sendo também testado na produção in vitro de embriões (PIVE). Os dados foram analisados utilizando o programa estatístico SAS (versão 9.3). Primeiro, verificou-se a normalidade e homogeneidade das variáveis e, quando necessário, foram aplicadas mudanças. Os dados foram primeiramente analisados através da ANOVA (análise de variância). Quando encontrada diferença para as variáveis paramêtricas utilizou-se o teste de médias Tukey e para as variáveis não paramétricas utilizou-se do teste Wilcoxon. Na análise de lipidômica utilizou-se de contrastes ortagonais (FC de 1.5, sem usar FDR- taxa de descoberta falsa). Utilizou-se 5% como nível de significância para todas as avaliações. Nas análises do sêmen fresco os animais dos grupos que receberam a suplementação contendo gordura de palma e gordura de soja protegidos, separados ou em associação apresentaram maior circunferência escrotal em comparação aos animais do grupo CO, porém os animais que consumiram dieta contendo oléo de Palma apresentaram maior volume do testículo direito (P= 0,0109) e tendência para menor volume do testículo esquerdo (P=0,0541), além disso apresentaram menor quantidade de túbulos seminíferos e números de células espermatogênica, menor densidade testicular. Por outro lado, apresentaram células com maior diâmetro máximo e maior relação núcleo-célula. Já a diferença estatística encontrada para a variável DAB -3, onde o grupo OP também apresentou diferença em relação aos outros grupos experimentais (P=0,0290) e é reflexo do maior número de espermatozoides DAB 1 nos animais que consumiram gordura de Palma. Já no sêmen pós-descongelamento a suplementação com a associação de gordura de palma e gordura de soja diminuiu a quantidade de espermatozoides com atividade mitocondrial reduzida (DAB 3) e melhorou a porcentagem da movimentação dos espermatozoides de touros Nelores jovens. Observou-se também a diminuição de 79 lipídeos (membrana e armazenamento) em todos os grupos que receberam suplementação com gordura protegida comparando os dias 0 e 86 do período experimental. Ademais, o grupo suplementado com gordura de Palma (OP) teve maior porcentagem de blastocistos (P= 0,0018) na produção in vitro de embriões. Dessa forma, conclui-se que a utilização de 285g de gordura de palma (protegido com sais de cálcio) pode ser uma alternativa de suplementação para melhorar os índices reprodutivos de touros jovens da raça Nelore.

The hypothesis of the present study was that testicular histology, fresh and post-thawed semen quality, sperm cells lipid profile and in vitro embryo production of young Nellore bulls could be affected after dietary supplementation with palm and soy oils protected with calcium salts. A review was carried out in order to answer the main questions of dietary supplementation with protected fat. In addition, two scientific papers were written aiming a better understanding of the results regarding dietary supplementation with protected fat in bulls. In the first chapter, the objective was to evaluate fresh semen quality and testicular histology of forty-eight young Nellore breeders. Therefore, the animals were randomly divided in four groups which were fed with the following supplements: Control (CO, corn and distillery grains supplement without the addition of protected fat); Palm oil (OP, control supplement + 285 g of protected palm fat); Soy oil (OS, control supplement + 285g of protected soy fat) or combination (OP + OS, control supplement + 145 g of protected soy fat + 145 g of protected palm). The supplementation lasted 84 days, and at the end this period semen collection and evaluation, as well as testicular sampling for histological analysis were performed. Scrotal circumference (SC), testicular consistency of the left testis (LTCONS), testicular volume on the left side (LTV), testicular consistency of the right testicle (RTCONS) and testicular volume of the right testicle (RTV) were assessed. Additionally, fresh semen swirling (SWIR), motility (MOT), vigor (VIG), concentration (CONC), seminal volume (VOL), mitochondrial cytochemical activity (DAB) and functional integrity of the plasma membrane (HIPO) were also evaluated. Histological analysis was performed by determining the number of seminiferous tubules (NTF), tubular surface density (DST) and germ cells morphological characteristics. Thus, for the latter: cell area (CAR), perimeter (CPT), circularity (CCIR), minimum diameter (CMiD) and maximum diameter (CMaD); and nucleus area (NAR), perimeter (NPT), circularity (NCIR), minimum diameter (NMiD), maximum diameter (NMaD), and nucleus/cell ratio (NC) were determined. The second chapter aimed to evaluate the sperm fertility of post-thawed semen on in vitro embryo production, and to determine if there was any correlation between this variable and changes in the sperm lipid profile. Therefore, post-thawed sperm movement characteristics were assessed by computer assisted sperm analysis (CASA). Sperm mitochondrial cytochemical activity and induced oxidative stress (induced TBARS) were also analyzed. Flow cytometry with the aid of fluorescent probes determined integrity of the plasma and acrosomal membranes (FITC-PI), mitochondrial membrane potential (JC-1), the amount of intracellular reactive oxygen species (CellROX) and DNA sperm denaturation through chromatin stability (LA). Sperm lipid profile (LIP) was performed and sperm was tested for fertility by in vitro embryo production (IVP). Normality and homogeneity of the variables were verified and, when necessary, changes were applied. Data were subjected to analysis of variance (ANOVA) and compared by the Tukey mean test and Wilcoxon test for parametric and non-parametric variables, respectivelly. Orthogonal contrasts were applied (FC 1.5, without using FDR- false discovery rate) for lipidomic analysis. Level of significance was set at 5%, using SAS statistical program (version 9.3). Fresh semen evaluations showed lower percentage of sperm with less than half of the active mitochondria - DAB 3 (P = 0.0267) in animals supplemented with palm and soy oils (OP + OS). In addition, this finding affected directly the percentage of sperm with total (MOT) and progressive (PROG) motilities. Additionally, animals receiving palm and/or soy oils supplements showed greater scrotal circumference compared to the ones that were not supplemented. Nevertheless, animals fed with an enriched palm oil diet presented a greater right testicle volume (P = 0.0109) and a tendency of a smaller left testicle volume (P = 0.0541). The statistical difference found for the variable DAB 3 on OP group compared to the others experimental groups (P = 0.0290) is a reflection of an increased percentage of DAB 1 sperm in animals that consumed palm oil. In the post-thaw semen, supplementation with palm and/or soybean oils decreased the amount of sperm with reduced mitochondrial activity (DAB 3) and improved the percentage of motile sperm in young Nellore bulls. Sperm lipid profile evaluation showed a decrease of 79 lipids (membrane and storage) in all groups supplemented with protected fat comparing days 0 86 of the experimental period. As for the sperm fertility tested by IVP, palm oil supplementation led to a higher percentage of blastocysts (P = 0.0018). In conclusion, the use of 285g of palm oil (protected with calcium salts) in the animal feed could be used as a supplement alternative to improve reproductive rates of young Nellore bulls.

Exposure to 50 Hz electromagnetic field changes the efficiency of the scorpion alpha toxin

Background Extremely low-frequency (50 Hz) electromagnetic field (ELF-EMF) is produced by electric power transmission lines and electronic devices of everyday use. Some phenomena are proposed as first effects of ELF-EMF: the discrete changes in the membrane potential and the increase of the calcium channel activity as well as the intracellular concentration of Ca 2+ . Interaction of the scorpion alpha toxin with the sodium channel depends on the orientation of the charges and may be perturbed by changes in the membrane polarization. The toxin induces overexcitability in the nervous system and an increase in the neurotransmitters released with different consequences, mainly the paralysis of muscles. We assumed that the exposure to ELF-EMF 0.7 mT will change the effects of the insect selective scorpion alpha toxin (recombinant LqhIT from Leiurus quinquestriatus hebraeus) at the level of the cercal nerve function, the synaptic transmission and on the level of entire insect organism. Taking into account the compensatory mechanisms in organisms, we tested in addition ten times higher ELF-EMF on whole insects.Methods Experiments were performed in vivo on cockroaches (Periplaneta americana) and in vitro on isolated cockroach abdominal nerve cord with cerci. In biotests, the effects of LqhIT (10 8 M) were estimated on the basis of the insect ability to turn back from dorsal to ventral side. Three groups were compared: the control one and the two exposed to ELF-EMF 0.7 and 7 mT. Bioelectrical activity of the cercal nerve and of the connective nerve that leaves the terminal abdominal ganglion was recorded using extracellular electrodes. LqhIT (5 × 10 8 M) induced modifications of neuronal activity that were observed in the control cockroach preparations and in the ones exposed to ELF-EMF (0.7 mT). The exposure to ELF-EMF was carried out using coils with a size appropriate to the examined objects.Results The exposure to ELF-EMF (0.7 mT) modified the effects of LqhIT (5 × 108 M) on activity of the cercal nerve and of the connective nerve. We observed a decrease of the toxin effect on the cercal nerve activity, but the toxic effect of LqhIT on the connective nerve was increased. Biotests showed that toxicity of LqhIT (10 8 M) on cockroaches was reduced by the exposure to ELF-EMF (0.7 and 7 mT).Conclusions The exposure to 50 Hz ELF-EMF modified the mode of action of the anti-insect scorpion alpha toxin LqhIT at cellular level of the cockroach nervous system and in biotests. Toxin appeared as a usefull tool in distinguishing between the primary and the secondary effects of ELF-EMF.(AU)

Exposure to 50 Hz electromagnetic field changes the efficiency of the scorpion alpha toxin

Background Extremely low-frequency (50 Hz) electromagnetic field (ELF-EMF) is produced by electric power transmission lines and electronic devices of everyday use. Some phenomena are proposed as first effects of ELF-EMF: the discrete changes in the membrane potential and the increase of the calcium channel activity as well as the intracellular concentration of Ca 2+ . Interaction of the scorpion alpha toxin with the sodium channel depends on the orientation of the charges and may be perturbed by changes in the membrane polarization. The toxin induces overexcitability in the nervous system and an increase in the neurotransmitters released with different consequences, mainly the paralysis of muscles. We assumed that the exposure to ELF-EMF 0.7 mT will change the effects of the insect selective scorpion alpha toxin (recombinant LqhIT from Leiurus quinquestriatus hebraeus) at the level of the cercal nerve function, the synaptic transmission and on the level of entire insect organism. Taking into account the compensatory mechanisms in organisms, we tested in addition ten times higher ELF-EMF on whole insects.Methods Experiments were performed in vivo on cockroaches (Periplaneta americana) and in vitro on isolated cockroach abdominal nerve cord with cerci. In biotests, the effects of LqhIT (10 8 M) were estimated on the basis of the insect ability to turn back from dorsal to ventral side. Three groups were compared: the control one and the two exposed to ELF-EMF 0.7 and 7 mT. Bioelectrical activity of the cercal nerve and of the connective nerve that leaves the terminal abdominal ganglion was recorded using extracellular electrodes. LqhIT (5 × 10 8 M) induced modifications of neuronal activity that were observed in the control cockroach preparations and in the ones exposed to ELF-EMF (0.7 mT). The exposure to ELF-EMF was carried out using coils with a size appropriate to the examined objects.Results The exposure to ELF-EMF (0.7 mT) modified the effects of LqhIT (5 × 108 M) on activity of the cercal nerve and of the connective nerve. We observed a decrease of the toxin effect on the cercal nerve activity, but the toxic effect of LqhIT on the connective nerve was increased. Biotests showed that toxicity of LqhIT (10 8 M) on cockroaches was reduced by the exposure to ELF-EMF (0.7 and 7 mT).Conclusions The exposure to 50 Hz ELF-EMF modified the mode of action of the anti-insect scorpion alpha toxin LqhIT at cellular level of the cockroach nervous system and in biotests. Toxin appeared as a usefull tool in distinguishing between the primary and the secondary effects of ELF-EMF.

Erliquiose canina revisão de literatura / Ehrlichiosis canine - literature review

A Erliquiose é uma das principais doenças infecto-contagiosas, causada por um hemoparasita da ordem Rickettsiales e do gênero Ehrlichia spp., são parasitas intracelulares obrigatórios de células hematopoiéticas maduras ou imaturas. A principal espécie que acomete os cães é a Ehrlichia canis. Sua transmissão pode ocorrer pela participação de um vetor, o carrapato Rhipicephalus sanguineus, ou por transfusão sanguínea. Os cães infectados com E. canis podem desenvolver sinais brandos a intensos ou mesmo não apresentar sinais, dependendo da fase da doença em que se encontram. O diagnóstico clínico geralmente não é o suficiente para confirmação da doença, devido aos sinais clínicos inespecíficos, portanto há a necessidade de diagnóstico complementar. Apesar de ser uma doença que pode ser bem severa, o tratamento é simples, e consiste na administração de antibióticos; sendo a doxiciclina o antibiótico de escolha. Sua incidência vem aumentando significativamente nos últimos anos, em todas as regiões do Brasil, por isso a necessidade de maior estudo e entendimento sobre esta enfermidade. (AU)

Ehrlichiosis is one of the major infectious diseases, caused by a hemoparasite of Rickettsiales order and genus Ehrlichia spp., Are obligate intracellular parasites of immature and mature hematopoietic cells. The main species that affects dogs is Ehrlichia canis. Transmission can occur through the participation of a vector, the tick Rhipicephalus sanguineus, or by blood transfusion. Infected with E. canis dogs may develop mild to intense signs or no signs, depending on the stage of the disease in which they are. Clinical diagnosis is usually not enough to confirm the disease, due to nonspecific clinical signs, so there is the need for additional diagnostic. Although a disease which can be very severe, treatment is simple and consists in the administration of antibiotics; Doxycycline is the antibiotic of choice. Its incidence has increased during the recent years in all regions of Brazil, so the need for more study and understanding of this disease. (AU)

Erliquiose canina – revisão de literatura / Ehrlichiosis canine - literature review

A Erliquiose é uma das principais doenças infecto-contagiosas, causada por um hemoparasita da ordem Rickettsiales e do gênero Ehrlichia spp., são parasitas intracelulares obrigatórios de células hematopoiéticas maduras ou imaturas. A principal espécie que acomete os cães é a Ehrlichia canis. Sua transmissão pode ocorrer pela participação de um vetor, o carrapato Rhipicephalus sanguineus, ou por transfusão sanguínea. Os cães infectados com E. canis podem desenvolver sinais brandos a intensos ou mesmo não apresentar sinais, dependendo da fase da doença em que se encontram. O diagnóstico clínico geralmente não é o suficiente para confirmação da doença, devido aos sinais clínicos inespecíficos, portanto há a necessidade de diagnóstico complementar. Apesar de ser uma doença que pode ser bem severa, o tratamento é simples, e consiste na administração de antibióticos; sendo a doxiciclina o antibiótico de escolha. Sua incidência vem aumentando significativamente nos últimos anos, em todas as regiões do Brasil, por isso a necessidade de maior estudo e entendimento sobre esta enfermidade.

Ehrlichiosis is one of the major infectious diseases, caused by a hemoparasite of Rickettsiales order and genus Ehrlichia spp., Are obligate intracellular parasites of immature and mature hematopoietic cells. The main species that affects dogs is Ehrlichia canis. Transmission can occur through the participation of a vector, the tick Rhipicephalus sanguineus, or by blood transfusion. Infected with E. canis dogs may develop mild to intense signs or no signs, depending on the stage of the disease in which they are. Clinical diagnosis is usually not enough to confirm the disease, due to nonspecific clinical signs, so there is the need for additional diagnostic. Although a disease which can be very severe, treatment is simple and consists in the administration of antibiotics; Doxycycline is the antibiotic of choice. Its incidence has increased during the recent years in all regions of Brazil, so the need for more study and understanding of this disease.

ÔMEGA-CONOTOXINA MVIIC E/OU DANTROLENE NO TRAUMA MEDULAR AGUDO DE RATOS

O trauma medular agudo (TMA) ocasiona danos aos tecidos neuronais por mecanismos primários e secundários. A hiperconcentração intracelular de íons cálcio é um elemento chave no desenvolvimento da lesão secundária e ocorre por diversos mecanismos, dentre eles, influxo de cálcio através dos canais de cálcio voltagem dependentes presentes na membrana celular e por mobilização de reservas intracelulares localizadas no retículo endoplasmático. O objetivo desse estudo foi avaliar o efeito neuroprotetor da associação de bloqueadores de canais de cálcio, a -conotoxina MVIIC e o dantrolene, em ratos submetidos ao TMA. Vinte ratos machos adultos, linhagem Wistar, foram divididos aleatoriamente em cinco grupos: CN (controle negativo), CP (controle positivo), T (-conotoxina MVIIC), D (dantrolene) e T+D (-conotoxina MVIIC + dantrolene). Os animais do grupo CN foram submetidos à laminectomia em T12 e injeção de PBS por via intratecal (i.t.). Os demais grupos foram submetidos à laminectomia em T12 e trauma medular compressivo com a queda da haste de 10 gramas a uma altura de 25 mm no sistema indutor de trauma espinhal MASCIS Impactor. O grupo CP recebeu injeção de PBS por via i.t., o grupo T recebeu 20 pmol/10L de -conotoxina MVIIC por via i.t., o grupo D recebeu 10 mg/kg de dantrolene por via intraperitoneal (i.p) e o grupo T+D recebeu 20 pmol/10L de -conotoxina MVIIC por via i.t. e 10 mg/kg de dantrolene por via i.p. Os animais foram submetidos à avaliação clínica durante os sete dias subsequentes ao trauma, para observação da função motora em campo aberto e classificação conforme escala proposta por Basso, Beatie e Bresnahan, 1995. Oito dias após o tratamento, os animais foram eutanasiados e foram coletadas amostras de sangue para avaliações hematológica e bioquímica plasmática, e de segmentos da medula espinhal para quantificação de espécies reativas de oxigênio, peroxidação lipídica e avaliação da expressão gênica de fatores relacionados à apoptose (Bax, caspase 3, 8 e 9). Observou-se que apenas o grupo D mostrou recuperação motora significativa após os sete dias de avaliação pelo teste BBB (p<0,05). Os valores dos perfis hematológico e bioquímico se mantiveram dentro dos limites fisiológicos em todos os grupos, com exceção dos valores de ureia que se apresentaram mais elevados. Não houve diferença significativa entre os grupos nas análises de espécies reativas de oxigênio, peroxidação lipídica e expressão gênica de fatores relacionados à apoptose. Conclui-se que o dantrolene promoveu recuperação motora e sua associação com a MVIIC não apresentou efeito sinérgico no tratamento do TMA

Acute spinal cord injury causes damage to the nervous tissue by primary and secondary mechanisms. Excessive intracellular calcium concentration is a key element to the development of secondary injury. It occurs by several mechanisms: influx of calcium through membrane voltage-gated calcium channels and mobilization of intracellular reserves from the endoplasmic reticulum. The purpose of this study was to evaluate the neuroprotective effect of the association of calcium channel blockers, -conotoxin MVIIC and dantrolene, on experimental acute spinal cord injury. Twenty male Wistar rats were randomly distributed into five groups: CN (negative control), CP (positive control), T (-conotoxina MVIIC), D (dantrolene) and T+D (-conotoxina MVIIC + dantrolene). Animals of the CN group underwent T12 laminectomy and intrathecal PBS injection. The other groups underwent T12 laminectomy and compressive spinal trauma due to a 10 gram stem drop from the height of 25 mm at the MASCIS Impactor spinal trauma induction system. The CP group received intrathecal PBS injection, the T group received intrathecal 20 pmol/10 l of -conotoxin MVIIC, group D received 10 mg/kg dantrolene intraperitoneally and the T + D group received 20 pmol/10 L of -conotoxin MVIIC intrathecally and 10 mg/kg dantrolene intraperitoneally. Neurological examination was performed daily for seven days and consisted with Basso, Beatie, Bresnahan test for the evaluation of motor function. Eight days after treatment, the animals were euthanized and the blood samples were collected for hematological and biochemistry analysis, and spinal cords samples were colleted for quantification of reactive oxygen species, lipid peroxidation and the evaluation of the gene expression of factors related to apoptosis (Bax, caspase 3, 8 and 9). It was observed that only group D improved motor function with significant difference after seven days evaluation by BBB test (p<0,05). Values for hematological and biochemistry profiles were within the reference intervals in all groups, with the exception of urea values that were higher in all animals. There was no statistical difference between the values of quantification of reactive oxygen species, lipid peroxidation and gene expression of related factors to apoptosis. It was conclude that dantrolene promoted motor recovery. There was not effect of pharmacological addition on the association MVIIC and dantrolene.

EFFECTS OF BOVINE SERUM ALBUMIN (BSA) AND BICARBONATE ON THE ACHIEVEMENT OF IN VITRO CAPACITATION AND SUBSEQUENT PROGESTERONEINDUCED ACROSOME REACTION IN BOAR SPERMATOZOA

Objetivou-se avaliar os efeitos de diferentes concentrações de bicarbonato e albumina sérica bovina (BSA) adicionados ao meio de capacitação in vitro sobre a indução da reação acrossômica no sêmen suíno. Oito diferentes tratamentos com diferentes concentrações de bicarbonato (0, 5, 15 e 38 mM) e BSA (0 e 5 mg/ml) foram testados: 1) controle negativo (No BSA / No Bic), o meio básico sem bicarbonato ou BSA; 2) meio básico suplementado apenas com 5 mg / mL de BSA (BSA / No Bic); 3) meio básico suplementado com 5 mM de NaHCO3 e 5 mg / mL de BSA (BSA + 5 mM Bic); 4) meio básico suplementado apenas com 5 mM de NaHCO3 (sem BSA + 5 mM Bic); 5) meio básico suplementado com 5 mM de NaHCO3 e 5 mg / mL de BSA (BSA + 15 mM Bic); 6) meio básico suplementado apenas com 15 mM de NaHCO3 (sem BSA + 15 mM Bic); 7) meio básico suplementado com NaHCO3 38 mM e 5 mg / mL de BSA (BSA + Bic 38 mM); e 8) meio básico suplementado apenas com NaHCO3 38 mM (sem BSA + Bic 38 mM). As células espermáticas foram submetidas aos meios e incubadas a 38,5 C com 5% de CO2 por 5h. A motilidade espermática, integridade de membrana, distúrbio lipídico de membrana, níveis de cálcio intracelular, atividade mitocondrial e níveis de fosforilação de tirosina de GSK3 e DARPP32 foram determinados após 0, 2 e 4 h de incubação e também após 30 e 60 min da adição de progesterona. Os meios sem BSA não se capacitaram, obtendo resultados semelhantes ao meio não capacitante (sem BSA e sem bicarbonato) (P<0,05). Os meios com BSA e bicarbonato a 15 ou 38 mM apresentaram (P<0,05) altos níveis de cálcio (Fluo-3 e Rhod 5), desordem lipídica (M540) e fosforilação de proteínas (GSK3). O meio com BSA e bicarbonato a 15 mM apresentou (P<0,05) maior motilidade e viabilidade que o meio com BSA e bicarbonato a 38 mM. O meio com BSA e sem bicarbonato ou com bicarbonato a 5 mM se capacitaram, porém com menores indices de capacitação que os meios que contiam BSA e bicarbonato a 15 ou 38 mM. Os resultados mostraram que a capacitação in vitro de sêmen suíno está relacionada com a presença de BSA no meio capacitante e a presença do bicarbonato não é um fator limitante para a capacitação. O meio que obteve melhor resultado de capacitação foi o BSA com bicarbonato a 15 mM.

The objective of the present work was to evaluate the effects of different concentrations of bicarbonate and bovine serum albumin (BSA), added to the in vitro capacitation medium, on the induction of the acrosome reaction in boar semen. Eight different treatments with different concentrations of bicarbonate (0, 5, 15 and 38 mM) and BSA (0 and 5 mg / ml) were tested: 1) negative control (No BSA / No Bic), which was the basic medium without bicarbonate or BSA; 2) basic medium supplemented with 5 mg/mL BSA only (BSA / No Bic); 3) basic medium supplemented with 5 mM NaHCO3 and 5 mg/mL BSA (BSA + 5 mM Bic); 4) basic medium supplemented with 5 mM NaHCO3 only (No BSA + 5 mM Bic); 5) basic medium supplemented with 5 mM NaHCO3 and 5 mg/mL BSA (BSA + 15 mM Bic); 6) basic medium supplemented with 15 mM NaHCO3 only (No BSA + 15 mM Bic); 7) basic medium supplemented with 38 mM NaHCO3 and 5 mg/mL BSA (BSA + 38 mM Bic); and 8) basic medium supplemented with 38 mM NaHCO3 only (No BSA + 38 mM Bic). Sperm cells were submitted to the medium and incubated at 38.5 C with 5% of CO2 for 5 h. Sperm motility, membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial activity and tyrosine phosphorylation levels of GSK3 and DARPP32 were determined after 0, 2 and 4 h of incubation and after 30 and 60 min of the addition of progesterone. The medium without BSA did not capacitated, generating similar results to the non-capacitive medium (without BSA and without bicarbonate) (P <0.05). Mediums with BSA and bicarbonate at 15 or 38 mM showed high (P <0.05) levels of calcium (Fluo-3 and Rhod 5), lipid disorder (M540) and protein phosphorylation (GSK3). The medium with BSA and 15 mM bicarbonate presented (P <0.05) greater motility and viability than the medium with BSA and bicarbonate at 38 mM. The medium with BSA and without bicarbonate or with 5 mM bicarbonate, were capacitated, however with lower indices than medium containing BSA and bicarbonate at 15 or 38 mM. The analyzes showed that the in vitro capacitation of boar semen is related to the presence of BSA in the capacitive medium and the presence of bicarbonate is not a limiting factor for the capacitation. The medium that obtained the best qualification result was BSA with bicarbonate at 15 mM.

ANFOTERICINA B CONVENCIONAL ASSOCIADA AO ÁCIDO GÁLICO E AO ÁCIDO ELÁGICO EM FORMULAÇÃO TÓPICA PARA TERAPIA ANTILEISHMANIA

As leishmanioses são zoonoses parasitárias causadas por protozoários do gênero Leishmania, pertencentes ao grupo de doenças tropicais negligenciadas. São de difícil controle, não há vacina eficaz e o seu tratamento é baseado em um número limitado de drogas que apresentam uma gama de efeitos indesejáveis, além de já existirem relatos de resistência à essas drogas. O desenvolvimento de novos medicamentos demanda tempo e grandes investimentos financeiros e, tendo em vista essa limitação, a associação farmacológica de fármacos convencionais e a introdução de novas moléculas e tecnologias no desenvolvimento de novas formulações tem despertado interesse de pesquisadores. Neste estudo, demonstramos o efeito de associação da anfotericina B (Anf B) convencional e ácido gálico (AG) ou ácido elágico (AE) em formulação tópica de Gel de Polaxamer 407®. O AG e o AE são dois imunomoduladores naturais anteriormente estudados por nosso grupo e já reportamos sua ação antileishmania e potencial em modular a resposta imune. Foram realizados testes de estabilidade preliminar e de liberação in vitro dos fármacos nas formulações de Anf B, AG, Anf B + AG, AE e Anf B + AE na composição e tratamento in vivo de camundongos BALB/c infectados por L. major. Passados 40 dias da infecção, os animais foram divididos em 6 grupos, tratados 2x/dia, durante 21 dias com os géis de Anf B, AG, Anf B + AG, AE e Anf B + AE e o grupo controle negativo tratado com o veículo (polaxamer 407®). Os animais foram avaliados clinicamente mensurando-se o tamanho das lesões 1x/semana. Ao termino do tratamento e 14 dias após, os animais foram eutanasiados para avaliação ex vivo quanto à redução da carga parasitária no local da lesão e ativação de resposta imune celular, por meio da retirada de macrófagos peritoneais para avaliar sua ativação por capacidade fagocítica, atividade lisossomal, quantificação de nitrito e cálcio intracelular. Também foi realizado a infecção in vitro dos macrófagos peritoneais dos animais tratados com as formulações para avaliar o índice de sobrevivência de amastigotas internalizadas e o percentual de macrófagos infectados. Todas as formulações apresentaram-se estáveis no tempo T0 nos testes preliminares, sendo a formulação de AG a que houve maior liberação in vitro, seguido de AE e das formulações combinadas de Anf B + AG e de Anf B + AE. Houve efeito da associação entre Anf B e AG e Anf B e AE em todos os ensaios realizados. Nos animais que receberam tratamento houve redução do tamanho da lesão e redução da carga parasitária. O tratamento com as formulações contendo AG e AE foram capazes de ativar macrófagos em todos os parâmetros avaliados, bem como reduziram a quantidade de macrófagos infectados e o número de amastigotas por macrófago, indicando, portanto, ação terapêutica e ativação da resposta imune, levando a cura e proteção. O AG e AE produziram efeito de associação com a Anf B, o que torna promissor no tratamento da leishmaniose.

Leishmaniasis are parasitic zoonoses caused by protozoa of the genus Leishmania belonging to the group of neglected tropical diseases. It is difficult to control, there is no effective vaccine and its treatment is based on a limited number of drugs that have a range of undesirable effects, and there are already reports of resistance to these drugs. The development of new drugs requires time and large financial investments, in view of this limitation, the pharmacological association of conventional drugs and the introduction of new molecules and technologies in the development of new formulations has aroused interest of researchers. In this study we demonstrated the synergistic effect of the combination of conventional amphotericin B (Anf B) and natural molecules, gallic acid (GA) and ellagic acid (EA) in topical formulation of Polaxamer 407® Gel. GA and AE are two natural immunomodulators previously studied by our group and have already reported their antileishmania action and potential in modulating the immune response. Preliminary stability tests and in vitro release of the drugs were formulated in the formulations of Anf B, GA, Anf B + GA, EA and Anf B + EA in the composition and in vivo treatment of L. major infected BALB/c mice. After 40 days of infection, the animals were divided into 6 groups, treated 2x/day for 21 days with the gels of Anf B, GA, Anf B + GA, EA and Anf B + EA and the negative control group treated with vehicle (polaxamer 407®). The animals were evaluated clinically by measuring the lesion size 1x/week. At the end of the treatment and 14 days later, the animals were euthanized for evaluation of the ex vivo assays for the reduction of the parasite load at the lesion site and activation of the cellular immune response, by the removal of peritoneal macrophages to evaluate its activation by phagocytic capacity , lysosomal activity, quantification of nitrite and intracellular calcium. In vitro infection of the peritoneal macrophages was also performed to evaluate the survival rate of internalized amastigotes and the percentage of infected macrophages. All formulations were stable at time T0 in the preliminary tests, the GA formulation being the most liberated in vitro, followed by AE and the combined formulations of Anf B + GA and Anf B + EA. There was synergism between Anf B and GA and Anf B and EA in all the tests performed. In the animals that received treatment there was reduction of the size of the lesion and reduction of the parasitic load. The formulations containing GA and EA activated macrophages in all evaluated parameters, as well as reduced the amount of infected macrophages and the number of amastigotes by macrophage, indicating, therefore, therapeutic action and activation of the immune response, leading to healing and protection. GA and EA produced a synergistic effect with Anf B, which makes this association promising in the treatment of leishmaniasis.

Pimobendan improves clinical signs in short term compared to digoxin or placebo in dogs with heart failure due to chronic degenerative mitral valve disease

Background: Chronic degenerative mitral valve disease (CDMVD) continues to be the most common cause of heart failure (HF) in small breed dogs. Pimobendan (PIMO) is a mixed action drug with inotropic and vasodilator properties and is widely used to treat heart disease in dogs. Therefore, PIMO increases cardiac output, reduces both preload and afterload and increases myocardial contractility without increasing energy consumption and myocardial oxygen. Digoxin (DIG) is a cardiac glycoside acting through inhibition of the sarcolemmal Na+/K+ ATPase pump, hence increasing intracellular calcium. It exerts beneficial effects on left ventricular function, symptoms and exercise tolerance. The purpose of this prospective, randomized, double blind clinical trial was to evaluate the clinical response and QoLQ in heart failure (HF) dogs treated with digoxin or pimobendan in addition to conventional therapy (furosemide and benazepril).Materials, Methods & Results: Inclusion criteria: dogs in class III or stabilized class IV (NYHA). Exclusion criteria: use of positive inotrope and antiarrhythmic, presence of atrial fibrillation, renal or hepatic disease or neoplasia. Thirty three dogs were included and randomly assigned to DIG (n = 11), PIMO (n = 14) and placebo (PL) (n = 8) and followed up weekly. Data was evaluated for days zero, 7, 14 and 28. Increasing score was assigned to...(AU)

Pimobendan improves clinical signs in short term compared to digoxin or placebo in dogs with heart failure due to chronic degenerative mitral valve disease

Background: Chronic degenerative mitral valve disease (CDMVD) continues to be the most common cause of heart failure (HF) in small breed dogs. Pimobendan (PIMO) is a mixed action drug with inotropic and vasodilator properties and is widely used to treat heart disease in dogs. Therefore, PIMO increases cardiac output, reduces both preload and afterload and increases myocardial contractility without increasing energy consumption and myocardial oxygen. Digoxin (DIG) is a cardiac glycoside acting through inhibition of the sarcolemmal Na+/K+ ATPase pump, hence increasing intracellular calcium. It exerts beneficial effects on left ventricular function, symptoms and exercise tolerance. The purpose of this prospective, randomized, double blind clinical trial was to evaluate the clinical response and QoLQ in heart failure (HF) dogs treated with digoxin or pimobendan in addition to conventional therapy (furosemide and benazepril).Materials, Methods & Results: Inclusion criteria: dogs in class III or stabilized class IV (NYHA). Exclusion criteria: use of positive inotrope and antiarrhythmic, presence of atrial fibrillation, renal or hepatic disease or neoplasia. Thirty three dogs were included and randomly assigned to DIG (n = 11), PIMO (n = 14) and placebo (PL) (n = 8) and followed up weekly. Data was evaluated for days zero, 7, 14 and 28. Increasing score was assigned to...