RESUMEN
Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.
RESUMEN
Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences.
Asunto(s)
Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Muramidasa/química , Fenilglioxal/química , Adsorción , Interacciones Hidrofóbicas e Hidrofílicas , Químicos de LaboratorioRESUMEN
Non-specific mechanisms involving ATP-binding cassette drug efflux transporters may play an important role in xenobiotic clearance in ovine gastro-intestinal nematodes. By using transporter inhibitors, the aim of this trial was to assess the possibility of increasing drug bioavailability in the host in an attempt to improve treatment efficacy. Thirty-six lambs were infected with 5000 multiple-drug resistant Haemonchus contortus third stage larvae and separated into six groups (n=6): ivermectin alone (IVM; 0.2 mg/kg body-weight, BW), ketoconazole alone (KET; 10 mg/kg BW), Pluronic 85 alone (P85; 4 mg/kg BW), IVM+KET, IVM+P85 or untreated control. Ivermectin was administered once on day 28 post-infection for all appropriate groups, whereas KET and P85 were administered as five separate doses on day 26-30 post-infection inclusive. The resultant data showed that concomitant administration of KET or P85 with IVM induced increases in plasma and tissue concentrations of IVM in treated animals, resulting in a two-fold increase in the area under the time-concentration curve (p<0.05). Faecal egg counts and worm burdens of the IVM+KET and IVM+P85 groups were lower than in the untreated, KET and P85 alone control animals. Worm burdens were reduced by between 16% and 51% with IVM+KET and IVM+P85 respectively compared to untreated control animals. The co-administration of P85 with IVM increased the efficacy by 34%, compared with IVM alone, in terms of worm count reduction of the multi-resistant isolate of H. contortus.
Asunto(s)
Hemoncosis/veterinaria , Haemonchus/efectos de los fármacos , Ivermectina/farmacocinética , Ivermectina/uso terapéutico , Cetoconazol/farmacocinética , Poloxaleno/farmacocinética , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Abomaso , Animales , Antihelmínticos/farmacocinética , Antihelmínticos/uso terapéutico , Área Bajo la Curva , Interacciones Farmacológicas , Heces/parasitología , Femenino , Hemoncosis/tratamiento farmacológico , Masculino , Recuento de Huevos de Parásitos , Ovinos , Enfermedades de las Ovejas/tratamiento farmacológico , Enfermedades de las Ovejas/parasitología , Distribución TisularRESUMEN
P-glycoprotein (P-gp) homologues, belonging to the ATP Binding Cassette (ABC) transporter family, are thought to play an important role in the resistance of gastro-intestinal nematode parasites against macrocyclic lactones. The aim of this study was to investigate the influence of various P-gp interfering compounds on the efficacy of ivermectin (IVM) in sensitive and resistant nematode isolates. The feeding of IVM resistant and sensitive Teladorsagia circumcincta and Haemonchus contortus first-stage larvae (L1) was assessed using a range of IVM concentrations (0.08-40 nm) with or without P-gp inhibitors: valspodar, verapamil, quercetin, ketoconazole and pluronic P85. The P-gp inhibitors were selected on the basis of their ability to interfere with P-gp transport activity in an epithelial cell line over-expressing murine P-gp. In the presence of P-gp interfering agents, the in vitro susceptibility to IVM of both sensitive and resistant isolates of T. circumcincta and H. contortus was increased. These results show that compounds interfering with P-gp transport activity could enhance IVM efficacy in sensitive isolates, and also restore IVM sensitivity in resistant nematodes. These results support the view that ABC transporters can play an important role in resistance to IVM, at least in the free-living stages of these economically important gastro-intestinal nematodes.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antihelmínticos/farmacología , Resistencia a Medicamentos , Ivermectina/farmacología , Trichostrongyloidea/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Larva/efectos de los fármacosRESUMEN
Like most drugs, macrocyclic lactone endectocides (MLs) exert their antiparasitic effects within the defined target tissues where parasites are located, and whose drug concentrations correlate with those in the plasma compartment. The process of drug distribution to the active site constitutes the link in the pharmacokinetic/pharmacodynamic relationship. In the past few years it has become evident that transporter proteins play a major role in regulating the distribution, elimination and metabolism of the antiparasitic macrocyclic lactones. The efflux transporter P-glycoprotein (P-gp) has received the most attention with regards to its strong interaction with ivermectin and other MLs. P-gp has been reported to be involved in restricting the absorption of these drugs, in enhancing their intestinal elimination, in the protection against their neurotoxicity and in the ML resistance mechanisms in parasites. This review focuses on the interaction of MLs with P-glycoprotein and with other multidrug resistance transporters. Given the structural and physicochemical diversity of these drugs, they constitute models of interest to study the major molecular determinants for the interaction with transporters. We will discuss the consequences of such interactions on ML pharmacokinetics and the possibility of benefiting from of drug/drug interaction to reverse multidrug resistance in several therapeutic fights such as against parasites and tumors.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Antihelmínticos/farmacología , Lactonas/farmacología , Compuestos Macrocíclicos/farmacología , Proteínas de Neoplasias/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Absorción , Animales , Resistencia a Múltiples Medicamentos , Humanos , Ivermectina/farmacología , Lípidos/química , Proteínas de Neoplasias/genéticaRESUMEN
The plasma kinetics disposition of moxidectin following a subcutaneous administration with a long-acting formulation (Cydectin) 10%, Fort Dodge Animal Health, France) at the recommended dose of 1 mg kg(-1) body weight was evaluated in Charolais cattle breed (five females weighing 425-450 kg) for 120 days. Furthermore, its concentration was measured in hair for the same period. After plasma extraction and derivatization, samples were analysed by HPLC with fluorescence detection. Moxidectin was first detected at 1 h after treatment for plasma (2.00+/-1.52 ng ml(-1)) and at 2 days for hair (446.44+/-193.26 ng g(-1)). The peak plasma concentration (C(max)) was 55.71+/-15.59 ng ml(-1) and 444.44+/-190.45 ng g(-1) for plasma and hair, respectively. The mean calculated time of peak occurrence (T(max)) was 3.40+/-3.36 and 2 days for plasma and hair, respectively. The mean residence time (MRT) was 28.93+/-2.87 and 13.32+/-2.48 days for plasma and hair cattle. The area under concentration-time curve (AUC) was 1278.95+/-228.92 ng day ml(-1) and 2663.82+/-1096.62 ng day g(-1) for plasma and hair, respectively. At the last sampling time (120 days), the concentration was 1.91+/-0.26 ng ml(-1) and 0.69+/-0.52 ng g(-1) for plasma and hair, respectively. The bioavailability of this long-acting formulation of moxidectin is similar to that registered after subcutaneous administration of moxidectin in cattle at 0.2 mg kg(-1) body weight. For the first time the moxidectin pharmacokinetics parameters in hair after a subcutaneous administration was described. The moxidectin profile concentrations in hair reflected that registered in plasma. The previous studies of efficacy have to be correlated to the extended period of absorption and distribution by the LA formulation due to the fivefold higher dose rate in comparison with the 1% injectable formulation (0.2 mg kg(-1) body weight).
Asunto(s)
Antihelmínticos/farmacocinética , Bovinos , Animales , Antihelmínticos/administración & dosificación , Antihelmínticos/sangre , Antihelmínticos/química , Preparaciones de Acción Retardada , Femenino , Cabello/química , Inyecciones Subcutáneas , Macrólidos/administración & dosificación , Macrólidos/sangre , Macrólidos/química , Macrólidos/farmacocinéticaRESUMEN
We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is co-purified with paraoxonase (PON1). The association between HPON1 and HPBP is modulated by phosphate and calcium concentrations. The HPBP X-ray structure solved at 1.9 A resolution is similar to the prokaryotic phosphate solute-binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation of genes between evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus it is thought to become a new predictor and a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.
Asunto(s)
Apolipoproteínas/química , Secuencia de Aminoácidos , Animales , Arildialquilfosfatasa/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatos/sangreRESUMEN
We have tested the ability of two compounds licensed in veterinary medicine: fumagillin and diminazene diaceturate to increase intracellular moxidectin quantity in rat hepatocytes. These compounds significantly increased the quantity of 14C-moxidectin (expressed as area under the time curve concentrations) in cultured rat hepatocytes by 44% and 65% for diminazene and fumagillin treatments respectively. In addition, we have tested these drugs for their interference with P-glycoprotein (P-gp) function in porcine kidney epithelial cells transfected with murine mdr1a (Mdr1a-LLCPK1). We examined the intracellular accumulation of rhodamine 123 (Rho 123) as a functional test to evaluate the effects of these two drugs on P-gp activity. In this model, only fumagillin led to a marked intracellular accumulation of Rho 123. After transforming the data to express the results as a percentage of the accumulation in the presence of the P-gp inhibitor valspodar (VSP), the maximal Rho 123 accumulation was 47% of that with VSP for 100 microm fumagillin. The EC50, the concentration needed to determine 50% of the maximal effect was 34 microm. Fumagillin interacts with P-gp function and appears as a promising compound among registered drugs available, which may optimize the therapeutic use of macrocyclic lactones (MLs).
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antihelmínticos/farmacocinética , Antibacterianos/farmacología , Ciclohexanos/farmacología , Ácidos Grasos Insaturados/farmacología , Hepatocitos/metabolismo , Animales , Área Bajo la Curva , Células Cultivadas , Interacciones Farmacológicas , Macrólidos/farmacocinética , Ratones , Ratas , Sesquiterpenos/farmacología , PorcinosRESUMEN
The interaction of moxidectin (a macrocyclic lactone, ML) with P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs) and breast cancer resistance protein (BCRP) was studied in primary cultures of rat hepatocytes by measuring the intracellular accumulation of [14C]-moxidectin over 72 h in the presence of specific inhibitors: for P-gp, verapamil (10 microM); for MRPs, MK571 (100 microM), indomethacin (10 microM) and probenecid (3.8 mM); and for BCRP, fumitremorgin C (5 microM). The P-gp and MRP inhibitors increased significantly (P < 0.01) by 48.7%, 49.8%, 49.9% and 57.2% the area under the time-intracellular concentration curve (AUC) of moxidectin in rat hepatocytes, while the BCRP inhibitor, fumitremorgin C, had no effect on the AUC compared with the control. In addition, the mRNAs of all the drug transporters studied were detected in rat hepatocytes from 0 to 72 h. Using this cellular model it has been shown that MRP inhibitors increase moxidectin intracellular concentrations to a similar extent as the P-gp inhibitor. The identification of all the transporters that interact with MLs remains a challenge, which currently concerns several important therapeutic fields.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antihelmínticos/farmacocinética , Hepatocitos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Área Bajo la Curva , Células Cultivadas , Macrólidos/farmacocinética , Masculino , Modelos Biológicos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Iron-regulatory proteins (IRPs) 1 and 2 are closely related molecules involved in animal iron metabolism. Both proteins can bind to specific mRNA regions called iron-responsive elements and thereby control the expression of proteins involved in the uptake, storage and utilization of iron. In iron-replete cells, IRP1, but not IRP2, binds a [4Fe-4S] cluster and functions as a cytoplasmic aconitase, with simultaneous loss of its RNA-binding ability. Whereas IRP2 is known to be involved in Fe homeostasis, the role of IRP1 is less clear; it may provide a link between citrate and iron metabolisms and be involved in oxidative stress response. Here, two crystal forms of the aconitase version of recombinant human IRP1 are reported. An X-ray fluorescence measurement performed on a gold-derivative crystal showed the unexpected presence of zinc, in addition to gold and iron. Both native and MAD X-ray data at the Au, Fe and Zn absorption edges have been collected from these crystals.
Asunto(s)
Aconitato Hidratasa/química , Proteína 1 Reguladora de Hierro/química , Aconitato Hidratasa/metabolismo , Cristalización , Humanos , Proteína 1 Reguladora de Hierro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difracción de Rayos XRESUMEN
Some pharmacokinetic parameters of selamectin were determined in male (n = 5) and female (n = 5) Beagle dogs following a topical application at a dose rate of 6 mg/kg. The plasma concentration versus time data for the drug were analysed using a one-compartment model. The maximum plasma concentrations of 12.72 +/- 5.13 ng/ml for males and 22.65 +/- 11.95 ng/ml for females occurred around 5 days after administration. The area under the concentration-time curve (AUC) was 192.08 +/- 63.85 ng.day/ml for males and 370.97 +/- 146.87 ng.day/ml for females. The mean residence time was the same in males and females (12.55 days). This study reveals a sex-influence on the disposition of selamectin in the plasma of dogs, which implies that further information will be needed for correlation with efficacy studies in dogs.
Asunto(s)
Perros/sangre , Ivermectina/análogos & derivados , Ivermectina/farmacocinética , Administración Tópica , Animales , Antiparasitarios/administración & dosificación , Antiparasitarios/sangre , Antiparasitarios/farmacocinética , Femenino , Semivida , Ivermectina/administración & dosificación , Ivermectina/sangre , Masculino , Tasa de Depuración MetabólicaRESUMEN
White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin.
Asunto(s)
Antibacterianos/metabolismo , Bivalvos/química , Virus ADN/patogenicidad , Palaemonidae/virología , Temperatura , Secuencia de Aminoácidos , Animales , Acuicultura , Virus ADN/genética , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The yak (Bos grunniens) belongs to the cattle family Bovidae and lives in the mountains of China and adjacent areas. Due to the physiological adaptations of yak to its environment and the lack of data, the ivermectin pharmacokinetic was studied following a single subcutaneous dose at the recommended dose for cattle (0.2 mg kg(-1)). The observed peak plasma concentration (Cmax) was 48.93 ng ml(-1) and the time to reach Cmax (Tmax) was 0.73 day. These results show a faster rate of absorption than in cattle. The values for the absorption half-life (t(1/2a)), the distribution half-life (t(1/2alpha)) and the terminal half-life (t(1/2beta)) were 0.31, 0.74 and 4.82 days, respectively. The calculated area under the concentration-time curve (AUC) was 146.2 ng day ml(-1) and the mean residence time (MRT) was 3.57 days. The availability of ivermectin appears low in yaks in comparison to cattle but equivalent to that reported in horses and is likely to be due to physiological characteristics of this species.
Asunto(s)
Antihelmínticos/farmacocinética , Bovinos/metabolismo , Ivermectina/farmacocinética , Absorción , Animales , Antihelmínticos/sangre , Área Bajo la Curva , Enfermedades de los Bovinos/tratamiento farmacológico , Enfermedades de los Bovinos/parasitología , Femenino , Inyecciones Subcutáneas/veterinaria , Ivermectina/sangreRESUMEN
Moxidectin is an antiparasitic drug widely used in cattle, sheep and companion animals. Due to the involvement of P-glycoprotein (P-gp) and cytochrome P450 3A in the metabolism of moxidectin, we studied the influence of various P-gp interfering agents (ivermectin, quercetin and ketoconazole) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. This in vitro study allowed selection of compounds which are able to increase the moxidectin bioavailability in lambs. From this, the modulation of moxidectin pharmacokinetics in plasma of lambs was studied after co-administration of 0.2 mg kg(-1) moxidectin (subcutaneously (SC)) and 0.2 mg kg(-1) ivermectin (SC), or 10 mg kg(-1) quercetin (SC), or 10 mg kg(-1) ketoconazole (orally). Ivermectin and quercetin increased significantly the quantity of 14C moxidectin in the rat hepatocytes. Ketoconazole co-administration led to a higher concentration of moxidectin in the rat hepatocytes. In vivo, only quercetin was able to modify the pharmacokinetics of moxidectin in plasma of lambs by increasing significantly the area under the plasma concentration-time curve. This study allowed the use of a natural agent, quercetin, to improve the bioavailability of moxidectin.
Asunto(s)
Antihelmínticos/metabolismo , Antihelmínticos/farmacocinética , Antibacterianos/metabolismo , Antibacterianos/farmacocinética , Quercetina/farmacología , Animales , Antihelmínticos/administración & dosificación , Antihelmínticos/sangre , Antibacterianos/administración & dosificación , Antibacterianos/sangre , Antifúngicos/administración & dosificación , Antifúngicos/farmacología , Área Bajo la Curva , Disponibilidad Biológica , Células Cultivadas , Interacciones Farmacológicas , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Ivermectina/administración & dosificación , Ivermectina/farmacología , Cetoconazol/administración & dosificación , Cetoconazol/farmacología , Macrólidos , Masculino , Quercetina/administración & dosificación , Ratas , Ratas Sprague-Dawley , Oveja Doméstica/sangreRESUMEN
A complete general unknown screening procedure was developed using liquid chromatography-mass spectrometry (LC-MS), a coupling that can increase the range of compounds amenable to MS. Sample preparation was by solid-phase extraction on a mixed-mode support in parallel with serum deproteination in order to recover the most hydrophilic compounds. Chromatography employed a reversed-phase narrow-bore column (150 x 1-mm i.d.) and a 50-min gradient elution at low flow-rate (50 microL/min), compatible with the electrospray source used without splitting nor heating. The single quadrupole LC-MS instrument used was operated in the 100 to 1100 mu mass range in both the positive and negative modes, with two different, alternated collision-induced dissociation voltages in the source, in order to obtain the molecular or pseudo-molecular ions as well as fragments for the compounds analyzed. The addition of spectra obtained at low and high fragmentation voltages gave reconstructed spectra for each polarity, representing library entries. Finally, a program was created in order to detect the peaks of interest in the chromatographic noise using a very efficient signal processing algorithm, compute their relative retention time with respect to the internal standard (glafenine), draw their reconstructed spectra, search them in the libraries, and edit a report.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Tamizaje Masivo/métodos , Preparaciones Farmacéuticas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Xenobióticos/sangre , Humanos , Preparaciones Farmacéuticas/análisis , Xenobióticos/análisisRESUMEN
Eprinomectin is only available as a topically applied anthelmintic for dairy cattle. To determine whether eprinomectin can be administered in the goat as an injectable formulation, it was subcutaneously delivered to six goats and measured in the plasma at different times after administration. The area under the concentration-time curve (AUC) reported after subcutaneous administration of 0.2 mg kg(-1) eprinomectin (68.5+/-23.2 ng day(-1) ml(-1)) was similar to the AUC previously reported for goats after a pour-on administration of 0.5 mg kg(-1) eprinomectin. Thus, our results clearly show that subcutaneous administration is 2.5 times more effective than pour-on administration, in terms of amount of drug present in the organism. This work should encourage the development of a subcutaneous formulation of eprinomectin and should contribute to defining optimal therapeutic conditions for goat anthelmintic treatment.
Asunto(s)
Antihelmínticos/administración & dosificación , Cabras/metabolismo , Ivermectina/análogos & derivados , Ivermectina/administración & dosificación , Animales , Antihelmínticos/farmacocinética , Femenino , Inyecciones Subcutáneas/veterinaria , Ivermectina/farmacocinética , Concentración Osmolar , Factores de TiempoRESUMEN
Apolipoprotein E knockout (apoEKO) mice have been shown to be impaired in the spatial Morris water maze (MWM). However, several groups failed to replicate this finding. One reason for this inconsistency may stem from variations in the experimental protocols and environment between laboratories. In the present study, we have tested if age and variations in protocol implementation that specifically affect salience of the visual extramaze cues influence performance and navigational strategies in the MWM. We tested three- and 12-month-old apoEKO and wild type mice in three versions of the MWM differing on the availability of visual extramaze cues: (1) salient cues, (2) diffuse cues, and (3) absence of cues. Our results show that the presence of salient cues enhances acquisition performance of wild type, but not apoEKO mice in the MWM. This effect was restricted to the acquisition phase since apoEKO mice reached a level of performance that was comparable to that of controls toward the end of the task. No significant differences were detected between apoEKO and controls in either the diffuse cues or absence of cues paradigms. Thigmotaxic tendencies were observed in apoEKO mice and correlated high latency scores. Thigmotaxis may have interfered with the initial ability to engage in a proficient navigational strategy. These findings suggest that, in contrast to what has been proposed in the past, apoEKO mice appear not to be impaired in spatial memory per se but are deficient in a procedural component of the MWM. Furthermore, the procedural deficit and corresponding thigmotaxic tendencies of apoEKO mice appeared to increase with age. Taken together, these findings confirm our hypothesis that age and variations in experimental protocols can influence MWM performances.
Asunto(s)
Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/metabolismo , Factores de Edad , Animales , Masculino , Trastornos de la Memoria/psicología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proyectos de Investigación/estadística & datos numéricosRESUMEN
The activity and expression of cytochromes P450 were determined in liver, kidneys, lungs, duodenum, jejunum, ileum, and caecum of adult Lacaune sheep. High expression of total P450, benzphetamine and erythromycin demethylase activities, and P450 2B isoforms, as two distinct proteins that were detected and called P4502 Bm and P4502 Bx, was found in the lungs (in addition to liver). By contrast, the P450 3A subfamily was only expressed in liver and duodenal mucosa of untreated sheep. Phenobarbital (PB) treatment led to significant increases in all measured hepatic parameters and in total P450 of each investigated organ with the exception of ileum and caecum. Benzphetamine demethylase activity increased in liver and kidneys, correlating with the expression of the two P450 2B proteins, which were also induced in duodenum and ileum. By contrast, benzphetamine demethylase activity and expression of the P450 2B isoforms in lungs were unchanged by PB treatment. Erythromycin demethylation activity and P450 3A subfamily expression was increased only in liver of PB-treated sheep.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/biosíntesis , Antagonistas de Aminoácidos Excitadores/farmacología , Oxidorreductasas N-Desmetilantes/biosíntesis , Fenobarbital/farmacología , Ovinos/metabolismo , Animales , Western Blotting , Citocromo P-450 CYP3A , Inducción Enzimática/efectos de los fármacos , Femenino , Isoenzimas/biosíntesis , Masculino , Microsomas/efectos de los fármacos , Microsomas/enzimologíaRESUMEN
A method is described for the determination of selamectin in dog plasma, using High-Performance Liquid Chromatography (HPLC) with fluorescence detection (excitation and emission wavelengths fixed at 355 and 465 nm, respectively). The fluorescent derivative was obtained by condensation reaction with trifluoroacetic anhydride and N-methylimidazole. The method employs 1 mL plasma samples and gives linear calibration graphs (r = 0.999) over the concentration range studied (0.5-50 ng mL(-1)). Automatic solid phase extraction using the benchmate procedure was used for sample preparation. This method permits the determination of selamectin at levels as low as 0.1 ng mL(-1) and its suitability was demonstrated by a pharmacokinetic study on a dog receiving the therapeutic dose (Spot-on administration).
Asunto(s)
Perros/sangre , Insecticidas/sangre , Ivermectina/análogos & derivados , Ivermectina/sangre , Animales , Calibración , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/veterinaria , Perros/metabolismo , Relación Dosis-Respuesta a Droga , Fluorescencia , Insecticidas/farmacocinética , Insecticidas/uso terapéutico , Ivermectina/farmacocinética , Ivermectina/uso terapéutico , Sensibilidad y EspecificidadRESUMEN
We studied the implication of cytochrome P450 enzymes in the in vitro metabolism of moxidectin (MXD) in homogenates of Haemonchus contortus adult stages (susceptible isolate, Weybridge, UK). After homogenisation in a phosphate buffer, 2 ml of homogenates (equivalent to 1 g of nematodes) were incubated with 5 microg [14C] MXD at 37 degrees C for 24 h. MXD and its metabolites were separated by HPLC with radiodetection on-line. Only one metabolite was detected and its production was inhibited by carbon monoxide. This result demonstrates that the cytochrome P450 system is implicated in the metabolisation of MXD in H. contortus susceptible to milbemycin. Furthermore, this metabolite did not match those previously described in vertebrates.