Effects on health, performance, and tissue residues of the ionophore antibiotic salinomycin in finishing broilers (21 to 38 d).

A study was conducted to evaluate the effects of feeding salinomycin at the recommended prophylactic level, and at 2 and 3 times this level, to finishing male broilers (d 21 to 38). Four treatment groups were given the experimental diets containing 0, 60, 120, or 180 parts per million (ppm) salinomycin from d 21 to 38. Performance, relative organ weights, selected serum enzymes, and salinomycin residues in liver, muscle, and serum were determined. Salinomycin supplementation had no effect on body weight, feed intake, or feed conversion, and caused no overt signs of toxicity. After a week of being fed the salinomycin diets, the serum activity of aspartate aminotransferase was significantly increased in chickens fed 180 ppm compared with controls. These birds also showed microscopic lesions in breast and thigh muscles, but not in cardiac muscle. Salinomycin residues were not detected by high-performance liquid chromatography coupled to tandem mass spectrometry in liver or muscle samples from the birds fed 0, 60, or 120 ppm salinomycin. However, chickens fed 180 ppm salinomycin had detectable levels in liver and muscle above the maximum residue level of 5 µg/kg established by the European Union. All birds fed salinomycin had salinomycin in their sera with levels ranging from N.D. (not detected) in the controls to 24.4 ± 7.9, 61.4 ± 18.9, and 94.5 ± 9.1 µg/L for salinomycin dietary levels of 60, 120, and 180 ppm, respectively. Serum salinomycin concentration was linearly related with salinomycin content in feed (y = 0.584x - 10, r2 = 0.999). The results showed that even at 3 times the prophylactic level, salinomycin does not induce clinical toxicosis or mortality. No salinomycin residues were found in edible tissues at the recommended dietary level or at 2 times this level. However, salinomycin was detected in serum regardless of the dietary level. A simple method for salinomycin determination in serum is described which can be used as a marker of exposure and/or to predict levels in the diet.

Transformation of ionophore antimicrobials in poultry litter during pilot-scale composting.

Ionophores are the second top selling class of antimicrobials used in food-producing animals in the United States. In chickens, ionophores are used as feed additives to control coccidiosis; up to 80% of administered ionophores are excreted in the litter. Because poultry litter is commonly used to fertilize agricultural fields, ionophore residues in litter have become contaminants of emerging concern. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantify ionophores, and identify their transformation products (TPs) in poultry litter after on-farm pilot-scale composting. The validation parameters of the optimized method showed good accuracy, ranging from 71 to 119% recovery and relative standard deviation (precision) of ≤19% at three different concentration levels (10, 50 and 100 µg/kg). Monensin, salinomycin and narasin, were detected in the poultry litter samples prior to composting at 290.0 ± 40, 426 ± 46, and 3113 ± 318 µg kg(-1), respectively. This study also aims to investigate the effect of different composting conditions on the removal of ionophores, such as the effect of turning or aeration. Results revealed a 13-68% reduction in ionophore concentrations after 150 d of composting, depending on whether the compost was aerated, turned, or subjected to a combination of both aeration and turning. Three transformation products and one metabolite of ionophores were identified in the composted litter using high-resolution liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QToF/MS).

Effect of monensin sodium on metabolic profile of ewes before and pospartum / Efeito da monensina sódica sobre o perfil metabólico de ovelhas antes e após o parto

The study was carried out to analyze the effect of monensin supplementation during 60 days prior to parturition, and 30 days postpartum, on the metabolic and hormonal profile of ewes. Pregnant ewes (n=13) were randomly divided into two groups: monensin group (n = 7; 30 mg/day) and control group (n = 6). Blood and ruminal fluid samples were collected 60, 50, 40, 30, 20 and 10 days prior to parturition, at the time of parturition and on 10, 20 and 30 days postpartum. The following variables were analyzed: glucose, fructosamine, non-esterfied fatty acids, ?-hydroxybutyrate, cholesterol, triglycerides, total protein, albumin, urea and ketone bodies in the urine. The hormonal determinations were cortisol and insulin. The analysis of the ruminal fluid involved pH and concentration of volatile fatty acids. The statistical analysis involved ANOVA and correlation studies (P 0,05). Monensin increased (P 0,05) the propionic acid concentration in the rumen and blood fructosamine and insulin. The administration of monensin improved some indicators of energy balance(AU)

O estudo foi realizado com o intuito de avaliar o efeito da monensina, suplementada a partir de 60 dias antes do parto (dap) e por 30 dias pós-parto, sobre o perfil metabólico e hormonal de ovelhas. As ovelhas prenhas (n=13), foram divididas, de forma aleatória, em dois grupos, um que recebeu a monensina (n=7) (30 mg/dia) e o controle (n=6). Amostras de sangue e fluido ruminal foram colhidas aos 60, 50, 40, 30, 20 e 10 dias antes do parto, no momento do parto e nos 10, 20 e 30 dias pós-parto. As variáveis mensuradas foram: glicose, frutosamina, ácidos graxos não esterificados (AGNEs), ?-hidroxibutirato, colesterol, triglicérides, proteína total, albumina, ureia e pesquisa de corpos cetônicos na urina. As determinações hormonais foram cortisol e a insulina. No fluido ruminal foi determinado o pH e a concentração dos ácidos graxos voláteis. Na análise estatística foi empregada a ANOVA e estudo de correlação (P 0,05). A monensina elevou (P 0,05) a concentração do propionato no rúmen e frutosamina e insulina no sangue. A administração da monensina promoveu melhora em alguns indicadores do balanço energético(AU)

Effect of monensin sodium on metabolic profile of ewes before and pospartum / Efeito da monensina sódica sobre o perfil metabólico de ovelhas antes e após o parto

The study was carried out to analyze the effect of monensin supplementation during 60 days prior to parturition, and 30 days postpartum, on the metabolic and hormonal profile of ewes. Pregnant ewes (n=13) were randomly divided into two groups: monensin group (n = 7; 30 mg/day) and control group (n = 6). Blood and ruminal fluid samples were collected 60, 50, 40, 30, 20 and 10 days prior to parturition, at the time of parturition and on 10, 20 and 30 days postpartum. The following variables were analyzed: glucose, fructosamine, non-esterfied fatty acids, ?-hydroxybutyrate, cholesterol, triglycerides, total protein, albumin, urea and ketone bodies in the urine. The hormonal determinations were cortisol and insulin. The analysis of the ruminal fluid involved pH and concentration of volatile fatty acids. The statistical analysis involved ANOVA and correlation studies (P 0,05). Monensin increased (P 0,05) the propionic acid concentration in the rumen and blood fructosamine and insulin. The administration of monensin improved some indicators of energy balance

O estudo foi realizado com o intuito de avaliar o efeito da monensina, suplementada a partir de 60 dias antes do parto (dap) e por 30 dias pós-parto, sobre o perfil metabólico e hormonal de ovelhas. As ovelhas prenhas (n=13), foram divididas, de forma aleatória, em dois grupos, um que recebeu a monensina (n=7) (30 mg/dia) e o controle (n=6). Amostras de sangue e fluido ruminal foram colhidas aos 60, 50, 40, 30, 20 e 10 dias antes do parto, no momento do parto e nos 10, 20 e 30 dias pós-parto. As variáveis mensuradas foram: glicose, frutosamina, ácidos graxos não esterificados (AGNEs), ?-hidroxibutirato, colesterol, triglicérides, proteína total, albumina, ureia e pesquisa de corpos cetônicos na urina. As determinações hormonais foram cortisol e a insulina. No fluido ruminal foi determinado o pH e a concentração dos ácidos graxos voláteis. Na análise estatística foi empregada a ANOVA e estudo de correlação (P 0,05). A monensina elevou (P 0,05) a concentração do propionato no rúmen e frutosamina e insulina no sangue. A administração da monensina promoveu melhora em alguns indicadores do balanço energético

In vitro metabolism of monensin A: microbial and human liver microsomes models.

1. Monensin A, an important antibiotic ionophore that is primarily employed to treat coccidiosis, selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. 2. In this study, we evaluated the fungi Cunninghamella echinulata var. elegans ATCC 8688A, Cunninghamella elegans NRRL 1393 ATCC 10028B and human hepatic microsomes as CYP-P450 models to investigate the in vitro metabolism of monensin A and compare the products with the metabolites produced in vivo. 3. Mass spectrometry analysis of the products from these model systems revealed the formation of three metabolites: 3-O-demethyl monensin A, 12-hydroxy monensin A and 12-hydroxy-3-O-demethyl monensin A. We identified these products by tandem mass spectrometry and through comparison with the in vivo metabolites. 4. This analysis demonstrated that the model systems produce the same metabolites found in in vivo studies, thus they could be used to predict the metabolism of monensin A. Furthermore, we verified that liquid chromatography coupled to mass spectrometry is a powerful tool to study the in vitro metabolism of drugs, because it allows the successful identifications of several derivatives from different metabolic models.

Anionic selectivity sequence of the Cl(-)-H+ symporter in the synaptosomal preparation from rat brain cortex.

The Na(+)/H(+) exchanger has been the only unequivocally demonstrated H(+)-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl(-)-H(+) symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl(-)-H(+) symporter is NO(3)(-) > Br(-) > Cl(-) >> I(-) = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 muM inhibits the gluconate-dependent alkalinization by 30 +/- 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization.

Characterizing plasma membrane H+-ATPase in two varieties of coffee leaf (Coffea arabica L.) and its interaction with an elicitor fraction from the orange rust fungus (H. vastatrix Berk and Br.) race II.

Early intercellular signaling in Coffea arabica L.-Hemileia vastatrix host-pathogen interaction was studied, using inside-out plasma membrane from two varieties of coffee leaf and a fungal fraction to determine the plant's biochemical responses. Microsomal pellets (100,000 x g) from the susceptible (Caturra) and resistant (Colombia) coffee leaf varieties were purified by partitioning in two-polymer DEX (6.3% w/w) and PEG (6.3% w/w) system aqueous phase. Fungal material was obtained from orange rust Hemileia vastatrix Berk and Br. race II urediospore germ tubes. Plasma membrane vesicles were preferentially localized to PEG phase, as indicated by its enzyme marker distribution. Both H(+)-ATPase activities displayed similar kinetic and biochemical characteristics, comparable to those described for P-type ATPases. Several enzymes may play pivotal roles in plants regarding early interaction with fungal elicitors. Studies of fungal fractions' effects on H(+)-ATPase and both varieties' proton pumping activities were thus carried out. Concentration as low as 0.1 Gluc eq. ml(-1) fungal fraction induced specific inhibition of H(+)-ATPase and the resistant variety's proton pumping activities. The present work describes characterizing the H(+)-ATPase plasma membrane from two Coffea arabica L. varieties (Caturra and Colombia) for the first time and the race specific inhibitory effect of a crude fungal fraction on both H(+)-ATPase and the resistant variety's proton pumping activities.

Myosin V and iNOS expression is enhanced in J774 murine macrophages treated with IFN-gamma.

Actin-based motor protein requirements and nitric oxide (NO) production are important features of macrophage activity during phagocytosis or microbicidal processes. Different classes of myosins contribute directly or indirectly to phagocytosis by providing mechanical force for phagosome closure or organelle movement. Recent data have shown the presence of myosins IC, II, V and IXb in phagosomes of bone marrow-derived murine macrophages. In our investigation we demonstrated the presence of different classes of myosins in J774 macrophages. We also analyzed the effect of gamma interferon (IFN-gamma), with or without calcium ionophore or cytochalasin B, on myosins as well as on inducible nitric oxide synthase (iNOS) expression and NO production. Myosins IC, II, Va, VI and IXb were identified in J774 macrophages. There was an increase of myosin V expression in IFN-gamma-treated cells. iNOS expression was increased by IFN-gamma treatment, while calcium ionophore and cytochalasin B had a negative influence on both myosin and iNOS expression, which was decreased. The increases in NO synthesis were reflected by increased iNOS expression. Macrophages activated by IFN-gamma released significant amounts of NO when compared to control groups. In contrast, NO production by calcium ionophore- and cytochalasin B-treated cells was similar to that of control cells. These results suggest that IFN-gamma is involved in macrophage activation by stimulating protein production to permit both phagocytosis and microbicidal activity.

Calcium and phospholipase A2 are both required for the acrosome reaction mediated by G-proteins stimulation in human spermatozoa.

G-proteins, calcium, and phospholipase A2 (PLA2) have all been implicated in the cascade of signaling events leading to the acrosome reaction in human spermatozoa. In order to study the role of Ca+2 and PLA2 during the acrosome reaction triggered by G-proteins, we treated human spermatozoa incubated for 3 hr under capacitating conditions with several reagents (GTPgammaS, A23187, ONO-RS-082, arachidonic acid, BAPTA-AM, and TPEN), alone or in different combinations. Our results suggest that GTP-binding proteins require Ca+2 and PLA2 to accomplish their stimulatory effect, and that Ca+2 is also required when the acrosome reaction--bypassing the action of PLA2--is stimulated by AA. Accordingly, when treated with GTPgammaS or AA, the cells loaded with Fura 2-AM showed a steady increase of [Ca+2]i. On the other hand, a massive influx of Ca+2 was completely unable to induce the acrosome reaction if PLA2 was inhibited, suggesting that both an increase of [Ca+2]i and PLA2 activation are required for the acrosome reaction to occur.

Lipid peroxidation: the role of Ca2+ and protection by calcinine.

When calcinine (A-23187) (2 microM), a known Ca2+ ionophore, is present, a significant protection is observed to a mitochondrial suspension undergoing lipid peroxidation by Fe(2+)-citrate complex. A-23187 can remove Ca2+, which seems to have an important role in the lipid peroxidation process, from its 'lesive sites' and consequently preventing the damage. This information has importance in terms of knowing the mechanisms and avoiding the damages of lipid peroxidation that occur in some pathological cases such as tumor promotion and hemochromatosis.

Production of bacteriocins and siderophore-like activity by Azospirillum brasilense.

Sixty Azospirillum strains were tested for their bacteriocin production ability; twenty-seven (45%) were able to produce bacteriocins and inhibited the growth of one or more indicator strains in solid medium. Mitomycin C treatment enhanced the proportion to 80%. Sometimes large growth inhibition zones were formed, but not when FeCl3 was added in the medium. These inhibition zones probably result from the activity of siderophores. Partially purified bacteriocins produced by four strains were inactivated at pH 4, but were very stable between pH 5 to 10; bacteriocins produced by three strains lost their activity between 55 and 80 degrees C. Loss or decrease in the bacteriocin activity was observed with pronase E treatment; trypsin, lysozyme and alpha-amylase did not have an effect on bacteriocin activity. These findings show that the antagonism among azospirilla was due principally to the bacteriocins and sometimes probably due to siderophores, but not to bacteriophages or other substances.

A culture medium for Paracoccidioides brasiliensis with high plating efficiency, and the effect of siderophores.

The plating efficiency of Paracoccidioides brasiliensis on standard mycological media is poor, impairing its isolation and recovery from various sources, particularly infected tissues. We describe a medium that markedly improves P. brasiliensis plating efficiency. It consists of a synthetic medium (modified McVeigh-Morton) supplemented with 4% (v:v) horse serum and 5% (v:v) culture filtrate from stationary phase P. brasiliensis cultures. A commercially available medium (brain-heart infusion), ordinarily inferior to unsupplemented McVeigh-Morton medium, is at least as efficacious as supplemented McVeigh-Morton medium when supplemented in this manner. We show that plating efficiency varies among P. brasiliensis isolates and can even vary with the isolate's history of passage in culture. In contrast, all isolates studied could produce the growth enhancing factors present in culture filtrate. Some siderophores produced by other fungi can be substituted for the culture filtrate, whereas others can be substituted for both the filtrate and serum. The enhancing effect of filtrate and/or serum could be removed by chelating iron. P. brasiliensis-produced siderophores are likely to be the growth enhancing moiety in culture filtrates.