RESUMEN
Lepeophtheirus salmonis is an ectoparasitic copepod that causes serious disease outbreaks in both wild and farmed salmonids. As the relationship between L. salmonis and its hosts is not well understood, the current investigation was undertaken to investigate whether any immunomodulatory compounds could be identified from secretions of L. salmonis. By incubating live L. salmonis adults with the neurotransmitter dopamine in seawater, we were able to obtain secretions from the parasite. These were analyzed by RP-HPLC column, as well as LC-MS. L. salmonis secretions contained a compound with the same retention time and mass of PGE(2). The identity of this compound as PGE(2) was confirmed by MS-in source dissociation. The concentrations of PGE(2) in L. salmonis secretions ranged from 0.2 to 12.3 ng/individual and varied with incubation temperature and time kept off the host. Prostaglandin E(2) is a potent vasodilator and thought to aid in parasite evasion from host immune responses. This is the first reported evidence of prostaglandin production in parasitic copepod secretions and its implications for the host-parasite relationship are discussed.
Asunto(s)
Copépodos/metabolismo , Dinoprostona/metabolismo , Infestaciones Ectoparasitarias/veterinaria , Enfermedades de los Peces/parasitología , Salmo salar/parasitología , Animales , Cromatografía Líquida de Alta Presión , Copépodos/inmunología , Dinoprostona/análisis , Dinoprostona/fisiología , Infestaciones Ectoparasitarias/inmunología , Infestaciones Ectoparasitarias/parasitología , Femenino , Enfermedades de los Peces/inmunología , Interacciones Huésped-Parásitos , Masculino , Espectrometría de MasasRESUMEN
Simultaneous detection and confirmation of 15 quinolone antibiotics was accomplished by fast short-column liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS). Several physiochemical parameters such as hydrophobicity and aqueous dissociation constants were calculated from the structural formulas of the quinolone drugs, and their impact on both chromatographic and mass spectrometric behavior was studied. Additionally, a possible influence of bulk solution pH on electrospray detection sensitivity of 4-quinolones was investigated and compared to predictions based on solution-phase equilibria. A signal intensity comparison of the MH+ ions at different pH values for all 15 compounds did not reveal any pH effect, despite variations by several orders of magnitude in equilibrium concentrations in bulk solution. To demonstrate the potential of the LC/MS/MS method, its application to trace analysis in several biological matrices such as milk, salmon, and human urine was investigated. The method was shown to be sensitive with detection limits down to 1 ppb in both milk and salmon tissue. The versatility of the method was also exhibited by utilizing it for rapid identification of urinary metabolites of ciprofloxacin. Finally, a new complementary approach is described for confirmatory analyses of 4-quinolones by means of a quasi-MS/MS/MS technique involving in-source collision-induced dissociation. It is shown that LC/quasi-MS/MS/MS can significantly enhance structural information and, thus, the specificity of analysis for the investigated 4-quinolones.
Asunto(s)
Antiinfecciosos/análisis , 4-Quinolonas , Animales , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/orina , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masas , Leche/químicaRESUMEN
A capillary electrophoresis (CE) method using acidic buffers and capillaries coated with Polybrene, a cationic polymer has been developed for the separation of glycoproteins and glycopeptides. Electrophoretic conditions have been optimized to provide resolution of individual glycoforms observed for different glycoprotein preparations. These conditions were found to be entirely compatible with the operation of electrospray mass spectrometry (ESMS), which facilitated the assignments of possible carbohydrate compositions of glycopeptides arising from digests of glycoproteins. By using operating conditions enhanced the formation of oxonium fragment ions prior to mass spectral analysis, selective identification of glycopeptides was achieved for complex samples such as those from proteolytic digests or chemical cleavages. Examples of applications are presented for ribonuclease B, ovalbumin, horseradish peroxidase, and a lectin from Erithrina corallodendron using both CE-ESMS and CE with ultraviolet detection (CE-UV).
Asunto(s)
Electroforesis Capilar/métodos , Glicoproteínas/análisis , Espectrometría de Masas/métodos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Hidrólisis , Datos de Secuencia Molecular , Ovalbúmina/análisis , Ribonucleasas/análisis , Espectrofotometría UltravioletaRESUMEN
The hepatic response to a fructose challenge for control rats, and rats subjected to an acute sublethal dose of carbon tetrachloride (CCl4) or bromobenzene (BB), was compared using dynamic in vivo 31P MRS. Fructose loading conditions were used in which control rats showed only a modest increase in hepatic phosphomonoester (PME), and a small decrease in ATP, Pi, and intracellular pH after fructose administration. Both CCl4 and BB-treated rats showed a much greater fructose-induced accumulation of PME than did controls. Trolox C, a free radical scavenger, prevented most of this PME increase. BB-treated rats, given sufficient time to recover from the hepatotoxic insult, responded to the fructose load similarly to controls. Liver aldolase activities of control, toxicant-treated rats, and toxicant plus Trolox C-treated rats correlated inversely with PME accumulation after fructose loading (correlation coefficient: -0.834, P < 0.05). Perchloric acid extracts of rat livers studied by in vitro 31P MRS confirmed that the PME accumulation after fructose loading is mainly due to an increase in fructose 1-phosphate. These studies are consistent with the aldolase-catalyzed cleavage of fructose 1-phosphate being rate-limiting in hepatic fructose metabolism, and that the CCl4 and BB treatment modify and inactivate the aldolase enzyme.
Asunto(s)
Bromobencenos/efectos adversos , Tetracloruro de Carbono/efectos adversos , Fructosa/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Espectroscopía de Resonancia Magnética , Adenosina Trifosfato/metabolismo , Animales , Antioxidantes/farmacología , Cromanos/farmacología , Fructosa-Bifosfato Aldolasa/metabolismo , Fructosafosfatos/metabolismo , Glucosa-6-Fosfato , Glucofosfatos/metabolismo , Glicerofosfatos/metabolismo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Hígado/enzimología , Masculino , Organofosfatos/metabolismo , Fenobarbital/farmacología , Fósforo/metabolismo , Fosforilcolina/metabolismo , Ratas , Ratas Wistar , Factores de Tiempo , Vitamina E/análogos & derivados , Vitamina E/farmacologíaRESUMEN
We have previously demonstrated that the subcellular distribution of the adhesion plaque protein, talin, changes dramatically in human platelets in response to platelet activation (Beckerle et al., J. Cell Biol. 109, 3333-3346, 1989). Talin is uniformly distributed throughout the cytoplasm of resting platelets. However, when platelets are stimulated to become activated and adhesive, a significant amount of the talin population rapidly redistributes to a peripheral, submembranous location. In the present study we have examined talin phosphorylation and proteolytic cleavage as possible mechanisms by which talin's subcellular distribution could be regulated in platelets. We have found that thrombin activation of platelets leads to a fourfold increase in talin phosphorylation. Proteolytic cleavage of talin, however, is not detected in washed platelets activated with thrombin for as long as 30 minutes. Because talin moves to a submembranous location upon platelet activation and has been shown to interact with integrins in vitro, we also investigated whether the major platelet integrin, GPIIb-IIIa, is required for talin redistribution. Using Glanzmann thrombasthenic platelets, which are deficient in GPIIb-IIIa, we found that talin redistribution occurs even in the absence of GPIIb-IIIa. Collectively, our studies suggest that neither proteolytic cleavage of talin nor interactions between talin and GPIIb-IIIa is required for the regulated redistribution of talin in thrombin-activated platelets. Phosphorylation of talin in response to thrombin activation may, however, be one mechanism utilized by platelets to regulate talin distribution and function in human platelets.
Asunto(s)
Activación Plaquetaria/fisiología , Talina/metabolismo , Transporte Biológico , Plaquetas/efectos de los fármacos , Calcio/farmacología , Compartimento Celular , Endopeptidasas/efectos de los fármacos , Humanos , Integrinas/metabolismo , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Trombastenia/metabolismo , Trombina/farmacologíaRESUMEN
Proton magnetic resonance imaging (MRI) and 31P magnetic resonance spectroscopy (MRS) have been used to study the response of the rat liver in situ to bromobenzene, a classic hepatotoxicant. A localized region of high proton signal intensity was seen in the perihilar region of the liver 24 hr after injection of a sublethal dose of bromobenzene. The signal intensity of the entire liver was increased at 48 hr with a gradual return approaching control values by 120 hr. These results are consistent with acute hepatic edema followed by repair of the damaged tissue. In vivo 31P MRS studies of the same rat livers were performed under conditions whereby localized, quantitative spectra could be obtained without surgical intervention. Initial concentrations of the major endogenous phosphorus-containing metabolites within the livers of control rats were 2.97 +/- 0.43 mM for the phosphomonoesters (PME), 2.92 +/- 0.56 mM for inorganic phosphate, 11.3 +/- 1.0 mM for phosphodiesters (PDE), 4.09 +/- 0.54 mM for ATP, and 0.56 +/- 0.50 mM for ADP and the intracellular pH was 7.39 +/- 0.14 (mean +/- SD, n = 10). Bromobenzene was found to cause statistically significant (p less than 0.05) changes in several of these metabolites: a decrease in hepatic ATP levels (20% at 24 hr; 27% at 48 hr), a decrease in PDE levels (15% at 24 hr; 18% at 48 hr), and an increase in the PME (63% at 24 hr; 84% at 48 hr). Both the proton MRI and the 31P MRS changes have an onset of 15-20 hr and maximum effect at 25-60 hr, but the MRS changes returned to normal well before the MRI changes. The decreased ATP levels indicate deleterious effects of bromobenzene on the bioenergetic status of the liver in situ, while the increase in PME, due to a selective increase in phosphocholine, suggests the activation of a phosphatidylcholine-specific phospholipase C in response to tissue damage. Trolox C, a potent inhibitor of lipid peroxidation, prevented the bromobenzene-induced hepatic edema (i.e., the increase in proton MRI signal intensity) and the bioenergetic deterioration (i.e., the decrease in ATP levels). However, the bromobenzene-induced increase in PME levels was not prevented by Trolox C. These results indicate that the process of lipid peroxidation plays a significant role in the hepatotoxicity of bromobenzene within the intact animal.
Asunto(s)
Bromobencenos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas , Hígado/efectos de los fármacos , Animales , Cromanos/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Edema/inducido químicamente , Inducción Enzimática , Peroxidación de Lípido/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Hepatopatías/enzimología , Hepatopatías/metabolismo , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Fenobarbital/efectos adversos , Fosfolípidos/metabolismo , Fósforo , Ratas , Ratas Endogámicas , Reproducibilidad de los Resultados , Factores de Tiempo , Transaminasas/sangreRESUMEN
Cholesterol, when sequestered in saturated liposomes of dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC), undergoes peroxidation thermally initiated either by a lipid-soluble or a water-soluble azo initiator and in both cases the reaction is inhibited effectively by the water-soluble antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox). Quantitative kinetic methods of autoxidation show that the oxidizability, kp/(2kt)1/2 (where kp and 2kt are the rate constants of radical chain propagation and termination, respectively) of cholesterol in DMPC or DPPC multilamellar liposomes, where kp/(2kt)1/2 is 3.0.10(-3) to 4.3.10(-3) M-1/2 s-1/2 at 37-45 degrees C, is similar to that measured in homogeneous solution in chlorobenzene, where kp/(2kt)1/2 is 3.32.10(-3). However, its oxidizability in smaller unilamellar vesicles of DMPC or DPPC increases by at least 3-times that measured in multilamellar systems. Autoxidation/antioxidant methods show that cholesterol partitions directly from the solid state into DMPC or DPPC liposomes by shaking and this is confirmed by 31P and 2H quadrupole NMR spectra of deuterated cholesterol when membrane bound. Analytical studies indicate that up to 21 mol% cholesterol will partition into the membranes by shaking.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Cromanos , Dimiristoilfosfatidilcolina/química , Peroxidación de Lípido , Liposomas , Antioxidantes , Radicales Libres , Cinética , Espectroscopía de Resonancia Magnética , Termodinámica , Factores de TiempoRESUMEN
Talin is a high molecular weight protein localized at adhesion plaques in fibroblasts. It binds vinculin and integrin and appears to participate in generating a transmembrane connection between the extracellular matrix and the cytoskeleton. We have recently shown that talin is an abundant protein in platelets, cells highly specialized for regulated adhesion. Although talin constitutes greater than 3% of the total protein in intact human platelets, its location within the cells had not been defined. In the work reported here, we have investigated the distribution of talin in resting and activated human platelets by immunofluorescence and immunoelectron microscopy. We have found that talin undergoes an activation-dependent change in its subcellular location. In resting platelets, which are nonadhesive, talin is uniformly distributed throughout the cytoplasm. In contrast, in thrombin- and glass-activated, substratum-adherent platelets, talin is concentrated at the cytoplasmic face of the plasma membrane. This dramatic, regulated redistribution of talin raises the possibility that talin plays a role in the controlled development of platelet adhesion.
Asunto(s)
Plaquetas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Activación Plaquetaria , Adhesividad Plaquetaria/fisiología , Plaquetas/ultraestructura , Calcio/fisiología , Proteínas del Citoesqueleto/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Técnicas Inmunológicas , Técnicas In Vitro , Péptido Hidrolasas , Glicoproteínas de Membrana Plaquetaria/metabolismo , Talina , TrombinaRESUMEN
The Total (Peroxyl) Radical-trapping Antioxidant Parameter (TRAP) of six freshly prepared human plasma samples and 45 frozen plasma samples has been determined. It is shown that contributions from urate (35-65%), plasma proteins (10-50%), ascorbate (0-24%) and vitamin E (5-10%) to TRAP account for all of the peroxyl radical-trapping antioxidant activity in the majority of the samples. The changes in concentrations of the plasma antioxidants during peroxyl radical attack show that the first line of defense is provided by the plasma sulfhydryl groups, even urate being spared during the initial stages of the reaction. The modes of action of all of these plasma antioxidants and possible interactions between them are discussed, with particular emphasis on the abilities of the water-soluble antioxidants to regenerate or spare the only lipid-soluble antioxidant, vitamin E.