RESUMEN
Rodent toxicity studies typically use water bottles to administer test chemicals via drinking water. However, water bottles provide inconsistent exposure of volatile chemicals due to varying headspace, and lead to excessive waste of test material. To refine drinking water toxicity studies in rodents by enhancing sample quality and consistency, and minimizing waste, we designed and implemented a novel water delivery system that keeps the water chilled, headspace free and protected from light. Materials used were resistant to chemical interaction. In this gravity-fed system, a 6-L Teflon water bag, stored in a polystyrene cooler on the cage rack, was connected to a stainless steel manifold delivering water to five cages via specialized drinking valves. Due to the absence of headspace in the water bag, this system allows consistent exposure of volatile chemicals. In addition, small diameter tubing throughout the system reduces the amount of test material residing in the system and minimizes chemical waste.
Asunto(s)
Crianza de Animales Domésticos/instrumentación , Ingestión de Líquidos/fisiología , Residuos Peligrosos/prevención & control , Ciencia de los Animales de Laboratorio/instrumentación , Pruebas de Toxicidad/instrumentación , Xenobióticos/toxicidad , Crianza de Animales Domésticos/métodos , Animales , Ciencia de los Animales de Laboratorio/métodos , Ratones , Ratas , Pruebas de Toxicidad/métodos , Volatilización , Abastecimiento de Agua , Xenobióticos/clasificaciónRESUMEN
The kinetics of the flash-induced photodissociation and rebinding of carbon monoxide in cytochrome aa3-CO have been studied by time-resolved infrared (TRIR) and transient ultraviolet-visible (UV-vis) spectroscopy at room temperature and by Fourier transform infrared (FTIR) spectroscopy at low temperature. The binding of photodissociated CO to CuB+ at room temperature is conclusively established by the TRIR absorption at 2061 cm-1 due to the C-O stretching mode of the CuB(+)-CO complex. These measurements yield a first-order rate constant of (4.7 +/- 0.6) x 10(5) s-1 (t1/2 = 1.5 +/- 0.2 microseconds) for the dissociation of CO from the CuB(+)-CO complex into solution. The rate of rebinding of flash-photodissociated CO to cytochrome a(3)2+ exhibits saturation kinetics at [CO] > 1 mM due to a preequilibrium between CO in solution and the CuB(+)-CO complex (K1 = 87 M-1), followed by transfer of CO to cytochrome a(3)2+ (k2 = 1030 s-1). The CO transfer from CuB to Fe alpha 3 was followed by CO-FTIR between 158 and 179 K and by UV-vis at room temperature. The activation parameters over the temperature range 140-300 K are delta H++ = 10.0 kcal mol-1 and delta S++ = -12.0 cal mol-1 K-1. The value of delta H++ is temperature independent over this range; i.e., delta Cp++ = 0 for CO transfer. Rapid events following photodissociation and preceding rebinding of CO to cytochrome a(3)2+ were observed. An increase in the alpha-band of cytochrome a3 near 615 nm (t1/2 ca. 6 ps) follows the initial femtosecond time-scale events accompanying photodissociation. Subsequently, a decrease is observed in the alpha-band absorbance (t1/2 approximately 1 microsecond) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the loss of CO by CuB+. We ascribe these observations to structural changes at the cytochrome a3 induced by the formation and dissociation of the CuB(+)-CO complex. We suggest that the picosecond binding of photodissociated CO to CuB triggers the release of a ligand L from CuB. We infer that L then binds to cytochrome a3 on the distal side and that this process is directly responsible for the observed alpha-band absorbance changes. We have previously suggested that the transfer of L produces a transient five-coordinate high-spin cytochrome a3 species where the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Monóxido de Carbono/química , Complejo IV de Transporte de Electrones/química , Cobre/química , Cinética , Oxidación-Reducción , Fotólisis , Análisis EspectralRESUMEN
Past studies have found that the laboratory rat placed in a temperature gradient prefers temperatures that are markedly below its lower critical ambient temperature (LCT), whereas other rodents (e.g., mouse, hamster, and guinea pig) generally select thermal environments associated with minimal metabolic expenditure. To further study the rat's thermoregulatory behavior, a temperature gradient was designed to monitor the selected ambient temperature (STa) and motor activity (MA) of food-deprived rats of the Long-Evans (LE), Fischer 344 (F344), and Sprague-Dawley (SD) strains over a 22-h period. All three rat strains selected relatively cool STas of 21-26 degrees C during the first 1-3 h in the temperature gradient. This was followed by a gradual increase in the STa that peaked at 4 (F344) to 6 h (SD and LE) after being placed in the gradient. The LE strain had a significantly lower STa during the initial period in the gradient. There were slight decreases in the STa during the nocturnal phase in the F344 and SD strains concomitant with marked increases in MA. These results indicate that the rat requires a relatively long adjustment period in a temperature gradient before it exhibits STas that are associated with minimal metabolic expenditure. Given adequate time for accommodation, behavioral thermoregulatory responses of the rat appear to be similar to those of other rodents.
Asunto(s)
Regulación de la Temperatura Corporal , Ritmo Circadiano , Actividad Motora , Ratas/fisiología , Análisis de Varianza , Animales , Temperatura Corporal , Métodos , Ratas Endogámicas F344/fisiología , Ratas Endogámicas/fisiología , Especificidad de la Especie , TemperaturaRESUMEN
The C-O stretching frequencies of fully reduced carbonmonoxy cytochrome ba3, a newly discovered terminal oxidase of the bacterium Thermus thermophilus (Zimmermann, B.H., Nitsche, C.I., Fee, J.A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 5779-5783), are studied by Fourier transform infrared spectroscopy. Multiple C-O frequencies are observed in the Fourier transform infrared spectra, indicating the presence of discrete interconverting conformers of the enzyme. Upon photolysis, the CO is shown to migrate exclusively to CuB+. Above 200 K, the CO returns to the heme a3 by a thermal process which follows simple first-order kinetics. The rate of the reaction was studied from 205 to 230 K and at 300 K, yielding the activation parameters delta H = 14.9 kcal/mol and delta S = -5 cal/mol/K. These are compared with previously determined activation parameters for CO recombination in mitochondrial cytochrome aa3 preparations (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 257, 1639-1650). We report the novel finding that CO remains bound to CuB+ at room temperature during continuous photolysis of cytochrome ba3, and we conjecture on the possible interference of copper-bound CO in "flow-flash" and "triple-trap" studies of cytochrome c oxidases.