RESUMEN
OBJECTIVE: To determine if a healthy newborn's age in hours (3, 6, or 9 hours after birth) affects thermoregulatory status after the first bath as indicated by axillary and skin temperatures. DESIGN: Quasi-experimental, mixed-model (between subjects and within subjects) design with hours of age as the nonrepeated variable and prebath and postbath temperatures as the repeated variables. SETTING: Family-centered care unit at an urban hospital in the southwestern United States. PARTICIPANTS: Healthy newborns (N = 75) 37 weeks or more completed gestation. METHODS: Mothers chose time of first bath based on available time slots (n = 25 newborns in each age group). Research nurses sponge bathed the newborns in the mothers' rooms. Axillary temperature, an index of core temperature, was measured with a digital thermometer, and skin temperature, an index of body surface temperature, was measured with a thermography camera. Temperatures were taken before the bath; immediately after the bath; and 5, 30, 60, and 120 minutes after the bath. Immediately after the bath, newborns were placed in skin-to-skin care (SSC) for 60 or more minutes. RESULTS: We found a difference (p = .0372) in axillary temperatures between the 3- and 9-hour age groups, although this difference was not clinically significant (0.18 °F [0.10 °C]). We found no statistically significant differences in skin temperatures among the three age groups. Regardless of age group, axillary and skin temperatures initially decreased and then recovered after the bath. CONCLUSION: For up to 2 hours postbath, axillary and skin temperatures were not different between healthy newborns bathed at 3, 6, or 9 hours of age. Thermography holds promise for learning about thermoregulation, bathing, and SSC.
Asunto(s)
Baños/métodos , Regulación de la Temperatura Corporal , Temperatura Corporal/fisiología , Cuidado del Lactante/métodos , Factores de Edad , Femenino , Voluntarios Sanos , Humanos , Recién Nacido , Masculino , Ensayos Clínicos Controlados no Aleatorios como Asunto , Temperatura , Factores de TiempoRESUMEN
Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable (77)Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate (77)Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, (77)Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.
Asunto(s)
Cisteína/metabolismo , Reductasas del Citocromo/química , Flavinas/metabolismo , Espectroscopía de Resonancia Magnética , Selenocisteína/metabolismo , Selenometionina/metabolismo , Catálisis , Cristalografía por Rayos X , Cisteína/química , Cisteína/genética , Reductasas del Citocromo/genética , Reductasas del Citocromo/metabolismo , Humanos , Mutación/genética , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Conformación Proteica , Pliegue de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The sulfhydryl oxidase augmenter of liver regeneration (ALR) binds FAD in a helix-rich domain that presents a CxxC disulfide proximal to the isoalloxazine ring of the flavin. Head-to-tail interchain disulfide bonds link subunits within the homodimer of both the short, cytokine-like, form of ALR (sfALR), and a longer form (lfALR) which resides in the mitochondrial intermembrane space (IMS). lfALR has an 80-residue N-terminal extension with an additional CxxC motif required for the reoxidation of reduced Mia40 during oxidative protein folding within the IMS. Recently, Di Fonzo et al. [Di Fonzo, A., Ronchi, D., Lodi, T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni, A., Fortunato, F., Nizzardo, M., Napoli, L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I., and Comi, G. P. (2009) Am. J. Hum. Genet. 84, 594-604] described an R194H mutation of human ALR that led to cataract, progressive muscle hypotonia, and hearing loss in three children. The current work presents a structural and enzymological characterization of the human R194H mutant in lf- and sfALR. A crystal structure of human sfALR was determined by molecular replacement using the rat sfALR structure. R194 is located at the subunit interface of sfALR, close to the intersubunit disulfide bridges. The R194 guanidino moiety participates in three H-bonds: two main-chain carbonyl oxygen atoms (from R194 itself and from C95 of the intersubunit disulfide of the other protomer) and with the 2'-OH of the FAD ribose. The R194H mutation has minimal effect on the enzyme activity using model and physiological substrates of short and long ALR forms. However, the mutation adversely affects the stability of both ALR forms: e.g., by decreasing the melting temperature by about 10 degrees C, by increasing the rate of dissociation of FAD from the holoenzyme by about 45-fold, and by strongly enhancing the susceptibility of sfALR to partial proteolysis and to reduction of its intersubunit disulfide bridges by glutathione. Finally, a comparison of the TROSY-HSQC 2D NMR spectra of wild-type sfALR and its R194H mutant reveals a significant increase in conformational flexibility in the mutant protein. In sum, these in vitro data document the major impact of the seemingly conservative R194H mutation on the stability of dimeric ALR and complement the in vivo observations of Di Fonzo et al.