RESUMEN
Isothermal titration calorimetry (ITC) was used to quantify the thermodynamics of Pb(2+) and Zn(2+) binding to metallothionein-3 (MT-3). Pb(2+) binds to zinc-replete Zn7MT-3 displacing each zinc ion with a similar change in free energy (ΔG) and enthalpy (ΔH). EDTA chelation measurements of Zn7MT-3 and Pb7MT-3 reveal that both metal ions are extracted in a tri-phasic process, indicating that they bind to the protein in three populations with different binding thermodynamics. Metal binding is entropically favoured, with an enthalpic penalty that reflects the enthalpic cost of cysteine deprotonation accompanying thiolate ligation of the metal ions. These data indicate that Pb(2+) binding to both apo MT-3 and Zn7MT-3 is thermodynamically favourable, and implicate MT-3 in neuronal lead biochemistry.
Asunto(s)
Plomo/metabolismo , Metalotioneína/metabolismo , Neuronas/metabolismo , Zinc/metabolismo , Humanos , Cinética , Unión Proteica , TermodinámicaRESUMEN
OBJECTIVES: To assess life expectancy and cardiovascular mortality in carriers of Duchenne and Becker muscular dystrophy. DESIGN: Family pedigrees of individuals affected with these conditions, held by the four genetics centres in Scotland, were examined to identify a cohort of definite carriers. Electronic death registration data, held by the General Register Office for Scotland, were used to identify death certificates of carriers who had died, to obtain age at death and cause of death. Survival and mortality data were obtained for the general population for comparison. PATIENTS: 397 definite carriers in 202 pedigrees were identified from which 94 deaths were identified by record linkage to death certificates. MAIN OUTCOME MEASURES: Observed numbers surviving to certain ages and numbers dying of cardiac causes were compared with expected numbers calculated from general population data. RESULTS: There were no significant differences between observed and expected numbers surviving to ages 40-90. The standardised mortality ratio for the 371 carriers alive in 1974 was 0.53 (95% confidence interval 0.32 to 0.82). CONCLUSIONS: Whereas female carriers may have clinical features of cardiomyopathy, this study does not suggest that this is associated with reduced life expectancy or increased risk of cardiac death. Routine cardiac surveillance of obligate carriers is therefore probably unnecessary.
Asunto(s)
Cardiomiopatía Dilatada/mortalidad , Esperanza de Vida , Distrofia Muscular de Duchenne/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Cardiomiopatía Dilatada/genética , Distrofina/genética , Femenino , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Distrofia Muscular de Duchenne/genética , Linaje , Sistema de Registros , Escocia/epidemiología , Factores Sexuales , Análisis de SupervivenciaRESUMEN
Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.
Asunto(s)
Proteína Adaptadora GRB7/química , Proteína Adaptadora GRB7/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Dominios Homologos src , Alanina/genética , Calorimetría , Humanos , Ligandos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/química , Fosforilación , Unión Proteica , Receptor EphB1/química , Receptor EphB1/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , TermodinámicaRESUMEN
Recent results on the oxidation of cysteine residues that bind zinc in transcription factors and their analogous peptides and in related proteins and model systems are reviewed. Two classes of oxidants, the transition metals and dioxygen, hydrogen peroxide, and related species, are considered, and the role of metal ions in suppressing or enhancing Cys oxidation is a major focus. Cysteines in the zinc-bound structures of transcription factors are less susceptible to oxidation than in the metal-free form, and this appears to correlate with reduced accessibility of the thiolates to oxidants. Substitution of other metal ions for Zn(II) increases the rate of Cys oxidation, apparently through increased oxidant accessibility. Reactions that result in reversible or irreversible oxidation of these zinc-binding cysteines under biological conditions are identified in the context of deleterious implications for gene expression.
Asunto(s)
Cisteína/química , Factores de Transcripción/química , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Antioxidantes/farmacología , Sitios de Unión , Cobalto/química , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Peróxido de Hidrógeno/farmacología , Hierro/química , Metaloproteínas/metabolismo , Metalotioneína/química , Metalotioneína/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Níquel/química , Oxidantes/farmacología , Oxidación-Reducción , Oxígeno/metabolismo , Oxígeno/farmacología , Conformación Proteica , Relación Estructura-Actividad , Reactivos de Sulfhidrilo/metabolismo , Reactivos de Sulfhidrilo/farmacología , Factores de Transcripción/metabolismo , Zinc/químicaRESUMEN
The activity of potato polyphenol oxidase (tyrosinase) toward DL-3,4-dihydroxyphenylalanine (K(M) 5.39 mM) was studied using a variety of carboxylate buffers at a common pH and ionic strength. Enzyme activity, greatest in citrate and least in oxalate, correlated with increasing carboxyl concentration and molecular mass. The lower activity in oxalate was attributed to more effective chelation of a copper(II) form of the enzyme by the oxalate dianion. Sodium halide salts inhibited the enzyme. Although there was little difference in inhibition between sodium and potassium salts, the degree and type of inhibition was anion dependent; K(is), values for NaCl and KCl, (competitive inhibitors) were 1.82 and 1.62 mM, whereas Na(2) SO(4) and K(2) SO(4) (mixed inhibitors) had K(is) and K(ii) values in the 250 to 450 mM range.
Asunto(s)
Ácidos Carboxílicos/farmacología , Catecol Oxidasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Solanum tuberosum/enzimología , Tampones (Química) , Cobre/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Concentración Osmolar , Oxalatos/farmacología , Cloruro de Potasio/farmacología , Cloruro de Sodio/farmacología , Sulfatos/farmacologíaAsunto(s)
Cationes/farmacología , Cisteína/análogos & derivados , Cisteína/química , Metales/farmacología , Nitrógeno/química , Compuestos Nitrosos/química , S-Nitrosotioles , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Sitios de Unión/efectos de los fármacos , Bovinos , Cistina/química , Inhibidores Enzimáticos/farmacología , Enzimas/química , Enzimas/efectos de los fármacos , Humanos , Hidroliasas/química , Hidroliasas/efectos de los fármacos , Hidrogenasas/química , Hidrogenasas/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Mamíferos/sangre , Metales/química , Modelos Moleculares , Oxidación-Reducción , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/efectos de los fármacos , Albúmina Sérica/química , Albúmina Sérica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de Transcripción/química , Factores de Transcripción/efectos de los fármacos , Vanadatos/farmacología , Dedos de Zinc/efectos de los fármacosRESUMEN
The binding of Ni(II) and Cu(II) to histidine, to the tripeptides GlyGlyHis and HisGlyHis, and to the protein bovine serum albumin has been studied by isothermal titration calorimetry (ITC) to determine the experimental conditions and data analysis necessary to reproduce literature values for the binding constants and thermodynamic parameters. From analysis of the ITC data, we find that there are two major considerations for the use of this method to accurately quantify metal ion interaction with biological macromolecules. First, to determine true pH-independent binding constants, ITC data must be corrected for metal ion competition with protons by accounting for the experimental pH and pKa values of the metal-binding residues. Second, metal interaction with the buffer (stability and enthalpy of formation of metal-buffer complex(es)) must be included in the analysis of the ITC data to determine the binding constants and the change in enthalpy. While it may be possible to use a buffer that forms only weak, and therefore negligible, complexes with the metal, a buffer that has a strong and well-characterized interaction has the benefit of suppressing metal ion hydrolysis and precipitation, and of allowing the quantification of high-affinity metal-binding sites on biological macromolecules. This study has also quantified the contribution of the N-terminal imidazole of HisGlyHis to the stability of the Cu(II) and Ni(II) complexes of this protein sequence and has provided new insight about Cu(II) binding to albumin.
Asunto(s)
Calorimetría/métodos , Cobre/metabolismo , Histidina/metabolismo , Níquel/metabolismo , Oligopéptidos/metabolismo , Albúmina Sérica Bovina/metabolismo , Estudios de Evaluación como Asunto , Unión ProteicaRESUMEN
Recent reports indicate that oxidized cobalamin, Cbl(III), can interfere with the biological effects of nitric oxide (NO) on vascular and visceral smooth muscle and in other systems. In attempting to elucidate the mechanism of these effects of Cbl(III), we reported that a Cbl(III)NO complex could be detected by electron paramagnetic resonance (EPR) spectroscopy, but not by ultraviolet/visible spectroscopy. Subsequently, others concluded that the alleged Cbl(III)NO complex is detectable by ultraviolet/visible, but not by EPR spectroscopy and provided ultraviolet/visible evidence for an alleged Cbl(III)NO complex. We report further investigation of the interaction of NO with Cbl, using both techniques, Fourier transform infrared (FTIR) spectroscopy and mass spectrometry. Our EPR results and the UV/VIS results of others appear to be experimental artifacts that can now, at least in part, be explained. Under conditions where FTIR measurements readily detect a N-O stretching frequency of NO bound to Fe(II), we do not detect a similar signal that can be ascribed to either Cbl(III)NO or Cbl(II)NO, indicating that neither Cbl(III) nor Cbl(II) form a stable complex with NO. Loss of the Cbl(II) EPR signal and mass spectral detection of N2O upon addition of NO to Cbl(II) solutions, demonstrates that Cbl(II), which is present in aerobic Cbl(III) solutions, reduces NO; however, this reaction does not appear to be fast enough to account for the observed biological effects in aerated media. Nitric oxide also reacts rapidly and irreversibly with the superoxo complex of Cbl(III), Cbl(III)O2-, which is always present in aerated solutions of Cbl(III). We believe that this latter reaction accounts for the observed inactivation of NO by Cbl(III) in biological systems. Because Cbl(III)O2- is spontaneously regenerated from Cbl(II) and O2 in aerated solutions, this may constitute a cyclic mechanism for the rapid elimination (oxidation) of NO. Thus, several physicochemical techniques fail to provide convincing evidence for the existence of stable Cbl(III)NO or Cbl(II)NO complexes but do provide evidence that Cbl species participate in redox reactions with NO under aerobic conditions, thereby inhibiting its physiological roles.
Asunto(s)
Óxido Nítrico/metabolismo , Vitamina B 12/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Glutatión/metabolismo , Hemoglobinas/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Superóxidos/metabolismoRESUMEN
Toxic and/or carcinogenic consequences may result from metal ion substitution for the Zn-(II) in transcription factors containing zinc fingers, and the small Cys-rich metal-binding protein metallothionein (MT) may play a role in this metal substitution. To begin to evaluate this hypothesis, with regard to the carcinogenic metal ion Ni(II), a peptide corresponding to the third finger of the transcription factor Sp1 (Sp1-3) has been synthesized and its metal binding and redox reactions have been studied. The peptide binds Zn(II), Co(II), and Ni(II), with spectroscopic data indicating a tetrahedral coordination for the latter two; metal ion affinities have been quantified (Kd = 6 (+/- 3) x 10(-10), 3 (+/- 1) x 10(-7), and 4 (+/- 1) x 10(-6), respectively) and found to be less than those of an optimized zinc finger peptide (Krizek, B. A., Merkle, D. L., and Berg, J. M. (1993) Inorg. Chem. 32, 937-940) but greater than those of the second finger of transcription factor IIIA (Berg, J. M., and Merkle, D. L. (1989) J. Am. Chem. Soc. 111, 3759-3761). Reactions of the peptide and its metal-bound forms with dioxygen or hydrogen peroxide did not produce oxygen radical species; however, oxidation of the two Sp1-3 cysteines was modulated by metal ions (Zn < Co = apo < Ni), suggesting a protective role for Zn(II) but an enhancing role for Ni(II). Metal binding competition between Sp1-3 and the alpha domain of human liver MT-2 (alpha-hMT2) indicates a similar affinity for Zn(II). However, alpha-hMT2 has a higher affinity for Ni(II), suggesting that MT may play a protective role by ensuring Zn(II), rather than Ni(II), coordination to zinc finger sequences of transcription factors.
Asunto(s)
Metalotioneína/metabolismo , Níquel/metabolismo , Factor de Transcripción Sp1/química , Dedos de Zinc , Zinc/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oxidación-Reducción , Factor de Transcripción Sp1/metabolismoRESUMEN
Interactions of nitric oxide (NO) with various cobalamin species have been examined, apparently for the first time, with both absorption and electron paramagnetic resonance spectroscopy. Only slight shifts in the absorption spectrum of hydroxocobalamin, B12a [Cb(III)], were produced by NO, but dramatic changes in the spectrum of B12r [Cb(III)] were found on addition of NO. The addition of NO shifted the spectrum of Cb(II) to one very similar to that of Cb(III), indicating the oxidation of Cb(II). The addition of NO to Cb(III) resulted in a novel, weak and previously undescribed electron paramagnetic resonance signal. Although it has not been fully characterized, this appears to represent a reversible complex in which NO is liganded to the Cb(III). When NO was added to Cb(II), its strong electron paramagnetic resonance spectrum was replaced by that of this novel species, consistent with oxidation of Cb(II) by NO and then binding of additional NO by the resulting Cb(III). Porcine, aortic endothelial cells were able to partially reduce Cb(III), and release to the supernatant a previously characterized superoxide cobalt(III) complex, but some Cb(II) remained with the cell fraction. These reactions of Cb species could play a role in altering intracellular and intratissue levels of NO.
Asunto(s)
Hematínicos/metabolismo , Hematínicos/farmacología , Hidroxocobalamina/metabolismo , Hidroxocobalamina/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacología , Vitamina B 12/metabolismo , Vitamina B 12/farmacología , Animales , Aorta/efectos de los fármacos , Borohidruros/farmacología , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Endotelio Vascular/efectos de los fármacos , Concentración de Iones de Hidrógeno , Hidroxocobalamina/química , Óxido Nítrico/química , Oxidación-Reducción , Nitrito de Sodio/farmacología , Espectrofotometría Ultravioleta , Porcinos , Vitamina B 12/químicaRESUMEN
Sodium nitroprusside (Na2[(CN)5FeNO], SNP), which is stable, diamagnetic, and not detectable by electron paramagnetic resonance (EPR) spectroscopy, can be activated by one-electron reduction. The initial product, which retains the five cyanides and is here called penta, has a distinctive EPR signal. Penta spontaneously dissociates the trans-cyanide ligand resulting in a second paramagnetic species called tetra, which has a different and distinctive EPR signal. Tetra is able to transfer its NO ligand to a suitable acceptor, and all four equatorial cyanides subsequently dissociate. However, excess free cyanide shifts the tetra-penta equilibrium in the direction of penta and prevents NO release. This study was an attempt to extend the above results on SNP reduction, which were obtained in a model hemoglobin system, to intact porcine cells by characterizing all EPR-detectable intermediates. When porcine aortic endothelial or smooth muscle cells in culture were incubated under anaerobic conditions with SNP, an EPR spectrum was obtained, which could be resolved into the signal for penta and a signal previously described as a nonheme iron-nitrosyl-sulfur complex, Fe-NOSR. Tetra was not detected. This FeNOSR has some differences in its stability and location from that described by others in activated macrophages. When incubations were carried out under air, penta could not be detected, but a somewhat diminished signal for FeNOSR was still detectable. When incubations were carried out in the presence of excess free cyanide, conditions under which reduced SNP does not nitrosylate hemoglobin, the penta signal became stronger and the FeNOSR signal, though decreased, was still observed. Depletion (95%) of intracellular reduced glutathione in endothelial cells had no effect on the FeNOSR signal strength. We conclude that SNP is activated in porcine endothelial cells by a one-electron reduction to penta, which apparently dissociates its trans-cyanide to form tetra which then goes on to form FeNOSR upon reaction with a membrane-bound thiol. Glutathione is not involved in any of these reactions.
Asunto(s)
Endotelio Vascular/metabolismo , Nitroprusiato/metabolismo , Animales , Células Cultivadas , Cianuros/análisis , Espectroscopía de Resonancia por Spin del Electrón , Endotelio Vascular/citología , Glutatión/metabolismo , Microesferas , Compuestos de Sulfhidrilo/metabolismo , PorcinosRESUMEN
N-Morpholino-N-nitrosoaminoacetonitrile (SIN-1), a nitrovasodilator metabolite of the drug, molsidomine, is widely used in studies on the pharmacology and toxicology of nitric oxide (NO) because solutions of SIN-1 'spontaneously' release NO in a pathway involving molecular oxygen. Preliminary results, however, suggested that SIN-1 could react with hemoglobin in anaerobic solutions to release NO and form NO-hemoglobin. Electron paramagnetic resonance (EPR) studies showed that heme(III) of methemoglobin was not being reduced, thereby not serving as the oxidant in the reaction generating NO-hemoglobin. When anaerobic solutions of SIN-1 and hemoglobin kept in the light and in the dark were compared, substantially more NO-hemoglobin was eventually generated in the dark, indicating that SIN-1 did not undergo photochemical decomposition to NO under the conditions used. Solutions of NO-hemoglobin were equally stable under these same conditions of light and dark. The initial pH (7.0) of stirred, unbuffered solutions of SIN-1 decreased at nearly the same rate whether or not oxygen was present. Anaerobic and aerobic solutions plateaued at the same pH, namely 5.4. Anaerobic solutions of SIN-1 in phosphate buffer, pH 7.4, released NO to the gas phase, where it was identified by trapping it with hemoglobin on agarose beads and deriving the characteristic NO-hemoglobin EPR spectrum. High pressure liquid chromatography revealed the presence of an unknown species with a retention time between that of SIN-1 and molsidomine. Samples from two different lots of SIN-1 contained this impurity which appears to oxidize SIN-1 to products that release NO in the absence of oxygen. This unknown impurity may be unstable toward light.
Asunto(s)
Contaminación de Medicamentos , Molsidomina/análogos & derivados , Óxido Nítrico/metabolismo , Oxígeno/farmacología , Anaerobiosis , Animales , Espectroscopía de Resonancia por Spin del Electrón , Hemoglobinas/efectos de los fármacos , Humanos , Molsidomina/metabolismo , Óxido Nítrico/toxicidad , Conejos , Soluciones/químicaRESUMEN
Nitric oxide (NO) has been discovered recently to be a ubiquitous, endogenous mediator, which is responsible for a variety of normal physiological functions. However, NO also has been implicated in several pathophysiological processes. For example, the pulmonary toxicity of various nitrogen oxides, including NO, found in photochemical smog has been studied for decades; endogenous NO also is associated with bleomycin-induced lung damage, as well as other adverse effects. Recently, a variety of xenobiotics have been shown to owe their biological activity in vivo to their biotransformation to NO. Thus, the therapeutic vasodilatation produced by drugs such as nitroglycerin and sodium nitroprusside is now believed to result from their release of NO, which then mimics the effects of endogenously synthesized NO. The toxic effects of NO prodrugs are, therefore, a matter of concern, especially the extent to which, if any, NO contributes to their toxicity. As reviewed here, NO does not appear to contribute importantly to the toxicity of the NO donors nitrite, hydroxylamine, or nitroprusside. However, it is by no means clear whether or not the NO generated in vivo from sodium azide contributes in a major way to its toxicity. Azide is almost as acutely toxic as cyanide, with which it shares a number of biological effects; yet, azide also has certain cardiovascular actions in common with nitrite. Unlike either cyanide or nitrite, some evidence suggests a tendency for azide to produce low-grade cumulative toxicity. In laboratory animals, azide frequently produces nonasphyxial convulsions, whereas most human deaths appear to be the result of cardiovascular collapse. Neither of these azide-induced syndromes appears to be due to the inhibition of cytochrome c oxidase. Azide is widely used as a preservative in aqueous laboratory reagents and as the propellant in automobile air bags and aircraft escape chutes. Both of these inflable systems are generally safe, and will prevent untold numbers of injuries and deaths. However, to protect workers who handle these devices and others who may come into contact with the sodium azide propellant in these systems, our rudimentary knowledge of azide toxicity needs to be expanded.
Asunto(s)
Azidas/toxicidad , Óxido Nítrico/química , Óxido Nítrico/toxicidad , Xenobióticos/toxicidad , Animales , Azidas/química , Humanos , Nitritos/toxicidad , Nitroprusiato/toxicidadRESUMEN
Nitrovasodilators react with hemoglobin (Hb) to form heme(III) and nitric oxide (NO)Hb. These reactions can be exploited as models for events that take place at the cellular level leading to the biological effects of the prodrugs. Sodium nitroprusside (SNP) is known to undergo a one-electron reduction in its reaction with heme(II), resulting in the labilization of the cyanide ligand trans to the NO ligand. This reduced form is here called "penta." Upon dissociation of the trans-cyanide, the resulting species is here called "tetra." Dissociation of the trans-cyanide is obligatory for transfer of the NO to a heme(II) group. NO release from penta is blocked by excess free cyanide in solution, which prevents the formation of tetra. As reported here, both penta and tetra had unique EPR signals when frozen at -196 degrees, but only tetra gave an EPR signal at 22 degrees. NOHb also has a unique EPR signal, but it could not be detected when SNP was incubated with Hb in air or 10 or 5% oxygen. NOHb was detected in similar incubations under 1% oxygen, but the levels were 3- to 10-fold lower than those found under 100% nitrogen. The concentration of tetra was also much lower under 1% oxygen and penta was not detectable, suggesting that oxygen may either shift the penta-tetra equilibrium towards tetra or that penta may be susceptible to oxidation by molecular oxygen. Nitroglycerin (GTN) also generated much less NOHb but more heme(III) under 1% oxygen than under nitrogen. Carbon monoxide (CO), which binds to heme(II), completely blocked the reactions of SNP and GTN with Hb, whereas N-ethylmaleimide (NEM) alkylation of globin sulfhydryl groups increased both NOHb and heme(III) formation. 13C NMR studies on uniformly 13C-labeled SNP suggested that oxygen had little effect on the concentrations of the NMR-detectable species in the reaction. In summary, the most oxygen-sensitive step in the nitrosylation of Hb by SNP was probably the transfer of NO to heme(II). However, the penta-tetra equilibrium was affected by oxygen, temperature and cyanide. No evidence was found for the involvement of the globin sulfhydryl groups in either the GTN or the SNP reaction with Hb.
Asunto(s)
Hemo/química , Hemoglobinas/química , Nitroglicerina/química , Nitroprusiato/química , Compuestos de Sulfhidrilo/química , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Oxígeno , TemperaturaRESUMEN
Blood specimens were obtained from 30 adult patients admitted to a coronary care unit after the decision to use nitroglycerin had been made by their physicians. The samples were drawn before nitroglycerin administration, within 1 hour after starting nitroglycerin, after several hours of therapy, and more than 4 hours after discontinuing therapy. One patient was admitted twice, accounting for 31 sets of blood specimens. A positive identification of nitric oxide-hemoglobin (NOHb), with electron paramagnetic resonance (EPR) spectroscopy could be made in the blood of 10 of the subjects after they had been receiving nitroglycerin for several hours (third blood sample). In seven subjects this third blood sample was not drawn, and they were dropped from the study. A final positive finding of NOHb was made in 10 of 24 patients. NOHb has not been identified previously in human subjects given nitroglycerin, and a significant dose-response relationship was observed between nitroglycerin and NOHb. We ascribe our inability to detect NOHb in all subjects before nitroglycerin (basal levels) and after nitroglycerin in 14 subjects to concentrations that were below the limits of detection of the technique as used. Subtraction of the EPR signal for plasma ceruloplasmin was necessary to detect the NOHb EPR signals. Thus we have shown EPR spectroscopy to be a highly specific and sensitive method for detecting and quantifying NOHb in human subjects. Further refinements in the technique to improve sensitivity are possible.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hemoglobinas/análisis , Óxido Nítrico/sangre , Nitroglicerina/uso terapéutico , Adulto , Anciano , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nitroglicerina/metabolismoRESUMEN
The Michigan Computer-Graphics Coordinate Measurement System (MCGCMS) was used to determine the dimensional accuracy of dentures processed by three different techniques: conventional heat compression, microwave, and visible-light activation. Standardized dentures were fabricated from casts made in an RTV silicone mold. All casts were duplicated with hydrocolloid and 42 dentures were made (ie, 14 dentures for each technique). The MCGCMS measured 22 points on two frontal planes to compare master casts to dentures. The results showed no significant difference in overall dimensional accuracy. At specific sites, however, the visible-light-activated technique produced significantly more flange distortion than did either the conventional or microwave techniques.
Asunto(s)
Resinas Acrílicas/química , Gráficos por Computador , Bases para Dentadura/normas , Dentadura Completa/normas , Análisis de Varianza , Estudios de Evaluación como Asunto , Calor , Luz , Microondas , Reproducibilidad de los ResultadosRESUMEN
A family is reported in which the father was affected by facioscapulohumeral muscular dystrophy FSHD. One son was affected by Duchenne muscular dystrophy (DMD). The second son died at the age of 3 yr of a severe primary muscle disease and it is suggested that this was the outcome of dual expression of the two conditions.
Asunto(s)
Ligamiento Genético/genética , Distrofias Musculares/genética , Cromosoma X , Adulto , Niño , Preescolar , Músculos Faciales , Femenino , Humanos , Húmero , Masculino , Persona de Mediana Edad , Distrofias Musculares/patología , Linaje , EscápulaRESUMEN
Recent studies using chemiluminescence and spectrophotometry have shown that cultured and native endothelial cells release nitric oxide (NO). Pharmacological and biochemical evidence argue for and against the proposal that endothelium-derived relaxing factor (EDRF) is identical with free NO. In an attempt to identify EDRF as free NO, a bioassay technique was combined with an NO trap (hemoglobin bound to agarose; Ag-Hb), and electron paramagnetic resonance (EPR) spectroscopy was used to detect the resultant nitrosylhemoglobin (NO-Hb). Canine femoral arteries with or without endothelium were perfused with physiological saline solution containing superoxide dismutase and ibuprofen and were stimulated with acetylcholine. The relaxing activity of the effluent was monitored in canine coronary artery rings without endothelium (bioassay tissue) half-maximally contracted with U46619. Acetylcholine stimulated the release of EDRF from intact femoral arteries (but not from segments without endothelium), which relaxed the bioassay tissue by 63 +/- 5%. NO (approximately 1 and approximately 10 nM) infused directly over the bioassay tissue produced 34 +/- 8% and 96 +/- 3% relaxation, respectively (ED50, approximately 2 nM). Effluents were collected under vacuum in the absence of oxygen through a column containing Ag-Hb, and the samples were assayed for NO-Hb by EPR. Samples containing NO produced the triplet EPR signal characteristic of NO-Hb, but the effluent containing EDRF did not. Infusion of NO through the donor tissue in the presence of acetylcholine gave an EPR signal similar to that observed when NO had no contact with the tissue. Nitrite anion (up to 2.7 x 10(-2) M) produced no detectable NO-Hb in analogous experiments. Thus, EDRF released from the native endothelium of canine femoral artery cannot be identified as free NO. The present findings support a concept that EDRF may be a labile precursor of NO.
Asunto(s)
Acetilcolina/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Óxido Nítrico/análisis , Animales , Bioensayo , Vasos Coronarios/efectos de los fármacos , Perros , Arteria Femoral/efectos de los fármacos , Arteria Femoral/metabolismoRESUMEN
Pairs of osmotic minipumps containing 400 mg/ml (6.15 M) sodium azide in distilled water were subcutaneously implanted in timed pregnancy Syrian golden hamsters. The total delivered dose was calculated as 6 X 10(-2) mmol kg-1 hr-1 at the maximal pumping rate. Most dams exhibited obvious signs of toxicity during the period of pump implantation which was Days 7 through 9 of gestation. After removal of the pumps the dams were euthanized on Day 13 of gestation, and the uteri were removed for counting of the number of living, malformed, and resorbed fetuses. This dose rate resulted in a significantly increased incidence of resorptions of embryos over that in a control group implanted with pumps delivering only distilled water. The incidence of gross malformations exclusively in the form of encephaloceles was not different between control and azide-infused groups. The extent of nitrosylation of circulating hemoglobin was followed with time and found to involve only about 0.1% of the total blood pigment. Thus, this commercially important and widely distributed chemical with high acute toxicity is not considered to be teratogenic in hamsters, and it produces embryotoxicity only at dose rates that result in toxic signs in the dams.