The interaction of yeast Complex III with some respiratory inhibitors.

We have examined the effects of eight inhibitors of the bovine-heart mitochondrial Complex III on the catalytic activity of the analogous complex from yeast mitochondria. All eight compounds were inhibitory, with potent inhibition being obtained with antimycin, myxothiazol and UHDBT (5-N-undecyl-6-hydroxy-4,7-dioxobenzothiazole). These three inhibitors, and also funiculosin, have been further studied by characterizing their effects on the visible absorbance, magnetic circular dichroism and EPR spectra of the complex and also on the potentiometric properties of the individual metal centers present in the complex. All four inhibitors had little or no effect on either the absorbance or magnetic circular dichroism spectra. Funiculosin produced a change in the EPR lineshape of the iron-sulfur cluster; EPR spectra recorded at 12 K also revealed complete reduction of cytochrome b-562 by ascorbate. UHDBT also changed the lineshape of the iron-sulfur cluster and this change could be partially reversed by myxothiazol. Neither antimycin nor myxothiazol affected the iron-sulfur cluster and produced only small changes in the EPR absorption envelope of the b cytochromes. Both funiculosin and UHDBT raised the midpoint potential of the iron-sulfur cluster, by about 150 and 70 mV, respectively. Only UHDBT changed the potential of c1, lowering it by about 30 mV. Funiculosin raised the potential of b-562 by about 30 mV, while myxothiazol had no effect; the other two compounds produced only small changes. All four compounds had only small effects on the midpoint potential of b-566. The relative contributions of the two b cytochromes to the magnetic circular dichroism amplitudes could be changed by the addition of inhibitors, even though the absolute magnetic circular dichroism spectra of oxidized and reduced complex were unaffected.

1H-NMR heme resonance assignments by selective deuteration in low-spin complexes of ferric hemoglobin A.

The heme methyl and vinyl alpha-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole pi bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.

The effect of nimodipine on high-energy phosphates and intracellular pH during cerebral ischemia.

Experimental and clinical studies suggest that the calcium channel blocker nimodipine may reduce cerebral ischemic injury. Using rapid acquisition phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy, we examined the effect of nimodipine on cerebral energy metabolism during severe ischemia in gerbils. High-energy phosphates and intracellular pH were characterized at baseline and at 2-min intervals following bilateral common carotid artery (CCA) ligation. Serial forebrain spectroscopy was continued until phosphocreatine (PCr) and adenosine triphosphate (ATP) resonances disappeared. Controls (n = 10) were compared to gerbils receiving intraperitoneal nimodipine 30 min prior to carotid ligation, at the following doses: 0.5 mg/kg (n = 8), 1.0 mg/kg (n = 10), 2.0 mg/kg (n = 8), or 4.0 mg/kg (n = 4). In the control group, PCr and ATP peaks were undetectable after a mean of 5.4 +/- 0.47 min following CCA ligation. Compared with controls, the mean time for depletion of high-energy phosphates following carotid ligation was prolonged at nimodipine doses of 0.5 mg/kg and 1.0 mg/kg, but the differences did not reach statistical significance. In the 2.0 mg/kg group, however, ATP was preserved until 9.8 +/- 1.0 min following the onset of ischemia, significantly longer than the control group (p = 0.005, Mann-Whitney test). Nimodipine had no effect on the time course or severity of intracellular acidosis. In this model of severe ischemia, relatively high doses of nimodipine slowed the depletion of high-energy phosphates without altering intracellular acidosis. This suggests that nimodipine may provide cerebral protection by directly altering ischemic cellular metabolism.

Dynamics of fluid/particulate mixtures in tube flow.

Magnetic resonance imaging (MRI) techniques have been utilized to experimentally measure the velocity profile of fluid/particulate mixtures as a function of flow rate, particle loading, and particle size. The experimental velocity profiles in tube flow were described by a power law model; the power law parameter decreased as flow rate, particle loading, and particle size increase. This work is relevant to aseptic processing of particulate foods.

Nuclear magnetic resonance as a noninvasive method of diagnosing intestinal ischemia: technique and preliminary results.

Despite the long-standing effort to identify a noninvasive method of diagnosing intestinal ischemia, no reliable biochemical or radiographic technique has evolved. We explored the use of phosphorus nuclear magnetic resonance (PNMR) as a method of detecting surgically induced intestinal ischemia. Using Lewis strain rats (250 to 300 g), small intestine ischemia was produced by ligation in succession from the ligament of Treitz to the ileocecal valve 1 of 2 (group I), 2 of 3 (group II), 3 of 4 (group III), and 4 of 5 (group IV) mesenteric terminal vessels. A sham-operated group was used as a control. Following the surgical procedure, the abdomen was closed and the rat positioned under the PNMR apparatus. Using phosphorus spectroscopy, data were analyzed using a computer program and plotted on a graph indicating relative peaks for the phosphate-based compounds. As a means of comparing groups, we devised an inorganic phosphate to phosphocreatine ratio ("ischemia index"), a qualitative measurement indicating trends used to evaluate ischemia. At the completion of the PNMR study, the abdomen was reopened and proximal, mid, and distal small intestine segments were harvested for histological evaluation using a previously established grading system for intestinal ischemia. Preoperatively, immediately postoperatively, and approximately 2 hours postoperatively, blood samples were obtained for hexosaminidase levels. With increasing vascular ligation, there was an upward trend in both the histological appearance of ischemia and the PNMR ischemia index indicative of increasing tissue ischemia. A similar trend was identified when the histological ischemia grade was directly correlated with the PNMR ischemia index. Hexosaminidase levels did not correlate with ischemia in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of gentamicin and ethacrynic acid on mammalian microsomal protein synthesis.

The ototoxic antibiotic gentamicin, and the ototoxic diuretic ethacrynic acid, both produce inhibitory effects on protein synthesis in microsomes isolated from rat brain. Inhibitory effects appear to be essentially independent of each other. The inhibitory dose-response curve for gentamicin is logarithmic, while that for ethacrynic acid is linear. The dose-response curves make gentamicin the predominant inhibitor when the drugs are combined at low concentrations and ethacrynic acid the predominant inhibitor when the drugs are combined at high concentrations. Accumulation of aminoglycoside by tissues of the inner ear may result in inhibition of protein synthesis in spite of low and transient plasma levels of the drug. Further inhibition of translation by ethacrynic acid could account, in part, for the ototoxic interaction of aminoglycosides and high ceiling diuretics.

The kinetics of reduction of yeast complex III by a substrate analog.

The kinetics of reduction of the cytochrome and quinone constituents of yeast complex III by the substrate homolog Q1H2 have been measured under a variety of conditions. The maximum rates of reduction of cytochromes b and c1 and of the endogenous Q6 by Q1H2 were sufficiently fast to support the Vmax for the reduction of cytochrome c by this substrate. The absorbance at 562 nm showed an initial increase which was subsequently followed by a decrease. This decrease was synchronous with the appearance of reduced cytochrome c1 and is interpreted as reflecting the absorbance contribution of c1 at 562 nm under conditions where the steady state level of the b cytochromes is constant. Prereduction of c1 and the Fe/S cluster did not affect the initial very rapid reduction of b, but the second phase was eliminated. Antimycin abolished the very rapid rate of reduction of cytochrome b in untreated complex III and completely inhibited the reduction of cytochrome b in complex III in which c1 and the Fe/S cluster had been prereduced. However, the reduction of the endogenous quinone was essentially unaffected by these treatments. Antimycin had no effect on the reduction of c1. Funiculosin also suppressed the very rapid reduction of b while both myxothiazol and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole did not modify this phase of the reaction; no secondary decrease in absorbance was observed in the presence of any of these inhibitors. Most of the observed kinetic changes could be reproduced by simulation of the Q-cycle; simple linear and branched schemes were unable to reproduce the data.

Redox changes in coenzyme Q in the millisecond time range: an approach using rapid quenching and high-performance liquid chromatography.

We have combined a rapid-quenching protocol with HPLC analysis to measure the kinetics of reduction of coenzyme Q in a mitochondrial enzyme complex. The method has a time resolution of several milliseconds and will readily measure 1-20 nmol of the Q derivatives under investigation. By changing the HPLC solvent, either Q6 or Q10 can be studied.

Inhibition of mammalian microsomal protein synthesis by aminoglycoside antibiotics.

The aminoglycoside antibiotics gentamicin, kanamycin and netilmicin produce a dose-dependent inhibition of amino acid incorporation in microsomes isolated from human liver and rat brain, kidney and liver. Inhibitory effects on microsomal protein synthesis occur at concentrations that have been shown to accumulate in rodent and human renal cortex and perilymph following therapeutic administration. Inhibition of translation in those tissues which specifically accumulate aminoglycoside antibiotics may, in part, explain toxicities observed following exposure to aminoglycosides.

Proton NMR characterization of isomeric sulfmyoglobins: preparation, interconversion, reactivity patterns, and structural features.

The preparations of sulfmyoglobin (sulf-Mb) by standard procedures have been found heterogeneous by 1H NMR spectroscopy. Presented here are the results of a comprehensive study of the factors that influence the selection among the three dominant isomeric forms of sperm whale sulf-Mb and their resulting detailed optical and 1H NMR properties as related to their detectability and structural properties of the heme pocket. A single isomer is formed initially in the deoxy state; further treatment in any desired oxidation/ligation state can yield two other major isomers. Acid catalysis and chromatography facilitate formation of a second isomer, particularly in the high-spin state. At neutral pH, a third isomer is formed by a first-order process. The processes that alter oxidation/ligation state are found to be reversible and are judged to affect only the metal center, but the three isomeric sulf-Mbs are found to exhibit significantly different ligand affinity and chemical stability. The present results allow, for the first time, a rational approach for preparing a given isomeric sulf-Mb in an optimally pure state for subsequent characterization by other techniques. While optical spectroscopy can distinguish the alkaline forms, only 1H NMR clearly distinguishes all three ferric isomers. The ring current shifts in the carbonyl complexes of reduced sulf-Mb complexes support saturation for a pyrrole in each isomer. The hyperfine shift patterns in the various oxidation/spin states of sulf-Mbs indicate relatively small structural alteration, and the proximal and distal sides of the heme suggest that peripheral electronic effects are responsible for the differentially reduced ligand affinities for the three isomeric sulf-Mbs. The first 1H NMR spectra of sulfhemoglobins are presented, which indicate a structure similar to that of the initially formed sulf-Mb isomer but also suggest the presence of a similar molecular heterogeneity as found for sulf-Mb, albeit to a smaller extent.

Nuclear magnetic resonance as a measure of cerebral metabolism: effects of hypertonic saline resuscitation.

UNLABELLED: Fears of central nervous system dysfunction from acute hypernatremia and hyperosmolarity with hypertonic saline resuscitation are often cited. We used high-energy phosphate nuclear magnetic resonance to investigate resuscitation effects on cerebral metabolism. Rats were instrumented for hemodynamic monitoring and fluid infusion and a phosphorus surface coil placed on their skulls. After shimming, baseline spectra were obtained. Animals were then bled for one hour to a mean arterial pressure (MAP) of 45 mm Hg, followed by resuscitation for one hour to a MAP of 75 mm Hg with lactated Ringer's (LR, n = 17) or 7.5% hypertonic saline (HS, n = 25). Spectra were obtained again and analyzed for the ratio of high-energy phosphocreatine (PCr) to low-energy inorganic phosphate (Pi). Intracellular hydrogen ion concentration [H+] was calculated from the PCr/Pi shift. [table: see text] CONCLUSIONS: (1) Hypertonic saline results in a decreased intracellular pH compared with LR without associated changes in high-energy phosphate metabolism. (2) Decreases in pH may be the result of cell dehydration rather than metabolic dysfunction.