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Biomedical subjects

C C Cunningham

Publications and source records attributed to C C Cunningham.

At least 73 records · Page 4Linked to original sources
PubMed1986

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside.

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.

Animals↗
PubMed1986

Effect of phospholipids on the catalytic subunits of the mitochondrial F0.F1-ATPase.

Beef heart mitochondrial F0.F1-ATPase was reconstituted into phospholipid liposomes using the octylglucoside solubilization, discontinuous sucrose gradient centrifugation procedure described in the preceding manuscript (Laird, D., Smith Eble, K., and Cunningham, C. (1986) J. Biol. Chem. 261, 14844-14850). The influence of individual phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), and diphosphatidylglycerol (DPG)) on the kinetic parameters related to ATPase activity were investigated. The specific activities for the PC, PE, and DPG reconstituted preparations were 9.8, 6.8, and 7.6 mumol of ATP hydrolyzed per min/mg of protein, respectively. The F0.F1-DPG complex demonstrated a 40% decrease in the Km for ATP. Both the F0.F1-PC and the F0.F1-PE complexes exhibited Ki values for adenyl-5'-yl imidodiphosphate and guanyl-5'-yl imidodiphosphate approximately 2.5 times lower than those obtained in the absence of exogenous phospholipid. The F0.F1-DPG complex displayed Ki values 11.7- and 1.8-fold lower for adenyl-5'-yl imidodiphosphate and guanyl-5'-yl imidodiphosphate, respectively, as compared to the lipid-depleted enzyme. The phospholipids with which F0.F1 were reconstituted also influenced the ATP-induced decrease in the fluorescence of enzyme-associated aurovertin. The rate of the ATP-elicited decrease in aurovertin fluorescence was accelerated in the presence of all three phospholipids with DPG having the most dramatic effect; the t1/2 for maximal decrease in aurovertin fluorescence was 4.3 s for lipid-deficient enzyme and 0.48 s with the F0.F1-DPG complex. The effects of phospholipids on these parameters associated with the catalytic center of the ATPase suggest that phospholipids can modulate catalytic events occurring in F1. In the intact mitochondrion the primary role of phospholipids may be to stabilize conformations of the enzyme consistent with its range of activities.

Animals↗
PubMed1986

The metabolic state of the rat liver in vivo measured by 31P-NMR spectroscopy.

Previous 31P nuclear magnetic resonance (NMR) studies have measured the concentrations of phosphates, free Mg2+, pH and flux through enzyme-catalyzed reactions in a variety of tissues. A surgically-implanted coil has been developed to measure these parameters in the rat liver in vivo, and to assess the effect of external perturbations on the concentrations and physiological environment of phosphorus metabolities in the liver. The sensitive volume and optimal pulse were determined for the coil, which was insulated to exclude signal from surrounding tissues. The metabolic stability of the liver during acquisition of spectra was demonstrated by normal values for [Pi], [ATP], [lactate], and [pyruvate] in livers which were freeze-clamped immediately after completion of the NMR experiment. The stability was also confirmed by constant values for intracellular pH (7.2), free [Mg2+] (0.7 mM), and NMR detectable [Pi]/[ATP]. The sensitivity of the 31P-NMR spectrum of the liver in vivo to the physiological state of the animals was illustrated by comparing spectra from fed and 48 h fasted rats. The major qualitative differences were an increase in the pyridine nucleotide/adenine nucleotide ratio, and a small, but consistent shift in the frequency of the composite phosphomonoester peak. The spin-lattice relaxation time of each major phosphate resonance was measured in vivo using a modified homospoil saturation recovery pulse sequence; the T1 of ATP gamma-phosphate was 0.17 s. Selective saturation experiments did not detect magnetization transfer between the ATP gamma-phosphate and inorganic phosphate.

Adenine Nucleotides↗
PubMed1986

Effect of fasting and acute ethanol administration on the energy state of in vivo liver as measured by 31P-NMR spectroscopy.

The effects of 48 h fasting, administration of ethanol or 2,4-dinitrophenol, on the phosphorus-containing metabolites in liver in vivo have been determined utilizing 31P nuclear magnetic resonance spectroscopy. These measurements were combined with determinations of metabolite concentrations in livers which were freeze-clamped immediately after the NMR measurements were completed. Administration of sub-lethal amounts of dinitrophenol dramatically decreased ATP and increased Pi concentrations in liver in vivo as indicated by a 2.7-fold increase in the NMR-derived [Pi]/[ATP] ratio. Ethanol administration to fed animals increased the NMR-derived [Pi]/[ATP] ratio 27%; in contrast, the same amount of ethanol administered to fasted animals decreased the NMR-derived [Pi]/[ATP] ratio 30%. The NMR visible Pi and ADP represent about 50% and 15% of the total Pi and ADP, respectively. The phosphorylation potentials calculated from the NMR visible Pi and ADP were an order of magnitude higher than those obtained from metabolite concentrations in freeze-clamped tissue. There was no apparent correlation between the phosphorylation potentials derived from either the NMR spectral analyses or from metabolite concentrations and the hepatic [NAD+]/[NADH] ratio. The chemical shift of Pi indicated that ethanol administration elicited a decrease in pH of 0.1 unit in liver in vivo. Hepatic free [Mg2+] was increased 21% in fasted animals, but was unaffected by ethanol administration.

Acetoacetates↗
PubMed1986

The effect of intensity of training on sensori-motor development in infants with Down's syndrome.

Twenty-four children with Down's syndrome involved in an early intervention programme were divided into matched intensive training (ITG) and control groups (CG), at a mean age of 42 weeks. Parents of children in the ITG were given exercises to carry out daily to train object permanence, imitation and span of attention. Parents of children on the CG were given general advice. All children were assessed on check lists in the three areas and on standard developmental tests. Results showed small short-term effects in favour of the ITG during the training but no long-term effects on development. It was suggested that the short-term effects were mainly due to improved stability of performance in the ITG.

Attention↗
PubMed1986

Use of nuclear magnetic resonance spectroscopy to study the effects of ethanol consumption on liver metabolism and pathology.

The application of NMR spectroscopy to studies of intact organs represents a powerful technology applicable for investigations of alcohol-induced liver disease in animal models. NMR-assisted studies of ethanol metabolism and its related pathology are now possible utilizing the 13C and 31P nuclei primarily. The major advantage of the technology includes the capacity to investigate biochemical processes at the level of the intact organ which is analyzed as a perfused preparation or in situ in an anesthetized animal model. Quantitative measurements of compounds containing either 31P or 13C nuclei are possible, and the kinetics of precursor incorporation into compounds associated with carbohydrate, lipid, and amino acid metabolism are readily followed. The concentrations of free Mg2+ and the pH of the intact liver can be monitored as a function of external perturbation and/or metabolic disturbances.

Adenine Nucleotides↗
PubMed1985

Phospholipid association with the bovine cardiac mitochondrial adenosine triphosphatase.

The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility.

Adenosine Triphosphatases↗
PubMed1985

The effects of mentally handicapped children on families--a conceptual review.

Recent research which examines the effects of mentally handicapped children upon families is reviewed. The studies are grouped into three categories based on the underlying conceptions which appear to guide them. The first category examines which families are most vulnerable to the presumed stress of a mentally handicapped child. The second category emphasises the material and practical problems families experience. The third category stresses the competence of families and describes the resources used to develop coping strategies. Links are drawn between these three categories and to parallel developments in related areas, and implications for future research are discussed.

Adaptation, Psychological↗
PubMed1985

Mental ability, symbolic play and receptive and expressive language of young children with Down's syndrome.

The relationship between measures of mental ability, symbolic play and expressive and receptive language was investigated in a sample of 73 children with Down's syndrome (mean CA 48.9 months, range 19-90 months; mean MA 30.4 months, range 13-73 months). Language delay became increasingly apparent with age, and more so in boys than girls. There was no significant difference between mental ages derived from standard ability tests and corresponding age equivalent scores of the Lowe and Costello symbolic play test. A developmental progression of play similar to that seen in non-handicapped groups was found. However, it is suggested that the use of a more detailed scoring system would be informative.

Child↗
PubMed1984

Selective auditory preferences and the use of automated equipment by severely, profoundly and multiply handicapped children.

Using automated equipment, contingent responding and auditory preferences were examined in 15 severely, profoundly and multiply handicapped children (CMAs 3-20 months) and 2 non-handicapped children at ages 5.0 and 9.5 months. 15 children showed contingent responding; prolonged responding was shown by the profoundly handicapped group. Where auditory preferences were apparent, they were the same as those seen in Down's syndrome and non-handicapped groups at the same developmental level. Children at the 5 months developmental level and less, had difficulty in making choice responses. Implications for intervention are discussed.

Association Learning↗
PubMed1984

Down's syndrome: is dissatisfaction with disclosure of diagnosis inevitable?

A survey of a representative sample of 62 parents of Down's syndrome infants revealed a high level (58 per cent) of dissatisfaction with the procedure for disclosing the diagnosis and with the immediate counselling services. From the replies and previous reports, a 'model procedure' was set up in one Area Health Authority to test the hypothesis that dissatisfaction is inevitable. Parents receiving the 'model procedure' reported 100 per cent satisfaction, compared with 20 per cent in a control group. It was concluded that dissatisfaction is not inevitable but results directly from organisational difficulties in not providing the necessary service.

Adaptation, Psychological↗
PubMed1983

Ethanol-related changes in liver microsomes and mitochondria from the monkey, Macaca fascicularis.

Four Macaca fascicularis monkeys were maintained 1 year on a liquid diet containing 26% of calories as ethanol. Four control animals were fed a liquid diet of equivalent calories with protein, carbohydrate, and fat being substituted for ethanol calories. In liver mitochondria prepared from ethanol-fed monkeys (ethanol mitochondria), respiratory control was lowered 20% due to a decrease in state 3 respiration (28%). This was also accompanied by a 20% decrease in ADP translocation into ethanol mitochondria. The major change was a 61% decrease in cytochrome oxidase activity. The respiratory rate in the presence of uncoupler was also lowered 14%, but the decrease was not statistically significant. In contrast with our earlier observations with Macaca nemestrina, no significant ethanol-induced changes were observed in enzyme activities associated with the microsomal electron transport system, and no ethanol-elicited fatty liver was evident. The major changes in fatty acid composition of microsomal and mitochondrial phospholipids were increased amounts of palmitoleic and oleic acids, and decreased amounts of linoleic and arachidonic acids.

Animals↗
PubMed1982

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils.

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.

Cytochrome b Group↗
PubMed1982

Effect of ethanol consumption on the phospholipid composition of rat liver microsomes and mitochondria.

Male Sprague-Dawley rats were maintained for 31 days on a liquid diet containing 36% of calories as ethanol. Pair-fed controls were administered a similar diet, but with maltose-dextrin isocalorically substituted for ethanol. A phospholipid analysis has been carried out in liver microsomes and mitochondria isolated from the two groups of animals. The phospholipid phosphorus/protein ratio was not significantly different in the organelles of the ethanol-fed animals as compared to the same organelles of liquid diet controls, which indicates that ethanol feeding did not influence the total phospholipid content of microsomes and mitochondria. The phospholipid distribution within organelles was not changed, except for a significant increase in the phosphatidylinositol content of microsomes from ethanol-fed animals. The fatty acid compositions of both microsomal and mitochondrial phospholipids were significantly altered by ethanol feeding. In microsomes from ethanol-fed rats, palmitic acid levels were lowered in the total phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine; oleic acid levels were elevated in microsomal phosphatidylethanolamine. In mitochondria from ethanol-fed animals, palmitic and arachidonic acid were lowered in phosphatidylcholine and phosphatidylethanolamine. Oleic and linoleic acid were elevated in the same phospholipids. In contrast, linoleic acid levels in cardiolipin were depressed significantly. These alterations in the fatty acid composition are suggestive of ethanol-induced changes in fatty acid desaturation activities.

Animals↗
PubMed1982

Control of adenine nucleotide metabolism in hepatic mitochondria from rats with ethanol-induced fatty liver.

Male rats developed fatty liver after being fed on an ethanol-containing diet for 31 days. Liver mitochondria from these animals catalysed ATP synthesis at a slower rate when compared with mitochondria from pair-fed control rats (control mitochondria), and demonstrated lowered respiratory control with succinate as substrate, owing to a decrease in the State-3 respiratory rate. Respiration in the presence of uncoupler was comparable in mitochondria from both groups of rats. Translocation of both ATP and ADP was decreased in mitochondria from ethanol-fed rats, with ADP uptake being lowered more dramatically by ethanol feeding. Parameters influencing adenine nucleotide translocation were investigated in mitochondria from ethanol-fed rats. Experiments performed suggested that lowered adenine nucleotide translocation in these mitochondria is not the result of inhibition of the translocase by either long-chain acyl-CoA derivatives or unesterified fatty acids. Analysis of endogenous adenine nucleotides in these mitochondria revealed lowered ATP concentrations, but no decrease in total adenine nucleotides. In experiments where the endogenous ATP in these mitochondria was shifted to higher concentrations by incubation with oxidizable substrates or defatted bovine serum albumin, the rate of ADP translocation was increased, with a linear correlation being observed between endogenous ATP concentrations and the rate of ADP translocation. The depressed ATP concentration in mitochondria from ethanol-fed rats suggests that the ATP synthetase complex is replenishing endogenous ATP at a slower rate. The lowered ATPase activity of the ATP synthetase observed in submitochondrial particles from ethanol-fed animals suggests a decrease in the function of the synthetase complex. A decrease in the rate of ATP synthesis in mitochondria from ethanol-fed rats is sufficient to explain the decreased ADP translocation and State-3 respiration.

Adenosine Diphosphate↗
PubMed1982

Negatively charged phospholipid requirement of the oligomycin-sensitive mitochondrial ATPase.

The highly-purified, oligomycin-sensitive mitochondrial adenosine triphosphatase has been reconstituted with phosphatidylserine. Treatment of the phosphatidylserine-reconstituted ATPase with phosphatidylserine decarboxylase produced a 3-fold decrease in the specific activity of the resulting phosphatidylethanolamine-enriched ATpase complex. Subsequent control experiments indicated that the resulting phosphatidylethanolamine was responsible for the lowered ATPase specific activity. These observations indicate that acidic phospholips do more than facilitate and interaction between the highly-purified, lipid-depleted ATPase and phospholipid. The negatively charged phospholipid appears to be essential for maintaining high levels of oligomycin-sensitive activity even after the initial interaction between phospholipid and the ATPase complex has occurred.

Adenosine Triphosphatases↗