PubMed Health⌕ Search

Biomedical subjects

G C Ford

Publications and source records attributed to G C Ford.

At least 19 recordsLinked to original sources
PubMed1992

Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86).

The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C.

Bacillus↗
PubMed1991

The location of exon boundaries in the multimeric iron-storage protein ferritin.

The nature of the amino acids whose codons border introns in ferritin genes is novel; the disposition of these intron boundaries within the three-dimensional structure of the 24-subunit molecule differs significantly from that of other proteins. These observations are discussed in relation to the functions of isoferritins.

Amino Acid Sequence↗
PubMed1990

Influence of glucagon on protein and leucine metabolism: a study in fasting man with induced insulin resistance.

The counter-regulatory hormones, including glucagon, may be involved in the generation of postoperative negative nitrogen balance. We examined the influence of glucagon on whole body and forearm muscle protein kinetics, determined by L-[1-13C, 15N]leucine, in two matched groups of healthy fasting subjects. In one study somatostatin alone was infused continuously (0.12 mg h-1) and in another with glucagon (0.04 mg h-1) to generate insulin resistance. Somatostatin infusion increased leucine oxidation (P less than 0.05) and reduced the negative protein balance (P less than 0.01) across the forearm; the 15 per cent decrease in protein breakdown was not significant. Whole body leucine kinetics showed increased flux (P less than 0.05) and synthesis (P less than 0.01) but reduced oxidation (P less than 0.05). Hyperglucagonaemia caused a threefold enhancement of leucine oxidation (P less than 0.02), while the negative protein balance further increased (P less than 0.05) across the forearm. Whole body leucine flux was unchanged; oxidation increased (P less than 0.01) and synthesis decreased (P less than 0.01). These studies confirm that physiological hyperglucagonaemia during insulin resistance is catabolic in the short-term and indicates, for the first time, that glucagon may influence muscle protein metabolism acutely in man. We suggest that therapeutic manoeuvres designed to reduce glucagon levels after surgery may ameliorate protein kinetic abnormalities.

Adult↗
PubMed1990

Sources of propionate in inborn errors of propionate metabolism.

Amino acids are widely regarded as the most important sources of propionate in disorders of propionate metabolism. Propionate production was measured in the fasting state by continuous infusion of sodium [1-13C]propionate in three children with methylmalonic acidemia (MMA) and three with propionic acidemia (PA). The contribution of isoleucine, valine, threonine, and methionine catabolism to total propionate production was estimated by extrapolation from the hydroxylation of phenylalanine determined by a continuous-infusion [2H5]phenylalanine technique. The contribution of gut bacterial propionate production was determined by measuring total propionate production before and after treatment with oral metronidazole (10 to 20 mg/kg/d for 1 week). Amino acid catabolism accounted for a mean of 51.7% (range, 24.5% to 66.4%) of total propionate production. The mean decrease in propionate production after metronidazole was 22.2% +/- 8.5 (P less than .02); this percentage is likely to represent the minimum propionate production attributable to gut bacteria. Approximately 30% of total propionate production was unaccounted for, and is likely to arise primarily from odd-chain fatty acid catabolism in the fasting state. These results indicate that sources of propionate other than from protein catabolism are important in disorders of propionate metabolism, and explain the generally disappointing response to dietary protein restriction.

Amino Acids↗
PubMed1990

Aspects of protein metabolism after elective surgery in patients receiving constant nutritional support.

1. The present study was designed in an attempt to resolve conflicting views currently in the literature relating to the effect of surgery on various aspects of protein metabolism. 2. Sequential post-operative (2, 4 and 6 days) changes in whole-body protein turnover, forearm arteriovenous difference of plasma amino acids, glucose, lactate and free fatty acids, muscle concentration of free amino acids, RNA and protein, urinary nitrogen and 3-methylhistidine, plasma concentrations of insulin, cortisol and growth hormone, and resting metabolic rate, were measured in six patients undergoing uncomplicated elective total abdominal hysterectomy. 3. All patients received a constant daily diet, either orally or intravenously, based on 0.1 g of nitrogen/kg and an energy content of 1.1 times the resting metabolic rate for 7 days before and 6 days after surgery. 4. Whole-body protein turnover, synthesis and breakdown increased significantly 2 days after surgery (P less than 0.05) and returned towards pre-operative levels thereafter. 5. Forearm release of branched-chain amino acids and alanine, and efflux of glucose and lactate, were enhanced 4 days after surgery (P less than 0.05). Muscle glutamine and alanine concentrations were decreased on the fourth and sixth days after surgery (P less than 0.05). The RNA/protein ratio (indicating the capacity for protein synthesis) was unaltered. 6. A significant increase in urinary nitrogen and 3-methylhistidine was observed on days 3 and 4 after surgery (P less than 0.05). Thereafter, these parameters remained elevated, although failing to reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Effect of general anaesthesia on whole body protein turnover in patients undergoing elective surgery.

To determine if general anaesthesia alone or in conjunction with surgery alters body protein turnover, we studied six healthy, unpremedicated females undergoing elective total abdominal hysterectomy. Changes in protein metabolism, synthesis and breakdown were estimated by an isotope dilution technique using a continuous infusion of the stable isotope tracer, L-[1-13C]leucine, before anaesthesia (4 h), during anaesthesia alone (1 h), during anaesthesia and surgery (1 h) and in the recovery period (2 h). General anaesthesia comprised thiopentone, pancuronium, enflurane (1 MAC) and oxygen-enriched air. An isotopic steady state in plasma 13C-alpha-ketoisocaproate (13C alpha-KIC) and expired 13C-carbon dioxide were obtained during the four periods. Collections of plasma and expired air were made during the steady state periods and plasma alpha-KIC enrichment measured to indicate precursor pool labelling from which leucine flux (equal to protein breakdown in the post-absorptive state) and oxidation were calculated, and whole body protein synthesis was derived. Whole body protein breakdown did not change with anaesthesia, but decreased with both surgery and during the acute recovery period (P less than 0.05). Protein synthesis did not change with anaesthesia and surgery, but decreased significantly after surgery (P less than 0.05).

Adult↗
PubMed1989

Molecular size and symmetry of the bacterioferritin of Escherichia coli. X-ray crystallographic characterization of four crystal forms.

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A.

Bacterial Proteins↗
PubMed1989

Practical considerations in the use of stable isotope labelled compounds as tracers in clinical studies.

Increasingly widespread usage of stable isotope tracers to aid clinical diagnosis and support basic research has stemmed from both advances in mass spectrometry and the availability of competitively priced labelled compounds. Stable isotopes have been used generally to investigate normal and abnormal metabolic pathways, to estimate energy expenditure and body composition and to quantitate substrate flux and oxidation rates. Despite the fact that the underlying principles relating to the use of stable isotopes for in vivo studies are straightforward, careful consideration must be given to all aspects of human studies. This review highlights some of these, including choice of label and tracer molecule, mode of tracer administration and sampling site, analytical instrumentation, interpretation of data and ethical constraints.

Carbon Isotopes↗
PubMed1989

Substrate disposal in metabolic disease: a comparison between rates of in vivo propionate oxidation and urinary metabolite excretion in children with methylmalonic acidemia.

The relative importance of endogenous metabolism and urinary metabolite excretion was assessed in vivo in six children with methylmalonic acidemia by examining the kinetics of the immediate precursor to methylmalonate, propionate. Total production and oxidation of propionate were measured by means of a continuous infusion of (1-13C)propionate and were compared with the urinary excretion of propionate metabolites. Propionate oxidation was substantial (mean 48.9 mumol/kg/hr +/- SD 18.0) and, in four children, exceeded urinary metabolite excretion (mean urinary excretion in all subjects 40 mumol/kg/hr +/- 25). The sum of urinary excretion and oxidation rates (88 mumol/kg/hr +/- 29) approximated the total propionate production (93.4 +/- 37.0), suggesting that these routes together constitute the major mechanisms of propionate disposal. These results suggest that propionate oxidation is an important route of disposal in methylmalonic acidemia. Variations in the relative proportions of propionate disposal through oxidation and urinary excretion may be one reason for the often poor correlation between clinical status and urinary metabolite excretion. Measurement of urinary metabolite concentration alone may not always reflect clinical status and responses to treatment accurately.

Humans↗
PubMed1989

Rapid measurement of whole body and forearm protein turnover using a [2H5]phenylalanine model.

Whole body protein turnover was measured in six normal adults using a model based on a primed constant infusion of [2H5]phenylalanine and, independently, by an established method of a primed constant infusion of [1-13C]leucine. Isotopic plateau in plasma was achieved within 2 h for [2H5]phenylalanine and, in four of the subjects who received a priming dose of [2H4]tyrosine, for [2H4]tyrosine. In all subjects whole body protein turnover measured with the phenylalanine model (mean protein synthesis, 2.65 +/- (SD) 0.16 g.kg-1.24 h-1; catabolism, 3.58 +/- 0.26 g.kg-1.24 h-1) was similar to that measured using the leucine model (synthesis, 3.09 +/- 0.27 g.kg-1.24 h-1; catabolism, 3.70 +/- 0.35 g.kg-1.24 h-1). Mean forearm fractional muscle protein synthesis calculated by the phenylalanine model was 0.06 +/- 0.03%/h, which compares closely with literature values derived by other methods. The phenylalanine model allows the rapid assessment of whole body and muscle protein turnover from plasma samples alone, obviating the need for measurement of expired air CO2 production or enrichment.

Adult↗
PubMed1989

Effect of triiodothyronine on leucine kinetics, metabolic rate, glucose concentration and insulin secretion rate during two weeks of fasting in obese women.

We investigated the effect of the fasting-induced reduction in triiodothyronine (T3) on metabolic changes that occur during fasting. In five obese women (T3 group) the serum T3 level during a 2200 kcal/day diet was 3.34 +/- 0.23 nmol/l while taking 20 micrograms T3 8-hourly, 0.65 +/- 0.04 after one week of fasting while not taking T3, and 2.9 +/- 0.3 after a second week of fasting while taking T3 20 micrograms 8-hourly. In three other obese women (control group) who did not take T3, T3 concentrations decreased during fasting and remained low at the end of both the first and second week of fasting. In the T3 group, resting metabolic rate (RMR) (O2 ml/min) decreased 11.7 percent at the end of one week of fasting, but then increased by 5.3 percent at the end of the second week of fasting, whereas in the control group RMR decreased by 13.1 percent after one week of fasting and a further fall of 13.3 percent occurred by the end of the second week of fasting. The RMR at the end of 2 weeks of fasting of the control group was significantly lower (P less than 0.05) than predicted using the equation of Dore, whereas that of the T3 group was as predicted using the same equation. The changes in leucine flux, leucine oxidation and non-oxidative portion of leucine flux were not different during fasting between the T3 group and the control group. Plasma concentrations of glucose, insulin and C-peptide increased from the end of one week of fasting to the end of 2 weeks of fasting in the T3 group, whereas they decreased in the control group. It is concluded that the changes in T3 concentration during fasting affected RMR, glucose metabolism and insulin secretion but failed to influence leucine metabolism.

Adult↗
PubMed1988

The identity of Escherichia coli bacterioferritin and cytochrome b1.

Two different procedures were used to prepare pure samples of 'cytochrome b1' (column chromatography) and 'bacterioferritin' (immunoprecipitation) from Escherichia coli K-12 strain CA265. Both were crystallized, and X-ray-crystallographic data were compared with those from the bacterioferritin of E. coli strain W3300 used as a standard. We conclude that 'cytochrome b1' and 'bacterioferritin' are identical.

Bacterial Proteins↗
PubMed1988

Influence of amino acid administration on whole-body leucine kinetics and resting metabolic rate in postabsorptive normal subjects.

1. We have investigated the effect of an amino acid mixture (Vamin 14; 57.4 +/- 10.2 mumol h-1 kg-1) on whole-body leucine kinetics, calculated by a steady-state reciprocal pool model, and resting metabolic rate in eight postabsorptive normal subjects. 2. Vamin 14 infusion increased whole-body leucine flux (P less than 0.001), leucine employed for protein synthesis (P less than 0.001), leucine oxidation (P less than 0.001), metabolic clearance rate of alpha-ketoisocaproic acid (P less than 0.05) and levels of all three branched-chain amino acids (P less than 0.001) compared with the basal situation. In contrast, whole-body proteolysis was reduced (P less than 0.05). 3. Resting metabolic rate was increased during Vamin 14 infusion (P less than 0.05) and was positively correlated with whole-body protein synthesis (n = 16, r = 0.6342, P less than 0.01; y = 0.605x + 173.7), as was the change in metabolic rate with the change in protein synthesis (n = 8, r = 0.772, P less than 0.05; y = 0.493x - 10.85). 4. Overall, Vamin 14 infusion was associated with increased blood glucose (P less than 0.001), although the observed increase in plasma glucagon (t = 2.012) and plasma insulin (t = 1.683) failed to reach statistical significance. 5. These data lend a measure of support to the hypothesis that the apparent increase in whole-body protein synthesis in insulin-dependent diabetic (type I) subjects during insulin withdrawal may be substrate related.

Adult↗
PubMed1988

Relationships between plasma isotope enrichments of leucine and alpha-ketoisocaproic acid during continuous infusion of labelled leucine.

The ratio at steady-state of alpha-ketoisocaproic acid (KIC) to leucine 13C enrichments in plasma during continuous infusion of [13C]leucine was examined in 58 studies under a variety of conditions. The ratio was lower (P less than 0.001) in 'arterialized' venous plasma (mean +/- SD 0.85 +/- 0.09) than in deep venous plasma (0.95 +/- 0.08), the sampling site enrichment differences being greater for leucine than for KIC. Variation in the ratio between the various conditions studied was largely explained by analytical variation. Three normal subjects were given an intravenous bolus dose of L-[1-13C, 15N]leucine. Both [13C]KIC and [13C]leucine appeared quantitatively in plasma within 10 min, indicating that both transamination of leucine and KIC, and their equilibration between intracellular fluid and plasma, is rapid. This study suggests that changes in sampling site, rather than in study conditions, are more likely to lead to variation in the KIC:leucine enrichment ratio. The lesser variation for KIC, and the rapid equilibration of the [13C]leucine bolus, favour KIC as tracee for [13C]leucine protein turnover studies.

Adult↗
PubMed1987

Effect of intravenous insulin treatment on in vivo whole body leucine kinetics and oxygen consumption in insulin-deprived type I diabetic patients.

In vivo leucine metabolism was studied after an overnight fast in nine type I diabetic patients and nine healthy control subjects using L-[1-13C] leucine as a tracer. In the insulin-deprived state, leucine flux (reflecting proteolysis), leucine oxidation, and plasma leucine concentrations were higher in the diabetic patients than in the control subjects (P less than .001). In 4 of the 9 insulin-deprived diabetic patients, a four-hour intravenous insulin treatment decreased plasma glucose and leucine concentrations and leucine flux, but failed to decrease leucine oxidation. In the remaining 5 of the 9 diabetic patients, uninterrupted insulin treatment prior to the study and a seven-hour intravenous insulin treatment during the study period decreased not only the concentrations of plasma glucose and leucine and leucine flux, but also leucine oxidation (P less than .01). In all 9 diabetic patients the nonoxidative portion of leucine flux (reflecting protein synthesis) decreased during insulin treatment (P less than .01), but this decrease was lower than that of leucine flux (reflecting proteolysis), and therefore protein was conserved during insulin treatment. We conclude that the effect of insulin on proteolysis (reflected by leucine flux) is more rapid than its effect on leucine oxidation, but on aggressive insulin treatment accelerated leucine oxidation also was decreased in type I diabetic patients.

Adult↗
PubMed1987

Influence of fasting on leucine and muscle protein metabolism across the human forearm determined using L-[1-13C,15N]leucine as the tracer.

1. We have used L-[1-13C,15N]leucine as the substrate tracer to study leucine and muscle protein metabolism across the forearm of eight normal fasting adults. 2. The rates of protein synthesis and breakdown, de- and re-amination of leucine, and the oxidative decarboxylation of its keto acid were calculated directly from the arteriovenous metabolite balances and isotope dilutions as described by the metabolic model. 3. The results were compared with those obtained previously when subjects were fed. The effects of fasting on protein and leucine metabolism were a significant decrease in protein synthesis from 127 (SEM 11; n = 6) to 70 (SEM 6; n = 12) nmol of leucine min-1 100 ml-1 of forearm tissue (P less than 0.001) and a marked decrease in leucine catabolism in the forearm muscle. 4. This model has demonstrated that each subject was in negative protein balance across the forearm during fasting while positive during feeding, the mean values being -29(SEM 5; n = 12) and +39(SEM 9; n = 6) nmol of leucine min-1 100 ml-1 of forearm tissue respectively. 5. These results are sufficiently encouraging to suggest a role for this model in future studies on muscle protein metabolism.

Adult↗