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Biomedical subjects

J F Burke

Publications and source records attributed to J F Burke.

At least 19 recordsLinked to original sources
PubMed1992

Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of xanthine dehydrogenase.

The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the xanthine dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of xanthine dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of xanthine dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant xanthine dehydrogenase variants in probing the structure and function of enzymes of this class.

Amino Acid Sequence↗
PubMed1992

Characterisation of a haem active-site mutant of horseradish peroxidase, Phe41----Val, with altered reactivity towards hydrogen peroxide and reducing substrates.

A horseradish peroxidase variant ([F41V] HRP-C*), in which Val replaces the conserved Phe at position 41 adjacent to the distal His, has been constructed. Its composition and spectroscopic, catalytic and substrate-binding properties were compared with those of the wild-type recombinant (HRP-C*) and plant (HRP-C) enzymes. Presteady-state kinetic measurements of the rate constant for compound I formation (k1) revealed an eightfold decrease in the reactivity of the Phe41----Val variant towards H2O2, in comparison with HRP-C or HRP-C*. Measurement of the remaining rate constants, k2 and k3, for the two single-electron reduction reactions of [F41V] HRP-C with para-aminobenzoic acid as reducing substrate, showed that they were 2.5-fold and 1.3-fold faster, respectively. In contrast, analysis of data from steady-state assays with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate) as reducing substrate, showed decreased reactivity of the mutant enzyme to this compound, indicating a change in substrate specificity. Over the substrate range studied, the data for HRP-C* and for [F41V] HRP-C conformed to a simple modification of the accepted peroxidase mechanism in which a first-order step (ku), assumed to be product dissociation, becomes rate-limiting under our standard assay conditions. Calculations of rate constants from steady-state data yielded values of k1 for both enzyme forms in adequate agreement with those from pre-steady state measurements. They showed, furthermore, that both k3 for 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate) and ku were substantially decreased, fivefold and tenfold, respectively, in the mutant. Analogous to the decrease in ku, we observed a twofold increase in the affinity of the mutant variant for the inhibitor benzhydroxamic acid. The coordination-state equilibrium of the haem iron also appeared shifted towards the hexacoordinate high-spin form. These observations indicate that in addition to affecting reactivity to H2O2, mutations in the distal region and close to the haem iron also affect reactivity towards different reducing substrates, inducing perturbations in the neighbourhood of the aromatic-substrate-binding site, known to be 0.8-1.2 nm from the haem iron.

Amino Acid Sequence↗
PubMed1992

Structural studies by proton-NMR spectroscopy of plant horseradish peroxidase C, the wild-type recombinant protein from Escherichia coli and two protein variants, Phe41----Val and Arg38----Lys.

Wild-type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41----Val and Arg38----Lys, have been characterised using both one- and two-dimensional NMR spectroscopy. Proton NMR spectra recorded in both resting and cyanide-ligated states of the proteins were compared with those of the corresponding plant peroxidase. The latter contains 18% carbohydrate in eight N-linked oligosaccharide side chains whereas the recombinant proteins are expressed in nonglycosylated form. The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydrate-linked resonances in the former, indicating that their solution structures are highly similar. This comparison also identifies classes of carbohydrate resonances in the plant enzyme which provides new information on the local environment and mobility of the oligosaccharide side chains. Comparison of the spectra of the cyanide-ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem-linked resonances. These could not be rationalised simply on the basis of the local perturbation expected from a single-site substitution. The two substitutions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group. Inspection of the spectra of the Phe41----Val variant also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly assigned to Phe41 in a previous study. A new assignment, based on additional information from two-dimensional nuclear Overhauser enhancement spectroscopy, was made to Phe152. The assignments made for the Phe41----Val variant were also used as a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid.

Arginine↗
PubMed1992

Pulmonary hypertension predicts mortality and morbidity in patients with dilated cardiomyopathy.

OBJECTIVE: To ascertain whether pulmonary hypertension, as assessed noninvasively by continuous-wave Doppler of tricuspid regurgitation, can be an important independent factor in the prognosis of patients with ischemic or idiopathic dilated cardiomyopathy. DESIGN: Cohort study of consecutive patients with dilated cardiomyopathy in whom follow-up was obtained on all survivors for 28 months. SETTING: Outpatient cardiology private practice office in a tertiary care center. PATIENTS: Consecutive sample of 108 patients who presented for a scheduled office visit during a 15-month period. MEASUREMENTS: M-mode, two-dimensional, and Doppler echocardiographic examinations were done on all patients at entry into the study and on survivors 1 year later. All examinations included extensive pulsed- and continuous-wave Doppler evaluation for tricuspid regurgitation. MAIN OUTCOME MEASURES: Overall mortality, mortality due to myocardial failure, and hospitalization for congestive heart failure. RESULTS: Twenty-eight patients had a high velocity of tricuspid regurgitation (greater than 2.5 m/s), and 80 patients had a low velocity (less than or equal to 2.5 m/s). After 28 months of follow-up, the mortality rate was 57% in patients with a high velocity compared with 17% in patients with a low velocity (difference of 40%, 95% CI, 20% to 60%). Hospitalization for congestive heart failure occurred in 75% and 26% of patients, respectively (difference of 49%, CI, 30% to 68%). Eighty-nine percent of patients with a high velocity either died or were hospitalized compared with only 32% of patients with a low velocity (difference of 57%, CI, 42% to 72%). The peak velocity of tricuspid regurgitation was the only prognostic variable selected using stepwise logistic regression models for the three outcome events. CONCLUSION: Noninvasive assessment of pulmonary hypertension using continuous-wave Doppler of tricuspid regurgitation can predict morbidity and mortality in patients with ischemic or idiopathic dilated cardiomyopathy.

Adult↗
PubMed1992

Uptake of alpha-aminoisobutyric acid and cycloleucine on skeletal muscles in burned rats.

The uptake of alpha-aminoisobutyric acid (AIB) and aminocyclopentane-carboxylic acid (cycloleucine) was studied in soleus muscles isolated from small rats (body weight 50-60 g) during the first 6 h after a major thermal injury, the so-called 'ebb phase' or 'hypometabolic phase'. Soleus muscles were dissected intact from rats at 0.5, 1, 3 and 6 h after extensive deep burn injury (30 per cent TBSA) and then incubated for 2 h in Krebs-Henseleit bicarbonate buffer (pH 7.4), 5.5 mM glucose, bovine serum albumin and radiolabelled AIB or cycloleucine. The results were expressed as the distribution ratio of AIB or cycloleucine between intracellular and extracellular fluid. The AIB uptake in vitro was found to be significantly increased (30 per cent) in the initial half hour postburn only, and then slowly reduced during the subsequent hours to near the value found in non-burn animals. Muscle cycloleucine uptake in vitro showed no significant change in these studies. In our second study, extensor digitorum longus leg muscle were incubated in Krebs-Henseleit bicarbonate buffer with different pH values (7.2-7.5). No significant difference was found in muscle AIB uptake. In summary, since muscle amino acid uptake remained relatively stable during this period, it is suggested that the alteration of amino acid transport across muscle cells may not be a contributing factor to the alteration of amino acid flux during the early phase of stress.

Aminoisobutyric Acids↗
PubMed1992

Epidemiology of infections with Pseudomonas aeruginosa in burn patients: the role of hydrotherapy.

Pseudomonas aeruginosa remains a cause of serious wound infection and mortality in burn patients. By means of restriction fragment length polymorphism analysis and a DNA probe for the pilin gene of Pseudomonas, a lethal strain of nosocomial P. aeruginosa was identified as the cause of an outbreak of wound infections among burn patients. Environmental surveys suggested an association of the outbreak with hydrotherapy provided to many patients in a common facility. In a trial of burn wound care without hydrotherapy, overall mortality was reduced significantly, mortality associated with pseudomonas sepsis was eliminated, and the strain of P. aeruginosa associated with earlier mortality was eradicated. Moreover, fewer nosocomial pseudomonas infections, lower levels of pseudomonas resistance to aminoglycoside antibiotics, significantly fewer pseudomonas infections of skin graft donor sites, and later appearance of Pseodomonas species in burn patients were found during the period when hydrotherapy was not used.

Adolescent↗
PubMed1992

Prophylaxis against wound infection following herniorrhaphy or breast surgery.

The effect of perioperative antibiotic prophylaxis on definite wound infections was assessed for 3202 herniorrhaphies or selected breast surgery procedures. Patients were identified preoperatively and monitored for greater than or equal to 4 weeks. Thirty-four percent of patients (1077/3202) received prophylaxis at the discretion of the surgeon; 86 definite wound infections (2.7%) were identified. Prophylaxis recipients were at higher risk for infection, with a higher proportion of mastectomies, longer procedures, and other factors. Patients who received prophylaxis experienced 41% fewer definite wound infections (odds ratio [OR], 0.59; 95% confidence interval [CI], 0.35-0.99; P = .04) and 65% fewer definite wound infections requiring parenteral antibiotic therapy (OR, 0.35; 95% CI, 0.15-0.88; P = .02) after adjustment for duration of surgery and type of procedure. Additional adjustment for age, body mass index, the presence of drains, diabetes, and exposure to corticosteroids did not change the magnitude of this effect meaningfully. The effect of prophylaxis was similar for all procedures studied. In the absence of formal guidelines, surgeons at these institutions administered prophylaxis preferentially to patients at highest risk.

Anti-Bacterial Agents↗
PubMed1992

Circulating interleukin-1 beta and tumor necrosis factor-alpha concentrations after burn injury in humans.

OBJECTIVES: To measure plasma interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) concentrations after burn injury and to determine if these concentrations relate to clinical status. DESIGN: Prospective assessment. SETTING: Hospital burn unit. PATIENTS: Thirty-one patients with second- or third-degree burns, covering 10% to 95% of body surface area. MEASUREMENTS AND MAIN RESULTS: Initial concentrations of IL-1 beta were increased (mean 188 +/- 31 pg/mL), and the concentrations for each patient correlated with body temperature at the time of the blood sample (rho = 0.51, p < .015) (rho is a nonparametric statistical measure; a nonparametric analysis is mandatory for data that is categorical [Acute Physiology and Chronic Health Evaluation, APACHE, scores] and data that are not normally distributed [IL-1 beta and tumor necrosis factor, TNF, data]). Mean TNF alpha concentrations were initially 264 +/- 132 pg/mL, and these concentrations were positively related to body temperature (rho = 0.41, p < .05) and inversely related to the total WBC count (rho = -0.45, p < .025). Through the course of hospitalization, plasma cytokine levels fluctuated, but transient increases (sometimes into the nanogram/mL range) did not consistently correspond to changes in clinical signs or severity of illness, as determined by APACHE II scores. The maximum plasma cytokine levels in any patient were not related to age, but maximum IL-1 beta concentrations were inversely related to burn size (rho = -0.46, p < .015). The final IL-1 beta concentrations measured in the patients who died (n = 7) were significantly less than measurements in surviving patients matched for burn size and age taken at approximately the same time after admission. CONCLUSIONS: These results indicate that early after burn injury there is a correspondence of IL-1 beta and TNF alpha with certain host responses, but these correlations disappear with the progression of illness. In general, IL-1 beta and TNF alpha appear to be poor indicators of prognosis during burn injury; however, the association of mortality with low circulating IL-1 beta values supports the concept of IL-1 beta as being an essential mediator of host defenses.

Adolescent↗
PubMed1992

Increased gut permeability early after burns correlates with the extent of burn injury.

OBJECTIVE: To determine if increased gut permeability within 48 hrs after burn injury correlates with the extent of injury, before sepsis and pulmonary disorders have complicated the clinical course. DESIGN: Nonrandomized, controlled study. PATIENTS: Consecutive patients admitted with burn injuries on > 20% of body surface area. INTERVENTIONS: Intestinal absorption and renal excretion of polyethylene glycol 3350 was used as the macromolecule to determine gut permeability; polyethylene glycol 400 intestinal absorption was used as an internal control for abnormal motility and malabsorption. Polyethylene glycol 3350 (40 g) and polyethylene glycol 400 (5 g) were administered enterally. MEASUREMENTS AND MAIN RESULTS: Gut permeability was significantly increased early after the injury. The patients excreted 0.56 +/- 0.34% (n = 11) of polyethylene glycol 3350, compared with the amount (0.12 +/- 0.04%) (p < .05) previously reported in normal volunteers. There was no significant difference in the excretion of polyethylene glycol 400 in the patients (27.0 +/- 4.6%, n = 11) vs. the normal volunteers previously reported (26.3 +/- 5.1%, n = 12), suggesting normal intestinal motility and absorption. The percentage of excretion of polyethylene glycol 3350 correlated with the percentage body surface burned; patients with smaller injuries excreted 0.32 +/- 0.17% (n = 6), which was greater than normal and less than those values from patients with larger injuries, 0.84 +/- 0.25% (n = 5) (p < .001 by Turkey test). CONCLUSIONS: Using our newly developed method to separate and quantify polyethylene glycols in urine, gut permeability was found to be increased early after burn injury, which confirms a previous study using lactulose as the permeability probe. Furthermore, this increased gut permeability to polyethylene glycol 3350 correlated with the extent of the burn injury.

Adult↗
PubMed1992

Splanchnic and whole body L-[1-13C,15N]leucine kinetics in relation to enteral and parenteral amino acid supply.

The effect of the route of administration of a complete amino acid solution (0.24 g.kg-1.h-1) on leucine (Leu) and alpha-ketoisocaproate (KIC) metabolism in the splanchnic region (Sp) was assessed in nine chronically catherized mongrel dogs receiving, for 6 h, amino acids by jugular vein (PN feeding). Results were compared with those obtained previously [Y. M. Yu, D. A. Wagner, E. E. Tredget, J.A. Waleszewski, J. F. Burke, and V. R. Young. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E36-E51, 1990] in eight dogs similarly studied but given amino acids by constant enteral feeding (EN). We used primed continuous intravenous infusions of L-[1-13C,15N]Leu and measurements of arteriovenous isotope and Leu balance across the gut, liver, and Sp to estimate parameters of whole body and organ Leu metabolism [Leu-N and Leu-C flux, Leu----KIC; KIC----Leu, Leu oxidation and rates of Leu appearance (B) from and disappearance (S) into proteins]. Whole body Leu kinetics were the same for both routes of amino acid administration. With PN, KIC----Leu, Leu----KIC, and total metabolic processing were lower than for EN in Sp, and overall Leu balance (S - B) was higher in Sp for EN. Leu appearance from protein breakdown in gut was higher with PN. The rate of KIC----Leu was higher in liver for EN. These findings reveal that route of amino acid (Leu) administration, under these acute feeding conditions, alters the processing and metabolic fate of Leu in Sp but that whole body parameters of Leu metabolism are stable. The implications of these metabolic findings in relation to the maintenance of intestinal function and integrity are discussed.

Amino Acids↗
PubMed1992

Cell-specific alternative RNA splicing of an FMRFamide gene transcript in the brain.

Individual neurons synthesize different peptide neurotransmitters and neuromodulators. In general, specificity is achieved by transcriptional regulation of neuropeptide-encoding genes. In Lymnaea, the FMRFamide and GDP/SDPFLRFamide neuropeptides are encoded by separate exons. Here we provide evidence that the two exons are part of the same gene and that in neurons expressing the gene the two exons are spliced onto a common upstream exon encoding a hydrophobic leader sequence. In addition, in situ hybridization data show that there is mutually exclusive cytoplasmic expression of each of the neuropeptide-encoding exons. Thus, differential neuropeptide synthesis is likely to be regulated by an alternative splicing mechanism. The cellular specificity of these splicing events is remarkable and suggests that cell-specific alternative splicing may be of major importance in establishing neuronal diversity in this system.

Animals↗
PubMed1979

Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system.

A comparative study of some commonly employed laboratory procedures for studying DNA synthesis in isolated nuclei was carried out. Nuclei isolated from baby-hamster kidney (BHK-21/C13) cells synthesize DNA for 30-60min at 37 degrees C in a reaction requiring uni- and bi-valent cations, ATP and all four deoxyribonucleoside 5'-triphosphates. The addition of either ribonucleotides or cytosol from S-phase cells had no effect, but DNA synthesis was stimulated by some dextrans (mol.wt. 5x10(6)). The extent of synthesis was influenced by apparently minor variations in experimental conditions. For example, DNA synthesis by nuclei in Tris/HCl, pH7.5, was only 50% of that observed in Hepes/NaOH, pH7.5; the presence of detergents Triton X-100, Triton N-101, Nonidet P-40, Brij 58 and Tween 80 in the incubation medium altered the amount of synthesis to different extents. Although most detergents inhibited synthesis, a stimulation occurred with Tween 80 (150% of controls). These effects were reversed on washing the nuclei, except that of Brij 58, which inhibited DNA synthesis by 90-95% irreversibly. Anomalous sucrose-density-gradient sedimentation behaviour of the DNA, and of precursor [(3)H]-dTTP, was observed when nuclei were lysed with solutions of sodium dodecyl sulphate/Mg(2+) or with Sarkosyl/Mg(2+), but consistent results, showing that the DNA synthesized in vitro sedimented exclusively at about 4S, were obtained when nuclei were lysed with sodium dodecyl sulphate (without Mg(2+))/EDTA, digested with proteinase K and heated at 100 degrees C with 11% (v/v) formaldehyde to prevent macromolecular association. These results, coupled with density-labelling studies with bromodeoxyuridine and CsCl-density-gradient analysis, showed that DNA synthesis in these nuclei was replicative and was restricted to a covalent extension of Okazaki pieces previously initiated in vivo. No new initiations were observed, and the DNA was not ligated into larger molecules. The cessation of DNA synthesis after about 60 min was due to the complete utilization of available primer/template DNA.

Animals↗