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Biomedical subjects

S Eaton

Publications and source records attributed to S Eaton.

At least 19 recordsLinked to original sources
PubMed2001

Glutamine and glutathione counteract the inhibitory effects of mediators of sepsis in neonatal hepatocytes.

BACKGROUND/PURPOSE: Surgical neonates are at risk of sepsis-associated liver dysfunction. Hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) are important mediators of sepsis, which impair neonatal hepatic metabolism. Glutamine has been shown to have beneficial effects on hepatocyte metabolism during neonatal sepsis. However, the molecular basis of these effects are unknown. The aim of this study was to test the hypotheses that (1) glutamine and its dipeptides counteract the inhibitory effect of septic mediators on neonatal hepatocyte oxygen consumption and (2) the effects of glutamine are specific and not shared by other amino acids. In addition, we wished to determine the metabolic pathways and mediators involved in the action of glutamine. METHODS: Hepatocytes were isolated from suckling rats, and O(2) consumption measured polarographically. Study A: the ability of 10 mmol/L glutamine to reverse the inhibitory effects of 1.5 mmol/L H(2)O(2) and 300 micromol/L S-Nitroso-N-acetylpenicillamine (SNAP; a nitric oxide donor) on O(2) consumption was examined. Study B: the ability of other amino acids and dipeptides of glutamine to reverse the effects of H(2)O(2) was examined. Study C: various concentrations of glutamine were tested for their ability to reverse the H(2)O(2) inhibition of O(2) consumption. Study D: the mechanism of action of glutamine was examined by incubating hepatocytes with either an inhibitor of entry into the Krebs cycle or an inhibitor of glutathione synthesis. Study E: the ability of glutathione to reverse the inhibitory effects of H(2)O(2) was examined. RESULTS: Study A: glutamine reversed the inhibition of hepatocyte O(2) consumption exerted by either H(2)O(2) or NO. Study B: glutamine dipeptides reversed the inhibition of hepatocyte O(2) consumption by H(2)O(2), but other amino acids did not. Study C: the counteracting effect of glutamine was proportional to the dose administered. Study D: blocking entry of glutamine into the Krebs cycle did not abolish the effects of glutamine, but blocking glutathione synthesis completely abolished the effect of glutamine. Study E: exogenous glutathione reversed the inhibitory effect of H(2)O(2) on hepatocyte O(2) consumption. CONCLUSIONS: This study found that glutamine and its dipeptides are unique in reversing the effects of septic mediators on neonatal rat liver oxidative metabolism. The effectiveness of glutamine appears to be mediated via glutathione synthesis. Addition of glutamine, glutamine dipeptides, or glutathione to total parenteral nutrition (TPN) may be beneficial in preventing liver damage in neonatal sepsis.

Animals↗
PubMed2001

Effect of lipopolysaccharide and cytokines on oxidative metabolism in neonatal rat hepatocytes.

BACKGROUND/PURPOSE: Lipopolysaccharide (LPS) and cytokines produced during neonatal sepsis trigger free radical production, which eventually results in inhibition of liver metabolism. Studies in adults have indicated a hypermetabolic response to sepsis; however, evidence for a hypermetabolic response in neonates is equivocal. This study was carried out to determine whether LPS and cytokines can cause liver hypermetabolism in neonates. METHODS: The initial bacterial insult and cytokine cascade were mimicked by the addition of lipopolysaccharide (Escherichia coli 055:B5), tumour necrosis factor (TNF-alpha), and interleukin-6 (IL6) during the isolation of hepatocytes by collagenase digestion from 11- to 13-day-old Wistar rats. Hepatocyte oxygen consumption was measured polarographically with cells respiring on palmitate (0.5 mmol/L). Myxothiazol, a specific inhibitor of mitochondrial respiration, was used to distinguish extra- and intramitochondrial oxygen consumption. Morphologic changes were assessed by electron microscopy. RESULTS: The addition of LPS, TNF-alpha and IL6 during hepatocyte isolation resulted in a 10% decrease in cell yield (P <.05) compared with untreated controls; however, cell viability was unchanged (n = 31). Both total and extramitochondrial oxygen consumption were significantly greater in treated cells compared with untreated controls (P <.05, Student's t test). Electron microscopy indicated that LPS, TNF-alpha, and IL6 did not cause ultrastructural changes to hepatocytes. CONCLUSIONS: The increase in oxygen consumption was predominantly extramitochondrial and likely to be caused by increased oxygen requirement for cytosolic detoxification and repair purposes. This study shows that liver hypermetabolism metabolism can occur in response to LPS and cytokines. However, during in vivo neonatal sepsis, additional free radical damage may blunt this hypermetabolic response.

Animals↗
PubMed2001

Energy metabolism of infants and children with systemic inflammatory response syndrome and sepsis.

OBJECTIVE: To evaluate whether critically ill children with systemic inflammatory response syndrome (SIRS) or sepsis have altered resting energy expenditure (REE) and substrate utilization. SUMMARY BACKGROUND DATA: Studies in adults with sepsis have shown increased energy expenditure and mobilization of endogenous fat. In infants and children, energy metabolism and substrate utilization during sepsis have not been characterized. METHODS: Metabolic studies were performed in 21 critically ill children with SIRS or sepsis. Twenty-one stable control children, matched for weight, were also studied. Seven patients required inotropic support and 17 received mechanical ventilation. Fifteen patients with SIRS had evidence of bacterial, fungal, or viral infection and were considered septic. Respiratory gas exchange was measured by computerized indirect calorimetry for 1 to 2 hours continuously. RESULTS: The REE of patients with SIRS or sepsis was not different from that of controls. Similarly, there were no differences in carbon dioxide production and oxygen consumption. Resting energy metabolism was not different between patients with SIRS and patients with sepsis. In addition, the presence of low platelet count or inotropic support did not affect resting energy metabolism. The median respiratory quotient of patients with SIRS or sepsis was 0.88 (range 0.75-1.12), indicating mixed utilization of fat and carbohydrate; this was not significantly different from that of controls. The Pediatric Risk of Mortality Score was not significantly correlated with REE or respiratory quotient. CONCLUSIONS: The energy requirements of children with SIRS or sepsis are not increased. Their resting metabolism is based on both carbohydrate and fat utilization. The authors speculate that these children divert the energy for growth into recovery processes.

Case-Control Studies↗
PubMed2000

Plasma coenzyme Q(10) in children and adolescents undergoing doxorubicin therapy.

The objective of this study was to test the hypothesis that doxorubicin treatment for cancer in childhood and adolescence causes a dose-related decrease in the concentration of plasma coenzyme Q(10). The concentration of plasma coenzyme Q(10) was measured before and after administration of doxorubicin in six patients, and before and after chemotherapy in six patients undergoing treatments that did not include doxorubicin. There was a significant increase in the concentration of plasma coenzyme Q(10) in post-treatment samples compared to pre-treatment samples in patients treated with doxorubicin (P=0.008; n=32), whereas there were no significant changes in plasma coenzyme Q(10) concentrations in patients treated with chemotherapy that did not include doxorubicin. (P=0.770; n=30). We hypothesise that the increase in plasma coenzyme Q(10) that was observed in patients undergoing doxorubicin treatment is due to release of coenzyme Q(10) from apoptotic or necrotic cardiac tissue. We conclude that the cardiotoxicity due to doxorubicin therapy does not involve acute myocardial depletion of coenzyme Q(10).

Adolescent↗
PubMed2000

Nitric oxide inhibits neonatal hepatocyte oxidative metabolism.

BACKGROUND/PURPOSE: Liver function is frequently impaired in neonates with sepsis. Nitric oxide (NO) is thought to be a mediator of organ dysfunction and liver oxidative metabolism during sepsis. The authors developed an in vitro model to investigate the effect of NO and the combined effect of NO plus H2O2 on neonatal hepatocyte oxidative metabolism. METHODS: Hepatocytes were isolated from neonatal rats. Oxygen consumption was measured polarographically. In Study A, cells were exposed to S-Nitroso-N-acetylpenicillamine (SNAP), an NO donor, at various concentrations. In study B, myxothiazol and oligomycin, inhibitors of mitochondrial respiration, were added to investigate the site of action of NO. In study C, hepatocytes were incubated in the presence of both SNAP (300 micromol/L) and H2O2 (1.5 mmol/L). In study D, morphological alterations induced by NO and NO plus H2O2 were investigated by hepatocyte electron microscopy. RESULTS: In study A, SNAP caused a dose-dependent decrease in oxygen consumption. A significant inhibition was reached at 300 micromol/L SNAP. In study B, the lack of further inhibition when SNAP was given together with myxothiazol indicates that NO acts intramitochondrially. Similarly, no further inhibition occurred when the NO donor was given together with oligomycin, suggesting that the effect of NO is mainly at the level of ATP synthase. In study C, concomitant addition of 300 micromol/L SNAP and 1.5 mmol/L H2O2 to hepatocytes caused further inhibition of oxygen consumption compared with either SNAP or H2O2 alone. In study D, mild alterations in hepatocyte morphology were noted in the presence of SNAP or SNAP plus H2O2. CONCLUSIONS: In neonatal hepatocytes, NO significantly inhibits mitochondrial oxygen consumption, possibly at the level of ATP synthase. The effect of NO is additive to that of H2O2. Morphological findings were consistent with these biochemical effects and suggest that NO and H2O2 are important mediators of liver damage during sepsis.

Adenosine Triphosphate↗
PubMed2000

Cholesterol in signal transduction.

Membrane cholesterol impinges on signal transduction in several ways, which is highlighted in particular by the Hedgehog signaling pathway. In Hedgehog signaling, cholesterol is important for ligand biogenesis, as well as for signal transduction in receiving cells. Hedgehog ligands are post-translationally modified by cholesterol, and the Hedgehog receptor, Patched, is structurally similar to the Niemann-Pick C1 protein, which functions in intracellular lipid transport. Although the exact role of cholesterol in Hedgehog signal transduction remains elusive and is probably multifaceted, studies over the past year have implicated raft membrane subdomains, cholesterol transport and a link between protein and lipid trafficking in endocytic compartments.

Animals↗
PubMed2000

The mitochondrial trifunctional protein: centre of a beta-oxidation metabolon?

The trifunctional enzyme comprises three consecutive steps in the mitochondrial beta-oxidation of long-chain acyl-CoA esters: 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. Deficiencies in either 3-hydroxyacyl-CoA dehydrogenase activity, or all three activities, are important causes of human disease. The dehydrogenase and thiolase have a requirement for NAD+ and CoA respectively, whose levels are conserved within the mitochondrion and thus provide possible means for control and regulation of beta-oxidation. Using analysis of the intact CoA ester intermediates produced by the complex, we have examined the sensitivity of the complex to NAD+/NADH and acetyl-CoA. We consider the evidence for channelling within the trifunctional protein and propose a model for a beta-oxidation 'metabolon'.

Amino Acid Sequence↗
PubMed2000

Amino acids counteract the inhibitory effect of fentanyl on hepatocyte oxidative metabolism.

BACKGROUND/PURPOSE: Hypothermia is common after major surgery in newborns and can be triggered by intraoperative fentanyl analgesia. Recent studies have found that fentanyl inhibits hepatocyte mitochondrial oxidative metabolism, which is proportional to thermogenesis. In adults it has been shown that amino acids have a thermogenic effect, although the biochemical basis of this phenomenon is not known. The aim of this study was to test the hypothesis that amino acids counteract the inhibition of neonatal hepatocyte oxygen consumption by fentanyl. METHODS: Hepatocytes were isolated from suckling rats, and O2 consumption was measured polarographically. In experiment A hepatocytes were incubated with (1) palmitate alone (control), (2) palmitate plus fentanyl, (3) palmitate plus fentanyl plus amino acids, and (4) palmitate plus amino acids. In experiment B the effects of essential and nonessential amino acids were tested separately. In experiment C, to investigate whether the effect of amino acids is intramitochondrial, hepatocytes were incubated with amino acids plus inhibitors of mitochondrial respiration. RESULTS: In experiment A, fentanyl significantly inhibited O2 consumption (P = .006). This inhibition was reversed by amino acids (P < .001). In experiment B, both essential and nonessential amino acids reversed the effect of fentanyl (P < .001). In experiment C, there was no difference in O2 consumption in the presence of myxothiazol among the groups indicating that amino acids affect intramitochondrial O2 consumption. CONCLUSIONS: (1) Amino acids abolish the inhibitory effect of fentanyl on hepatocyte oxidative metabolism. (2) Amino acids affect intramitochondrial O2 consumption and therefore thermogenesis. (3) Perioperative administration of amino acids in neonates may help to prevent hypothermia and its deleterious effects.

Adjuvants, Anesthesia↗
PubMed2000

Prediction of violence on a psychiatric intensive care unit.

The aim of the present study was to identify patients who were at particular risk of violent behaviour, over a period of one month in a psychiatric high dependency unit, and to examine whether particular patients could be identified at admission who would subsequently be violent. Medical records, and other documents for 52 inpatients were included. Demographic, historical and clinical data available at admission were noted. All violent incidents were recorded and classified. Seventeen of 52 patients committed physical violence, usually to the staff. Most incidents were carried out by a small number of individuals. Identifying the violent, using variables available at admission, was not possible. The ward admitted a group with a subsequent high rate of violence. Prediction within this high-risk group was not possible. Measures aimed at preventing violence on a local secure ward should apply to all patients and should not be targeted just at those that would appear to be the most likely to be violent.

Adult↗
PubMed1999

Characterization of the self association of Avian sarcoma virus integrase by analytical ultracentrifugation.

Retroviral integration protein (IN) has been shown to be both necessary and sufficient for the integration of reverse-transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-assembly of IN is essential for proper function. Analytical ultracentrifugation was used to determine the stoichiometry and free energy of self-association of a full-length IN in various solvents at 23.3 degrees C. Below 8% glycerol, an association stoichiometry of monomer-dimer-tetramer is observed. At salt concentrations above 500 mM, dimer is the dominant species over a wide range of protein concentrations. However, as physiological salt concentrations are approached, tetramer formation is favored. The addition of glycerol to 500 mM NaCl, 20 mM Tris (pH 8.4), 2 mM beta-mercaptoethanol significantly enhances dimer formation with little effect on tetramer formation. Furthermore, as electrostatic shielding is increased by increasing the ionic strength or decreasing the cation size, dimer formation is strengthened while tetramer formation is weakened. Taken together, the data support a model in which dimer formation includes favorable buried surface interactions which are opposed by charge-charge repulsion, while favorable electrostatic interactions contribute significantly to tetramer formation.

Avian Sarcoma Viruses↗
PubMed1999

The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling.

The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn.

Actins↗
PubMed1999

Rotational and translational motion of troponin C.

Time resolved fluorescence anisotropy and sedimentation velocity has been used to study the rotational and translational hydrodynamic behavior of two mutants of chicken skeletal troponin C bearing a single tryptophan residue at position 78 or 154 in the metal-free-, metal-bound-, and troponin I peptide (residues 96-116 of troponin I)-ligated states. The fluorescence anisotropy data of both mutants were adequately described by two rotational correlation times, and these are compared with the theoretically expected values based on the rotational diffusion of an idealized dumbbell. These data imply that the motion of the N- and C-terminal domains of troponin C are independent. They also suggest that in the metal-free, calcium-saturated and calcium-saturated troponin I peptide-bound states, troponin C is elongated, having an axial ratio of 4-5. Calcium or magnesium binding to the high affinity sites alone reduces the axial ratio to approximately 3. However, with calcium bound to sites III and IV and in the presence of a 1:1 molar ratio of the troponin I peptide, troponin C is approximately spherical. The metal ion and troponin I peptide-induced length changes in troponin C may play a role in the mechanism by which the regulatory function of troponin C is effected.

Animals↗
PubMed1999

Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains.

In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction. To establish a system in which raft-dependent processes could be studied genetically, we have analyzed the protein and lipid composition of these microdomains in Drosophila melanogaster. Using mass spectrometry, we identified the phospholipids, sphingolipids, and sterols present in Drosophila membranes. Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved. Consistent with this, we have identified a detergent-insoluble fraction of Drosophila membranes that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins. We show that the sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction. Our findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures. They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association.

Animals↗
PubMed1999

Intermediates of myocardial mitochondrial beta-oxidation: possible channelling of NADH and of CoA esters.

Adult rat heart mitochondria were isolated and incubated with [U-14C]hexadecanoyl-CoA or unlabelled hexadecanoyl-CoA. The accumulating CoA and carnitine esters and [NAD+]/[NADH] ratio were measured by HPLC or tandem mass spectrometry. Despite minimal changes in the intramitochondrial [NAD+]/[NADH] ratio, 2, 3-unsaturated and 3-hydroxyacyl esters were observed as well as saturated acyl-CoA and acylcarnitine esters. In addition to acetylcarnitine, significant amounts of butyryl-, hexanoyl-, octanoyl- and decanoylcarnitines were detected and measured. Rat myocardial beta-oxidation is subject to control at the level of 3-hydroxyacyl-CoA dehydrogenase but this control is not due to a simple lack of oxidised NAD. We hypothesise a pool of NAD in contact between the trifunctional protein of beta-oxidation and complex I of the respiratory chain, the turnover of which is responsible for some of the control of beta-oxidation flux. In addition, short- and medium-chain acylcarnitine esters were detected whereas only small amounts of long-chain acylcarnitines were present. This may imply the presence of a mitochondrial carnitine octanoyl transferase or may reflect channelling of long-chain CoA esters so that they are not available for carnitine palmitoyl transferase II activity.

Animals↗