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S Eaton

Publications and source records attributed to S Eaton.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1999

Lipid peroxidation can be reduced in infants on total parenteral nutrition by promoting fat utilisation.

BACKGROUND/PURPOSE: Increased oxygen-derived free radical activity has been reported during total parenteral nutrition (TPN) in infants and has been specifically linked to the fat infusion. The aim of this study was to test the hypothesis that during TPN, oxygen-derived free radical production can be reduced by increasing the utilisation of fat. METHODS: In experiment A (17 patients) the fat infusion was kept constant (3 g/kg/d) and the carbohydrate infusion was changed from 18 g/kg/d on day 1 to 10 g/kg/d on day 2. In experiment B (six patients) the carbohydrate infusion remained constant and the fat infusion was changed from 3 g/kg/d on day 1 to 0 g/kg/d on day 2. Fat utilisation was measured by indirect calorimetry. Plasma malondialdehyde, an index of lipid peroxidation resulting from increased oxygen-derived free radical activity, was measured by a colorimetric assay. RESULTS: In both experiments there was no significant change between the two study phases in oxygen consumption, carbon dioxide production, and resting energy expenditure demonstrating that the patients were metabolically stable. In experiment A there was a significant (P = .0005) increase in fat utilisation and a significant (P = .009) decrease in malondialdehyde (MDA) concentration between the two phases. In experiment B there was also a significant (P = .007) decrease in MDA concentration. The decrease in MDA concentration was similar between the two experiments. CONCLUSIONS: It is not necessary to stop the infusion of fat to reduce free radical production. Promoting fat utilisation by reducing the carbohydrate-fat ratio of the TPN reduces free radical activity to a similar extent as fat exclusion. These findings have important implications for the composition of TPN.

Calorimetry, Indirect↗
PubMed1999

Analgesic doses of fentanyl impair oxidative metabolism of neonatal hepatocytes.

BACKGROUND/PURPOSE: Studies in human surgical neonates have shown that intraoperative fentanyl analgesia results in greater fall in perioperative body core temperature compared with morphine analgesia. The aim of the study was to compare in a neonatal animal model the biochemical effect of fentanyl and morphine on hepatocyte oxidative metabolism. METHODS: Hepatocytes were isolated from suckling rats and the oxygen consumption from palmitate was measured polarographically. In experiment A, fentanyl and morphine within the respective analgesic serum ranges were added to hepatocytes to assess the effect on oxygen consumption. In experiment B, fentanyl was added to hepatocytes in the presence of inhibitors of mitochondrial respiration to investigate its site of action. In experiment C, hepatocytes were incubated with either fentanyl or morphine, centrifuged, and then examined ultrastructurally by electron microscopy. RESULTS: In experiment A, fentanyl inhibited oxygen consumption by up to 40% (P < .01). Morphine inhibited oxygen consumption to a maximum of 25% (P < .01). In experiment B, in the presence of oligomycin, fentanyl increased the inhibition of oxygen consumption; however, in the presence of myxothiazol, no further inhibition by fentanyl occurred. In experiment C, mild ultrastructural alterations to hepatocytes were observed after incubation with fentanyl but not with morphine. CONCLUSIONS: This study demonstrates that therapeutic doses of two commonly used analgesic drugs impair neonatal hepatic oxidative metabolism. Fentanyl exerts a greater effect than morphine by diminishing liver oxygen consumption by up to 40%. The inhibitory effect of fentanyl occurs directly on the mitochondrial respiratory chain, either on substrate oxidation or on the thermogenic proton leak. The findings of this study are relevant to the perioperative management of surgical neonates.

Analgesics, Opioid↗
PubMed1999

Free radical formation in infants: the effect of critical illness, parenteral nutrition, and enteral feeding.

BACKGROUND/PURPOSE: An increase in free radical activity has been observed in patients suffering from a variety of illnesses and has been correlated with disease severity. Free radical production is increased by the administration of total parenteral nutrition (TPN) and may be linked to its adverse effects. Some of the complications of TPN can be ameliorated by partial enteral feeding. The aim of this study was to investigate free radical activity during critical illness and during the administration of parenteral nutrition. METHODS: Three groups of surgical infants were studied: (1) control infants (n = 8) before minor surgery, (2) stable infants on the ward recovering from a major operation (n = 24), (3) critically ill infants in the neonatal intensive care unit (NICU, n = 28). Fourteen patients in the ward and 17 patients in NICU were receiving parenteral nutrition. Of the 31 patients on TPN, 9 were also receiving minimal enteral feeding (3% to 24% of total calorie intake). Plasma malondialdehyde (MDA), an index of free radical activity, was measured in all 60 infants. The Paediatric Risk of Mortality (PRISM) score was obtained on NICU patients. The cytokines tumor necrosis factor (TNF-alpha) and interleukin 6 (IL-6) were measured in 25 patients. RESULTS: Plasma MDA was significantly higher in (1) the stable patients on the ward compared with control patients (P < .001) and (2) patients in NICU compared with stable patients in the ward (P < .001). Parenteral nutrition was associated with higher levels of plasma MDA both in stable patients in the ward and critically ill infants in NICU. There was no correlation between the PRISM score and MDA. In patients not receiving TPN there is a correlation between MDA and TNF-alpha (r = 0.54, P = .02) and between MDA and IL-6 (r = 0.74, P = .001). The level of free radical activity in patients on TPN is not changed by partial enteral feeding. CONCLUSIONS: Critical illness causes a rise in free radical production. Parenteral nutrition causes a significant elevation in free radical activity in both stable infants in the ward and critically ill infants in NICU. The addition of minimal enteral feeding to parenteral nutrition does not reduce free radical activity. We hypothesize that the parenteral nutrition solution directly initiates free radical production.

Analysis of Variance↗
PubMed1999

Neonatal oxidative liver metabolism: effects of hydrogen peroxide, a putative mediator of septic damage.

BACKGROUND/PURPOSE: Surgical neonates are at risk for sepsis and liver dysfunction. These complications are more common in preterm neonates and in those who receive total parenteral nutrition. Elevated levels of reactive oxygen species (eg, hydrogen peroxide) have been reported in these "at-risk" patients and may be the mediators of liver impairment via their effect on oxidative energy metabolism. The aim of this study was to test the hypothesis that elevated levels of hydrogen peroxide (H2O2) impair neonatal liver oxidative energy metabolism. METHODS: An in vitro model to test this hypothesis was developed in hepatocytes isolated from neonatal (11-day to 15-day) rats. The cells, respiring on palmitate (0.5 mmol/L in 2% bovine serum albumin), were exposed to H2O2. Oxygen consumption was measured polarographically. In experiment A, H2O2 was added to the cell preparation at different concentrations (0.5 mmol/L, 1 mmol/L, 1.5 mmol/L, 2 mmol/L) to assess the effect on oxygen consumption. In experiment B, H2O2 (2 mmol/L) was added to hepatocytes in the presence of inhibitors of mitochondrial respiration to define the site of action of H2O2. In experiment C, electron microscopy was performed on hepatocytes after incubation with 1 mmol/L and 2 mmol/L of H2O2. RESULTS: In experiment A, H2O2 significantly reduced hepatocyte oxygen consumption at 1.5 and 2 mmol/L. In experiment B, in the presence of inhibitors of mitochondrial respiration, myxothiazol (inhibitor of substrate oxidation), and oligomycin (inhibitor of adenosine triphosphate (ATP) synthase), no further inhibition by H2O2 occurred, indicating that the effect of H2O2 was intramitochondrial and affecting the synthesis of ATP. In experiment C, microscopic alterations of mitochondria were noticed exclusively in hepatocytes incubated with 2 mmol/L H2O2. CONCLUSIONS: Results of this study demonstrate that H2O2 impairs neonatal liver oxidative metabolism. H2O2 probably directly inhibits ATP synthase. The authors hypothesize that H2O2 may play a role in the biochemical pathogenesis of liver dysfunction associated with sepsis. Identification of the precise target site of H2O2 may be valuable in directing therapy in septic neonates.

Adenosine Triphosphate↗
PubMed1999

Digital imaging: an accurate and easy method of measuring foot ulcers.

AIMS: A progressive reduction in the area of foot ulcer on serial measurement is traditionally done by tracing the margin of the ulcer on a transparent film and counting the number of squares on a graph paper underneath. We set out to use and validate the measurement of foot ulcers using a digital imaging technique and compare this with the traditional method. METHODS: Thirty diabetic foot ulcers (18 patients) were studied over 10 weeks. Each ulcer was traced by three independent observers with a pen over a flexigrid Opsite film and digital photographs were taken. Each observer calculated the area of an ulcer first using a 1-mm2 graph paper and then with the computer software. For each ulcer we calculated the mean area using measurements from all the observers. We then calculated the deviation from this mean for each observer. RESULTS: There was significantly less interobserver variation using the digital image than the traditional method with mean coefficient of variation (CV) 16% vs. 27%; P = 0.05. CONCLUSIONS: The digital imaging method was faster and easier to use and the patients preferred it, as it was a noncontact method. In addition it also provides a photographic record for comparison.

Diabetic Foot↗
PubMed1999

Combined microlightguide spectrophotometry and microendoscopy for measurement of oxygen saturation in peripheral nerves.

Microlightguide measurements of the spectral composition of backscattered light may be used to determine local tissue oxygen saturation and monitor tissue perfusion using intravenous injection of fluorescein dye as a contrast agent. We have used a combination of microlightguide spectrophotometry and microendoscopy to measure intravascular oxygen saturation (HbSaO2%) and monitor blood flow in the sciatic nerve of 12 healthy male Sprague-Dawley rats. The microlightguide and endoscope combination is a relatively new measurement technique. The aims of this study were to determine whether microlightguide spectrophotometry and microendoscopy could be used to measure HbO2 and blood flow in peripheral nerves and to compare the measurements made using the flexible lightguide with the endoscope-lightguide combination. We found no significant difference between the two types of measurement over similar regions of the nerve. mean SaO2% values 77.1% (95% CI = 75.4-78.8) and 78.8% (95% CI = 77.5-80.1) respectively. During a period of hypoxia there was a similar fall in both arterial and nerve oxygen saturation. Following injection of fluorescein, the rate of increase in nerve fluorescence was used as a measure of perfusion. The combination of microlightguide spectrophotometry and microendoscopy allows the exact site of measurement to be directly visualized. The minimally invasive nature of this technique may allow its application to the study of peripheral nerves in human subjects in conditions such as diabetic neuropathy where vascular factors are thought to have an important role in aetiology.

Animals↗
PubMed1998

Control of mitochondrial beta-oxidation: sensitivity of the trifunctional protein to [NAD+]/[NADH] and [acetyl-CoA]/[CoA].

Isolated human mitochondrial trifunctional protein was incubated with 2-hexadecenoyl-CoA, CoA and NAD+ and the resultant CoA esters measured. Steady state with respect to the concentrations of the intermediates 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA and the rate of formation of the product tetradecanoyl-CoA was reached within 4 min. Flux was greatly enhanced by the addition of Tween 20 (0.2% v/v) which stimulated 3-ketoacyl-CoA thiolase activity by over 7-fold. When 3-ketoacyl-CoA thiolase was not stimulated, 3-hydroxyhexadecanoyl-CoA was the prominent CoA ester accumulated, presumably due to inhibition of 3-hydroxyacyl-CoA dehydrogenase activity by accumulated 3-ketoacyl-CoA, analogous to the inhibition of short-chain 3-hydroxyacyl-CoA dehydrogenase by 3-ketoacyl-CoA. When [NAD+]/[NADH] was varied at a fixed total [NAD++NADH], the overall flux was only inhibited by [NAD+]/[NADH] less than 1. In contrast, when [acetyl-CoA]/[CoA] was varied at a fixed total [CoA], much greater sensitivity was observed.

Acetyl Coenzyme A↗