Biomedical subjects

T Clausen

Publications and source records attributed to T Clausen.

At least 19 recordsLinked to original sources

PubMed2006

Beta3-adrenoceptor agonist stimulation of the Na+, K+ -pump in rat skeletal muscle is mediated by beta2- rather than beta3-adrenoceptors.

BACKGROUND AND PURPOSE: In cardiac muscle, BRL 37344, a selective beta3-adrenoceptor agonist, activates the Na+, K+ -pump via NO signalling. This study investigated whether BRL 37344 also activates the Na+, K+ -pump via beta3-adrenoceptors in skeletal muscle. EXPERIMENTAL APPROACH: Isolated rat soleus muscles were incubated between 1 and 60 min in buffer. Intracellular Na+, K+ content and Na+, K+ -pump activity were measured using flame photometry and ouabain-suppressible 86Rb+ uptake, respectively. Additional muscles were mounted on force transducers and stimulated (60 Hz for 2 s) every 10 min. KEY RESULTS: BRL 37344 (10(-8) -10(-5) M) induced a concentration- and time-dependent reduction in intracellular Na+, and increased ouabain-suppressible 86Rb+ uptake by up to 112%. BRL 37344-induced reductions in intracellular Na+ were blocked by the beta1/beta2-adrenoceptor antagonist, nadolol (10(-7) M), and the beta2-adrenoceptor antagonist, ICI 118,551 (10(-7) -10(-5) M), but not by beta3- or beta1-adrenoceptor antagonists, SR 59230A (10(-7) M) and CGP 20712A (10(-7) -10(-5) M), respectively. Another beta3-adrenoceptor agonist, CL 316,243, did not alter intracellular Na+. BRL 37344-induced reductions in intracellular Na+ were not blocked by L-NAME, an NOS inhibitor, or ODQ, a guanylyl cyclase inhibitor. The NO donors, SNP and SNAP, did not alter intracellular Na+. BRL 37344 rapidly recovered force in muscles depressed by high [K+]o, an effect that was blocked by nadolol, but not L-NAME. CONCLUSIONS AND IMPLICATIONS: In rat soleus muscle, the beta3-adrenoceptor agonist BRL 37344 stimulated the Na+, K+ -pump via beta2-adrenoceptors. A more selective beta3-adrenoceptor agonist did not affect Na+, K+ homeostasis in skeletal muscle. NO did not seem to mediate Na+, K+ -pump stimulation in skeletal muscle.

Adrenergic beta-Agonists↗

PubMed2006

Less strain energy despite fewer misfit dislocations: the impact of ordering.

The average strain state of Ge films grown on Si(111) by surfactant mediated epitaxy has been compared to the ordering of the interfacial misfit dislocation network. Surprisingly, a smaller degree of average lattice relaxation was found in films grown at higher temperature. On the other hand, these films exhibit a better ordered dislocation network. This effect energetically compensates the higher strain at higher growth temperature, leading to the conclusion that, apart from the formation of misfit dislocations, their ordering represents an important channel for lattice-strain energy relaxation.

Journal Article↗

PubMed2006

Local and systemic hemorheological effects of cerebral hyper- and hypoperfusion in a porcine model.

Using a well defined pig model, we investigated whether cerebral hypertension and hypotension influence hemorheological factors. After surgical preparation and stabilization, periods of hyperventilation, controlled periods of cerebral perfusion pressure increases and decreases were utilized. After each period, blood samples were collected from the cannulated femoral artery and vein, and from the superior sagittal sinus. Erythrocyte deformability, whole blood and plasma viscosity and hematological parameters were determined. Erythrocyte deformability significantly worsened in arterial samples after hypertension and hypotension, and in sinus samples it was impaired after hypotension period. Hematocrit significantly increased in arterial and sinus samples during hypertensive period, accompanied by similar alterations in whole blood viscosity. We conclude that hemodynamic changes caused by hyperventilation, hyper- or hypotension can influence hemorheological factors, and suggest that the rheological alterations can affect local hemodynamic and metabolic conditions.

Analysis of Variance↗

PubMed2005

Ionic mechanisms of excitation-induced regulation of Na+-K+-ATPase mRNA expression in isolated rat EDL muscle.

This study investigated the effects of electrical stimulation on Na+-K+-ATPase isoform mRNA, with the aim to identify factors modulating Na+-K+-ATPase mRNA in isolated rat extensor digitorum longus (EDL) muscle. Interventions designed to mimic exercise-induced increases in intracellular Na+ and Ca2+ contents and membrane depolarization were examined. Muscles were mounted on force transducers and stimulated with 60-Hz 10-s pulse trains producing tetanic contractions three times at 10-min intervals. Ouabain (1.0 mM, 120 min), veratridine (0.1 mM, 30 min), and monensin (0.1 mM, 30 min) were used to increase intracellular Na+ content. High extracellular K+ (13 mM, 60 min) and the Ca2+ ionophore A-23187 (0.02 mM, 30 min) were used to induce membrane depolarization and elevated intracellular Ca2+ content, respectively. Muscles were analyzed for Na+-K+-ATPase alpha1-alpha3 and beta1-beta3 mRNA (real-time RT-PCR). Electrical stimulation had no immediate effect on Na+-K+-ATPase mRNA; however at 3 h after stimulation, it increased alpha1, alpha2, and alpha3 mRNA by 223, 621, and 892%, respectively (P = 0.010), without changing beta mRNA. Ouabain, veratridine, and monensin increased intracellular Na+ content by 769, 724, and 598%, respectively (P = 0.001) but did not increase mRNA of any isoform. High intracellular K+ concentration elevated alpha1 mRNA by 160% (P = 0.021), whereas A-23187 elevated alpha3 mRNA by 123% (P = 0.035) but reduced beta1 mRNA by 76% (P = 0.001). In conclusion, electrical stimulation induced subunit-specific increases in Na+-K+-ATPase mRNA in isolated rat EDL muscle. Furthermore, Na+-K+-ATPase mRNA appears to be regulated by different stimuli, including cellular changes associated with membrane depolarization and increased intracellular Ca2+ content but not increased intracellular Na+ content.

Animals↗

PubMed2005

N-Benzyl-p-toluene sulphonamide allows the recording of trains of intracellular action potentials from nerve-stimulated intact fast-twitch skeletal muscle of the rat.

In skeletal muscle, the intracellular recording of trains of action potentials is difficult owing to the movement of the muscle upon stimulation. A potential tool for the removal of muscle movement is the cross-bridge cycle blocker, N-benzyl-p-toluene sulphonamide (BTS), although the effects of BTS on the passive and active membrane properties of intact muscle fibres are not known. Rat extensor digitorum longus (EDL) muscle was used to show that 50 mum BTS reduced tetanic force to approximately 10% of control force, without markedly altering muscle excitability. Incubation with BTS did not alter intracellular K+ content or Na+-K+ pump activity, but produced minor decreases in intracellular Na+ content (7%), resting 22Na+ influx (14%) and excitation-induced 22Na+ influx (29%). Despite these alterations to Na+ fluxes, BTS did not impair muscle excitability, since membrane conductance, resting membrane potential (RMP), rheobase current and the amplitude, overshoot and maximum rate of depolarization of the action potential were all unaltered. However, BTS did induce a small (8%) decrease in the maximum rate of repolarization of the action potential and an increase in the refractory period. The minor effects of BTS on muscle membrane properties did not compromise the ability of the muscle to propagate action potentials, even during tetanic stimulation. In conclusion, BTS can be used successfully to reduce contractility, allowing the intracellular recording of action potentials during both twitch and tetanic contraction of nerve-stimulated muscle, thus making it an excellent tool for the study of electrophysiology in fast-twitch skeletal muscle.

Action Potentials↗

PubMed2005

Na+-K+ pump stimulation restores carbacholine-induced loss of excitability and contractility in rat skeletal muscle.

Intense exercise results in increases in intracellular Na+ and extracellular K+ concentrations, leading to depolarization and a loss of muscle excitability and contractility. Here, we use carbacholine to chronically activate the nicotinic acetylcholine (nACh) receptors to mimic the changes in membrane permeability, chemical Na+ and K+ gradients and membrane potential observed during intense exercise. Intact rat soleus muscles were mounted on force transducers and stimulated electrically to evoke short tetani at regular intervals. Carbacholine produced a 2.6-fold increase in Na+ influx that was tetrodotoxin (TTX) insensitive, but abolished by tubocurarine, resulting in a significant 36% increase in intracellular Na+, and 8% decrease in intracellular K+ content. The mid region, near the motor end plate, had much larger alterations than the more distal regions of the muscle, and showed a larger membrane depolarization from -73 +/- 1 to -60 +/- 1 mV compared with -64 +/- 1 mV. Carbacholine (10(-4) M) significantly reduced tetanic force to 31 +/- 3% of controls, which underwent significant recovery upon application of Na+-K+ pump stimulators: salbutamol (10(-5) M), adrenaline (10(-5) M) and calcitonin gene-related peptide (CGRP; 10(-7) M). The force recovery with salbutamol was accompanied by a recovery of intracellular Na+ and K+ contents, and a small but significant 4-5 mV recovery of membrane potential. Similar results were obtained using succinylcholine (10(-4) M), indicating that Na+-K+ pump stimulation may prevent or restore succinylcholine-induced hyperkalaemia. The stimulation of the Na+-K+ pump allows muscle to partially recover contractility by regaining excitability through electrogenically driven repolarization of the muscle membrane.

Animals↗

PubMed2005

Role of Na,K pumps in restoring contractility following loss of cell membrane integrity in rat skeletal muscle.

BACKGROUND AND AIM: In skeletal muscles, electrical shocks may elicit acute loss of force, possibly related to increased plasma membrane permeability, induced by electroporation (EP). We explore the role of the Na(+),K(+) pumps in force recovery after EP. METHODS: Isolated rat soleus or extensor digitorum longus (EDL) muscles were exposed to EP paradigms in the range 100-800 V cm(-1), and changes in tetanic force, Na(+),K(+) contents, membrane potential, (14)C-sucrose space and the release of the intracellular enzyme lactic acid dehydrogenase (LDH) were characterized. The effects of Na(+),K(+) pump stimulation or inhibition were followed. RESULTS: Electroporation caused voltage-dependent loss of force, followed by varying rates and degrees of recovery. EP induced a reversible loss of K(+) and gain of Na(+), which was not suppressed by tetrodotoxin, but associated with increased (14)C-sucrose space and release of LDH. In soleus, EP at 500 V cm(-1) induced complete loss of force, followed by a spontaneous, partial recovery. Stimulation of active Na(+),K(+) transport by adrenaline, the beta(2)-agonist salbutamol, calcitonin gene-related peptide (CGRP) and dibutyryl cyclic AMP increased initial rate of force recovery by 183-433% and steady-state force level by 104-143%. These effects were blocked by ouabain (10(-3) m), which also completely suppressed spontaneous force recovery. EP caused rapid and marked depolarization, followed by a repolarization, which was accelerated by salbutamol. Also in EDL, EP caused complete loss of force, followed by a spontaneous partial recovery, which was markedly stimulated by salbutamol. CONCLUSION: Electroporation induces reversible depolarization, partial rundown of Na(+),K(+) gradients, cell membrane leakage and loss of force. This may explain the paralysis elicited by electrical shocks. Na(+),K(+) pump stimulation promotes restoration of contractility, possibly via its electrogenic action. The major new information is that the Na(+),K(+) pumps are sufficient to compensate a simple mechanical leakage. This may be important for force recovery in leaky muscle fibres.

Animals↗

PubMed2005

Maternal anthropometric and metabolic factors in the first half of pregnancy and risk of neonatal macrosomia in term pregnancies. A prospective study.

OBJECTIVE: The prevalence of maternal overweight and fetal macrosomia is increasing. Fetal macrosomia is associated with increased risk of maternal and neonatal complications. The objective of the present study was to investigate if maternal metabolic parameters associated with maternal overweight were independent determinants of macrosomia (birth weight > 4500 g or above the 95 percentile of the z-score for standardized birth weight). DESIGN: Prospective population based cohort study of 2050 pregnancies and nested case control study. METHODS: Outcome measures were adjusted risks for macrosomia in relation to early second trimester maternal serum lipids, glucose and insulin (cohort study) and leptin and insulin-like growth factor (73 cases and 146 matched controls). RESULTS: Gestational diabetes was not independently associated with fetal macrosomia. First trimester body mass index (BMI), gestational weight gain and placental weight were associated with macrosomia. High serum insulin and non-high density lipoprotein (HDL)-cholesterol and low serum HDL-cholesterol were associated with increased risk of macrosomia independent of BMI, weight gain, placental weight and gestational diabetes. Slim women with macrosomic infants had higher insulin compared with those with normal weight infants. This relation was not found among obese women. Leptin was not associated with macrosomia after adjusting for maternal BMI. CONCLUSIONS: Blood parameters known to be associated with the metabolic syndrome were risk factors for macrosomia independent of maternal BMI.

Adult↗

PubMed2004

Evidence that the Na+-K+ leak/pump ratio contributes to the difference in endurance between fast- and slow-twitch muscles.

AIM: Muscles containing predominantly fast-twitch (type II) fibres [ext. dig. longus (EDL)] show considerably lower contractile endurance than muscles containing mainly slow-twitch (type I) fibres (soleus). To assess whether differences in Na+-K+ fluxes and excitability might contribute to this phenomenon, we compared excitation-induced Na+-K+ leaks, Na+ channels, Na+-K+ pump capacity, force and compound action potentials (M-waves) in rat EDL and soleus muscles. METHODS: Isolated muscles were mounted for isometric contractions in Krebs-Ringer bicarbonate buffer and exposed to direct or indirect continuous or intermittent electrical stimulation. The time-course of force decline and concomitant changes in Na+-K+ exchange and M-waves were recorded. RESULTS: During continuous stimulation at 60-120 Hz, EDL showed around fivefold faster rate of force decline than soleus. This was associated with a faster loss of excitability as estimated from the area and amplitude of the M-waves. The net uptake of Na+ and the release of K+ per action potential were respectively 6.5- and 6.6-fold larger in EDL than in soleus, which may in part be due to the larger content of Na+ channels in EDL. During intermittent stimulation with 1 s 60 Hz pulse trains, EDL showed eightfold faster rate of force decline than soleus. CONCLUSION: The considerably lower contractile endurance of fast-twitch compared with slow-twitch muscles reflects differences in the rate of excitation-induced loss of excitability. This is attributed to the much larger excitation-induced Na+ influx and K+ efflux, leading to a faster rise in [K+]o in fast-twitch muscles. This may only be partly compensated by the concomitant activation of the Na+-K+ pumps, in particular in fibres showing large passive Na+-K+ leaks or reduced content of Na+-K+ pumps. Thus, endurance depends on the leak/pump ratio for Na+ and K+.

Action Potentials↗

PubMed2003

Decreased muscle strength and contents of Mg and Na,K-pumps in chronic alcoholics occur independently of liver cirrhosis.

OBJECTIVES: To evaluate the influence of established liver cirrhosis on muscle strength and muscle contents of magnesium (Mg), potassium (K) and sodium, potassium pumps (Na,K-pumps) in chronic alcoholic patients. DESIGN: An open cross-sectional study. SETTING AND SUBJECTS: Forty consecutive chronic alcoholics (18 with cirrhosis and 22 without cirrhosis) admitted to the Department of Hepatology, Aarhus University Hospital, Denmark, or to a collaborating alcoholism treatment centre, and 36 healthy control subjects. MAIN OUTCOME MEASURES: Evaluation of participant's subjective physical ability and measurement of maximum isokinetic muscle strength and muscle mass, as well as measurements of Mg, K and Na,K-pumps in skeletal muscle. RESULTS: Maximum isokinetic muscle strength and muscle mass were equally reduced in patients with and without cirrhosis (P < 0.01 all). In keeping with this, both groups of patients felt equally physically restricted. Muscle Mg was reduced to the same extent in the two groups of patients (by 12 and 9%, P < 0.001, both), whereas the muscle K content was only significantly lower in the cirrhotic patients (10%, P < 0.001). The muscle content of Na,K-pumps was reduced by 14%, (P < 0.01) in the cirrhotic patients and by 8% (P < 0.05) in the noncirrhotic patients. CONCLUSION: Our alcoholic patients complained of physical disability, had reduced skeletal muscle mass, isokinetic muscle strength, content of muscle Mg and content of Na,K-pumps. There was no difference between patients with and without cirrhosis. It appears that it is the heavy alcohol intake, and not the cirrhosis per se, that is responsible for the observed defects.

Adult↗

PubMed2003

Brain tissue oxygen monitoring for assessment of autoregulation: preliminary results suggest a new hypothesis.

Brain tissue oxygen monitoring (P(ti)O2 (Neurotrend, Codman, Germany) was employed in addition to standard intracranial pressure (ICP) and cerebral perfusion pressure (CPP) monitoring in seven patients with severe neuronal damage of heterogeneous etiology. The correlation between P(ti)O2 changes and CPP fluctuations during periods of 30 minutes were analyzed, when CPP was above 70 mmHg and lower than 100 mmHg. A new ratio, the CPP-oxygen-reactivity (COR) index was calculated as COR=delta p(ti)O2 %/delta CPP%. The patient COR values were compared to those found in the brain of six noninjured anesthetized piglets. The analysis was performed to determine the significance of synchronous fluctuations of CPP and P(ti)O2, when CPP is above the lower threshold of autoregulation. The correlation between CPP variations and p(ti)O2 variations was found to be strong (R(mean)) = 0.74 +/- 0.17) in the patients and was weak in the uninjured animals (R(mean)) =0.38 +/- 0.43). The COR (mean) was 2.05 +/- 0.57 in patients and 0.78 +/- 0.6 in the animals. In the injured brain of our patients, we observed an unexpectedly close correlation between P(ti)O2 and CPP variations when CPP levels were within a therapeutically targeted range (70 to 100 mmHg). In a porcine model, we could not find this relationship in the noninjured brain. We speculate that an increased COR might be indicative for an impaired local pressure autoregulation. The preliminary data suggest that COR values above "1" might be pathologic. However, the reported sample sizes are too small to provide sufficient statistical power to justify inferential statistical analyses. As such, results are presented with descriptive statistics only, and should be regarded as a hypothesis.

Algorithms↗

PubMed2002

Muscle strength, Na,K-pumps, magnesium and potassium in patients with alcoholic liver cirrhosis -- relation to spironolactone.

OBJECTIVES: To evaluate the muscle strength in relation to muscle contents of magnesium (Mg), potassium (K) and sodium, potassium (Na,K)-pumps in patients with alcoholic cirrhosis. DESIGN: An open cross-sectional study. SETTING AND SUBJECTS: Fifty-one consecutive patients with liver cirrhosis admitted to the Department of Hepatology, Aarhus University Hospital, Denmark, and 28 age- and sex-matched healthy control subjects. MAIN OUTCOME MEASURES: Biopsies of skeletal muscle were performed in patients and controls for measurements of Mg, K, and Na,K-pumps. Furthermore, maximum isokinetic knee extension and skeletal muscle mass were evaluated. RESULTS: Muscle mass, muscle strength, muscle Mg and muscle K were substantially reduced in the patients (P < 0.01, all), and fell with increasing severity of the liver disease reflected in the Child-Pugh (C-P) class. Patients treated with spironolactone for 2 weeks or more, had increased muscle strength, muscle Mg and content of Na,K-pumps, compared with the rest of the patients (P < 0.05, all). In a multivariate analysis of the patients, skeletal muscle mass, muscle Mg and daily alcohol consumption (g) were independent predictors of isokinetic muscle strength (P < 0.05, all). CONCLUSIONS: Patients with alcoholic liver cirrhosis showed considerably reduced muscle strength and muscle Mg was an independent predictor of muscle strength. Surprisingly, in the spironolactone treated patients, muscle weakness was less pronounced, possibly because of the action of spironolactone on muscle Mg, K and Na,K-pump content.

Adult↗

PubMed2002

Altered plasma concentrations of leptin, transforming growth factor-beta(1) and plasminogen activator inhibitor type 2 at 18 weeks of gestation in women destined to develop pre-eclampsia. Circulating markers of disturbed placentation?

BACKGROUND: In many pre-eclamptic women the placentation process seems to be disturbed. Our objective was to investigate if disturbed placentation in pre-eclamptic women may be recognized in early second trimester as altered plasma levels of factors involved in the formation of the uteroplacental unit. METHODS: In a prospective study of 2190 pregnant women we compared plasma leptin, transforming growth factor-beta(1) (TGF-beta(1)) and plasminogen activator inhibitor type 2 (PAI-2) concentrations at 18 weeks' gestation in 71 women with subsequent pre-eclampsia and 71 controls matched for age, parity and first trimester body mass index. RESULTS: Leptin and TGF-beta(1) concentrations were lower and PAI-2 concentration higher in women destined to develop pre-eclampsia relative to controls (leptin: median (25-75 percentiles): 19.0 (14.5-29.0) vs 25.0 (16.0-35.0) ng/ml (p =0.03), TGF-beta(1): 3.2 (2.0-6.1) vs 5.3 (3.8-7.1) ng/ml (P=0.01) and PAI-2: 78.8 (65.1-118.1) vs 67.6 (61.6-79.6) ng/ml (P=0.002)). OR (95 per cent CI) for pre-eclampsia for women in the upper quartile compared to women in the lower quartile were: leptin: 0.2 (0.03-0.7), TGF-beta(1): 0.2 (0.08-0.7) and PAI-2: 3.1 (1.2-8.2). CONCLUSIONS: Altered plasma concentrations levels of factors involved in the process of placentation in women destined to develop pre-eclampsia, indicate that disturbed formation of the uteroplacental unit is reflected in the maternal circulation before 20 weeks' gestation.

Adult↗

PubMed2001

Crystal structures of cystathionine gamma-synthase inhibitor complexes rationalize the increased affinity of a novel inhibitor.

Cystathionine gamma-synthase catalyzes the committed step of methionine biosynthesis. This pathway is unique to microorganisms and plants, rendering the enzyme an attractive target for the development of antimicrobials and herbicides. We solved the crystal structures of complexes of cystathionine gamma-synthase (CGS) from Nicotiana tabacum with inhibitors of different compound classes. The complex with the substrate analog dl-E-2-amino-5-phosphono-3-pentenoic acid verifies the carboxylate-binding function of Arg423 and identifies the phosphate-binding pocket of the active site. The structure shows the function of Lys165 in specificity determination and suggests a role for the flexible side-chain of Tyr163 in catalysis. The importance of hydrophobic interactions for binding to the active-site center is highlighted by the complex with 3-(phosphonomethyl)pyridine-2-carboxylic acid. The low affinity of this compound is due to the non-optimal arrangement of the functional groups binding to the phosphate and carboxylate-recognition site, respectively. The newly identified inhibitor 5-carboxymethylthio-3-(3'-chlorophenyl)-1,2,4-oxadiazol, in contrast, shows the highest affinity to CGS reported so far. This affinity is due to binding to an additional active-site pocket not used by the physiological substrates. The inhibitor binds to the carboxylate-recognition site, and its tightly bent conformation enables it to occupy the novel binding pocket between Arg423 and Ser388. The described structures suggest improvements for known inhibitors and give guidelines for the development of new lead compounds.

2-Amino-5-phosphonovalerate↗

PubMed2001

Induced mitochondrial failure in the feline brain: implications for understanding acute post-traumatic metabolic events.

OBJECTIVE: Recently, evidence has become available implicating mitochondrial failure as a crucial factor in the pathogenesis of acute brain damage following severe traumatic brain injury (TBI). However, it remains unclear how mitochondrial dysfunction affects cerebral metabolism. Therefore the aim of the study was to evaluate the impact of 'isolated' mitochondrial failure on local cerebral metabolism. METHODS: Cerebral mitochondrial metabolism was blocked by local microdialysis perfusion with cyanide in seven cats. Local brain tissue oxygen tension (p(tiO(2))), carbon dioxide tension (p(tiCO(2))) and pH, as well as extracellular cerebral fluid, glucose, lactate, pyruvate and glutamate were monitored, using a Neurotrend sensor and microdialysis, respectively. Tissue oxygen consumption was measured in a microrespirometric system, and ultrastructural changes evaluated via electron microscopy. RESULTS: Brain tissue oxygen tension increased from a baseline of 31+/-9 mmHg to 84+/-30 mmHg after 60 min of cyanide perfusion (P<0.05), concomitant a decrease in oxygen consumption from 14.45+/-3.91 microl/h/mg to 10.83+/-1.74 microl/h/mg (P<0.05). Brain tissue pH was decreased after 60 min of cyanide perfusion (6.83+/-0.16) compared to baseline (7.07+/-0.39) (P<0.05), whereas p(tiCO(2)) did not show significant changes. Lactate massively increased from a baseline of 599+/-270 micromol/l to 2609+/-1188 micromol/l immediately after cyanide perfusion (P<0.05). The lactate:glucose ratio increased from 0.79+/-0.15 before cyanide perfusion to 6.40+/-1.44 at 40 min after cyanide perfusion (P<0.05), while no significant changes in the lactate:pyruvate ratio could be observed. Glutamate increased from a baseline of 11.6+/-7.2 micromol/l to 61.4+/-44.7 micromol/l after cyanide perfusion (P<0.05). CONCLUSION: The results of this study show that 'isolated' cerebral mitochondrial failure initiates changes in cerebral substrates and biochemistry, which are very similar to most of the changes seen after severe human head injury, except for the early fall in p(tiO(2)), further indicating a crucial involvement of mitochondrial impairment in the development of brain damage after TBI.

Animals↗

PubMed2001

X-ray structure of 12-oxophytodienoate reductase 1 provides structural insight into substrate binding and specificity within the family of OYE.

BACKGROUND: 12-Oxophytodienoate reductase (OPR) is a flavin mononucleotide (FMN)-dependent oxidoreductase in plants that belongs to the family of Old Yellow Enzyme (OYE). It was initially characterized as an enzyme involved in the biosynthesis of the plant hormone jasmonic acid, where it catalyzes the reduction of the cyclic fatty acid derivative 9S,13S-12-oxophytodienoate (9S,13S-OPDA) to 1S,2S-3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoate. Several isozymes of OPR are now known that show different stereoselectivities with regard to the four stereoisomers of OPDA. RESULTS: Here, we report the high-resolution crystal structure of OPR1 from Lycopersicon esculentum and its complex structures with the substrate 9R,13R-OPDA and with polyethylene glycol 400. OPR1 crystallizes as a monomer and folds into a (betaalpha)(8) barrel with an overall structure similar to OYE. The cyclopentenone ring of 9R,13R-OPDA is stacked above the flavin and activated by two hydrogen bonds to His187 and His190. The olefinic bond is properly positioned for hydride transfer from the FMN N(5) and proton transfer from Tyr192 to Cbeta and Calpha, respectively. Comparison of the OPR1 and OYE structures reveals striking differences in the loops responsible for binding 9R,13R-OPDA in OPR1. CONCLUSIONS: Despite extensive biochemical characterization, the physiological function of OYE still remains unknown. The similar catalytic cavity structures and the substrate binding mode in OPR1 strongly support the assumption that alpha,beta-unsaturated carbonyl compounds are physiological substrates of the OYE family. The specific binding of 9R,13R-OPDA by OPR1 explains the experimentally observed stereoselectivity and argues in favor of 9R,13R-OPDA or a structurally related oxylipin as natural substrate of OPR1.

Binding Sites↗

PubMed2001

Structural basis for the function of pyridoxine 5'-phosphate synthase.

BACKGROUND: Pyridoxal 5'-phosphate is the active form of vitamin B(6) that acts as an essential, ubiquitous coenzyme in amino acid metabolism. In Escherichia coli, the pathway of the de novo biosynthesis of vitamin B(6) results in the formation of pyridoxine 5'-phosphate (PNP), which can be regarded as the first synthesized B(6) vitamer. PNP synthase (commonly referred to as PdxJ) is a homooctameric enzyme that catalyzes the final step in this pathway, a complex intramolecular condensation reaction between 1-deoxy-D-xylulose-5'-phosphate and 1-amino-acetone-3-phosphate. RESULTS: The crystal structure of E. coli PNP synthase was solved by single isomorphous replacement with anomalous scattering and refined at a resolution of 2.0 A. The monomer of PNP synthase consists of one compact domain that adopts the abundant TIM barrel fold. Intersubunit contacts are mediated by three additional helices, respective to the classical TIM barrel helices, generating a tetramer of symmetric dimers with 422 symmetry. In the shared active sites of the active dimers, Arg20 is directly involved in substrate binding of the partner monomer. Furthermore, the structure of PNP synthase with its physiological products, PNP and P(i), was determined at 2.3 A resolution, which provides insight into the dynamic action of the enzyme and allows us to identify amino acids critical for enzymatic function. CONCLUSION: The high-resolution structures of the free enzyme and the enzyme-product complex of E. coli PNP synthase suggest essentials of the enzymatic mechanism. The main catalytic features are active site closure upon substrate binding by rearrangement of one C-terminal loop of the TIM barrel, charge-charge stabilization of the protonated Schiff-base intermediate, the presence of two phosphate binding sites, and a water channel that penetrates the beta barrel and allows the release of water molecules in the closed state. All related PNP synthases are predicted to fold into a similar TIM barrel pattern and have comparable active site architecture. Thus, a common mechanism can be anticipated.

Bacterial Proteins↗

PubMed2001

Crystal structure of transcription factor MalT domain III: a novel helix repeat fold implicated in regulated oligomerization.

BACKGROUND: MalT from Escherichia coli, the best-studied member of the MalT family of ATP-dependent transcriptional activators, regulates the genes for malto-oligosaccharide utilization. The active form of this 4 domain protein is a homooligomer, and its multimerization is induced by the binding of maltotriose. Domains II and III of MalT were suggested to mediate the oligomerization process, but its molecular mechanism and the specific functions of these domains remain to be identified. RESULTS: We solved the crystal structure of MalT domain III at 1.45 A resolution by multiple isomorphous replacement phasing. The structure reveals eight copies of a two-helix bundle motif arranged in a novel, right-handed superhelix fold with closed walls, followed by a small C-terminal subdomain. The MalT superhelix contains a potential maltotriose binding site and forms a large hydrophobic protein-protein interaction interface that mediates the contact between two MalT domain III molecules. Structure-based analysis of the two-helix bundle motifs revealed a novel degenerated sequence pattern, and repeats of this pattern could be identified in other regulator proteins. CONCLUSIONS: MalT domain III contains a novel superhelix fold. Its protein-protein interaction interface, however, resembles protein binding sites of other superhelical proteins, suggesting a model with domain III mediating MalT oligomerization. Maltotriose seems to modulate the interaction interface and MalT oligomerization by occupying the ligand binding site inside the superhelix. Similar structural and mechanistic features in other MalT protein-family members and unrelated regulator proteins are indicated by the reappearance of a novel sequence motif derived from the MalT domain III structure.

Amino Acid Motifs↗