[Back pain--time for a little science?].
Explore the source record for details and available documents.
Back Pain↗
Biomedical subjects
T Clausen
Publications and source records attributed to T Clausen.
[Cisatracurium in patients with compromised kidney function. Pharmacodynamic and intubation conditions under isoflurane-nitrous oxide anesthesia].
OBJECTIVES: The choice of cisatracurium, especially for patients with organ dysfunction, seems to be beneficial, because of organ-independent Hofmann-elimination and less histamine release propensity. This study was designed to investigate pharmacodynamics and intubating conditions after bolus administration of 0.15 mg/kg cisatracurium (3 x ED95) in patients with renal failure and maintained with isoflurane/N2O in oxygen. METHODS: 20 patients with renal failure and 19 patients with normal renal function were studied. Anaesthesia was induced with fentanyl (2-3 micrograms/kg) and thiophentone (4-7 mg/kg). After rapid bolus administration of 0.15 mg/kg cisatracurium (3 x ED95), onset time and intubating conditions were assessed. Clinical duration (DUR 25%), recovery index and duration 90% were investigated by acceleromyography. Changes of mean arterial blood pressure and/or heart rate > or = 20% were defined as clinically significant. RESULTS: The onset time (3.1 +/- 0.8 min) was shorter in patients without renal failure (Cis-1) than in patients with normal renal function (3.6 +/- 0.8 min), but without statistical significance. Intubating conditions, scored according to a 3-step scale, were slightly better in patients with normal renal function. Other pharmacodynamic parameters did not differ significantly. However, a small tendency to a prolonged recovery with a wide inter-individual variety was characteristic for patients with renal failure. Regarding the hemodynamic actions, only minor individual cardiovascular changes occured. No clinical evidence of histamine release was observed in any patient. CONCLUSIONS: The result of this clinical study suggest, that cisatracurium is a suitable choice for patients with renal failure. The necessity for an intraoperative neuromuscular monitoring is given by the marked heterogeneity in the recovery parameters in patients with renal failure.
Adolescent↗
Jens Christian Skou awarded the Nobel prize in chemistry for the identification of the Na+, K(+)-pump.
Explore the source record for details and available documents.
Animals↗
The Na+,K+ pump and muscle excitability.
In most types of mammalian skeletal muscles the total concentration of Na+,K+ pumps is 0.2-0.8 nmol g wet wt(-1). At rest, only around 5% of these Na+,K+ pumps are active, but during high-frequency stimulation, virtually all Na+,K+ pumps may be called into action within a few seconds. Despite this large capacity for active Na+,K+ transport, excitation often induces a net loss of K+, a net gain of Na+, depolarization and ensuing loss of excitability. In muscles exposed to high [K+]o or low [Na+]o, alone or combined, excitability is reduced. Under these conditions, hormonal or excitation-induced stimulation of the Na+,K+ pump leads to considerable force recovery. This recovery can be blocked by ouabain and seems to be the result of Na+,K+ pump induced hyperpolarization and restoration of Na+,K+ gradients. In muscles where the capacity of the Na+,K+ pump is reduced, the decline in the force developing during continuous electrical stimulation (30-90 Hz) is accelerated and the subsequent force recovery considerably delayed. The loss of endurance is significant within a few seconds after the onset of stimulation. Increased concentration of Na+ channels or open-time of Na+ channels is also associated with reduced endurance and impairment of force recovery. This indicates that during contractile activity, excitability is acutely dependent on the ratio between Na+ entry and Na+,K+ pump capacity. Contrary to previous assumptions, the Na+,K+ pump, due to rapid activation of its large transport capacity seems to play a dynamic role in the from second to second ongoing restoration and maintenance of excitability in working skeletal muscle.
Animals↗
Thyroid hormone-induced upregulation of Na+ channels and Na(+)-K+ pumps: implications for contractility.
We have previously observed in rat soleus muscle that endurance is a function of the ratio between the concentration of Na+ channels and Na(+)-K+ pumps [Harrison, A. P., O. B. Nielsen, and T. Clausen. Am. J. Physiol. 272 (Regulatory Integrative Comp. Physiol. 41): R1402-R1408, 1997]. In this study we explore this relationship further by comparing the changes in Na+ channel and Na(+)-K+ pump concentrations induced by injections of 3,5,3'-triiodothyronine (T3) with endurance. T3 induced upregulation of the concentration of Na+ channels and Na(+)-K+ pumps, which was associated with a progressive loss of contractile endurance. The increase in Na+ channels preceded that of the Na(+)-K+ pumps and amounted to 49 and 52% (both P < 0.01) after 48 and 72 h of T3 treatment, respectively. Concomitantly, during 90-Hz stimulation, the initial rate of force decline increased by 42 and 45% after 48 and 72 h of T3 treatment, respectively (both P < 0.001). These observations are important for an understanding of the fatigue associated with hyperthyroidism and add further support to the hypothesis that muscle endurance depends on the leak-to-pump ratio for Na+.
Age Factors↗
Micronutrients, minerals and growth control.
(1) Dietary deficiencies of Zn, Mg and K lead to a rapid drop in serum concentrations, with no change (Zn) or a slow decline (Mg and K) in the concentrations in skeletal muscle. (2) These deficiencies all lead to inhibition of growth and protein synthesis in muscle. (3) The inhibition of protein synthesis is faster in onset than the loss of Zn, Mg and K from muscle and therefore unlikely to result from cellular mineral deficiency. (4) The deficiencies are likely to be detected by the early drop in serum concentration, but the mechanism is unknown. (5) Possible mediators of growth inhibition are anorexia, GH and IGF-I. (6) Early detection of mineral deficiencies allows the organism to minimize wasteful protein synthesis and the formation of functionally inadequate tissues.
Animals↗
Reduced levels of skeletal muscle Na+K+ -ATPase in McArdle disease.
We evaluated the hypothesis that impaired sarcolemmal function associated with exaggerated potassium release, impaired potassium uptake, or both may contribute to exertional fatigue and abnormal circulatory responses to exercise in McArdle disease (MD). The cellular mechanism of exertional fatigue and muscle injury in MD is unknown but likely involves impaired function of the ATPases that couple ATP hydrolysis to cellular work, including the muscle sodium potassium pump (Na+K+-ATPase). However, the concentration of muscle Na+K+ pumps in MD is not known, and no studies have related exercise increases in blood potassium concentrations to muscle Na+K+ pump levels. We measured muscle Na+K+ pumps (3H-ouabain binding) and plasma K+ in response to 20 minutes of cycle exercise in six patients with MD and in six sex-, age-, and weight-matched sedentary individuals. MD patients had lower levels of 3H-ouabain binding (231 +/- 18 pmol/g w.w., mean +/- SD, range, 210 to 251) than control subjects (317 +/- 37, range, 266 to 371, p < 0.0004), higher peak increases in plasma potassium in response to 45 +/- 7 W cycle exercise (MD, 1.00 +/- 0.15 mmol/L; control subjects, 0.48 +/- 0.09; p < 0.0001), and mean exercise heart rate responses to exercise that were 45 +/- 12 bpm greater than control subjects. Our results indicate that Na+K+ pump levels are low in MD patients compared with healthy subjects and identify a limitation of potassium reuptake that could result in sarcolemmal failure during peak rates of membrane activation and may promote exaggerated potassium-activated circulatory responses to submaximal exercise. The mechanism of the low Na+K+ pump concentrations in MD is unknown but may relate to deconditioning or to disruption of a close functional relationship between membrane ion transport and glycolysis.
Adult↗
Clinical and therapeutic significance of the Na+,K+ pump*.
1. The Na+,K+-ATPase or Na+,K+-pump, mediating the active transport of Na+ and K+, which was first identified 40 years ago, is a central target for acute and long-term regulation, as well as for therapeutic intervention. Acute stimulation of the Na+,K+-pump in skeletal muscle by insulin, catecholamines, beta2-agonists or theophylline increases the intracellular uptake of K+ and accounts for the hypokalaemia elicited by these agents. Conversely, digitalis intoxication elicits hyperkalaemia via acute inhibition of the Na+, K+-pump. 2. Simple and accurate methods have been developed for the quantification of the total concentration of Na+,K+-pumps in small (0.5-5 mg) fresh or frozen biopsies of human skeletal muscle, myocardium or other tissues. This has allowed the identification of several long-term regulatory changes in the concentration of this transport system in human tissues. In skeletal muscle, upregulation is induced by training, thyroid hormones or glucocorticoids. Downregulation is seen in hypothyroidism, cardiac insufficiency, myotonic dystrophy, McArdle disease, K+ deficiency and after muscle inactivity. 3. Since the skeletal muscles contain one of the major pools of Na+,K+-pumps, these changes are important for the ability to counterregulate the hyperkalaemia elicited by exercise or the ingestion of K+. Moreover, downregulation or inhibition of the Na+, K+-pumps in skeletal muscle interferes with contractile performance. Since digitalis glycosides bind to the Na+,K+-pump, the muscles constitute a large distribution volume for these agents and are therefore an important determinant for their plasma level. 4. In cardiac insufficiency, the decrease in the concentration of Na+, K+-pumps in the myocardium is over a wide range correlated to the concomitant reduction in ejection fraction. The regulatory and pathophysiological changes in the activity and concentration of Na+, K+-pumps are important for the contractile function of skeletal muscle and heart as well as for K+ homoeostasis and the response to digitalization.
Animals↗
Slow-binding inhibition of Escherichia coli cystathionine beta-lyase by L-aminoethoxyvinylglycine: a kinetic and X-ray study.
The pyridoxal 5'-phosphate (PLP)-dependent cystathionine beta-lyase (CBL) was previously found to be inhibited by the natural toxins rhizobitoxine and l-aminoethoxyvinylglycine (AVG). The present study characterizes the interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG) by a combination of kinetic methods and X-ray crystallography. Upon AVG treatment, time-dependent, slow-binding inhibition [Morrison, J. F. (1982) Trends Biochem. Sci. 7, 102-105] was observed due to the generation of a long-lived, slowly dissociating enzyme-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (CBL + AVG --> CBLAVG, Ki = 1.1 +/- 0.3 microM) with an association rate constant (k1) of 336 +/- 40 M-1 s-1. This value is several orders of magnitude lower than typical bimolecular rate constants of ES formation, suggesting that additional steps occur before formation of the first detectable CBLAVG complex. Loss of activity is paralleled by the conversion of the pyridoxaldimine 426 nm chromophore to a 341 nm-absorbing species. On the basis of the recently solved structure of native CBL [Clausen, T., et al. (1996) J. Mol. Biol. 262, 202-224], it was possible to elucidate the X-ray structure of the CBLAVG complex and to refine it to an R-factor of 16.4% at 2.2 A resolution. The refined structure reveals the geometry of the bound inhibitor and its interactions with residues in the active site of CBL. Both the X-ray structure and the absorbance spectrum of the CBLAVG complex are compatible with a ketimine as the reaction product. Thus, the inhibitor seems to bind in a similar way to CBL as the substrate, but after alpha-proton abstraction, the reaction proceeds in a CBL nontypical manner, i.e. protonation of PLP-C4', resulting in the "dead-end" ketimine PLP derivative. The CBLAVG structure furthermore suggests a binding mode for rhizobitoxine and explains the failure of MVG to inhibit CBL.
Crystallography, X-Ray↗
Cloning, purification, crystallization, and preliminary X-ray diffraction analysis of cystathionine gamma-synthase from E. coli.
The Escherichia coli metB gene has been PCR-extracted from genomic DNA and placed under the control of a tac and a T7 promoter in plasmids pCYB1 and pET22b(+), respectively, to produce overexpressing bacterial strains for the gene product, cystathionine gamma-synthase. Efficient purification procedures have been developed for a C-terminally intein-tagged version and the wild-type target protein, yielding the product in a quantity and homogeneity amenable to high-resolution single-crystal X-ray analysis. Crystals have been obtained in space group P1 with unit cell constants a=82.2 A, b=84.2 A, c=116.2 A, alpha=107.0 degrees, beta=96.3 degrees, gamma=108.0 degrees, suggesting eight monomers per asymmetric unit (V[M]=2.23 A3/Da). Crystals diffract to beyond 2.6 A resolution and a data set complete to 2.8 A resolution has been collected using a rotating anode X-ray source. A cryogenic buffer system has been developed to allow synchrotron data collection. Patterson self rotation searches reveal the presence of two independent tetramers with local 222 symmetry in an asymmetric unit. The crystallographic results corroborate and extend previous solution studies regarding the quaternary organization of the enzyme.
Carbon-Oxygen Lyases↗
Regulation of Na(+)-K+ pump activity in contracting rat muscle.
1. In rat soleus muscle, high frequency electrical stimulation produced a rapid increase in intracellular Na+ (Na+i) content. This was considerably larger in muscles contracting without developing tension than in muscles contracting isometrically. During subsequent rest a net extrusion of Na+ took place at rates which, depending on the frequency and duration of stimulation, approached the maximum transport capacity of the Na(+)-K+ pumps present in the muscle. 2. In isometrically contracting muscles, the net extrusion of Na+ continued for up to 10 min after stimulation, reducing Na+i to values 30% below the resting level (P < 0.001). This undershoot in Na+i, seen in both soleus and extensor digitorum longus muscles, could be maintained for up to 30 min and was blocked by ouabain or cooling to 0 degree C. 3. The undershoot in Na+i could be elicited by direct stimulation as well as by tubocurarine-suppressible stimulation via the motor endplate. It could not be attributed to a decrease in Na+ influx, to effects of noradrenaline or calcitonin gene-related peptide released from nerve endings, to an increase in extracellular K+ or the formation of nitric oxide. 4. The results indicate that excitation rapidly activates the Na(+)-K+ pump, partly via a change in its transport characteristics and partly via an increase in intracellular Na+ concentration. This activation allows an approximately 20-fold increase in the rate of Na+ efflux to take place within 10 s. 5. The excitation-induced activation of the Na(+)-K+ pump may represent a feed-forward mechanism that protects the Na(+)-K+ gradients and the membrane potential in working muscle. Contrary to previous assumptions, the Na(+)-K+ pump seems to play a dynamic role in maintenance of excitability during contractile activity.
Albuterol↗
Glutaconate CoA-transferase from Acidaminococcus fermentans: the crystal structure reveals homology with other CoA-transferases.
BACKGROUND: Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition. Members of this group of enzymes are found in anaerobic fermenting bacteria, aerobic bacteria and in the mitochondria of humans and other mammals, but so far none have been crystallized. A defect in the human gene encoding succinyl-CoA: 3-oxoacid CoA-transferase causes a metabolic disease which leads to severe ketoacidosis, thus reflecting the importance of this family of enzymes. All CoA-transferases share a common mechanism in which the CoA moiety is transferred from a donor (e.g. acetyl CoA) to an acceptor, (R)-2-hydroxyglutarate, whereby acetate is formed. The transfer has been described by a ping-pong mechanism in which CoA is bound to the active-site residue of the enzyme as a covalent thiol ester intermediate. We describe here the crystal structure of glutaconate CoA-transferase (GCT) from the strictly anaerobic bacterium Acidaminococcus fermentans. This enzyme activates (R)-2-hydroxyglutarate to (R)-2-hydroxyglutaryl-CoA in the pathway of glutamate fermentation. We initiated this project to gain further insight into the function of this enzyme and the structural basis for the characteristics of CoA-transferases. RESULTS: The crystal structure of GCT was solved by multiple isomorphous replacement to 2.55 A resolution. The enzyme is a heterooctamer and its overall arrangement of subunits can be regarded as an (AB)4tetramer obeying 222 symmetry. Both subunits A and B belong to the open alpha/beta-protein class and can be described as a four-layered alpha/alpha/beta/alpha type with a novel composition and connectivity of the secondary structure elements. The core of subunit A consists of seven alpha/beta repeats resulting in an all parallel central beta sheet, against which helices pack from both sides. In contrast, the centre of subunit B is formed by a ninefold mixed beta sheet. In both subunits the helical C terminus is folded back onto the N-terminal domain to form the third layer of helices. CONCLUSIONS: The active site of GCT is located at the interface of subunits A and B and is formed by loops of both subunits. The funnel-shaped opening to the active site has a depth and diameter of about 20 A with the catalytic residue, Glu54 of subunit B, at the bottom. The active-site glutamate residue is stabilized by hydrogen bonds. Despite very low amino acid sequence similarity, subunits A and B reveal a similar overall fold. Large parts of their structures can be spatially superimposed, suggesting that both subunits have evolved from a common ancestor.
Amino Acid Sequence↗
Concentrations of lysosomal cysteine proteases are decreased in renal cell carcinoma compared with normal kidney.
Renal cell carcinoma contains significantly lower concentrations of the lysosomal cysteine proteases, cathepsins B, C, H, L and S, than does normal kidney, as shown by several methods, such as activity determination, enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry. The same low levels of enzyme activity and concentration have been determined in renal cell carcinoma metastases in the lung. Our results on the decreased concentration of cysteine peptidases at the protein level would seem to conflict with earlier results on an increased concentration of the cathepsin L mRNA in renal cell carcinoma.
Carcinoma, Renal Cell↗
Effects of reduced electrochemical Na+ gradient on contractility in skeletal muscle: role of the Na+-K+ pump.
Continued excitation of skeletal muscle may induce a combination of a low extracellular Na+ concentration ([Na+]o) and a high extracellular K+ concentration ([K+]o) in the T-tubular lumen, which may contribute to fatigue. Here, we examine the role of the Na+-K+ pump in the maintenance of contractility in isolated rat soleus muscles when the Na+, K+ gradients have been altered. When [Na+]o is lowered to 25 mM by substituting Na+ with choline, tetanic force is decreased to 30% of the control level after 60 min. Subsequent stimulation of the Na+-K+ pump with insulin or catecholamines induces a decrease in [Na+]i and hyperpolarization. This is associated with a force recovery to 80-90% of the control level which can be abolished by ouabain. This force recovery depends on hyperpolarization and is correlated to the decrease in -Na+-i (r = 0. 93; P<0.001). The inhibitory effect of a low -Na+-o on force development is considerably potentiated by increasing [K+]o. Again, stimulation of the Na+-K+ pump leads to rapid force recovery. The Na+-K+ pump has a large potential for rapid compensation of the excitation-induced rundown of Na+, K+ gradients and contributes, via its electrogenic effect, to the membrane potential. We conclude that these actions of the Na+-K+ pump are essential for the maintenance of excitability and contractile force.
Animals↗
Role of Na(+)-K+ pump and Na+ channel concentrations in the contractility of rat soleus muscle.
The dependence of contractile performance on the leak-to-pump ratio for Na+ has been examined. In isolated rat soleus muscle the concentration of Na(+)-K+ pumps was shown to decrease with age (-57%) or K+ deficiency (-69%), whereas Na+ channel concentration remained constant. This relative increase in the ratio between Na+ channels and Na(+)-K+ pumps was associated with a markedly faster rate of force decline (58 and 97%, respectively; both P < 0.001) during stimulation at 90 Hz and reduced subsequent force recovery (-34 and -38%, respectively; both P < 0.001). Similar effects were elicited by acute inhibition of Na(+)-K+ pump activity with ouabain. Preincubation with aconitine and veratridine, resulting in a 91 and 118% increase in Na+ influx per contraction, respectively (both P < 0.05), significantly hastened the initial rate of force decline (19%; P < 0.05 for aconitine and 69%; P < 0.001 for veratridine) and slowed recovery (-59 and -86%, respectively, both P < 0.001). It is concluded that the ratio between excitation-induced Na+ influx and Na(+)-K+ pump capacity is an important determinant for endurance and rate of recovery of force in skeletal muscle.
Aconitine↗
Effects of adrenal steroids on the concentration of Na(+)-K+ pumps in rat skeletal muscle.
Since adrenal steroids have been shown to upregulate the concentration of Na(+)-K(+)-ATPase in cardiac muscle, similar effects could be expected in skeletal muscle. Following infusion of dexamethasone (0.02-0.1 mg/kg per day) for 7 days in 10-week-old rats, the total concentration of [3H]ouabain-binding sites rose by up to 22-42% in soleus, extensor digitorum longus, gastrocnemius and diaphragm muscle. Dexamethasone produced no or minute changes in the Na(+)-K+ contents of skeletal muscle. In contrast, infusion with aldosterone (0.02-0.5 mg/kg per day) for 7 days produced hypokalemia and a graded reduction in the K+ content of skeletal muscle, which was closely correlated to a downregulation of the [3H]ouabain-binding site concentration (r = 0.65-0.70; P < 0.001). The results indicate that in skeletal muscle high doses of glucocorticoids upregulate the concentration of Na(+)-K+ pumps whereas mineralocorticoids induce a downregulation, which is secondary to the concomitant K+ deficiency. Since adrenalectomy produced no significant change in [3H]ouabain-binding site concentration, basal levels of endogenous adrenal steroids seem to be of minor importance for the regulation of Na(+)-K+ pump concentration in skeletal muscle.
Adrenal Cortex Hormones↗
Simple guidelines for experimental reports, documentation and storage of data. A new initiative to further research integrity.
Since 1992, the Danish Medical Research Council has taken several initiatives to oppose scientific misconduct. One preventive measure has been the development of concise guidelines for the presentation of experimental reports and data documentation. After revisions and accept by the major user institutions, these guidelines have been well-received by a large number of research units and are made available to all Ph.D. students and their supervisors. We here describe the motives for the choice of guidelines and how they are adapted to the requirements of ongoing medical research in Denmark.
Clinical Medicine↗
Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase from Escherichia coli at 1.83 A.
Cystathionine beta-lyase (CBL) is a member of the gamma-family of PLP-dependent enzymes, that cleaves C beta-S bonds of a broad variety of substrates. The crystal structure of CBL from E. coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 15.2% at 1.83 A resolution using synchroton radiation diffraction data. The asymmetric unit of the crystal cell (space group C222(1)) contains two monomers related by 2-fold symmetry. A homotetramer with 222 symmetry is built up by crystallographic and non-crystallographic symmetry. Each monomer of CBL can be described in terms of three spatially and functionally different domains. The N-terminal domain (residues 1 to 60) consists of three alpha-helices and one beta-strand. It contributes to tetramer formation and is part of the active site of the adjacent subunit. The second domain (residues 61 to 256) harbors PLP and has an alpha/beta-structure with a seven-stranded beta-sheet as the central part. The remaining C-terminal domain (residues 257 to 395), connected by a long alpha-helix to the PLP-binding domain, consists of four helices packed on the solvent-accessible side of an antiparallel four-stranded beta-sheet. The fold of the C-terminal and the PLP-binding domain and the location of the active site are similar to aminotransferases. Most of the residues in the active site are strongly conserved among the enzymes of the transsulfuration pathway. Additionally, CBL is homologous to the mal gamma gene product indicating an evolutionary relationship between alpha and gamma-family of PLP-dependent enzymes. The structure of the beta, beta, beta-trifluoroalanine inactivated CBL has been refined at 2.3 A resolution to an R-factor of 16.2%. It suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma.
Alanine↗