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Biomedical subjects

M Wikström

Publications and source records attributed to M Wikström.

At least 109 records · Page 6Linked to original sources
PubMed1995

The action of 5-HT on calcium-dependent potassium channels and on the spinal locomotor network in lamprey is mediated by 5-HT1A-like receptors.

5-HT has a powerful modulatory action on the firing properties of single neurons as well as on locomotor activity. In lamprey, 5-HT increases the neuronal firing frequency in spinal neurons by reducing the conductance in Ca(2+)-dependent K+ channels (KCa) underlying the slow afterhyperpolarization (sAHP), and it also lowers the burst frequency of the spinal locomotor network. To elucidate which type of 5-HT receptor mediates these effects, different specific receptor agonists and antagonists were applied during intracellular current clamp recordings and during NMDA-induced fictive locomotion in the lamprey spinal cord in vitro preparation. The 5-HT1A receptor agonist 8-OH-DPAT ((+/-)-8-hydroxy-dipropylaminotetralin hydrobromide), the 5-HT1 receptor agonist 5-CT (5-carboxyamidotryptamine maleate) and the 5-HT2 receptor agonist alpha-CH3-5-HT (alpha-methylserotonin maleate) all reproduced the actions of 5-HT at both the cellular and the network levels. The effects of all agonists were completely or partially blocked by the 5-HT1A and 5-HT2 receptor antagonist spiperone (spiroperidol hydrochloride) while selective 5-HT2 receptor antagonists were ineffective. The selective 5-HT1A receptor antagonist S(-)-UH301 (S(-)-5-fluoro-8-hydroxy-dipropylaminotetralin hydrochloride) also counteracted the effect of 5-HT on the sAHP. 5-HT3 and 5-HT4 receptor agonists and antagonists were without effects. The intracellular coupling mechanism was not sensitive to pertussis toxin nor to the cAMP dependent protein kinase blocker (Rp)-cAMPS.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1995

A novel antiporter activity catalyzing sodium and potassium transport from right-side-out vesicles of E. coli.

Downhill sodium efflux from right-side-out E. coli membrane vesicles was found to be stimulated by negative electric potential, as has been reported earlier [Bassilana et al., Biochemistry 23 (1984) 1015-1022], and in agreement with the concept of electrogenic Na+/nH+ antiporters with n > 1. However, sodium efflux was much more accelerated by positive electric potential, indicating the operation of another sodium transport system. delta pH (alkaline inside), created by a pH shift from 8.5 to 6.8 in the medium was found to drive sodium efflux against its concentration gradient, but only when the vesicles had been loaded with both Na+ and K+. Efflux of K+ against the concentration gradient was also observed under these conditions. When the vesicles were loaded separately with sodium tricine or potassium tricine, no K+ efflux and insignificant Na+ efflux were observed. We propose that there are at least two different mechanisms responsible for Na+ efflux in E. coli vesicles. One is the Na+/nH+ antiporter previously described, and the other is a novel Na+,K+/mH+ antiporter.

Antiporters↗
PubMed1995

Proton transfer in cytochrome bo3 ubiquinol oxidase of Escherichia coli: second-site mutations in subunit I that restore proton pumping in the mutant Asp135-->Asn.

The ubiquinol oxidase, cytochrome bo3, of Escherichia coli is a member of the respiratory heme-copper oxidase family and conserves energy from the reduction of dioxygen to water by translocation of protons across the bacterial membrane. Mutation of an aspartic acid residue (Asp135) to asparagine in subunit I of this enzyme was previously found to impair proton translocation [Thomas et al. (1993) Biochemistry 32, 10923-10928]. This residue is located in an interhelical "loop" between transmembranous helices II and III, which contains six well-conserved residues (Asn124, Pro128, Gly132, Asp135, Pro139, and Asn142). Site-directed mutagenesis was performed to study the function of this entire domain. Nonconservative mutations of Asn124 and Asn142 also resulted in a loss of proton translocation, whereas their conservative substitution to glutamine had no effect. Mutations in eight other positions within this domain did not affect proton translocation. Introduction of an acidic group at positions 139 or 142, but not at eight other tested positions, restored proton pumping in the Asp135-->Asn mutated protein. These results suggest that the C-terminal part of the domain may be alpha-helical and that the entire "loop" plays an important structural and functional role as part of an input channel of the proton translocation machinery.

Amino Acid Sequence↗
PubMed1995

Control of lamprey locomotor neurons by colocalized monoamine transmitters.

Neurons in the central nervous system (CNS) often store more than one neurotransmitter, but as yet the functional significance of this type of coexistence is poorly understood. 5-Hydroxytryptamine (5-HT) modulates calcium-dependent K+ channels (KCa) responsible for the postspike afterhyperpolarization in different regions of the CNS. In lamprey, 5-HT neurons control apamine-sensitive KCa channels in spinal locomotor network interneurons, thereby in addition regulating the duration of locomotor bursts. We report here that these spinal 5-HT neurons also contain dopamine. Like 5-HT, dopamine causes a reduction of the afterhyperpolarization, but in this case it is due to a reduction of calcium entry during the action potential, which results in a reduced activation of KCa. 5-HT and dopamine are both released from these midline neurons, and both reduce the afterhyperpolarization through two distinctly different, but complementary cellular mechanisms. The net effect of dopamine (10-100 microM) on the locomotor network is similar to that of 5-HT, and the effects of dopamine and 5-HT are additive at the network level.

Action Potentials↗
PubMed1995

Measurements of magnetic field variations in the human brain using a 3D-FT multiple gradient echo technique.

A magnetic resonance 3DFT multiple gradient-echo technique was used for measurements of the proton spectrum for each voxel in the measured slice. Water, fat, magnetic field and T2 distributions in the head of a normal volunteer and a patient with intracerebral hematoma were computed. Magnetic field variations caused by the head were calculated after correction for the static magnetic field inhomogeneity. Large local magnetic field variations up to 3 ppm were found in the human brain near interfaces between air or bone and brain tissues and 0.5 ppm between hematoma and brain tissue. Information about magnetic field variations could be useful for shimming procedures in vivo and for correcting artifacts in imaging and spectroscopy.

Aged↗
PubMed1995

Structural features of a polypeptide carrier promoting secretion of a beta-lactamase fusion protein in yeast.

Escherichia coli beta-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150 delta-carrier. The hsp150 delta-carrier is an N-terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 times in tandem. Here we expressed the hsp150 delta-carrier fragment alone in S. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150 delta-carrier were synthesized. About half of the de novo synthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre-Golgi compartment. The extensively O-glycosylated carrier fragment was purified from the culture medium under non-denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary structure. Nuclear magnetic resonance spectroscopy showed that a non-glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure. The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologous proteins attached to it.

Amino Acid Sequence↗
PubMed1995

Occurrence of enteric rods, staphylococci and Candida in subgingival samples.

The frequency and percentage of enteric rods, staphylococci and Candida were determined in 973 subgingival samples collected from 535 patients subjected to different periodontal treatment procedures. The analysis was performed with culture technique using selective and nonselective media. One or more organisms were detected in 65.5% of the samples and in 76.7% of the patients. In most samples enteric rods, staphylococci and/or Candida constituted a small amount of the total microbial viable count. Enteric rods exceeded 10% of the total viable count in 30 samples. Staphylococci occurred in more than 10% in only 3 samples. In these 3 samples, enterics constituted more than 10% of the total viable count. Candida was not found to exceed 10% of the total viable count in any of the samples. No statistically significant correlation was found between the presence of any of the target microorganisms and kind of periodontal treatment procedure received, antibiotic administration or sample transport time.

Adolescent↗
PubMed1995

Dy-DTPA-BMA as an indicator of tissue viability in MR imaging. An experimental study in the pig.

The aim of this study was to investigate whether dysprosium (Dy) induced signal intensity (SI) loss in infarcted tissue in MR imaging. Myocardial infarction was induced in 12 pigs and Dy-DTPA-BMA (1.0 mmol/kg b.w.) was administered i.v. to 6 pigs 4 hours after occlusion and allowed to accumulate in the infarctions for 2 hours. Dy was analysed by inductively coupled plasma atomic emission spectrometry in infarcted and non-ischaemic tissue samples. The remaining 6 pigs, not administered contrast medium, served as controls. The infarctions demonstrated a high SI in the proton density- and T2-weighted sequences in both groups (ex vivo), although the Dy-DTPA-BMA group demonstrated a 3-fold greater concentration of Dy in infarcted compared with non-ischaemic myocardium. The lack of SI loss after Dy accumulation indicates that susceptibility effects are minor or absent in infarcted myocardium.

Analysis of Variance↗
PubMed1995

MR imaging of double-contrast enhanced porcine myocardial infarction. Correlation with microdialysis.

MR imaging was performed to investigate whether Gd-DTPA-BMA-induced contrast enhancement of myocardial infarction is counteracted by Dy-DTPA-BMA. Myocardial infarction was induced in 5 pigs. Microdialysate probes were inserted in ischemic and nonischemic myocardium. Gd-DTPA-BMA (0.3 mmol/kg b.w.) and Dy-DTPA-BMA (1.0 mmol/kg b.w.) were administered i.v. 4 hours post occlusion. The microdialysate was collected every 10 min and measured for Gd and Dy using inductively coupled plasma atomic emission spectrometry. The pigs were sacrificed 2 hours after administration of contrast media. The concentration of both contrast agents was 3 times higher in infarcted myocardium than in nonischemic myocardium. The infarctions displayed high signal intensity in spin-echo sequences ex vivo. This lack of detectable susceptibility effects from Dy may be caused by loss of cell membrane integrity in infarcted tissue as shown by our microdialysate and biopsy data.

Analysis of Variance↗
PubMed1995

MR imaging of gadolinium-DTPA-BMA-enhanced reperfused and nonreperfused porcine myocardial infarction.

To investigate whether Gd-DTPA-BMA-enhanced MR imaging permits differentiation between reperfused and nonreperfused myocardial infarction, myocardial infarction was induced in 12 domestic pigs. In 6 pigs, Gd-DTPA-BMA, 0.3 mmol/kg b.w. was administered i.v. 60 min after the occlusion. In 6 other pigs, the infarctions were reperfused 80 min after the occlusion, followed by injection of Gd-DTPA-BMA after 20 min of reperfusion. Radiolabeled microspheres were used to confirm zero-flow during the occlusion period and reperfusion in the infarcted myocardium. All pigs were killed 20 min after injection of contrast medium, and the hearts were excised and imaged with MR. The Gd concentration was measured in infarcted and nonischemic myocardium by ICP-AES. In the reperfused hearts, the infarctions were strongly highlighted, corresponding to a 5-fold higher Gd concentration in infarcted vis-à-vis nonischemic myocardium. In the hearts subjected to occlusion without reperfusion, there was only a rim of enhancement in the peripheral part of the infarctions.

Animals↗
PubMed1994

Three-dimensional solution structure of an immunoglobulin light chain-binding domain of protein L. Comparison with the IgG-binding domains of protein G.

Protein L is a multidomain protein expressed at the surface of some strains of the anaerobic bacterial species Peptostreptococcus magnus. It has affinity for immunoglobulin (Ig) through interaction with framework structures in the variable Ig light chain domain. The Ig-binding activity is located to five homologous repeats called B1-B5 in the N-terminal part of the protein. We have determined the three-dimensional solution structure of the 76 amino acid residue long B1 domain using NMR spectroscopy and distance geometry-restrained simulated annealing. The domain is composed of a 15 amino acid residue long disordered N-terminus followed by a folded portion comprising an alpha-helix packed against a four-stranded beta-sheet. These secondary structural elements are well determined with a backbone atomic root mean square deviation from their mean of 0.54 A. The B domains of protein L show very limited sequence homology to the domains of streptococcal protein G interacting with the heavy chains of IgG. However, despite this fact, and their different binding properties, the fold of the B1 domain was found to be similar to the fold of the IgG-binding protein G domains [Wikström, M., Sjöbring, U., Kastern, W., Björck, L., Drakenberg, T., & Forsén, S. (1993) Biochemistry 32, 3381-3386]. In the present study, the solution structure of the B1 domain enabled a more detailed comparison which can explain the different Ig-binding specificities of these two bacterial surface proteins. Among the differences observed, the alpha-helix orientation is the most striking.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Isolation and characterisation of subcomplexes of the mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Enzymically active subcomplexes were purified from bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I) by sucrose-gradient centrifugation in the presence of detergents. These subcomplexes, named I lambda, IS, and I lambda S, catalyse ferricyanide and ubiquinone-1 (Q-1) reduction by NADH at similar rates to complex I, but do not catalyse the reduction of decylubiquinone. In addition, the Q-1 reductase activity of all the subcomplexes is insensitive to rotenone. Chemical and EPR analyses of the subcomplexes show that FMN and all the Fe-S clusters of complex I are present, but that the line shape of cluster 2 is modified. The smallest subcomplex, I lambda S, contains only approximately 13 subunits, as compared to approximately 22 in the previously described subcomplex I alpha [Finel, M., Skehel, J. M., Albracht, S. J. P., Fearnley, I. M. & Walker, J. E. (1992) Biochemistry 31, 11425-11434], but it retains the 75-, 51-, 49-, 30-, 24-, 23- (TYKY) and 20-kDa (PSST) subunits, which are suggested to form a functional core that comprises the EPR-detectable Fe-S clusters 1-4, and FMN. The structural and functional implications of such an arrangement are discussed.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis of residues within helix VI in subunit I of the cytochrome bo3 ubiquinol oxidase from Escherichia coli suggests that tyrosine 288 may be a CuB ligand.

The heme-copper oxidase superfamily contains all of the mammalian mitochondrial cytochrome c oxidases, as well as most prokaryotic respiratory oxidases. All members of the superfamily have a subunit homologous to subunit I of the mammalian cytochrome c oxidases. This subunit provides the amino acid ligands to a low-spin heme component as well as to a heme-copper binuclear center, which is the site where dioxygen is reduced to water. The amino acid sequence of transmembrane helix VI of subunit I is the most highly conserved within the superfamily. Previous efforts have demonstrated that one of the residues in this region, H284, is critical for oxidase activity and for the assembly of CuB. This paper presents the analysis of additional site-directed mutants in which other highly conserved residues in helix VI (P285, E286, Y288, and P293) have been substituted. Most of the mutants are enzymatically inactive. Structural perturbations reported by Fourier transform infrared absorption difference spectroscopy of CO adducts of the mutant oxidases confirm the previous suggestion that this region is adjactent to CuB. Furthermore, the analysis of five different substitutions for Y288 indicates that all lack CuB. On the basis of these data, it is proposed that Y288 may be a CuB ligand along with H333, H334, and H284, and a plausible molecular model of the CuB site is presented.

Amino Acid Sequence↗
PubMed1994

Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (Complex I).

Diphenyleneiodonium (DPI) inhibits the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) on the substrate side of the Fe-S clusters. In the inhibited NADH-supplemented state all of the Fe-S clusters are oxidized, whereas the reduced minus oxidized difference spectrum of the protein-bound FMN can be visualized. It is characterized by troughs at 370 and 450 nm and a small increase of absorbance in the 500-700-nm region. DPI probably reacts irreversibly with FMN, because oxidation of FMN is blocked even after its extraction from the enzyme. Inhibition requires preincubation of enzyme in the presence of NADH and DPI. The lower the NADH/NAD+ ratio or the pH, or the higher the NAD+/DPI ratio, the more DPI is required for inhibition. NAD+ and DPI apparently compete for a common site. Both ubiquinone and dichlorophenolindophenol reductase activities are fully blocked by DPI, whereas the ferricyanide reductase activity is inhibited by 75%. Similar results were found with Complex I and two rotenone-insensitive preparations, subcomplex I lambda and the flavoprotein fraction. DPI also inhibits NADH oxidation by bacterial NADH-ubiquinone oxidoreductase-1 (NDH-1) in membranes of Paracoccus denitrificans and Escherichia coli.

Electron Spin Resonance Spectroscopy↗
PubMed1994

Oxygen binding and activation: early steps in the reaction of oxygen with cytochrome c oxidase.

We have studied the flow-flash reaction of fully reduced cytochrome c oxidase with a high concentration of oxygen (1 mM), recording the first 200 microseconds of the reaction at a number of wavelengths between 400 and 455 nm. This approach has allowed us to observe kinetic phases with time constants of 8 and 32 microseconds and to separate their spectra. The spectrum of the first phase is comparable to that of oxygen binding to myoglobin, while the spectrum of the second phase appears to contain a contribution from a peroxy intermediate. The results are discussed in the context of a model in which the 8-microseconds phase reflects the establishment of an equilibrium of oxygen-bound states, while the 32-microseconds phase arises when this system is trapped as a peroxy intermediate, by inter-heme electron transfer.

Animals↗
PubMed1994

Structure of the binuclear heme iron-copper site in the quinol-oxidizing cytochrome aa3 from Bacillus subtilis.

Cytochrome aa3-600 is a terminal quinol oxidase of Bacillus subtilis, belonging to the large family of structurally and functionally related respiratory enzymes to which the mitochondrial cytochrome c oxidase also belongs. However, the CuA center typical of the cytochrome c oxidases is lacking from cytochrome aa3-600. The presence of only one copper, viz. CuB of the binuclear heme iron-copper site, makes cytochrome aa3-600 especially suitable for XAS analysis of this structure. Cu and Fe XAS data for fully oxidized cytochrome aa3-600 indicate a structure for the binuclear site similar to that previously reported for mitochondrial cytochrome c oxidase (see Powers et al. (1981) Biophys. J. 34, 465-468). Heme Fea3 has a proximal histidine nitrogen ligand 2.10 +/- 0.02 A from the iron, and a distal S or Cl ligand at 2.36 +/- 0.03 A. The latter is also a ligand of CuB (2.21 +/- 0.02 A), and apparently forms a bridge between the two metals which are 3.70 +/- 0.06 A apart. CuB has two more close-lying ligands at 1.95 +/- 0.02 A, which are likely histidine nitrogens. The similarity between EXAFS of CuB and type 1 'blue' copper is contrasted to EPR and optical spectroscopic properties of CuB, and the nature of the bridging ligand is discussed.

Bacillus subtilis↗