Biomedical subjects

M Wikström

Publications and source records attributed to M Wikström.

At least 145 records · Page 8Linked to original sources

PubMed1992

pH dependence of proton translocation by Escherichia coli.

Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.

Cytochrome b Group↗

PubMed1992

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.

Animals↗

PubMed1992

Oxygen activation and the conservation of energy in cell respiration.

Many of the membrane-associated oxidases that catalyse respiratory reduction of O2 to water simultaneously couple this exergonic reaction to the translocation of protons across the inner mitochondrial membrane, or the cell membrane in prokaryotes, a process by which metabolic energy is conserved for subsequent synthesis of ATP. The molecular mechanism of O2 reduction and its linkage to H+ translocation are now emerging. The bimetallic haem iron-copper reaction centre in this family of enzymes is the critical structure for catalysis of both these processes.

Animals↗

PubMed1992

Determination of catecholamines in urine by liquid chromatography and electrochemical detection after on-line sample purification on immobilized boronic acid.

Norepinephrine, epinephrine and dopamine in urine were measured by an automated liquid chromatographic method. After sample purification on a column containing silica-immobilized boronic acid, which showed great affinity for catecholamines at neutral pH, the catecholamines were eluted by backflushing with an acidic mobile phase and transferred to a cation exchanger for separation. Detection was performed electrochemically and the relative standard deviation was 2% for the analysis of endogenous concentrations in human urine.

Boronic Acids↗

PubMed1992

Reflectance method for simple determination of proteinase activity in microliter samples of a complex serum-like fluid.

A technique using an optical instrument, a reflectometer, for quantitative determination of proteinase activity in microliter samples of complex serum-like fluids, e.g., crevicular exudate from single sites, was developed. The technique allowed the use of various proteins as enzyme substrate. The reflectometer measures the mass of a layer, such as protein, adsorbed to a reflecting surface. This is done by measuring the reflected light intensity of the p-polarized light beam on a surface. We used methylized silicon surfaces that were coated with fibrinogen, alpha 2-macroglobulin, or hemoglobin as enzyme substrates. The test solution was incubated overnight in a basin made in an agar gel applied on the top of the protein-coated surface. In 82 exudates from periodontitis sites, with pocket depths greater than or equal to 6 mm, fibrinogenolytic activity corresponding to 1 microgram ml-1 of trypsin and pronase P was found in 20% of the samples.

Endopeptidases↗

PubMed1992

Laminin binding to Prevotella intermedia.

The interaction of laminin (Lm), a basement membrane protein abundant in the periodontium, with 66 strains of Prevotella intermedia isolated from diseased pockets, was tested in a 125I-labeled protein binding assay. The mean binding value was 28% of the total protein added. The binding significantly increased to 35% when the environmental pH decreased from 7 to 6. The Lm interaction was characterized in a highly binding (about 65%) strain, OMGS105. The binding was rapid and required about 1 min and 1-2 h for 50% and 100% equilibrium respectively. The 125I-Lm binding was maximum in the pH interval 3.0 to 6.5 and could not be displaced by unlabeled Lm or inhibited by other proteins and carbohydrates. The interaction was stable in the presence of NaCl or urea (concentrations up to 4 M) but was dissociated by > or = 1 M KSCN. The Lm-binding component was thermolabile and sensitive to proteolytic enzymes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed a approximately 62 kDa Lm-binding protein, both in the whole cell extract and the outer membrane preparation. Weaker binding was also observed to other proteins. These data establish the ability of P. intermedia to interact with Lm via certain cell surface proteins, a property that might contribute to the colonization of this bacterium in the periodontal pocket.

Bacterial Adhesion↗

PubMed1992

Comparative study of subgingival microbiological sampling techniques.

The presence of specific bacteria in subgingival plaque has been used as an indicator of active periodontal disease. The technique of subgingival sampling may conjecturally influence the identification and enumeration of microorganisms reported. In this study, paper point sampling and scaler sampling are compared. Subgingival samples using both methods were taken from three surfaces in each of 12 patients at the following time points: at each of two appointments one week apart before treatment and at each of two appointments 12 and 13 weeks following treatment. Microbiological analyses were undertaken to determine the total number of colony forming units, the proportions of suspected periodontal pathogens, and the number of spirochetes using phase contrast microscopy. Significantly higher numbers of colony forming units and spirochetes were found for paper point sampling both before and after treatment.

Adult↗

PubMed1992

MR imaging of experimental myocardial infarction.

The signal enhancement in MR imaging of normal, infarcted and reperfused myocardium was investigated using different types of contrast agents. To investigate the organ distribution of a macromolecular contrast agent, normal rats were imaged before, and serially after injection of dextran-(Gd-DTPA)15. Dextran-(Gd-DTPA)15 had an essentially intravascular distribution, and significantly enhanced various organs in normal rats, including the heart. There was a linear relationship between the injected dose and observed enhancement. To investigate myocardial signal enhancement, acute myocardial infarction was induced in pigs by ligating a coronary artery. In two groups of pigs, dextran-(Gd-DTPA)15 or Gd-DPTA was administered i.v. after approximately 4 h of occlusion. Imaging was performed repeatedly in vivo. The animals were sacrificed about 2-2.5 h after injecting the contrast medium. The hearts were excised and re-imaged ex vivo. In three other groups of pigs, imaging was only carried out on excised hearts with 6-hour-old infarctions (a) without injecting contrast medium, (b) with injection of Dy-DTPA-BMA 3 min before sacrifice, and (c) with injection of Gd-DTPA-BMA 2 h before sacrifice followed by injection of Dy-DTPA-BMA 3 min before sacrifice. Without contrast medium, the infarctions were visualized as regions with a high signal intensity in the proton density- and T2-weighted images of excised hearts. Injection of dextran-(Gd-DTPA)15 resulted in infarct visualization also in the T1-weighted images ex vivo, due to a pronounced enhancement in parts of the infarct periphery and moderate enhancement in normal myocardium. Gd-DTPA accumulated in the infarctions, resulting in a better infarct visualization in the T1- and proton density-weighted images of excised hearts, compared with the control and dextran-(Gd-DTPA)15 groups. In vivo, however, the infarctions could not be visualized either before or after injection of dextran-(Gd-DTPA)15 or Gd-DTPA. Injection of Dy-DTPA-BMA improved infarct visualization mainly in the proton density- and T2-weighted images of excised hearts, due to susceptibility-induced reduction of signal intensity in the nonischemic myocardium. With T2-weighting, the infarct visualization was markedly better in the Dy-DTPA-BMA group than in the controls, or in the dextran-(Gd-DTPA)15 and Gd-DPTA groups. The double-contrast technique yielded an excellent infarct visualization ex vivo in all sequences, superior to that obtained in all other groups of pigs, due to Gd-DTPA-BMA-induced enhancement of the infarctions combined with Dy-DTPA-BMA-induced reduction of signal intensity in normal myocardium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

MR imaging of acute myocardial infarction in pigs using Gd-DTPA-labeled dextran.

Myocardial infarctions were induced in 12 pigs. In 6 pigs, dextran-(Gd-DTPA)15 (approximately 0.1 mmol Gd/kg b.w.) was injected i.v. 4 to 4.5 hours after coronary artery occlusion. ECG gated MR images were obtained repeatedly before (n = 4) and after (n = 6) contrast medium injection. Relaxation times in blood samples were measured repeatedly. The animals were sacrificed 2 hours after contrast medium administration. The hearts were excised, reexamined in the MR equipment and stained with triphenyltetrazolium chloride (TTC) in order to define areas of infarction. The remaining 6 pigs were sacrificed 6 hours after occlusion without administration of contrast medium. These hearts were only imaged ex vivo. In vivo, the infarctions could not be identified with or without dextran-(Gd-DTPA)15. Ex vivo, without contrast medium, the infarctions had an increased signal intensity, most pronounced in the T2-weighted images. Dextran-(Gd-DTPA)15 caused a prolonged, pronounced shortening of T1 and T2 in blood samples. The infarct demarcation improved in the T1-weighted images after injection of dextran-(Gd-DTPA)15, due to a moderate enhancement in normal myocardium and a stronger enhancement at the periphery of the infarctions, while the central parts of the infarctions were only weakly enhanced.

Animals↗

PubMed1991

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON).

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.

DNA, Mitochondrial↗

PubMed1991

The heme groups of cytochrome o from Escherichia coli.

Cytochrome o, one of the two terminal ubiquinol oxidases of Escherichia coli, is structurally and functionally related to cytochrome c oxidase of mitochondria and some bacteria. It has two heme groups, one of which binds CO and forms a binuclear oxygen reaction center with copper. The other heme is unreactive toward ligands, exhibits strong interactions with the binuclear center, and is mainly responsible for the reduced-minus-oxidized alpha band. Protoheme has been thought to be the prosthetic group of b-type cytochromes, including cytochrome o. However, the hemes of cytochrome o are of a different kind, for which we propose the name heme O. Its pyridine hemochrome spectrum is blue-shifted by 4 nm relative to that of protoheme, and chromatographic behavior showed that it is much more hydrophobic than protoheme. Fast atom bombardment mass spectrometry yielded a molecular mass of 839 Da. Heme O is proposed to be a heme A-like molecule, containing a 17-carbon hydroxyethylfarnesyl side chain, but with a methyl residue replacing the formyl group.

Carbon Monoxide↗

PubMed1991

Determination of vanilmandelic acid in urine by coupled-column liquid chromatography combining affinity to boronate and separation by anion exchange.

An automated liquid chromatographic method for assaying vanilmandelic acid in urine is described. Vanilmandelic acid and potential interfering substances, such as catechol compounds and their metabolites, have been tested for affinity to boronic acid-substituted silica at various pH values. Vanilmandelic acid and the internal standard, isovanilmandelic acid, were bound to the boronate matrix at an acidic pH, whereas for instance catecholamines were unretained and passed through the column. The alpha-hydroxycarboxylic acids were then desorbed by another mobile phase (pH 6.0) and transferred to an anion exchanger for chromatography and electrochemical detection. A relative standard deviation of 2.8% was obtained for the analysis of human urine samples containing 6.6 microM vanilmandelic acid.

Boron Compounds↗

PubMed1991

Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases.

The expression of two different aa3-type cytochrome oxidases is demonstrated in Bacillus subtilis. One of them (denoted caa3-605), was predicted by DNA-sequencing of Bacillus cytochrome oxidase genes, but has not been found previously. It contains covalently bound haem C in subunit II and is very similar to the enzyme previously described in the thermophilic bacterium PS3. The other oxidase (denoted aa3-600) deviates from most known oxidases of aa3 type, and is probably identical with the oxidase described by de Vrij et al. [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103]. It shows no immunological cross-reactivity to the PS3 enzyme and differs from this spectroscopically; it contains no CuA and does not oxidise cytochrome c despite of its haem-A chromophores. It catalyses oxidation of quinols, which is proposed to be its physiological function.

Bacillus subtilis↗

PubMed1991

Properties of the two terminal oxidases of Escherichia coli.

Proton translocation coupled to oxidation of ubiquinol by O2 was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter. O2 pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump. In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation. E. coli membranes and isolated cytochrome bo lack the CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe. Optical spectra indicate that high-spin heme o contributes less than 10% to the reduced minus oxidized 560-nm band of the enzyme. Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes. Proteoliposomes with cytochrome bo exhibited good respiratory control, but H+/e- during quinol oxidation was only 0.3-0.7. This was attributed to an "inside out" orientation of a significant fraction of the enzyme. Possible metabolic benefits of expressing both cytochromes bo and d in E. coli are discussed.

Cardiolipins↗

PubMed1991

Steady-state redox behavior of cytochrome c, cytochrome a, and CuA of cytochrome c oxidase in intact rat liver mitochondria.

We have examined the steady-state redox behavior of cytochrome c (Fec), Fea, and CuA of cytochrome c oxidase during steady-state turnover in intact rat liver mitochondria under coupled and uncoupled conditions. Ascorbate was used as the reductant and TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) as the redox mediator. After elimination of spectroscopic interference from the oxidized form of TMPD, we found that Fea remains significantly more oxidized than previously thought. During coupled turnover, CuA always appears to be close to redox equilibrium with Fec. By increasing the amount of TMPD, both centers can be driven to fairly high levels of reduction while Fea remains relatively oxidized. The reduction level at Fea is close to a linear function of the enzyme turnover rate, but the levels at Fec and CuA do not keep pace with enzyme turnover. This behavior can be explained in terms of a redox equilibrium among Fec, CuA, and Fea, where Fea is the electron donor to the oxygen reduction site, but only if Fea has an effective Em (redox midpoint potential) of 195 mV. This is too low to be accounted for on the basis of nonturnover measurements and the effects of the membrane potential. However, if there is no equilibrium, the internal CuA----Fea electron-transfer rate constant must be slow in the time average (about 200 s-1). Other factors which might contribute to such a low Em are discussed. In the presence of uncoupler, this situation changes dramatically. Both Fec and CuA are much less reduced; within the resolution of our measurements (about 10%), we were unable to measure any reduction of CuA. Fea and CuA remain too oxidized to be in redox equilibrium with Fec during steady-state turnover. Furthermore, our results indicate that, in the uncoupled system, the (time-averaged) internal electron-transfer rate constants in cytochrome oxidase must be of the order of 2500 s-1 or higher. When turnover is slowed by azide, the relative redox levels at Fea and Fec are much closer to those predicted from nonturnover measurements. In presence of uncouplers, Fea is always more reduced than Fec, but in the absence of uncouplers, the two centers track together. Unlike the uninhibited, coupled system, the redox behavior here is consistent with the known effect of the electrical membrane potential on electron distribution in the enzyme. Interestingly, in these circumstances (azide and uncoupler present), Fea behaves as if it were no longer the kinetically controlling electron donor to the bimetallic center.

Animals↗

PubMed1991

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Subunit III (COIII) is one of the three core subunits of the aa3-type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD-binding glutamic acid) and D259 of COIII were changed by site-directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.

Amino Acid Sequence↗

PubMed1991

Variance in recovery of periodontitis-associated bacteria caused by sampling technique and laboratory processing.

The influence of sampling procedure and of laboratory processing on the recovery of Bacteroides gingivalis, Bacteroides intermedius, and Actinobacillus antinomycetemcomitans from periodontitis sites was evaluated. Thirty-three adult subjects with severe periodontitis participated in the study. In all, 462 samples from 81 sites were examined. The samples were taken using the paper point technique. The cultivations were performed by use of enriched Brucella agar for determination of total colony-forming units and Bacteroides species, and trypticase soy bean agar for determination of A. actinomycetemcomitans. The risk of getting a false negative result was 4% for B. gingivalis, 20% for B. intermedius, and 38% for A. actinomycetemcomitans. It was considerably reduced if duplicate samples were taken. The sampling procedure alone explained up to 98% of the false-negative results.

Adult↗

PubMed1991

Stimulation of proteinase and amidase activities in Porphyromonas (Bacteroides) gingivalis by amino acids and dipeptides.

Proteolytic enzymes from the organism Porphyromonas gingivalis are believed to be involved in the development of periodontitis. Studies on both crude extracts and purified trypsinlike enzymes from this organism indicate that substantial stimulation of both amidase and proteinase activities can be obtained during incubation with glycine-containing compounds. We postulate that P. gingivalis may have developed this unusual property to take advantage of the glycine-rich environment which occurs during the periodontitis-associated degradation of gingival collagen. The finding of such a stimulation in crevicular fluids from discrete periodontal sites has been correlated with the presence of P. gingivalis and could be utilized for the early detection of infection by this organism during the onset of periodontitis.

Amidohydrolases↗