PubMed Health⌕ Search

Biomedical subjects

M Wikström

Publications and source records attributed to M Wikström.

At least 73 records · Page 4Linked to original sources
PubMed1997

Mutations in subunit 6 of the F1F0-ATP synthase cause two entirely different diseases.

A lowered efficiency of oxidative phosphorylation was recently found in a Leber hereditary optic neuropathy (LHON) proband carrying a mutation in the mtDNA gene for subunit 6 of the membrane-bound F0 segment of the F1F0-ATP synthase [9]. This phenotype was transferred to cytoplasmic hybrid cells together with the mutation, proving its functional significance. Increasing the respiratory rate in the mitochondria from this mutant raised the ATP/2e- ratio back to normal values. A different mutation in the same mtDNA gene has been found in patients with the NARP syndrome [10]. Although the ATP/2e- ratio is also decreased in this mutant, in this case an increase in the respiratory rate could not compensate for it. Whilst both mutations affect subunit 6 of the proton-translocating F0 segment, the LHON mutation induces a proton leak whereas the NARP mutation blocks proton translocation. Hence, the latter will have much more destructive metabolic consequences in agreement with the large clinical differences between the two diseases.

Adenosine Triphosphate↗
PubMed1997

NMR analysis of the interaction between protein L and Ig light chains.

Protein L is a cell wall protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. It binds to immunoglobulin (Ig) light chains predominantly of the kappa subtype from a wide range of animal species. This binding is mediated by five highly homologous repeats designated as B1-B5, each of which comprises 72 to 76 amino acid residues. The fold of the Ig light chain-binding B1 domain of protein L has previously been shown to comprise an alpha-helix packed against a four-stranded beta-sheet. The Ig-binding region of the protein L domain involves most of the residues in the second beta-strand, the C-terminal residues of the alpha-helix, and residues in the loop connecting the alpha-helix with the third beta-strand. In the present study, we have identified the protein L-binding site of an Ig light chain by use of stable isotope-assisted NMR spectroscopy. The light chain of a murine monoclonal anti-17alpha-hydroxy-progesterone Fab fragment (IgG2b, kappa) was selectively labeled with 13C at carbonyl groups of Ala, Arg, Cys, Ile, Lys, Met, Phe, Trp, or Tyr. The residues in which the carbonyl 13C chemical shift was significantly perturbed upon binding of the protein L B1 domain were preferentially found in the second beta-strand of the variable kappa domain and parts of its flanking beta-strands. None of these residues were affected by the addition of the antigen against which the monoclonal Fab fragment is directed. Therefore, we conclude that protein L binds to the outer surface of the framework region of the V(L) domain, primarily involving the V(L) second strand, and that this binding is independent of antigen-binding. The present NMR data, in combination with sequence comparisons between kappa light chains with and without protein L affinity, suggest that the amino acid substitutions at positions 9, 20, and/or 74 of the kappa light chains could crucially affect the interaction between protein L and the V(L) domain.

17-alpha-Hydroxyprogesterone↗
PubMed1997

Solution structure of the albumin-binding GA module: a versatile bacterial protein domain.

The albumin-binding GA module is found in a family of surface proteins of different bacterial species. It comprises 45 amino acid residues and represents the first known example of contemporary module shuffling. Using 1H NMR spectroscopy we have determined the solution structure of the GA module from protein PAB, a protein of the anaerobic human commensal and pathogen Peptostreptococcus magnus. This structure, the first three-dimensional structure of an albumin-binding protein domain described, was shown to be composed of a left-handed three-helix-bundle. Sequence differences between GA modules with different affinities for albumin indicated that a conserved region in the C-terminal part of the second helix and the flexible sequence between helices 2 and 3 could contribute to the albumin-binding activity. The effect on backbone amide proton exchange rates upon binding to albumin support this assumption. The GA module has a fold that is strikingly similar to the immunoglobulin-binding domains of staphylococcal protein A but it shows no resemblance to the fold shared by the immunoglobulin-binding domains of streptococcal protein G and peptostreptococcal protein L. When the gene sequences, binding properties and thermal stability of these four domains are analysed in relation to their global folds an evolutionary pattern emerges. Thus, in the evolution of novel binding properties mutations are allowed only as long as the energetically favourable global fold is maintained.

Amino Acid Sequence↗
PubMed1997

[Infectious spondylitis. A retrospective evaluation of MRI markers].

AIM AND METHODS: The aim of the present study was to evaluate the MRI criteria of infectious spondylitis (spondylodiscitis). The MR images of 23 patients suffering from spondylodisitis (78% unspecific, 22% specific) were retrospectively analyzed. RESULTS: The height of the intervertebral discs involved was normal in 40%, reduced in 43% and increased in 17% of the cases. The most common findings can be summarized in an MR triad: 1) The vertebral bodies involved are hypointense in T1-weighted images (100%) with a lack of delineation of the intervertebral discs (53%). 2) The injection of Gd-DTPA yields an enhancement of the vertebral bodies involved and intervertebral discs (95% and 74% respectively). 3) The vertebral bodies and intervertebral discs are hyperintense in T2-weighted sequences (76% and 90% respectively). When present, a paravertebral or intraspinal extension of the infection was isointense compared with the adjacent involved vertebral body in the majority of the patients. A differentiation between unspecific and specific etiology based on the MR images was not possible. CONCLUSIONS: The vertebral bodies affected were usually hypointense in T1-W with enhancement after the administration of Gd-DTPA and hyperintense in T2-W. The discs involved were usually hyperintense in T2-W and demonstrated an inhomogeneous enhancement.

Contrast Media↗
PubMed1997

Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.

AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in periodontitis, owing to the release of proteinases from infecting P gingivalis and neutrophils, with a contribution to the tissue destruction seen in periodontitis as a probable consequence.

Bacteria↗
PubMed1996

The "ferrous-oxy" intermediate in the reaction of dioxygen with fully reduced cytochromes aa3 and bo3.

We have studied the reactions with oxygen of two terminal oxidases, cytochrome c oxidase from mitochondria and cytochrome bo3 from Escherichia coli. In each case, flow-flash methodology was used to react the fully reduced enzyme with a high concentration of oxygen (1 mM), and absorbance changes were recorded for a number of separate wavelengths in the alpha-band (visible) region. In both enzymes, an early kinetic phase could be resolved, corresponding to the binding of oxygen to produce a ferrous-oxy heme intermediate. In cytochrome c oxidase, this intermediate appears with a time constant of 10 microseconds; its spectrum has a peak at 595 nm (relative to the unliganded reduced enzyme). In cytochrome bo3, the ferrous-oxy intermediate, resolved by optical absorbance spectroscopy for the first time, appears with a time constant of 11 microseconds and has a broad maximum near 570 nm.

Animals↗
PubMed1996

Redox transitions between oxygen intermediates in cytochrome-c oxidase.

Some intermediates in the reduction of O2 to water by cytochrome-c oxidase have been characterized by optical, Raman, and magnetic circular dichroism spectroscopy. The so-called "peroxy" (P) and "ferryl" (F) forms of the enzyme, which have been considered to be intermediates of the oxygen reaction, can be generated when the oxidized enzyme reacts with H2O2, or when the two-electron reduced ("CO mixed-valence") enzyme reacts with O2. The structures as well as the overall redox states of P and F have recently been controversial. We show here, using tris(2,2'-bipyridyl)ruthenium(II) as a photoinducible reductant, that one-electron reduction of P yields F, and that one-electron reduction of F yields the oxidized enzyme. This confirms that the overall redox states of P and F differ from the oxidized enzyme by two and one electron equivalents, respectively. The structures of the P and F states are discussed.

Animals↗
PubMed1996

A highly variable region in members of the streptococcal M protein family binds the human complement regulator C4BP.

Strains of Streptococcus pyogenes express one or more molecules that are members of the M protein family, a group of surface proteins implicated in virulence. A characteristic property of the molecules in this family is the presence of a highly variable N-terminal region, whose function is unknown. Here we show that human C4b-binding protein (C4BP), a regulatory component of the complement system, binds to the highly variable region of many members of the M protein family. Chimeric molecules, in which the N-terminal regions of four different C4BP-binding proteins were combined with the C-terminal part of the non-binding M5 protein, had intact C4BP-binding ability, as judged by binding assays and Scatchard analysis with highly purified molecules. Moreover, work with the C4BP-binding Arp4 protein showed that an N-terminal 52-residue fragment retained binding ability, and that a 21-residue synthetic peptide derived from the variable region completely inhibited the binding of C4BP. Computer-assisted analysis of the four C4BP-binding regions studied here (45-66 amino acid residues) indicated that they lack residue identities that could explain their ability to bind the same ligand, but differ from the nonbinding M5 protein in their lower propensity to form a coiled-coil. Thus, the variable C4BP-binding regions have an extraordinary capacity for sequence variation, while retaining the ability to bind C4BP. These data indicate that an important function of the variable region in members of the M protein family is to bind a host protein that down-regulates the complement system.

Amino Acid Sequence↗
PubMed1996

Observation and assignment of peroxy and ferryl intermediates in the reduction of dioxygen to water by cytochrome c oxidase.

The reaction of fully reduced cytochrome c oxidase with oxygen has been studied in flowflash experiments at -25 degrees C. Under these conditions the time course of the reaction at 445 nm is qualitatively similar to that recorded at room temperature. In addition to heme redox events, three intermediates in the oxygen reaction are observed: a ferrous-oxy species (A), a 607-nm species (P), and a 580-nm ferryl species (F). Formation of A is not resolved. Conversion of the ferrous-oxy intermediate (A) into the 607-nm species (P) takes place at the same time that an electron is transferred from the low-spin heme to the oxygen reduction center (k approximately 1500 s-1). Subsequently, P decays into the 580-nm species F at the same time that the low-spin heme becomes partially re-reduced by CuA (k approximately 280 s-1). Although the 607-nm species (P) has been produced in other reactions of the enzyme, this is the first time that it has been observed as a transient in the forward reaction of the fully reduced enzyme with its natural substrate, demonstrating that it is a true catalytic intermediate. The structures of both P and F are discussed in the light of these findings.

Animals↗
PubMed1996

Catalytic activity of complex I in cell lines that possess replacement mutations in the ND genes in Leber's hereditary optic neuropathy.

Short-chain ubiquinone analogues act as electron acceptors and as inhibitors in the lymphoblast mitochondria of ND1/3460 mutants, which indicates structural changes in the ubiquinone-binding domain of Complex I in this mutant. The ND4/11778 mutant and two secondary ND5 mutants studied are associated with reductions of at least 50, 35 and 30% in the catalytic rate constant, respectively. However, the efficiency of oxidative phosphorylation is unaffected in all these ND mutants. The rate of respiration is only slightly limited by Complex I in lymphoblast mitochondria. Consequently, there is a 30-35% reduction in the electron flow through Complex I compared with that through Complex II, and an increased lactate/pyruvate ratio, in the ND1 and ND4 mutants, but these factors were unaffected in the secondary ND5 mutants. Energy metabolism is thus less severely affected in the secondary mutants than in the primary mutants, which supports the division into these two categories. An increased ubiquinone-10 content in the mitochondrial membrane of all the mutants, and enhanced succinate dehydrogenase and citrate synthase activities in the ND4 mutant, are proposed to be compensatory changes. The efficiency of these changes and the level of kinetic limitation of respiration by Complex I in each tissue are proposed to determine the clinical development of the disease.

Catalysis↗
PubMed1996

The respiration-driven active sodium transport system in E. coli does not function with lithium.

Comparison of respiration-driven active transport of alkali cations from E. coli cells loaded with Na+ or Li+ showed that Li+ could not be expelled from the cells like Na+. K+ accumulation, which was fast in Na+-loaded cells, was strongly inhibited in Li+-loaded cells, despite high membrane potential and respiratory rate. When Li+-loaded cells were placed into medium containing Na+ instead of Li+, Li+/Na+ exchange took place initially, while K+ accumulation was delayed. Only after almost all inside Li+ was replaced by Na+ did active Na+ and K+ transport commence. These data confirm that it is a distinct active sodium transport system (AST) with Na+,K+/H+ antiporter activity, and not the Na+/H+ antiporters, that is responsible for active Na+ transport in E. coli [Verkhovskaya, M.L., Verkhovsky, M.I. and Wikstrom, M. (1996) Biochim. Biophys. Acta 1273, 207-216]. In contrast to the Na+/H+ antiporters, the AST system is inhibited by Li+.

Biological Transport, Active↗
PubMed1996

Molecular characterization of a saline-soluble lectin from a parasitic fungus. Extensive sequence similarities between fungal lectins.

It has been proposed that the interactions between several parasitic and pathogenic fungi and their hosts are mediated by soluble lectins present in the fungus. We have cloned and analyzed a gene encoding such a lectin (AOL) from the nematophagous fungus Arthrobotrys oligospora (deuteromycete). The deduced primary structure of the AOL gene displayed an extensive similarity (identity 46.3%) to that of a gene encoding a lectin (ABL) recently isolated from the mushroom Agaricus bisporus (basidiomycete), but not to any other fungal, microbial, plant or animal lectins. The similarities between AOL and ABL were further demonstrated by the observation that an antibody specific for AOL cross-reacted with ABL. Together with data showing that AOL has a binding specificity that is similar to that of ABL [Rosen, S., Bergström, J., Karlsson, K.-A. & Tunlid, A. (1996) Eur. J. Biochem. 238, 830-837], these results indicate that AOL and ABL are members of a novel family of saline soluble lectins present in fungi. Southern blots indicated that there is only one AOL gene in the genome encoding a subunit (monomer) of the lectin. The primary structure of AOL did not show the presence of a typical N-terminal signal sequence. Comparison of the deduced primary structure with the molecular mass of AOL as determined by electrospray mass spectrometry (16153 Da), indicated that AOL has an acetylated N-terminal but no other post-translational modifications, and that a minor isoform is formed by deamidation. Circular dichroism (CD) spectroscopy suggested that the secondary structure of AOL contains 34% beta-sheets, 21% alpha-helix, and 45% turns and coils.

Agaricus↗
PubMed1996

K+-dependent Na+ transport driven by respiration in Escherichia coli cells and membrane vesicles.

Respiration-driven Na+ transport from Escherichia coli cells and right-side-out membrane vesicles is strictly dependent on K+. Cells from an E. colic mutant deficient in three major K+ transport systems were incapable of accumulating K+ or expelling Na+ unless valinomycin was added. Membrane vesicles from an E. coli mutant from which the genes encoding the two known electrogenic Na+/nH+ antiporters nhaA and nhaB were deleted transported Na+ as well as did vesicles from wild-type cells. Quantitative analysis of Delta psi and Delta pH showed a high driving force for electrogenic Na+/nH+ antiport whether K+ was present or not, although Na+ transport occurred only in its presence. These results suggest that an Na+/nH+ antiporter is not responsible for the Na+ transport. Respiration-driven efflux of Na+ from vesicles was found to be accompanied by primary uphill efflux of K+. Also, no respiration-dependent efflux of K+ was observed in the absence of Na+. Such coupling between Na+ and K+ fluxes may be explained by the operation of an Na+, K+/H+ antiporter previously described in E. coli membrane vesicles (Verkhovskay, M.L., Verkhovsky, M.I. and Wikström, M. (1995) FEBS Lett. 363, 46-48). Active Na+ transport is abolished when delta mu H+ is eliminated by a protonophore, but at low concentrations the protonophore actually accelerated Na+ transport. Such an effect may be expected if the Na+, K+/H+ antiporter normally operates in tight conjunction with respiratory chain complexes, thus exhibiting some phenomenological properties of a primary redox-linked sodium pump.

Biological Transport↗
PubMed1996

Kinetic trapping of oxygen in cell respiration.

Cell respiration in eukaryotes is catalysed by mitochondrial enzyme cytochrome c oxidase. In bacteria there are many variants of this enzyme, all of which have a binuclear haem iron-copper centre at which O2 reduction occurs, and a low-spin haem, which serves as the immediate electron donor to this centre. It is essential that the components of the cell respiratory system have a high affinity for oxygen because of the low concentration of dissolved O2 in the tissues; however, the binding of O2 to the respiratory haem-copper oxidases is very weak. This paradox has been attributed to kinetic trapping during fast reaction of O2 bound within the enzyme's binuclear haem iron-copper centre. Our earlier work indicated that electron transfer from the low-spin haem to the oxygen-bound nuclear centre may be necessary for such kinetic oxygen trapping. Here we show that specific decrease of the haem-haem electron transfer rate in the respiratory haem-copper oxidase from Escherichia coli leads to a corresponding decrease in the enzyme's operational steady-state affinity for O2. This demonstrates directly that fast electron transfer between the haem groups is a key process in achieving the high affinity for oxygen in cell respiration.

Cytochrome b Group↗