Biomedical subjects
Publications and source records attributed to K Christiansen.
One of the most common reasons for prescribing antibiotics is an infection in the respiratory tract. There are a large number of antimicrobial agents available, some of long standing and others relatively new to the market. The rationale for the choice of agent, including emerging resistances, spectrum of activity, antibiotic pharmacokinetics, adverse reactions and cost, is discussed for the various sites of infection.
Eukaryotic topoisomerase I catalysis was characterized by the use of a DNA substrate system, which allows uncoupling of cleavage and ligation half-reactions. Covalent topoisomerase I-DNA intermediates formed by cleavage without concomitant ligation were able to catalyze hydrolysis of the 3'-phosphotyrosyl bond in the pH range 7.5-10, with a broad pH optimum between pH 8.5 and 9.5. In comparison, the DNA cleavage and ligation activity of topoisomerase I were found to be independent of pH in the pH range 7-10 and strongly impaired at higher pH values. Moreover, different polyhydric alcohol compounds were found to function as nucleophiles at pH 9 to facilitate the release of topoisomerase I. The hydrolysis and alcoholysis activities of topoisomerase I were specific for the 3'-phosphotyrosyl bond and blocked by enzyme denaturation or proteolysis. Taken together the data suggest that site-specific hydrolysis or alcoholysis mediated by topoisomerase I-DNA complexes reflects the ability of the enzyme to direct the activation of the 3'-phosphotyrosyl bond or the incoming nucleophile. Analysis of enzyme-directed coupling of non-DNA nucleophiles to the cleaved DNA strand may provide a useful tool for elucidation of the biochemical mechanism of type I DNA topoisomerases.
In this report, the nature of the protein components involved in the functioning of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5 Ac) hydroxylase in high-speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH-cytochrome b5 reductase, cytochrome b5 and a 56-kDa terminal electron acceptor having the CMP-Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction; however, the amphipathic, usually membrane-associated, forms of cytochrome b5 and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylase in vivo. Although the majority of cellular cytochrome b5 and cytochrome b5 reductase is membrane-bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent-solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochrome b5. Accordingly, in reconstitution experiments, pure hydrophilic cytochrome b5 interacts more effectively with the hydroxylase than isolated amphipathic cytochrome b5. Studies on the CMP-Neu5 Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containing N-glycolylneuraminic acid.
The capacity of eukaryotic topoisomerase I to catalyze intra- and intermolecular DNA strand transfer via a two-step cleavage/ligation reaction was investigated by use of purified enzyme and defined DNA substrates. Topoisomerase I-mediated cleavage requires separate interaction with a duplex region encompassing the cleavage site (region A) and a duplex region located on the side holding the 5'-OH end generated by cleavage (region B). Cleaved topoisomerase I-DNA complexes containing enzyme covalently attached at internal and terminal positions were employed to characterize the intra- and intermolecular ligation reactions. Enzyme attached covalently at an internal position of a partially single-stranded DNA molecule is able to catalyze ligation of a complementary dinucleotide within region A in the absence of interaction with region B. Moreover, the dinucleotide confines the minimal DNA acceptor for intramolecular ligation. Topoisomerase I attached covalently to DNA at a terminal position can ligate the cleaved strand to heterologous duplex DNA regardless of sequence, whereas ligation does not proceed with single-stranded DNA. When these features are considered together with the observation that intermolecular ligation is inhibited by 1 M NaCl, it suggests that the reaction requires bipartite DNA interaction. A model is proposed that relates the bipartite DNA binding of eukaryotic topoisomerase I to the catalytic functions.
Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation was confirmed by immunogold labeling of the alpha- and the beta-subunit of the receptor. Immunogold labeling of the C-terminus of the beta-subunit indicates that it resides about 6 nm off the membrane, while two alpha-subunit epitopes were labeled at about twice this distance, confirming that the alpha-subunit is harbored in the cross-bar of the T-structure.
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Topoisomerase I-mediated cleavage has previously been demonstrated to require interaction of the enzyme with a DNA duplex region encompassing the cleavage site (Svejstrup, J. Q., Christiansen, K., Andersen, A. H., Lund, R., and Westergaard, O (1990) J. Biol. Chem. 265, 12529-12535). The required region, designated region A, includes positions -5 through -1 on the noncleaved strand and positions -7 through +2 on the scissile strand, relative to the cleavage site. Utilizing defined DNA substrates in topoisomerase I cleavage assays we show that efficient cleavage within region A requires additional interaction of the enzyme with duplex DNA on the side holding the 5'-OH end generated by cleavage. By analyzing the interaction of topoisomerase I with DNA substrates varying by single nucleotides on either strand outside region A, an additional duplex region, designated region B, was delimited to positions 6-11. The ability of topoisomerase I to interact separately with regions A and B was assayed on sets of DNA substrates containing a nested series of single-stranded branch sites. The obtained results demonstrate that the normal reversible cleavage/religation equilibrium established by topoisomerase I on continuous duplex DNA is replaced by irreversible cleavage on DNA substrates containing branch sites between the cleavage site and region B as these DNA substrates allow cleavage but prevent religation due to release of the incised strands. The intramolecular bipartite interaction mode of topoisomerase I during the cleavage reaction is thus indicated by both the absence of enzyme-mediated duplex stabilization and the wide tolerance for protruding strands between the cleavage site and region B. Since the irreversibly cleaved topoisomerase I-DNA complexes are kinetically competent to ligate added DNA fragments carrying free 5'-OH ends, the results suggest a role of topoisomerase I in illegitimate recombination.
We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.
The relation between hair growth and levels of sex hormones in serum and saliva was investigated in 256 !Kung San and Kavango men (ages 18 to 39 years) from Namibia/Southern Africa. Serum concentrations of total testosterone (Tser), 5 alpha-dihydrotestosterone (DHT), and estradiol (E2) as well as the level of bioavailable non-SHBG-bound testosterone in the saliva (Tsal) were determined by radioimmunoassay. The distribution and density of scalp and facial hair as well as the development of terminal hair on the chest, abdomen, pubic area, arms, fingers, and legs were categorized using objective criteria. Covariance analyses revealed marked differences in the distribution of body hair in the San and the Negro sample. This is partly explained by a significant influence of androgen and estrogen levels on the growth of terminal hair. DHT and the ratio DHT/Tser are significantly positively related to midphalangeal hair growth and negatively to pubic hair development. Tsal, the bioavailable fraction of total testosterone, exerts a weak positive influence on the degree of arm and leg hair growth; the most significant positive effect on the growth of abdominal, arm, and leg hair in our samples is caused by E2. The ratio Tser/E2 correlates significantly negatively with the arm and leg hair development and the ratio DHT/E2 with the degree of abdominal, pubic, arm, and leg hair, whereas lower DHT concentrations occur in men with stronger hair development.
This clinical trial, which was composed of 1,031 adults undergoing cardiac operations, compared the efficacy of a single dose of 1 g of ceftriaxone with a 48-our regimen consisting of flucloxacillin and gentamicin. There was no significant difference (p = 0.89) in the overall incidence of major infections: 30 of 515 patients (5.8%; 95% confidence interval, 5.4% to 6.2%) taking ceftriaxone and 29 of 516 patients (5.6%; 95% confidence interval, 5.2% to 6.0%) taking flucloxacillin and gentamicin. Subgroup analyses, with a lower statistical power, failed to show a significant difference between patients who received ceftriaxone and those who received flucloxacillin/gentamicin: major sternal wound infections arose in 2.7% of the patients taking ceftriaxone versus 1.6% in those on the 48-hour regimen (p = 0.20) and major limb wound infections arose in 4.2% and 5.4%, respectively (p = 0.44). Single-dose prophylaxis was associated with fewer intravenous administrations (864 doses versus 9,570 doses) and cost less (A$17,248 versus A$78,510). Although the regimen that included gentamicin was associated with the greatest biochemical impairment of renal function, the overall toxicity for both groups was low. We conclude that a single dose of ceftriaxone provided cost-efficient prophylaxis for adults undergoing cardiac operations when compared with a 48-hour regimen of gentamicin and flucloxacillin. The general principle revealed by our data is that the short-term administration of an appropriate antibiotic regimen represents optimal prophylaxis for patients undergoing cardiac procedures.
The relation of circulating sex hormones and cognitive abilities was investigated in 114 healthy !Kung San men ('bushmen') of Namibia/Southern Africa who lived mainly as traditional hunter-gatherers. Blood and saliva samples were analysed by use of radioimmunoassays in order to determine the serum concentrations of total testosterone (Tser), 5 alpha-dihydrotestosterone (DHT), and estradiol (E2), as well as the level of bioavailable, non-SHBG-bound 'free' testosterone in the saliva. The cognitive ability was assessed with two verbal (verbal fluency) and three nonverbal tests (tactual-spatial functioning, field independence/field dependence). As to be expected from previous research on Western samples, all three androgens but not E2 are of significance to the !Kung San men's cognitive performance. Tser exhibits a positive relation to tactual-spatial abilities and to the degree of lateralization of this task; on the other hand, Tser is negatively correlated with verbal fluency. Testosterone in the saliva is also significantly positively correlated to tactual-spatial test scores and, in addition, to field independence. DHT and the ratio DHT/Tser are positively related to verbal fluency and negatively to the degree of lateralization of tactual-spatial performance.
In Denmark, formal services are viewed as a right to be used by any member of that society who is in need of assistance, premised upon a societal model of mutual self-help. The focus here is on the dual themes of philosophical consistency and of transition in the formal system of services and delivery of care in Denmark. Denmark's system centers on meeting the basic needs of the elderly while enabling individuals to retain control over decisions regarding their own lives. It was effective during the economically expansive period of the 1960s and 1970s, but became less effective during the 1980s. Because of the economic necessity of cutting back on budgets, the national government has in fact attempted to define and redirect available choices in service delivery. The dual themes of consistency and dynamism are demonstrated through the case of a rural municipality in northwest Jutland during the prosperous period into the more economically limited period of the last decade and the present. Implications for informal social support and relationships between the aged and their families, friends, and neighbors in Denmark are explored.
In this paper, we present the results of an investigation into the catalytic properties of CMP-Neu5Ac hydroxylase (Neu5Ac: N-acetylneuraminic acid) in high-speed supernatants of mouse liver. The enzyme was most active in Hepes/NaOH pH 7.4 and was markedly inhibited by relatively small increases in ionic strength, though the inhibition was abolished by desalting procedures. Several nonionic detergents could activate the hydroxylase to various degrees in a concentration-dependent manner. Ionic detergents and a number of phospholipids were, however, generally inert or inhibitory. The lack of inhibitory influence of a wide range of nucleotides revealed that CMP-Neu5Ac hydroxylase binds its sugar-nucleotide substrate with a high degree of specificity. Thus, even millimolar concentrations of several cytidine nucleotides elicited virtually negligible inhibition, though the reaction product, CMP-Neu5Gc (Neu5Gc: N-glycoloylneuraminic acid), was a weak inhibitor. The results also indicate that the enzyme is not regulated by any nucleotides or sugar-nucleotides. Dilution of high-speed supernatants with buffer gave rise to a decrease in the specific activity of the hydroxylase, implicating the involvement of more than one component in catalysis. Activity could be restored by the addition of a heat extract of the supernatant. The active principle in this extract was found to be a heat-stable protein with a molecular mass of about 17 kDa. Immunochemical studies allowed this protein to be identified as cytochrome b5 and it was shown that this electron carrier is essential for the activity of CMP-Neu5Ac hydroxylase. Inhibition studies using iron ligands and activation by exogenous iron salts suggest the involvement of a non-haem iron cofactor in the catalytic cycle of this hydroxylase. Cytochrome b5 may thus serve as an electron donor for this postulated cofactor.
We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase II and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug.
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Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.
Total testosterone and dihydrotestosterone in blood serum as well as free testosterone in saliva were determined by radioimmunoassay in 110 healthy young men. The results were compared with the development of terminal hair on the trunk and limbs, with the disposition to balding and with the disposition to acne. No significant correlations were found between terminal hair development and absolute androgen levels; however, some significant values were observed in the case of the metabolic rate of dihydrotestosterone/testosterone and the proportion of free to total testosterone. The disposition to balding also correlates positively with the latter ratio. Yet the absolute serum androgen concentrations in men with a disposition to balding is lower than in men with no reduction of scalp hair. The widespread assumption that androgen levels are in general elevated in bald-trait men must therefore be rejected. In accordance with this finding, men with a disposition to balding are morphologically (with regard to anthropometric measures) no more masculine than those with good scalp hair growth. When body build and age are taken into consideration, the relations between terminal hair and androgen ratio are also problematical. No relationship could be found between acne and androgens.
Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptor-transferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w)). Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5 x 10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2-3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.