Biomedical subjects

U Nilsson

Publications and source records attributed to U Nilsson.

At least 19 recordsLinked to original sources

PubMed1998

Asp477 is a determinant of the enantioselectivity in yeast transketolase.

The conserved residue Asp477 in yeast transketolase is located in the substrate channel of the enzyme and forms a hydrogen bond with the C2-hydroxyl group of the acceptor substrate. The significance of this interaction for the recognition of the preferred acceptor substrates, D-alpha-hydroxyaldehydes was investigated by site-directed mutagenesis. In the wild-type enzyme the kcat/KM values are by three to four orders of magnitude lower for 2-deoxyaldoses or substrates with L-configuration at the C2-atom. In the Asp477 Ala mutant, the kcat/KM values for D-alpha-hydroxyaldehydes are decreased by a thousandfold, while the kcat/KM values for substrates with L-configuration or 2-deoxyaldoses are similar to wild-type enzyme. These results indicate that Asp477 is involved in determining the enantioselectivity of transketolase.

Models, Molecular↗

PubMed1997

Identification of catalytically important residues in yeast transketolase.

The possible roles of four histidine residues in the active site of yeast transketolase were examined by site-directed mutagenesis. Replacement of the invariant His69 with alanine yielded a mutant enzyme with 1.5% of the specific activity of the wild-type enzyme and with an increased KM for the donor. This residue is located at the bottom of the substrate cleft close to the C1 hydroxyl group of the donor substrate, and the side chain of His69 might be required for recognition of this hydroxyl group and possibly for maintenance of the proper orientation of the reaction intermediate, (alpha, beta-dihydroxyethyl)thiamin diphosphate. Amino acid replacements of His481 by alanine, serine, and glutamine resulted in mutant enzymes with significantly increased KM values for the donor substrate and specific activities of 4.4%, 1.9%, and 5.5% of the wild-type enzyme. The kinetic data suggest that this residue, although close to the C2 carbonyl oxygen of the substrate, is not absolutely required for stabilization of the negative charge that develops at this oxygen in the transition state. This points toward the 4'-NH2 group of the pyrimidine ring of thiamin diphosphate as the major source of charge stabilization. Mutations at positions His30 and His263 result in mutant enzymes severely impaired in catalytic activity (1.5% and less of the activity of wild-type transketolase). The KM value for the donor substrate was increased for the His30Ala mutant but remained unchanged in the His263Ala enzyme. The side chains of both residues interact with the C3 hydroxyl group of the donor substrate, and the results indicate that the two residues act in concert during proton abstraction of the C3 hydroxyl proton during catalysis.

Binding Sites↗

PubMed1997

Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis.

The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

Amino Acid Sequence↗

PubMed1997

Examination of substrate binding in thiamin diphosphate-dependent transketolase by protein crystallography and site-directed mutagenesis.

The three-dimensional structure of the quaternary complex of Saccharomyces cerevisiae transketolase, thiamin diphosphate, Ca2+, and the acceptor substrate erythrose-4-phosphate has been determined to 2.4 A resolution by protein crystallographic methods. Erythrose-4-phosphate was generated by enzymatic cleavage of fructose-6-phosphate. The overall structure of the enzyme in the quaternary complex is very similar to the structure of the holoenzyme; no large conformational changes upon substrate binding were found. The substrate binds in a deep cleft between the two subunits. The phosphate group of the substrate interacts with the side chains of the conserved residues Arg359, Arg528, His469, and Ser386 at the entrance of this cleft. The aldehyde moiety of the sugar phosphate is located in the vicinity of the C-2 carbon atom of the thiazolium ring of the cofactor. The aldehyde oxygen forms hydrogen bonds to the side chains of the residues His30 and His263. One of the hydroxyl groups of the sugar phosphate forms a hydrogen bond to the side chain of Asp477. The preference of the enzyme for donor substrates with D-threo configuration at the C-3 and C-4 positions and for alpha-hydroxylated acceptor substrates can be understood from the pattern of hydrogen bonds between enzyme and substrate. Amino acid replacements by site-directed mutagenesis of residues Arg359, Arg528, and His469 at the phosphate binding site yield mutant enzymes with considerable residual catalytic activity but increased Km values for the donor and in particular acceptor substrate, consistent with a role for these residues in phosphate binding. Replacement of Asp477 by alanine results in a mutant enzyme impaired in catalytic activity and with increased Km values for donor and acceptor substrates. These findings suggest a role for this amino acid in substrate binding and catalysis.

Circular Dichroism↗

PubMed1997

Monitoring of polychlorinated biphenyls in human blood plasma: methodological developments and influence of age, lactation, and fish consumption.

Human plasma samples from 50 wives of fishermen have been analyzed with respect to PCBs. The non-ortho-substituted PCB congeners CB-126 and CB-169 were determined by mass spectrometry in negative ion chemical ionization mode, which demonstrated a limit of detection of 30 fg. The recoveries of the internal standards used for determination of ortho-substituted CBs were approximately 95%. Two methods, one gravimetric and the other based on enzymatic determinations of triglycerides, cholesterol and phospholipids, were compared for the determination of total amount of lipids in the plasma samples; the correlation coefficient was 0.82 and the slope 0.98. For practical reasons, enzymatic determinations are recommended for further use. The total, lipid-adjusted concentrations of PCBs in plasma were influenced by age, total lactation time and consumption of fatty fish from the Baltic Sea.

Adult↗

PubMed1997

Effects of pretreatment with an indeno-indole compound on lipid peroxidation in the cortex and medulla of rabbit kidneys after ischaemia-reperfusion.

The effects of 60 min of ischaemia with or without reoxygenation in vivo or in vitro on lipid peroxidation in cortical and medullary tissue from rabbit kidneys were measured as production of thiobarbituric acid-reactive substances (TBARS). Lipid peroxidation was more pronounced in medullary tissue compared with cortical tissue. The highest TBARS production was found in medullary slices subjected to reoxygenation in vitro immediately after 1 h of ischaemia. Reperfusion in vivo before reoxygenation in vitro attenuated the TBARS formation during subsequent in vitro incubation. Pretreatment of the rabbits with an indeno-indole compound (code name H 290/51) reduced the TBARS formation after 60 min of ischaemia and reoxygenation in vitro towards control values.

Animals↗

PubMed1997

Sexual behavior risk factors associated with bacterial vaginosis and Chlamydia trachomatis infection.

BACKGROUND AND OBJECTIVES: Few studies have demonstrated that bacterial vaginosis (BV) is associated with sexual behavior risk factors similar to those for other sexually transmitted diseases. The objective of this study was to determine the prevalence of these factors in a multivariate analysis of data from women infected with BV and Chlamydia trachomatis, and noninfected control subjects. GOALS: To study detailed sexual behavior risk factors reported by women with BV versus genital C. trachomatis infection (CT) and by non-BV-infected controls. STUDY DESIGN: A cross-sectional study was conducted with 1,011 women (Swedish Women's Health Study) recruited from family planning and youth clinics in Eskilstuna and Stockholm, Sweden (November, 1989-January, 1991). Participants were evaluated for the presence of BV, CT, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, and human immunodeficiency virus, and interviewed in detail with respect to sexual behaviors. Statistical comparisons were made using chi-square test (Pearson and likelihood ratio), t test, and logistic regression multivariate analysis. RESULTS: Of 956 women eligible for analysis, the prevalence of BV and CT was 13.7% and 8.9%, respectively. The comparison group consisted of the remaining 825 women without BV. After excluding those with concomitant CT infection, there were 118 women with BV who were compared with 72 women with CT infection only. Sexual factors associated with BV versus the comparison group were a short-term relationship before and after sexual debut, high number of lifetime sexual partners, multiple partners during the last month, high orgasm ability, and more frequent history of group sex, sexual abuse, and rape. When the BV group was compared with the CT group, there were no significant differences in sexual activity risk factors, except for a higher frequency of experience of casual sex in the CT group. CONCLUSIONS: Bacterial vaginosis is associated with sexual behavior risk factors similar to those associated with genital CT infection.

Adult↗

PubMed1996

Tubing loops as a model for cardiopulmonary bypass circuits: both the biomaterial and the blood-gas phase interfaces induce complement activation in an in vitro model.

We describe here a model for the study of blood/surface and blood/air interaction as encountered in cardiopulmonary bypass (CPB) circuits. Polyethylene tubing was filled with serum or blood and closed end to end into loops whereby the volume of the remaining air bubble was inversely varied with respect to that of the fluid. The loops were rotated vertically in a water bath at 37 degrees C. The profiles of C3a, iC3, and TCC generation were similar to those observed at surgery, involving CPB. Soluble heparin and heparan sulfate inhibited both C3a and TCC formation, but surface-conjugated heparin had only a minor effect. Binding of C3 and/or C3 fragments to the heparin surface was much reduced compared to the amine matrix to which heparin was linked, but compared with the polyethylene surface the effect was less pronounced. These data suggest that, in addition to the biomaterial surface, the blood-gas interface seems to play an important role in the activation of complement and that this activation is inhibitable by high concentrations of soluble glucose aminoglycans.

Biocompatible Materials↗

PubMed1996

Release of histamine in whole blood by oxygen radicals: division between specific and unspecific processes.

Oxygen derived free radicals are involved in many pathological processes such as postischemic reperfusion injuries, hepatotoxicity of drugs and inflammatory processes. Thereby these oxygen radicals induce lipid peroxidation and perturbation of cellular membranes. The aim of our present study was to determine whether oxygen radicals generated by the xanthine oxidase/ hypoxanthine system cause a release of histamine in human blood cell cultures. Stimulation of blood cell cultures with oxygen radicals induced a histamine liberation which was mainly due to calcium independent processes during the first 30 min, whereas then calcium requiring processes took part in the release of histamine. The regulation of the leukocyte selection LECAM-1 was altered by oxygen radicals whereas histamine, which is known to modulate vascular selectin expression, did not affect the expression of LECAM-1. Our data indicate that oxygen radicals induce a direct calcium independent release of histamine which is due to membrane pertubating processes during the first phase but also induce a specific reaction leading to a further indirect histamine liberation which is probably mediated by PAF.

Cell Adhesion Molecules↗

PubMed1996

PapG adhesin from E. coli J96 recognizes the same saccharide epitope when present on whole bacteria and as isolated protein.

Purified PapG adhesin from the genetically well-defined uropathogenic Escherichia coli strain J96, as well as whole bacteria, were bound to microtiter plates that carried covalently bound globotetraose and galabiose. The binding was inhibited by soluble saccharide derivatives corresponding to the glycolipids, including all di-, tri-, tetra-, and pentasaccharide fragments of the Forssman antigen and all monodeoxy analogues of galabiose. Analysis of the inhibition pattern showed no significant difference between purified adhesin and whole bacteria. The glucose unit at the reducing end of the natural saccharides was detrimental to PapG binding since deletion of the glucose unit increased the inhibitory power 10-20 fold. The five hydroxyl groups HO-6, -2', -3', -4', -6' of the galabiose unit were shown to be important for PapG binding, presumably via intermolecular hydrogen bonds.

Adhesins, Escherichia coli↗

PubMed1996

Effect of a new inhibitor of lipid peroxidation on kidney function after ischaemia-reperfusion. A study on rat and rabbit kidneys.

Lipid peroxidation of mitochondrial and cell membrane structures is the final step in the oxygen radical-induced damage observed at reperfusion of kidneys after ischaemia. We compared the ability of an indeno-indol compound (code name H290/51) with that of alpha-tocopherol to inhibit lipid peroxidation in reoxygenated isolated rat renal tissue in vitro measured as production of TBARS (thiobarbituric acid reactive substances). H290/51 was 100 times more efficient than alpha-tocopherol. Treatment of rats in vivo with H290/51 in a dosage giving a plasma concentration of 500 nmol L-1 inhibited TBARS production measured in vitro by 80%. Treatment of rabbits with H290/51 almost completely inhibited radical production at reperfusion after 60 min of ischaemia measured with spin trap technique using OXANOH (2-ethyl-3-hydroxy-2,4,4-trimethyloxazolidine) as a spin trap. Furthermore, such pretreatment significantly improved kidney function and survival of rabbits subjected to 60 min of ischaemia to the left kidney and contralateral nephrectomy. These studies stress the importance of inhibiting lipid peroxidation to prevent the ischaemia-reperfusion damage and furthermore suggest a role for treatment with antioxidants like H290/51 in clinical practice, e.g. at reconstructive renal surgery and transplantation.

Animals↗

PubMed1996

Increased amounts of C3a and the terminal complement complex at high dialysis blood-flow: the relation with dialysis efficiency.

To estimate the influence of blood-flow on complement generation and the relation with dialysis efficacy (KT/V) 10 patients underwent cuprophan hemodialyses for 6 h using low (200 ml/min) or high (400 ml/min) blood-flow (n = 40). Dialysis with high blood-flow compared to low induced a more rapid drop in leukocyte count and a more pronounced leukocyte rebound. Net generation of C3a (microgram/min) was also larger at all 15 measuring points during high blood-flow dialysis and there was significantly larger total generation of C3a (after 3 h p < 0.05, after 6 h p < 0.05) as compared to low blood-flow. Reaching a KT/V of 1.2-1.4 with high blood-flow induced a 40% larger total net generation of C3a and 21% more of TCC than with low blood-flow. Net generation of TCC (AU/min) was higher during the first and last parts of high blood-flow dialysis compared to low. In conclusion, increased blood-flow in order to increase dialysis efficiency (KT/V 1.2-1.4) results in larger net infusion of complement products with possible impact on immune response and complement-associated pathophysiological mechanisms.

Complement C3a↗

PubMed1995

Major histocompatibility complex class I binding glycopeptides for the estimation of 'empty' class I molecules.

Different forms of major histocompatibility complex (MHC) class I heavy chains are known to be expressed on the cell surface, including molecules which are functionally 'empty'. Direct peptide binding to cells is obvious during sensitization of target cells in vitro for cytotoxic T lymphocyte killing and 'empty' MHC-I molecules are comparatively abundant on TAP-1/2 peptide transporter mutant cells. In the present work we have estimated the fraction of 'empty' MHC class I molecules using glycosylated peptides and cellular staining with carbohydrate specific monoclonal antibodies. Synthetic Db and Kb binding peptides were coupled at different positions with different di- or trisaccharides, using different spacing between the carbohydrate and the peptide backbone. Binding of sugar specific mAbs was compared in ELISA and cellular assays. An optimal Db binding glycopeptide was used for comparative staining with anti-Db and anti-carbohydrate monoclonal antibodies to estimate fractions of 'empty' molecules on different T lymphoid cells. On activated normal T cells, a large fraction of Db molecules were found to be 'empty'. The functional role of such 'empty' MHC class I molecules on T cells is presently unclear. However, on antigen presenting cells they might participate in the antigen presentation process.

Amino Acid Sequence↗

PubMed1995

His103 in yeast transketolase is required for substrate recognition and catalysis.

Crystallographic studies of thiamin-diphosphate-dependent transketolase from Saccharomyces cerevisiae suggested the invariant active-site residue H103 as a possible enzymic group binding the C1 hydroxyl group of the donor substrate and stabilizing the reaction intermediate. To test this hypothesis, H103 was replaced by alanine, asparagine and phenylalanine using site-directed mutagenesis. The crystallographic analysis of the mutant transketolases verified that no structural changes occurred as a consequence of the side-chain replacements. The residual catalytic activities of the mutant enzymes were 4.3% for the H103A, 2.4% for the H103N and 0.1% for the H103F mutant transketolase. Further kinetic analysis of the H103A and H103N mutant enzymes showed that the Km values for the coenzyme were increased by about eightfold. The Km values for the acceptor substrate ribose 5-phosphate were similar to the Km value for wild-type transketolase. However, the Km value for the donor substrate, xylulose 5-phosphate is increased more than tenfold in these two mutants. Circular dichroism spectra of the mutant enzymes also indicated a weaker binding of the donor substrate and/or a less stable reaction intermediate. These observations provide further evidence in support of the proposed role for this invariant residue in recognition of the donor substrate by forming a hydrogen bond between the side chain of H103 and the C1 hydroxyl group of the sugar phosphate. The significant decrease in catalytic activity suggests that this residue also facilitates catalysis, possibly by maintaining the optimal orientation of the donor substrate and reaction intermediates.

Binding Sites↗

PubMed1995

Synthesis of the saccharide moiety of galactosylgloboside (SSEA-3) and its conjugation to bovine serum albumin and Sepharose.

The pentasaccharide glycoside corresponding to galactosylgloboside (SSEA-3), beta-D-Gal p-(1-->3)-beta-D-Gal pNAc-(1-->3)-alpha-D-Gal p-(1-->4)-beta-D-Gal p-(1-->4)-beta-D-Glc p-1-OCH2CH2Si-(CH3)3 (4), was synthesized via glycosylation (87%) of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-[2,3,6-tri-O-benzyl-4-O-(2,4, 6-tri-O-benzyl-alpha-D-galactopyranosyl)-beta-D-galactopyranosyl]-beta-D - glucopyranoside (2) with the glycosyl donor methyl 4,6-di-O-acetyl-2-deoxy-2-phthalimido-3-O-(2,3,4, 6-tetra-O-acetyl-beta-D-galactopyranosyl)-1-thio-beta-D-galactopyranosid e (1), followed by removal of protecting groups. Compound 4 was transformed into the spacer glycoside beta-D-Gal p(1-->3)-beta-D-Gal pNAc-(1-->3)-alpha-D-Gal p-(1-->4)-beta-D-Gal p-(1-->4)-beta-D-Glc p-1-SCH2CH2COOH (10), which was coupled to bovine serum albumin (BSA), and Sepharose beads, to give the corresponding neoglycoprotein (11, 6 mol of saccharide/mol of BSA), and glycosylated Sepharose (12, 2.7 mumol of saccharide/mL of sedimented beads), respectively. An improved synthesis of a protected globotetraoside beta-D-Gal pNAc-(1-->3)-alpha-D-Gal p-(1-->4)-beta-D-Gal p-(1-->4)-beta-D-Glc p-1-OCH2CH2SiMe3 is also reported.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed1995

Cadmium in kidneys in Swedes measured in vivo using X-ray fluorescence analysis.

An X-ray fluorescence (XRF) technique using plane polarized X-rays for excitation was used for in vivo measurements of cadmium in the kidney cortex among non-occupationally exposed members of the general population in southern Sweden. The measured concentrations of cadmium in the kidney cortex of smokers (median 28 micrograms/g, n = 10) were significantly higher (P = 0.0036) as compared to those in non-smokers (median 8 micrograms/g, n = 10), and so were the cadmium concentrations in blood and urine. The results show that smoking considerably increases the cadmium concentration in the kidney cortex and that smoking is a major source of cadmium exposure in the general population of Sweden. Except in the presence of very deeply situated kidneys, where the minimum detectable concentration is high, non-invasive in vivo XRF analysis of kidney cadmium should be a useful tool for evaluating the effects of long-term low-level exposure to cadmium and the risk of kidney damage.

Adult↗

PubMed1995

15-hydroperoxydehydroabietic acid--a contact allergen in colophony from pinus species.

A new hydroperoxide, 15-hydroperoxydehydroabietic acid (15-HPDA), with contact allergenic properties has been detected in rosin obtained from Pinus species. Detection was facilitated using a synthetic preparation of 15-HPDA for reference purposes. The synthesis and the detection (HPLC and GC) of 15-HPDA in rosin are described. The allergenic activity of 15-HPDA was studied in an experimental sensitization test on guinea-pigs.

Abietanes↗

PubMed1995

Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli.

The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human-specific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Gal alpha 4Gal-containing glycolipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Gal alpha 4Gal moiety, but parts of the GalNAc beta and glucose residues, which flank the Gal alpha 4Gal in globoside (GbO4), were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of Gal alpha 4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human-specific PapG in binding the GbO4 that dominates in the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG-GbO4 interaction will make it possible to design antiadherence therapeutics.

Adhesins, Escherichia coli↗