Biomedical subjects

M Strömqvist

Publications and source records attributed to M Strömqvist.

At least 19 recordsLinked to original sources

PubMed2001

Reduction of experimental adhesion formation by inhibition of plasminogen activator inhibitor type 1.

BACKGROUND: Adhesion formation is a common cause of complications following surgery. A reduction in peritoneal fibrinolytic capacity during operation is a key mechanism in the early formation of adhesions. An increase in the main inhibitor of fibrinolysis, plasminogen activator inhibitor type 1 (PAI-1), is a major factor in the loss of fibrinolytic activity. The aim of this study was to investigate if inhibition of PAI-1 could reduce the formation of adhesions after surgery. METHODS: Mice (n = 53) were subjected to a standard surgical procedure in order to induce adhesion formation to the abdominal side wall. At the conclusion of the operation, fragments for antigen binding of polyclonal rabbit antibody against PAI-1 (PRAP-1) were injected intraperitoneally, at two different concentrations. Control animals received an equal volume of the vehicle (saline). One week after operation adhesion formation was quantified. RESULTS: Both doses of PRAP-1 significantly reduced adhesion formation compared with the saline control (P = 0.003 and P = 0.002). There were no signs of bleeding in the postoperative period or at reoperation. CONCLUSION: The present observations lend further support to the hypothesis of a pivotal role of fibrinolysis in the early formation of adhesions, and open up new possibilities for adhesion reduction by inhibiting PAI-1.

Animals↗

PubMed2001

Immunological assay for the determination of procarboxypeptidase U antigen levels in human plasma.

The importance of carboxypeptidase U as a novel regulator of the fibrinolytic rate has attracted a lot of interest recently. In the present work, an ELISA was developed using polyclonal antibodies raised against recombinant proCPU, expressed in DON cells. The assay determines the antigen concentration of the zymogen of carboxypeptidase U, procarboxypeptidase U, in human citrated plasma or EDTA plasma. No interference is observed with plasma carboxypeptidase N. The assay is very reproducible (within-run: 4.6% CV, between-run: 6.8% CV). In a group of 479 healthy individuals the mean proCPU antigen concentration is 13.4 microg/ml (SD 2.5 microg/ml). A good correlation is found with the functional procarboxypeptidase U assay described earlier (r = 0.82, p < 0.0001) (Schatteman K, Goossens F, Scharpé S, Neels H, Hendriks D Clin Chem 1999: 45: 807-813). The significant correlation between the proCPU antigen concentration and the 50% clot lysis time stresses its importance as a player in fibrinolysis control.

Adult↗

PubMed2000

Changes in glycosylation of human bile-salt-stimulated lipase during lactation.

Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption-ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.

Amidohydrolases↗

PubMed2000

Plasma procarboxypeptidase U in men with symptomatic coronary artery disease.

Procarboxypeptidase U (proCPU) is the plasma precursor of carboxypeptidase U (CPU, carboxypeptidase R. plasma carboxypeptidase B or activated thrombin-activatable fibrinolysis inhibitor, TAFIa). CPU removes C-terminal lysine residues that act as plasminogen binding sites from partially degraded fibrin, thereby down-regulating plasminogen activation and fibrinolysis. The present study was carried out as a pilot study to examine whether the plasma proCPU concentration is related to the presence of coronary artery disease (CAD) and/or to levels of established risk indicators for CAD, in a case-control study of 110 men requiring coronary artery bypass grafting (CABG) because of stable angina pectoris. The preoperative plasma proCPU level in the CABG patients was significantly higher than in population-based controls (1029 +/- 154 vs. 974 +/- 140 U/L, p <0.05). In addition, in a subset of the patients (n = 31 ) the proCPU concentration, which was significantly lower on the third postoperative day (-17 +/- 10%), had increased significantly on the sixth day (+14 +/- 12%) after surgery, compared with the preoperative level. In both patients and controls, proCPU concentration was strongly and positively associated with factor VII amidolytic activity and protein C activity, suggesting a common mechanism modulating the plasma levels of these proteins. Otherwise, statistically significant correlations with proCPU were group-specific. In the patients, proCPU correlated significantly with plasma fibrinogen and protein S. In the controls, proCPU correlated significantly with concentrations of cholesterol in plasma. VLDL and LDL. In addition, proCPU correlated significantly with C-reactive protein and haptoglobin levels in the controls only, indicating that also inflammatory mechanisms are involved in the regulation of plasma proCPU. These results suggest that a mechanism exists by which fibrinolytic function is impaired in a manner that is likely to result in more stable fibrin deposits and increase the risk of precocious CAD as well as early occlusion of venous bypass grafts.

Acute-Phase Proteins↗

PubMed1998

Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis.

A novel low-molecular-weight inhibitor, AR-H029953XX, was developed from a known fibrinolytic compound, flufenamic acid, which prevented complex formation of human plasminogen activator inhibitor type 1 (PAI-1) with tissue plasminogen activator (tPA) by inhibition of PAI-1. To explore the binding site for AR-H029953XX, mutants of human PAI-1 were constructed by site-directed mutagenesis and were then expressed in CHO cells, purified, activated, and characterized. (1) PAI-1 with mutations in the reactive center loop: L1-PAI-1 (P10, Ser337Glu) had stability and activity similar to those of wild-type PAI-1 (wt-PAI-1), and L2-PAI-1 (P12, Ala335Glu) was highly stable but was a substrate for tPA. (2) PAI-1 with mutations near the binding epitope for the strongly inhibiting monoclonal antibody CLB-2C8: C1-PAI-1 (Phe114Glu), C2-PAI-1 (Val121Phe), C3-PAI-1 (Arg76Glu/Arg115Glu/Arg118Glu), and C4-PAI-1 (Arg115Glu) were all comparable in activity and stability to wt-PAI-1. AR-H029953XX (Ki = 25 microM) prevented complex formation between tPA and active wt-PAI-1 as well as that with mutants L1-, L2-, C1-, C2-, and C4-PAI-1. AR-H029953XX also inhibited binding of these PAI-1 variants to the antibody CLB-2C8, as measured by surface plasmon resonance. In contrast, AR-H029953XX had almost no inhibitory effect on the complex formation of tPA with C3-PAI-1. Moreover, AR-H029953XX had no effect on the binding rate of CLB-2C8 to C3-PAI-1, or on the binding to latent PAI-1 or to cleaved L2-PAI-1. The binding site of AR-H029953XX thus appears to be located in the neighborhood of the postulated epitope for CLB-2C8, near residues Arg76 and/or Arg118. This specific domain of the PAI-1 molecule might thus also be important for the mechanism of inhibitory activity toward tPA. Moreover, the structure of this region in active PAI-1 has to be different from the corresponding regions in latent and cleaved PAI-1.

Animals↗

PubMed1998

Bile salt-stimulated lipase (BSSL) distribution in rat, mouse and transgenic mouse expressing human BSSL.

In some species, including man and mouse, bile salt-stimulated lipase (BSSL) in milk catalyzes the hydrolysis of triacylglycerides into glycerol and free fatty acids, a reaction that is of particular importance during suckling. The enzyme is also secreted by the pancreas (referred to as carboxyl-ester hydrolase, CEH). We wished to localize sources and storage sites for BSSL/CEH in rats, in wild-type mice, and in transgenic mice producing recombinant human BSSL in milk. Immunoreactivity against several BSSL fragments was strong in the pancreatic acinar cells and moderate in the absorptive cells of the small intestine and in salivary duct cells of the mice, as well as in rats. Sections from lactating mammary glands of mouse, but not rat, also showed immunoreactivity for BSSL; the signal was strongest in the transgenic mice. Radioactive riboprobes for BSSL mRNA hybridized on sections of rat and mouse pancreatic acinar cells, and mouse mammary glands (both wild-type and transgenic). Using RT-PCR, it was possible to amplify BSSL mRNA from wild-type mouse pancreas and mammary gland, from rat submandibular glands, and, in a few cases, from rat liver. In transgenic mice, the BSSL mRNA was highly expressed only in lactating mammary gland, but could be detected in a few other organs as well.

Animals↗

PubMed1997

Naturally occurring variants of human milk bile salt-stimulated lipase.

Analysis of milk samples from a number of lactating women revealed molecular variants of bile salt-stimulated lipase (BSSL) of both lower and higher molecular mass than that commonly occurring. In contrast to previous observations, we report on individuals having only a variant of lower mass, both one of lower and one of common mass, or both one of lower and one of higher mass of the lipase. From two individuals we purified the lower molecular mass BSSL variant and characterized it. The amount of lipase in the milk of these two individuals was considerably less than average (mean of 10 women with BSSL of the most common molecular mass). The BSSL variant of lower mass showed the same bile salt activation, pH dependency, temperature stability as those most commonly occurring. We could localize the difference in mass to the large O-glycosylated repeat sequence close to the C-terminus of the protein. With respect to all characteristics studied, the BSSL variant of higher mass was also similar to that most commonly ocurring. Again, the difference in mass could be localized to the repeat region of the protein. Hence, it appears as if the repeat region, normally carrying 16 repeats of 11 amino acids each, varies in size between individuals.

Amino Acids↗

PubMed1997

Glycosylation of bile-salt-stimulated lipase from human milk: comparison of native and recombinant forms.

Bile-salt-stimulated lipase (BSSL) is an enzyme present in human milk. BSSL is important for fat digestion in infants. It contains one site for N-glycosylation and a serine/threonine-rich domain which is highly O-glycosylated. Both N- and O-linked sugar chains were studied on native BSSL from three donors and compared to the glycosylation of recombinant BSSL produced in Chinese hamster ovary or mouse fibroblast (C-127) cell lines. The carbohydrate composition of oligosaccharides was mapped using sugar and methylation analyses, enzyme-linked immunosorbant assay, and different separation techniques. Native BSSL was found to be highly glycosylated (19-26%). It contained a high amount of fucosylated oligosaccharides and expressed both Lewis a and Lewis b blood group antigens. None of the recombinant BSSL forms contained fucose. N-linked structures on native BSSL were identified as mainly mono- and disialylated biantennary complex type structures with or without fucose substitution. High-pH anion-exchange chromatography analysis indicated that the recombinant forms of BSSL contained similar types of N-glycan structures differing mainly in their content of sialic acid and by the absence of fucose residues. Native BSSL contained predominantly large O-linked oligosaccharides. This was in contrast to the recombinant forms of BSSL which contained mainly short type O-glycans with a high content of sialic acid. Interestingly, the estimated number of O-glycans attached to native BSSL was lower than that for the recombinant forms.

Antigens↗

PubMed1997

Expression and characterization of phosphorylated recombinant human beta-casein in Escherichia coli.

Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombinant protein. The phosphorylation of recombinant human beta-casein was confirmed by immunoblots, fast protein liquid chromatography, urea-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and liquid chromatography-mass spectrometry. The results indicate that the substrate specificity of casein kinase II in vivo was unaffected in its recombinant form. This is the first demonstration of in vivo phosphorylation of specific residues of a multiphosphorylated protein produced in E. coli with a single plasmid.

Casein Kinase II↗

PubMed1997

Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits.

Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml-1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones.

Animals↗

PubMed1996

Characterisation of the complex of plasminogen activator inhibitor type 1 with tissue-type plasminogen activator by mass spectrometry and size-exclusion chromatography.

Glycosylated human plasminogen activator inhibitor type 1 (PAI-1), produced in Chinese hamster ovary (CHO) cells, showed a variety of compounds with different molecular weights when subjected to electrospray mass spectrometry (ES-MS), owing to the heterogeneity of the carbohydrate chains. However, non-glycosylated human PAI-1, produced in E. coli, gave rise to a prominent species with a molecular weight of 42,774, consistent with the amino-acid sequence. A non-glycosylated mutant of the proteinase domain (B-chain) of tissue-type plasminogen activator (tPA) produced in C 127 cells, had a molecular weight of 28,168. Full-length, glycosylated, tPA showed a large heterogeneity in molecular mass. For a mass study, a tPA-PAI-1 complex was formed, composed of non-glycosylated PAI-1 and non-glycosylated B-chain. This complex was remarkably stable at room temperature in buffer with a neutral pH. The mass spectrum of the complex provided two main species, a peptide with a mass of 3803 and a dominating species of 67,133. These masses are consistent with a complex where PAI-1 is cleaved at the P1-P1' position. A trace of a species with a molecular mass of 70,942 was also found, corresponding to the complete, non-dissociated complex with PAI-1. Separation of the cleaved peptide, corresponding to the hydrophobic C-terminal 33 amino-acid residues of PAI-1, from the complex, was achieved by size-exclusion chromatography in the presence of 30% acetonitrile. Thus, in the complex between tPA and PAI-1, the proteins are held together by a tight covalent bond, but the C-terminal cleaved peptide of PAI-1 is only bound to the complex by hydrophobic forces. To assess whether this is specific to the tPA B-chain alone, experiments with the complex of full-length, glycosylated tPA and glycosylated PAI-1 were also performed, and it was possible to demonstrate the release of the C-terminal PAI-1 peptide by chromatography, mass spectrometry, as well as by SDS-PAGE.

Amino Acid Sequence↗

PubMed1996

Recombinant human bile salt-stimulated lipase: an example of defective O-glycosylation of a protein produced in milk of transgenic mice.

The expression of recombinant human bile salt-stimulated lipase (bssl) was targeted to the lactating mammary gland of transgenic mice. Expression of recombinant genes comprising bsslcDNA, or alternatively genomic bssl DNA, under control of regulatory elements derived from the murine whey acidic protein (wap) gene was achieved and evaluated. Constructs containing genomic bssl sequences mediated high levels (0.5-1 mg ml-1) of recombinant human BSSL in the milk. The recombinant BSSL produced was purified, biochemically characterized and compared to native BSSL and recombinant BSSL produced in mouse C127 and hamster CHO cells. Recombinant BSSL derived from transgenic mice showed a different migration and distribution after SDS-PAGE electrophoresis, lower apparent molecular mass on size-exclusion chromatography and no detectable interactions with a panel of lectins. These results indicate a significantly lower degree of O-glycosylation of recombinant BSSL in milk from transgenic mice than was found for the native enzyme or recombinant CHO- or C127 cell-produced BSSL. Despite these differences, mouse-milk-derived recombinant BSSL exhibited similar lipase activity, the same stability to low pH and similar sensitivity to elevated temperatures as the native enzyme. The observation that mouse-C127-cell-produced recombinant BSSL is heavily O-glycosylated makes species-related restrictions less attractive as an explanation for the reduced O-glycosylation.

Animals↗

PubMed1996

Anti-thrombotic effect of a PAI-1 inhibitor in rats given endotoxin.

The aim of this study was to investigate the anti-thrombotic effects of an inhibitor of the plasminogen activator inhibitor-1 (PAI-1) in rats given endotoxin. In studies in vitro, PRAP-1, a Fab-fragment of a polyclonal antibody against human PAI-1, was shown to inhibit PAI-1 activity in rat plasma as well as to stimulate clot-lysis of the euglobulin fraction derived from rat plasma. Endotoxin administered to anaesthetised rats produced a marked increase in plasma PAI-1 activity. To study fibrin formation and lysis in vivo after intravenous (i.v.) injection of the coagulant enzyme batroxobin, 125I-fibrinogen was administered to the animals. The thrombi formed by batroxobin were rapidly lysed in control animals, while the rate of lysis was markedly attenuated in rats given endotoxin. PRAP-1 was administered i.v. (bolus+infusion) to rats given endotoxin and batroxobin and the PAI-1 inhibitor caused a dose-dependent decrease in the 125I-fibrin deposition in the lungs. An immunohistochemical technique was used to confirm this decrease in density of fibrin clots in the tissue. Furthermore, PRAP-1 decreased plasma PAI-1 activity in the rats and this reduction was correlated to the decrease in lung 125I-fibrin deposition at the corresponding time point. It is concluded that in this experimental model the PAI-1 antibody PRAP-1 may indeed inhibit thrombosis in animals exposed to endotoxin.

Animals↗

PubMed1995

Differences in the glycosylation of recombinant and native human milk bile salt-stimulated lipase revealed by peptide mapping.

The milk of some mammals contains a bile salt-stimulated lipase (BSSL). Human milk BSSL is heavily glycosylated (30-40% carbohydrate) and present at a concentration of approximately 100-200 mg/l, thereby being one of the most abundant human whey proteins. BSSL has been shown to have an important role in the uptake of energy from human milk. The risk of HIV contamination has restricted the use of banked human milk for nutritional purposes. This has evoked an interest in the production of a recombinant form of the protein for supplementation of formula. We have produced BSSL in mouse C127 and hamster CHO cells, and used chromatographic methods for the characterization of the products. This study was focused on study of the glycosylation of the protein by using peptide mapping and isolation of glycosylated fragments. The results show how human BSSLs from different sources differ both in extent of glycosylation, in glycan heterogeneity, and in lectin binding.

Animals↗

PubMed1995

Primary substrate specificity of recombinant human stratum corneum chymotryptic enzyme.

Stratum corneum chymotryptic enzyme (SCCE) is a new human serine proteinase expressed by keratinocytes in the epidermis. Its function may be to catalyze the degradation of intercellular cohesive structures in the cornified layer of the skin in the continuous sheeding of cells from the skin surface. In this work the primary substrate specificity of recombinant SCCE was determined with oxidized bovine insulin B chain as substrate. Cleavage products were separated with high performance liquid chromatography and analyzed by amino acid sequence analysis and mass spectrometry. Cleavage sites were localized to Leu 6-cysteic acid 7, Tyr 16-Leu 17, Phe 25-Tyr 26, and Tyr 26-Thr 27 in insulin B chain. It is concluded that SCCE belongs to the family of serine endoproteinases specific for amino acid residues with aromatic side chains in the P1 position.

Amino Acid Sequence↗

PubMed1995

Carbohydrate specificity of the Escherichia coli P-pilus papG protein is mediated by its N-terminal part.

The adherence of pyelonephritic Escherichia coli isolates to mammalian host cells is mediated by the P-pili structures on the bacterial surface. The protein constituting the distal part of the pili structure, papG, interacts with glycan receptors on the host cell. Variation in specificity for different glycoconjugates between the isolates, that may reflect variation in host tropism, has been correlated to three different classes of papG. Truncated variants of the class I, II and III papG adhesins were produced as fusion protein in E. coli and analysed for carbohydrate binding. The results showed that both carbohydrate binding and specificity of the papG adhesin resided in a linear part of the N-terminus of the protein.

Adhesins, Escherichia coli↗

PubMed1995

Recombinant human-milk bile-salt-stimulated lipase. Functional properties are retained in the absence of glycosylation and the unique proline-rich repeats.

Human milk bile-salt-stimulated lipase ensures efficient utilization of milk lipid in breast-fed infants. The N-terminal two-thirds of the peptide chain is highly conserved and shows striking similarities to typical esterases. In contrast, the remaining C-terminal part consists of a unique sequence of 16 proline-rich O-glycosylated repeats of 11 residues each. Recently we could show, using recombinant lipase variants, that neither these repeats nor the single N-linked sugar chain are essential for catalytic efficiency. In the present study, we report on the lack of importance of glycosylation and the unique repeats for other important functional properties, i.e. bile-salt activation, heparin binding, heat stability, stability at low pH and resistance to proteolytic inactivation. Compared to native enzyme, recombinant full-length lipase produced in two mammalian cell lines differed slightly in glycosylation pattern with no effects on the functional properties. Moreover, a variant lacking all repeats and the C-terminal tail following the last repeat exhibited the same functional characteristics as purified native milk enzyme. Thus, the structural basis for all the typical and functionally important properties reside in the N-terminal conserved part, in spite of the fact that none of these properties are shared by typical esterases. We could however, demonstrate that the C-terminal repeats are responsible for the unusual behaviour of the enzyme in size-exclusion chromatography, resulting in a considerably higher than expected apparent molecular mass.

Animals↗

PubMed1995

Expression of truncated and full-length forms of the Lyme disease Borrelia outer surface protein A in Escherichia coli.

The lipidated major outer surface protein, OspA, of the Lyme disease spirochaete may be important in the pathogenesis during Lyme borreliosis. To produce sufficient amounts of purified OspA variants to perform pathogenesis studies in vivo and in vitro, different recombinant OspA expression systems in Escherichia coli were constructed. Recombinant OspA variants were produced as a full-length molecule, as a truncated variant lacking the N-terminal lipidated cysteine, or as a fusion protein with the synthetic dimer of Staphylococcus aureus protein A IgG binding domain (ZZ). In order to produce the full-length protein, four different promoters were evaluated. These were combined with either the OspA original signal sequence or the E. coli Brauns lipoprotein signal sequence, lpp. The most efficient production of the full-length lipidated OspA was mediated by the constitutive beta-lactamase promoter in combination with lipoprotein signal sequences. For production of truncated nonlipidated OspA the S. aureus protein A signal sequence was ligated to the OspA open reading frame. Alternatively, truncated OspA was produced intracellularly using expression vectors that lack signal sequences. Production of nonlipidated protein with a heterologous signal peptide resulted in a soluble protein located mainly in the periplasm and in the culture medium. The full-length lipidated OspA, on the other hand, was associated mainly with the membrane fraction. The production level of the lipidated recombinant OspA was much lower than the level obtained with the truncated ZZ-OspA fusion protein.

Antigens, Surface↗