Biomedical subjects

L A Svensson

Publications and source records attributed to L A Svensson.

At least 19 recordsLinked to original sources

PubMed2001

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k).

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.

Amino Acids↗

PubMed2001

Crystal structure of a superantigen bound to MHC class II displays zinc and peptide dependence.

The three-dimensional structure of a bacterial superantigen, Staphylococcus aureus enterotoxin H (SEH), bound to human major histocompatibility complex (MHC) class II (HLA-DR1) has been determined by X-ray crystallography to 2.6 A resolution (1HXY). The superantigen binds on top of HLA-DR1 in a completely different way from earlier co-crystallized superantigens from S.aureus. SEH interacts with high affinity through a zinc ion with the beta1 chain of HLA-DR1 and also with the peptide presented by HLA-DR1. The structure suggests that all superantigens interacting with MHC class II in a zinc-dependent manner present the superantigen in a common way. This suggests a new model for ternary complex formation with the T-cell receptor (TCR), in which a contact between the TCR and the MHC class II is unlikely.

Amino Acid Sequence↗

PubMed2001

The impact of Haemophilus ducreyi cytolethal distending toxin on cells involved in immune response.

The Haemophilus ducreyi cytolethal distending toxin (HdCDT) induces cell cycle arrest and thereby inhibits cell proliferation of many cultured mammalian cell-lines. We investigated the effect of HdCDT on circulating human hematopoietic cells, including T- and B-cells, monocytes and polymorphonuclear cells (PMN). Lymphocytes were stimulated with T- and B-cell specific mitogens, whereas monocytes and PMN with endotoxin. HdCDT inhibited the mitogen-induced proliferation of T-cells in a dose-dependent manner as assayed by [(3)H]-thymidine incorporation and MTT assays. Similarly to T-cells, HdCDT also inhibited the proliferation of B-cells and consequently the immunoglobulin production, measured by ELISPOT and ELISA assays. In contrast, the HdCDT did not affect monocytes or PMN, as measured by MTT assay. The TNF-alpha production by monocytes and the phagocytic ability of PMN were neither affected. The monocytic cell line THP-1 was, however, sensitive to the toxin, seen as a reduction of proliferation and viability after exposure to HdCDT. In conclusion, exposure to HdCDT significantly affects the proliferation and other biological activities of stimulated human T- and B-cells, while circulating monocytes and PMN are not sensitive to HdCDT. The sensitivity of cells of the acquired immune system to HdCDT may hamper specific host response to H. ducreyi and contribute to persistence of chancroid lesions.

B-Lymphocytes↗

PubMed2001

Cooperative zinc binding in a staphylococcal enterotoxin A mutant mimics the SEA-MHC class II interaction.

The structure of a mutant form of staphylococcal enterotoxin A (SEA) has been determined to 2.1 A resolution. The studied SEA substitution H187-->A187 (SEAH187A) leads to an almost 10-fold reduction of the binding to major histocompatibility complex (MHC) class II. H187 is important for this interaction since it coordinates Zn2+. The zinc ion is thought to hold MHC class II and SEA together in a complex. Interestingly, only one of two molecules in the asymmetric unit binds Zn2+. H225, D227, a water molecule, and H44 from a symmetry-related molecule ligate Zn2+. The symmetry-related histidine is necessary for this substituted Zn2+ site to bind to Zn2+ at low zinc concentration (no Zn2+ added). Since a water molecule replaces the missing H187, H44 binds Zn2+ at the position where betaH81 from MHC class II probably will bind. Dynamic light scattering analysis reveals that in solution as well as in the crystal lattice the SEA(H187A) mutant forms aggregates. The substitution per se does not cause aggregation since wild-type SEA also forms aggregates. Addition of EDTA reduces the size of the aggregates, indicating a cross-linking function of Zn2+. In agreement with the biological function, the aggregation is weak (i.e. not revealed by gel filtration) and non-specific.

Crystallography, X-Ray↗

PubMed2001

Direct separation of captopril diastereoisomers including their rotational isomers by RP-LC using a teicoplanin column.

A direct reversed-phase liquid chromatography (LC) method has been developed for the separation and analysis of captopril and its 2R,2S diastereoisomer using a teicoplanin stationary phase. The proline containing diastereoisomers, which are known to form conformers in aqueous solution, were also separated from their rotational isomers. The influence of temperature, different organic modifiers and buffer type, concentration and pH were optimised to obtain a working resolution between the two diastereoisomers and their respective rotational isomers. The diastereoisomeric purity of several commercial captopril batches was subsequently evaluated using a 0.05% triethylammonium acetate (TEAA) buffer (pH 3.8) run at 1.0 ml/min with mobile phase reservoir and column temperature controlled at 0 degrees C. Throughout the study online UV diode array and mass spectrometry detection was carried out simultaneously to confirm that peaks eluting from the teicoplanin column were in fact captopril and not its readily converted disulphide dimer. Additionally, as a result of the greater detection sensitivity of mass spectrometry, it also facilitated a more accurate optimisation study where trace amounts of the rotational isomers were found to be present in the baseline at temperatures higher than optimum.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2001

Prevalence of cdtABC genes encoding cytolethal distending toxin among Haemophilus ducreyi and Actinobacillus actinomycetemcomitans strains.

The aim of this study was to investigate the presence of the three cdtABC genes responsible for production of cytolethal distending toxin (CDT) in Haemophilus ducreyi and Actinobacillus actinomycetemcomitans strains. Of 100 H. ducreyi strains from the culture collection of the University of Göteborg (CCUG), 27 strains with low or intermediate cytotoxic titre (< 1 in 10(4)) and 23 of the remaining isolates with a high cytotoxic titre (> or = 1 in 10(4)) were selected. Twenty-nine strains of H. ducreyi were isolated recently from patients with chancroid and 50 A. actinomycetemcomitans strains from patients with periodontitis. The cytotoxic activity on HEp-2 cells and the presence of cdtABC genes were studied by cytotoxicity assay of bacterial sonicates and PCR with primers specific for individual cdtA, B, and C genes of H. ducreyi in bacterial DNA preparations, respectively. All strains that manifested a cytotoxic titre in sonicate > or = 1 in 100 possessed all the three cdt genes. Eighteen of the 50 strains selected from the culture collection were negative and 32 positive for cdt genes. As all strains with a high cytotoxic titre gave positive PCR results, it can be assumed that the remaining 50 strains, which have high cytotoxic titre, would have been positive as well. Thus, it can be estimated that 82% of the culture collection strains had cdtABC genes. Similarly, 24 (83%) of 29 recent H. ducreyi isolates expressed the CDT activity and displayed all cdtABC genes. Forty-three (86%) of 50 strains of the closely related A. actinomycetemcomitans, expressing a cytotoxic activity > or = 1 in 100, also possessed all three genes. Furthermore, the nucleotide sequence of the cdtABC genes was highly conserved among H. ducreyi strains from different geographic areas. These results indicate that the majority of pathogenic H. ducreyi and A. actinomycetemcomitans strains express a CDT activity encoded by all three cdtABC.

Actinobacillus Infections↗

PubMed2000

Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26.

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.

Binding Sites↗

PubMed2000

The crystal structure of staphylococcal enterotoxin H: implications for binding properties to MHC class II and TcR molecules.

The X-ray structure of the superantigen staphylococcal enterotoxin H (SEH) has been determined at 1.69 A resolution. In this paper we present two structures of zinc-free SEH (apoSEH) and one zinc-loaded form of SEH (ZnSEH). SEH exhibits the conventional superantigen (SAg) fold with two characteristic domains. In ZnSEH one zinc ion per SEH molecule is bound to the C-terminal beta-sheet in the region implicated for major histocompatibility complex class II (MHC class II) binding in SEA, SED and SEE. Surprisingly, the zinc ion has only two ligating amino acid residues His206 and Asp208. The other ligands to the zinc ion are two water molecules. An extensive packing interaction between two symmetry-related molecules in the crystal, 834 A(2)/molecule, forms a cavity that buries the zinc ions of the molecules. This dimer-like interaction is found in two crystal forms. Nevertheless, zinc-dependent dimerisation is not observed in solution, as seen in the case of SED. A unique feature of SEH as compared to other staphylococcal enterotoxins is a large negatively charged surface close to the Zn(2+) site. The interaction of SEH with MHC class II is the strongest known among the staphylococcal enterotoxins. However, SEH seems to lack a SEB-like MHC class II binding site, since the side-chain properties of structurally equivalent amino acid residues in SEH and those in SEB-binding MHC class II differ dramatically. There is also a structural flexibility between the domains of SEH. The domains of two apoSEH structures are related by a 5 degrees rotation leading to at most 3 A difference in C(alpha) positions. Since the T-cell receptor probably interacts with both domains, SEH by this rotation may modulate its binding to different TcR Vbeta-chains.

Amino Acid Sequence↗

PubMed2000

The crystallography beamline I711 at MAX II.

A new X-ray crystallographic beamline is operational at the MAX II synchrotron in Lund. The beamline has been in regular use since August 1998 and is used both for macro- and small molecule diffraction as well as powder diffraction experiments. The radiation source is a 1.8 T multipole wiggler. The beam is focused vertically by a bendable mirror and horizontally by an asymmetrically cut Si(111) monochromator. The wavelength range is 0.8-1.55 A with a measured flux at 1 A of more than 10(11) photons s(-1) in 0.3 mm x 0.3 mm at the sample position. The station is currently equipped with a Mar345 imaging plate, a Bruker Smart 1000 area CCD detector and a Huber imaging-plate Guinier camera. An ADSC 210 area CCD detector is planned to be installed during 2000.

Journal Article↗

PubMed2000

Immobilisation and evaluation of a vancomycin chiral stationary phase for capillary electrochromatography.

The macrocyclic antibiotic, vancomycin, is covalently bonded to LiChrospher diol silica packed columns and evaluated in capillary electrochromatography (CEC) both in the reversed-phase and polar organic mode. Initially, capillaries were packed with 5 microm LiChrospher 100 A diol silica and evaluated in CEC with a reversed-phase biphenyl-pyrene achiral test resulting in resolution and efficiency values of ca. 2.5 and 100000 plates meter(-1), respectively. Repeatability for this test (resolution and efficiency) was also examined and found to be acceptable for both run-to-run (n=5, 0.74% and 1.5%) and column-to-column (n=5, 3.4% and 9.0%), respectively. Similar results were obtained when the 10 microm LiChrospher 1000 A diol silica was examined with the exception of efficiency, where a reduced plate height value of four times lower was obtained compared to the 100 A material. A simple three step in-situ vancomycin immobilisation procedure was subsequently carried out on these packed diol columns. Selectivity was obtained for thalidomide enantiomers on this vancomycin chiral stationary phase in reversed-phase CEC with resolution and efficiency values of ca. 2.5 and 80000 plates meter(-1), with acceptable repeatability (n=8) 0.9% and 3.0%, respectively. Selectivity was also obtained for thalidomide enantiomers on this phase in the polar organic mode with resolution and efficiency values of ca. 2.5 and 120000 plates meter(-1), with acceptable repeatability (n=7) 0.9% and 2.0%, respectively. It was possible to deduce from a chemometric design carried out for evaluating the mobile phase component effects that organic modifier ratio, MeOH/MeCN, played a significant role in controlling both resolution and efficiency. It was also possible to separate a number of basic analytes including four beta-adrenergic blocking agents in the polar organic mode albeit with lower resolution and efficiency values, ca. 1.5 and 45000 plates meter(-1), respectively.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2000

Enantioselective supercritical fluid chromatography using ristocetin A chiral stationary phases.

Racemic mixtures of five acidic drugs have been successfully separated by supercritical fluid chromatography (SFC) using macrocyclic antibiotic chiral stationary phases (CSPs). A ristocetin A CSP has been prepared 'in-house' and effectively applied in packed capillary SFC to separate the enantiomers of dichlorprop (R(s) = 1.4), ketoprofen (R(s) = 0.9) and warfarin (R(s) = 0.9). The commercial ristocetin A CSP (Chirobiotic R) was subsequently studied in packed column SFC with similar results where the enantiomers of warfarin (R(s) = 2.2), coumachlor (R(s) = 2.5) and thalidomide (R(s) = 0.6) were separated. Interestingly, differences were observed between the two differently immobilised CSPs where the enantiomers of dichlorprop and ketoprofen, which were separated on the 'in-house' CSP, could not be separated on the commercial phase.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1999

Orientational order and dynamics of hydration water in a single crystal of bovine pancreatic trypsin inhibitor.

The orientational order and dynamics of the water molecules in form II crystals of bovine pancreatic trypsin inhibitor (BPTI) are studied by (2)H NMR in the temperature range 6-50 degrees C. From the orientation dependence of the single crystal quadrupole splitting and linewidth, the principal components of the motionally averaged quadrupole interaction tensor and the irreducible linewidth components for the orthorhombic crystal are determined. With the aid of water orientations derived from neutron and x-ray diffraction, it is shown that the NMR data can be accounted for by a small number of highly ordered crystal waters, some of which have residence times in the microsecond range. Most of these specific hydration sites must be located at intermolecular contacts. The surface hydration layer that is also present in dilute solution is likely to be only weakly ordered and would then not contribute significantly to the splitting and linewidth from the protein crystal. To probe water dynamics on shorter time scales, the (2)H longitudinal relaxation dispersion is measured for a polycrystalline BPTI sample. The observed dispersion is dominated by rapidly exchanging deuterons in protein side chains, undergoing restricted rotational motions on a time scale of 10 ns.

Animals↗

PubMed1999

Active suppression in orally tolerized rats coincides with in situ transforming growth factor-beta (TGF-beta) expression in the draining lymph nodes.

Adult rats were fed pellets containing ovalbumin (OvA) during 4 weeks, and were 2 weeks thereafter immunized subcutaneously with a mixture of OvA and human serum albumin (HSA) in Freund's complete adjuvant (day 0). As a result of the immunization, the draining lymph nodes of the nontolerized (control) rats were heavily enlarged from day 10 to day 18; however, this size increase was absent in the OvA-fed rats. This manifestation of active suppression in the tolerized rats was preceded by the appearance of scattered CD4+ TGF-beta-expressing T cells in the T cell area of their lymph nodes (days 5-8); correspondingly, the levels of TGF-beta mRNA in the nodes were elevated in the tolerant rats compared with the control rats. The anti-OvA antibody levels in sera from the rats revealed that there was an initial B cell priming in the OvA-fed group, with levels higher than in the control group during the first week. Thereafter, suppression governed the response, and from day 10 onwards the anti-OvA levels were considerably lower than in the controls. When other groups of animals were pretreated with neutralizing anti-TGF-beta antibodies 1 day before the immunization, the anti-OvA response of the OvA-fed rats was restored to the levels of the control group, demonstrating the importance of TGF-beta in the maintenance of suppression. In conclusion, we demonstrate that TGF-beta-producing cells appear in the draining lymph nodes shortly after immunization in rats made orally tolerant using a relatively high-dose feeding regime; these cells are probably responsible for the down-regulation of the immune response observed in the OvA-fed rats.

Administration, Oral↗

PubMed1999

Crystallization and preliminary X-ray diffraction analysis of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26.

Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18-20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris-HCl pH 8.9. The crystals diffract to at least 1.60 A using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 A. The asymmetric unit contains one molecule of the enzyme.

Crystallization↗

PubMed1999

Crystallization and preliminary X-ray analysis of birch-pollen allergen Bet v 1 in complex with a murine monoclonal IgG Fab' fragment.

The human type I allergic response is characterized by the presence of allergen-specific serum immunoglobulin E (IgE). Allergen-mediated cross-linking of receptor-bound IgE on the surface of mast cells and circulating basophils triggers the release of mediators, resulting in the development of the clinical symptoms of allergy. In order to study the structural basis of allergen-antibody interaction, a complex between the major birch-pollen allergen Bet v 1 and a Fab' fragment isolated from the murine monoclonal Bet v 1 antibody BV16 has been crystallized. Complex crystals belong to space group P1, with unit-cell parameters a = 91.65, b = 99.14, c = 108.90 A, alpha = 105.7, beta = 98.32, gamma = 97.62 degrees, and diffract to 2.9 A resolution when analyzed at 100 K using synchrotron-generated X--rays.

Allergens↗

PubMed1997

A structural and functional comparison of staphylococcal enterotoxins A and C2 reveals remarkable similarity and dissimilarity.

Staphylococcal enterotoxins and toxic shock syndrome toxin-1 are known as superantigens due to their ability to activate a large number of T-cells by crosslinking the major histocompatibility complex class II molecules with the T-cell receptor. Although superantigens seem to act by a common mechanism, they vary in many of their specific interactions and biological properties. A structural comparison of staphylococcal enterotoxins A and C2, members of the staphylococcal superantigens, has shown large conformational differences at the putative TcR interaction site (loops between alphaN-alpha2, alpha4-beta9 and beta10-alpha5 in staphylococcal enterotoxin A) that could explain the variability in their T-cell receptor specificity. A common Zn2(+)-binding site was identified in both staphylococcal enterotoxin A and C2 that is superimposable but differs somewhat in its coordination geometry between the two molecules.

Amino Acid Sequence↗

PubMed1997

X-ray structure of a ribonuclease A-uridine vanadate complex at 1.3 A resolution.

The X-ray crystal structure of a uridine vanadate-ribonuclease A complex has been determined at 1.3 A resolution. The resulting structure includes all 124 amino-acid residues, a uridine vanadate, 131 water molecules, and a single bound 2-methyl-2-propanol. Side chains of 11 surface residues showing discrete disorder were modeled with multiple conformations. The final crystallographic R factor is 0.197. Structures obtained from high-level ab initio quantum calculations of model anionic oxyvanadate compounds were used to probe the effects of starting structure on the refinement process and final structure of the penta-coordinate phosphorane analog, uridine vanadate. The least-squares refinement procedure gave rise to the same final structure of the inhibitor despite significantly different starting models. Comparison with the previously determined complex of ribonuclease A with uridine vanadate obtained from a joint X-ray/neutron analysis (6RSA) [Wlodawer, Miller & Sjölin (1983). Proc. Natl Acad. Sci. USA, 80, 3628-3631] reveals similarities in the overall enzyme structure and the relative position of the key active-site residues, Hisl2, His119 and Lys41, but significant differences in the V-O bond distances and angles. The influence of ligand binding on the enzyme structure is assessed by a comparison of the current X-ray structure with the phosphate-free ribonuclease A structure (7RSA) [Wlodawer, Svensson, Sjölin & Gilliland (1988). Biochemistry, 27, 2705-2717]. Ligand binding alters the solvent structure, distribution and number of residues with multiple conformations, and temperature factors of the protein atoms. In fact, the temperature factors of atoms of several residues that interact with the ligand are reduced, but those of the atoms of several residues remote from the active site exhibit marked increases.

Journal Article↗

PubMed1997

Structural basis for the negative allostery between Ca(2+)- and Mg(2+)-binding in the intracellular Ca(2+)-receptor calbindin D9k.

The three-dimensional structures of the magnesium- and manganese-bound forms of calbindin D9k were determined to 1.6 A and 1.9 A resolution, respectively, using X-ray crystallography. These two structures are nearly identical but deviate significantly from both the calcium bound form and the metal ion-free (apo) form. The largest structural differences are seen in the C-terminal EF-hand, and involve changes in both metal ion coordination and helix packing. The N-terminal calcium binding site is not occupied by any metal ion in the magnesium and manganese structures, and shows little structural deviation from the apo and calcium bound forms. 1H-NMR and UV spectroscopic studies at physiological ion concentrations show that the C-terminal site of the protein is significantly populated by magnesium at resting cell calcium levels, and that there is a negative allosteric interaction between magnesium and calcium binding. Calcium binding was found to occur with positive cooperativity at physiological magnesium concentration.

Allosteric Regulation↗