Biomedical subjects

T Stehle

Publications and source records attributed to T Stehle.

At least 37 records · Page 2Linked to original sources

PubMed1996

Structure determination of simian virus 40 and murine polyomavirus by a combination of 30-fold and 5-fold electron-density averaging.

BACKGROUND: Simian virus 40 (SV40) and murine polyomavirus (polyoma) are non-enveloped DNA tumor viruses. Their structurally similar capsids, about 500 degrees in diameter, are formed by 72 pentamers of the major coat protein VP1. RESULTS: We describe in this paper the structure determination of SV40 and polyoma at 3.8 degree resolution, focusing particularly on methodological issues, and on a comparison of the overall molecular organization in the two related virus particles. Initial phases for SV40 were obtained by single isomorphous replacement at 6.5 degree. Phases were refined and the resolution extended to 3.8 degree by a combination of strict 5-fold and partial 30-fold electron-density averaging. The structure of polyoma was subsequently determined by systematically translating and rotating the individual VP1 pentamers, in order to find the maximum correlation between calculated and observed structure factors. The resolution was then extended to 3.8 degree, also by phase refinement through electron-density averaging. CONCLUSION: The strategies for density averaging and for molecular replacement, used to determine the SV40 and polyoma structures, are likely to be generally useful. The individual building blocks, the VP1 pentamers, are essentially identical in both cases, as are the local details of their interactions with neighboring pentamers. Nevertheless, the arrangement of the pentamers with respect to each other is somewhat different in the two viruses. Whereas SV40 is almost spherical, with all pentamers at identical radii, the pentamers in polyoma that lie on icosahedral fivefold axes are displaced outward by about 5 degree.

Capsid↗

PubMed1996

The structure of simian virus 40 refined at 3.1 A resolution.

BACKGROUND: The structure of simian virus 40 (SV40), previously determined at 3.8 degree resolution, shows how its pentameric VP1 assembly units are tied together by extended C-terminal arms. In order to define more precisely the possible assembly mechanisms, we have refined the structure at 3.1 degree resolution. RESULTS: New data from a high-intensity synchrotron source have been used for phase extension by electron-density averaging and refinement, exploiting only the strict 5-fold non-crystallographic symmetry for the real-space averaging steps. The accurate model enables us to study important structural features of the virus particle in detail. The remarkably invariant core of the VP1 pentamer bears the docking sites for the C-terminal arms from other pentamers. These contacts are the principal way in which pentameric assembly units are linked together in the capsid. Only at the interface between five-coordinated and six-coordinated pentamers do the pentamer cores appear to interact strongly. There are two cation-binding sites per VP1 monomer, seen in a soaking experiment with gadolinium nitrate. These sites are quite close to each other at the interfaces between pentamers. CONCLUSION: We propose that the contact between five-coordinated and six-coordinated pentamers may help to generate a six-pentamer nucleus, with which further pentamers can assemble to generate the complete particle. Calcium ions probably stabilize the structure of the assembled particle, rather than direct its assembly.

Amino Acid Sequence↗

PubMed1996

Crystal structures of murine polyomavirus in complex with straight-chain and branched-chain sialyloligosaccharide receptor fragments.

BACKGROUND: Murine polyomavirus recognizes (alpha2,3)-linked alpha-5-N-acetylneuraminic acid (sialic acid) on the surface of susceptible cells. While all strains bind to straight-chain receptors terminating in (alpha2,3)-linked sialic acid, some strains also bind to branched oligosaccharides that carry a second, (alpha2,6)-linked sialic acid. The ability to bind to these branched-chain receptors correlates with a single amino acid mutation at position 91 on the outer surface of the major capsid protein, VP1, and with a significant decrease in tumorigenicity. RESULTS: We have determined the structures of polyomavirus strain P16, which bears a glycine at position 91, in complex with model compounds for both straight-chain and branched-chain sialoglycoconjugates. The structures have been refined to a resolution of 3.65 degree. The ligands bind to a shallow groove on the surface of VP1. The sialic acid-(alpha2,3)-galactose moiety, which is common to both compounds, has specific and identical contacts. The additional (alpha2,6)-linked sialic acid moiety of the branched-chain receptor fragment fits into a surface pocket, but it has high thermal factors and does not form hydrogen bonds to groups on VP1. Data collected from crystals soaked at different oligosaccharide concentrations establish that both receptor fragments have similar, low affinities (dissociation constants in the range 5-10 mM) for the P16 virus, consistent with the interactions seen in the two complexes. CONCLUSION: The oligosaccharide-binding groove is complementary to the shape of the bound glycan, but there are relatively few hydrogen bonds between glycan and protein. Thus, the nature of the glycosidic linkages appears to be the principal determinant of specificity, rather than the position of particular hydroxyl groups. The low receptor affinity may be important for avoiding inhibition of viral release by retention on surface receptors of infected cells. Evidence suggests that strains with still greater pathogenicity are likely to have even weaker affinity.

Amino Acid Sequence↗

PubMed1996

Prednisolone reduces experimental arthritis, and inflammatory tissue destruction in SCID mice infected with Borrelia burgdorferi.

Glucocorticosteroids (GC) are widely used as anti-inflammatory agents. The effects of Prednisolone on the development of Borrelia (B.) burgdorferi-induced clinical arthritis and organ inflammation was studied in severe combined immunodeficiency (SCID) mice. The drug was administered orally at a dose of 3, 10 and 30 mg/kg, starting shortly before experimental infection of the mice. A dose dependent inhibition of arthritic joint swelling was observed. Full protection was obtained with 30 mg/kg until 21 days after infection, subsequently, mild joint swelling developed but progression and severity of the disease was considerably less than in the other treated as well as in the untreated mice. Inhibition of clinical arthritis coincided with reduction of inflammatory cell infiltration in the joints, liver and muscle. Prednisolone was ineffective when application was initiated after arthritis was fully developed, i.e., 22 days after infection. Since the activated endothelium plays a critical role in development of inflammatory lesions, the expression of the cellular adhesion molecules (CAMs) E-selectin, P-selectin, ICAM-1 and VCAM-1 was determined in vitro using the bEnd3 endothelial cell line. Stimulation with a sonicated B. burgdorferi preparation in the presence of the water-soluble compound Prednisolone-21-hemisuccinate considerably reduced expression of ICAM-1, and marginally also of E-selectin, whereas the level of P-selectin and VCAM-1 remained unaltered. Thus, downregulation of ICAM-1 might be a critical factor in Prednisolone-mediated inhibition of B. burgdorferi-induced inflammation; the flare up of the disease after the initial protection indicates that additional therapy, e.g. with antibiotics, is necessary.

Animals↗

PubMed1995

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.

Amino Acid Sequence↗

PubMed1995

Genetic and structural analysis of a virulence determinant in polyomavirus VP1.

The LID strain of polyomavirus differs from other laboratory strains in causing a rapidly lethal infection of newborn C3H/Bi mice. This virulent behavior of LID was attenuated by dilution, yet at sublethal doses LID was able to induce tumors at a high frequency, like its parent virus PTA. By constructing and assaying LID-PTA recombinant viruses and by DNA sequencing, the determinant of virulence in LID was mapped to the major viral capsid protein, VP1. The VP1s of LID and PTA differed at two positions: at 185, LID has phenylalanine and PTA has tyrosine, and at 296, LID has alanine and PTA has valine. Results obtained with viruses constructed by site-directed mutagenesis showed that alanine at position 296 is sufficient to confer a fully virulent phenotype regardless of which amino acid is at position 185. However, with valine at position 296, an effect of phenylalanine at position 185 is apparent, as this virus possesses an intermediate level of virulence. A crystal structure of polyomavirus complexed with 3'-sialyl lactose previously indicated van der Waals contacts between the side chain of valine 296 and the sialic acid ring (T. Stehle, Y. Yan, T. L. Benjamin, and S. C. Harrison, Nature [London] 369:160-163, 1994). When this interaction was modeled with alanine, these contacts were greatly reduced. Direct confirmation that the substitutions in VP1 affected receptor binding was obtained by studying virus hemagglutination behavior. The ensemble of results are discussed in terms of the idea that a lower affinity of the virus for its receptor can result in more rapid spread and increased pathogenicity.

Animals↗

PubMed1994

Structure of murine polyomavirus complexed with an oligosaccharide receptor fragment.

The polyomaviruses are non-enveloped, icosahedrally symmetrical particles with circular double-stranded DNA genomes. The outer shell of the virion contains 360 copies of viral protein VP1 (M(r) approximately 42K) arranged in pentamers. We report here the structure at 3.65 A resolution of murine polyomavirus ('polyoma') complexed with an oligosaccharide receptor fragment. This structure has been determined using the previously described model of simian virus 40 (SV40). Although very similar in structure to SV40, polyoma has interesting biological differences. Cell-surface N-acetyl neuraminic acid (sialic acid) is required for polyoma infectivity, but not for SV40. Polyoma attaches to the surface of susceptible cells by stereospecific recognition of oligosaccharides terminating in (alpha 2,3)-linked sialic acid. Studies of pathogenicity show that the specificity of viral binding to such oligosaccharides is an important determinant of the virus' ability to establish a disseminated infection and to induce tumours in the natural host. The complex described here show how polyoma recognizes the receptor fragment and how strains with different receptor specificities can distinguish between alternative ligands. The results also suggest an explanation for the large disparity in pathogenicity exhibited by strains differing in only one amino-acid residue of VP1.

Capsid↗

PubMed1994

Protection against Borrelia burgdorferi infection in SCID mice is conferred by presensitized spleen cells and partially by B but not T cells alone.

Borrelia burgdorferi induces spirochetemia, arthritis, carditis and myositis in SCID mice but not in immunocompetent co-isogenic C.B-17 mice. The contribution of naive or presensitized B and T cells in the control of spirochetal infection has now been analysed in SCID mice reconstituted with unselected spleen cells or enriched B or T cell populations thereof and subsequently challenged with B. burgdorferi. It is shown that SCID mice were protected (i) completely against disease (arthritis, carditis, myositis) by unselected spleen cells previously sensitized either to intact spirochetes or to recombinant outer surface protein A (OspA), (ii) to a large extent by mixtures of enriched spirochete-specific B and T cells, (iii) partially by equivalent preparations of presensitized B cells or by naive spleen or B cells, and (iv) not at all by presensitized or naive T cells alone. The degree of protection transferred was similar for the corresponding lymphocyte populations presensitized either to viable spirochetes or to recombinant OspA and correlated mainly with serum levels of B. burgdorferi-specific antibodies, in particular those to OspA/OspB. The capacity of enriched presensitized or naive B cells alone to generate specific antibodies of the isotypes IgM, IgG2b and IgG3, and to confer partial protection to SCID mice upon challenge with B. burgdorferi is most probably due to a B cell mitogen(s) associated with the spirochetes. These data further emphasize the important role of B cells and antibodies in the control of B. burgdorferi infection in mice, and suggest that T cells are critically involved in the optimal generation of protective antibody responses but not in the direct elimination of spirochetes from the host.

Animals↗

PubMed1994

Borrelia burgdorferi upregulates the adhesion molecules E-selectin, P-selectin, ICAM-1 and VCAM-1 on mouse endothelioma cells in vitro.

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd 3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (approximately 50 h), bEnd3 cells were found to bind cells of a VLA-4+ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.

Animals↗

PubMed1994

Expression of endothelial cell adhesion molecules in joints and heart during Borrelia burgdorferi infection of mice.

The expression of adhesion molecules on endothelia was examined during chronic arthritis and carditis in SCID and immunocompetent susceptible AKR/N mice infected with Borrelia burgdorferi (B. burgdorferi). All stages of disease were associated with the upregulation or new expression of ICAM-1 and P-selectin and of VCAM-1 and E-selectin, respectively, on blood vessels of affected joint tissues of SCID and AKR/N mice as well as on heart tissue of SCID mice but not in other tissues. Moreover, ICAM-1 was also found on infiltrating mononuclear cells. The overall staining intensity for each of the four adhesion molecules on individual tissue sections of joint and heart increased with time of infection and was associated with the presence of spirochetes in the tissue. In addition it is shown that in both mouse strains inflammation of joints but not heart is accompanied by vascular proliferation. Synovial but not heart tissues of infected SCID mice were found to express both peripheral- (PNAd) and mucosal (MAdCAM-1) lymph node high endothelia venule associated vascular addressins as detected by mAb Meca-79 and Meca-367, respectively, but only at later stages of the disease and only on newly generated small venules. However, neither of the two addressins were evident in synovial lesions of AKR/N mice. Together the data suggest that the concomittant induction of ICAM-1, VCAM-1, E-selectin and P-selectin in lesions of infected mice provide a means for enhanced cellular infiltration into affected organs and that the regulation of these structures is conserved in the absence of a functional immune system. Furthermore, the differential induction of vascular proliferation in joint and heart tissues as well as the restricted expression patterns of vascular addressins indicate that the pathogenetic processes induced by B. burgdorferi are distinct for joint and heart.

Animals↗

PubMed1993

NADH binding site and catalysis of NADH peroxidase.

The structure of the complex between cofactor NADH and the enzyme NADH peroxidase from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been determined by crystal soaking, X-ray data collection, model building of NADH and refinement at 0.24-nm resolution based on the known enzyme structure [Stehle, T., Ahmed, S. A., Claiborne, A. & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325-1344]. Apart from NADH, the catalytic center of the enzyme contains FAD and a cysteine that shuttles between thiolate and sulfenic acid states. Unfortunately, this cysteine was irreversibly oxidized to a cysteine sulfonic acid in the established enzyme structure. Based on the geometry of the catalytic center, we discuss the stabilization of the oxidation-sensitive sulfenic acid and propose a reaction mechanism.

Binding Sites↗

PubMed1992

Refined structure of the complex between guanylate kinase and its substrate GMP at 2.0 A resolution.

The crystal structure of guanylate kinase from Saccharomyces cerevisiae complexed with its substrate GMP has been refined at a resolution of 2.0 A. The final crystallographic R-factor is 17.3% in the resolution range 7.0 A to 2.0 A for all reflections of the 100% complete data set. The final model has standard geometry with root-mean-square deviations of 0.016 A in bond lengths and 3.0 in bond angles. It consists of all 186 amino acid residues, the N-terminal acetyl group, the substrate GMP, one sulfate ion and 174 water molecules. Guanylate kinase is structurally related to adenylate kinases and G-proteins with respect to its central beta-sheet with connecting helices and the giant anion hole that binds nucleoside triphosphates. These nucleotides are ATP and GTP for the kinases and GTP for the G-proteins. The chain segment binding the substrate GMP of guanylate kinase differs grossly from the respective part of the adenylate kinases; it has no counterpart in the G-proteins. The binding mode of GMP is described in detail. Probably, the observed structure represents one of several structurally quite different intermediate states of the catalytic cycle.

Adenosine Triphosphate↗

PubMed1992

Biosynthesis and excretion of gangliosides by the isolated perfused rat liver.

De novo synthesis and excretion into perfusate and bile fluid of hepatic gangliosides were studied in isolated perfused rat livers. Addition of N-acetyl-[6-3H(n)]D-mannosamine to the perfusate resulted in radioactive synthesis of at least eight gangliosides labeled in their sialic acid residues. About 10% of total de novo synthesized gangliosides were excreted into the perfusate, less than 1% into the bile fluid. Labeled gangliosides were tentatively identified by cochromatography with known standards. All of them are known to occur in rat liver and sera. The results indicate that most, if not all, normal serum gangliosides are synthesized in the liver; excretion with bile fluid is negligible. They explain previous observations, and indicate clinical implications, which are discussed.

Animals↗

PubMed1991

Structure of NADH peroxidase from Streptococcus faecalis 10C1 refined at 2.16 A resolution.

The crystal structure of NADH peroxidase (EC 1.11.1.1) from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been refined to a resolution of 2.16 A using the simulated annealing method. The final crystallographic R-factor is 17.7% for all data in the resolution range 7 to 2.16 A. The standard deviations are 0.015 A in bond lengths and 3.0 degrees in bond angles for the final model, which includes all 447 amino acid residues, one FAD and 369 water molecules. The enzyme is a symmetrical tetramer with point group D2; the symmetry is crystallographic. The redox center of the enzyme consists of FAD and a cysteine (Cys42), which forms a sulfenic acid (Cys-SOH) in its oxidized state. A histidine (His10) close to Cys42 is likely to act as an active-site base. In the analyzed crystal, the enzyme was in a non-native oxidation state with Cys42 oxidized to a sulfonic acid Cys-SO3H. The chain fold of NADH peroxidase is similar to those of disulfide oxidoreductases. A comparison with glutathione reductase, a representative of this enzyme family, is given.

Amino Acid Sequence↗

PubMed1991

Myositis in mice inoculated with Borrelia burgdorferi.

The authors describe the appearance of myositis in immunocompetent and immunodeficient mice after subcutaneous inoculation with Borrelia burgdorferi by histology and immunohistology. Experimental infection of mice 1) causes inflammation of striated but not smooth muscles, 2) affects the entire musculoskeletal system, and 3) is characterized by perivascular and interfacicular infiltration of mononuclear leukocytes in the striated muscle leading to necrosis as well as disruption of muscle fibers. The lesions found in striated muscle specimens were most pronounced in immunodeficient (SCID), less severe in T-cell-deficient nu/nu (BALB/c, C57BL/6) and marginal to moderate or almost not present in immunocompetent AKR/N and C.B-17 mice, respectively.

Animals↗

PubMed1990

The structure of NADH peroxidase from Streptococcus faecalis at 3.3 A resolution.

NADH peroxidase (EC 1.11.1.1) previously isolated from Streptococcus faecalis 10C1 has been crystallized. The crystal structure has been solved by multiple isomorphous replacement and solvent-flattening at 3.3 A (1 A = 0.1 nm) resolution. The enzyme forms a tetramer consisting of 4 crystallographically related subunits. The monomer chain fold is in general similar to those of glutathione reductase and lipoamide dehydrogenase. FAD binds in the same region and in a similar conformation as in glutathione reductase. The unusual cysteine-sulfenic acid participating in catalysis is located at the isoalloxazine of FAD.

Crystallization↗

PubMed1990

Three-dimensional structure of the complex of guanylate kinase from yeast with its substrate GMP.

The enzyme guanylate kinase was isolated from baker's yeast and crystallized as a complex with its substrate GMP. The crystal structure was solved by multiple isomorphous replacement, solvent-flattening, restrained least-squares refinement, and simulated annealing. The current R-factor is 28.9% at a resolution of 2.0 A. The model is given as a backbone tracing, the GMP binding site is shown in atomic detail. In its major domain (residues 1 to 32 and 82 to 186), the chain fold is closely similar to the adenylate kinases, while the minor domain (residues 33 to 81) differs grossly from the 3-helix fold of the adenylate kinases. Structural homology and mechanistical similarity allow us to assign the AMP site of the adenylate kinases on the basis of the GMP site.

Binding Sites↗

PubMed1990

Volume regulation in liver: further characterization by inhibitors and ionic substitutions.

The present study has been performed to elucidate the mechanisms of volume regulation in isolated perfused liver. Reduction of extracellular osmolarity by 80 mOsm/L leads to a release of potassium and a sustained alkalinization of effluent. Reexposure to isotonic perfusate leads to reuptake of potassium by the liver and acidification of effluent. Part of the alkalinization could be due to release of bicarbonate parallel to potassium release. Carboanhydrase inhibition and replacement of bicarbonate/CO2 by HEPES buffer, however, do not significantly modify volume regulatory potassium release or reuptake. Reduction of perfusate chloride to 37 mmol/L by replacement of NaCl with raffinose leads to a decrease of liver weight indicative of shrinkage of liver cells. Subsequent omission of 180 mmol/L raffinose leads to potassium and chloride release and to alkalinization of effluent. Volume regulatory release of potassium is impaired in 1 mmol/L quinidine, 1 mmol/L SITS and 5 mmol/L barium. Volume regulatory reuptake of potassium is impaired by 1 mmol/L amiloride. Volume regulatory release of potassium is not appreciably affected by either; 1 mmol/L furosemide, 1 mumol/L verapamil, 1 mmol/L amiloride or 1 mmol/L barium and volume regulatory potassium reuptake proved insensitive to 1 mmol/L furosemide or 1 mmol/L barium. The data suggest that the cells release potassium and chloride during regulatory volume decrease by quinidine, SITS and weakly barium-sensitive transport systems and that regulatory volume increase is accomplished by activation of Na/H exchange.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗