PubMed Health⌕ Search

Biomedical subjects

R Ladenstein

Publications and source records attributed to R Ladenstein.

At least 109 records · Page 6Linked to original sources
PubMed1993

Impact of megatherapy on survival after relapse from stage 4 neuroblastoma in patients over 1 year of age at diagnosis: a report from the European Group for Bone Marrow Transplantation.

PURPOSE: Relapse from stage 4 neuroblastoma usually carries a poor prognosis. A retrospective study using the European Bone Marrow Transplant (EBMT) Solid Tumor Registry was undertaken to define the role of megatherapy (MGT) in relapsed patients. PATIENTS AND METHODS: After relapse, 33 boys and 15 girls with previous stage 4 neuroblastoma received intensification by MGT followed by either autologous (n = 42) or allogeneic (n = 6) bone marrow rescue in 11 European institutions. The median age at diagnosis was 47 months (range, 14 to 134) and the median interval from diagnosis to relapse was 16 months (range, 4 to 94). Thirty patients had received only conventional-dose primary treatments (group A), whereas 18 patients had previously received intensification with MGT (group B). The median follow-up time of the total group is 95 months (range, 25 to 185). RESULTS: The actuarial overall survival rate at 2 years after MGT for relapse is 27% for group A and 0% for group B (P = .02). Three adverse, independent prognostic factors were confirmed by multivariate analysis using the Cox proportional hazards regression model: an interval of less than 12 months between diagnosis and relapse (P < .0001), nonresponding or untreated relapse (P = .0002), and previous MGT during primary treatments (P = .055). None of the other variables analyzed, such as sex, age, bone or bone marrow involvement at diagnosis or at relapse, and type of MGT at relapse, influenced outcome in this patient cohort. CONCLUSION: Responding patients who relapse more than 12 months from diagnosis who had not received previous MGT appear to benefit from consolidation MGT. Relapse patients who do not fulfill these criteria gain no advantage from this cost-intensive procedure and should be treated differently.

Actuarial Analysis↗
PubMed1992

Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution.

The three-dimensional structure of human class pi glutathione S-transferase from placenta (hGSTP1-1), a homodimeric enzyme, has been solved by Patterson search methods and refined at 2.8 A resolution to a final crystallographic R-factor of 19.6% (8.0 to 2.8 A resolution). Subunit folding topology, subunit overall structure and subunit association closely resembles the structure of porcine class pi glutathione S-transferase. The binding site of a competitive inhibitor, S-hexylglutathione, is analyzed and the locations of the binding regions for glutathione (G-site) and electrophilic substrates (H-site) are determined. The specific interactions between protein and the inhibitor's glutathione peptide are the same as those observed between glutathione sulfonate and the porcine isozyme. The H-site is located adjacent to the G-site, with the hexyl moiety lying above a segment (residues 8 to 10) connecting strand beta 1 and helix alpha A where it is in hydrophobic contact with Tyr7, Phe8, Val10, Val35 and Tyr106. Catalytic models are discussed on the basis of the molecular structure.

Amino Acid Sequence↗
PubMed1992

Crystallization and preliminary crystallographic characterization of GTP cyclohydrolase I from Escherichia coli.

GTP cyclohydrolase I of Escherichia coli has been purified from a recombinant bacterial strain. The enzyme was crystallized from 0.6 M-sodium citrate and from 0.8 M-sodium/potassium phosphate, respectively. Crystals grown in citrate showed X-ray diffraction extending to a resolution better than 3 A. The space group was P2(1) with cell dimensions a = 204.8 A, b = 210.1 A, c = 72.2 A, alpha = gamma = 90 degrees and beta = 95.8 degrees.

Biopterins↗
PubMed1992

Electron microscopy of decorated crystals for the determination of crystallographic rotation and translation parameters in large protein complexes.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a core of 3 alpha subunits. The preparation of reconstituted hollow capsids consisting of 60 beta subunits and their crystallization in a hexagonal (space group P6(3)22) and in a monoclinic (space group C2) modification have been described. The rotational and translational parameters of the protein molecules in both crystal forms were studied by electron microscopy of freeze-etch replicas and by Patterson correlation techniques. Decoration with silver and image processing provided images with the positions of the 3-fold and 5-fold molecular axes being labelled by metal clusters. This allowed the unequivocal determination of the orientation and translational position of the protein molecules with respect to the crystallographic axes in the hexagonal modification. From inspection of the decoration images it was immediately obvious that the hexagonal crystal forms of alpha 3 beta 60 and of beta 60 are isomorphous. In the monoclinic crystals, a local icosahedral 2-fold coincides with the crystallographic 2-fold axis. The exact solution of the particle orientation was determined by interpretation of Patterson self-rotation functions for the icosahedral symmetry axes. Rotational and translational parameters for the monoclinic modification are given. A rational procedure for the efficient application of freeze-etching techniques in order to elucidate the packing in crystals of large proteins is described.

Bacillus subtilis↗
PubMed1992

Tumor necrosis factor as an autocrine growth factor for neuroblastoma.

Recombinant tumor necrosis factor (TNF) stimulates the proliferation of two neuroblastoma cell lines, SKNFI and SKNBE, in both serum-free medium and fetal calf serum-supplemented medium but has no effect in medium without insulin. This effect is very similar with TNF doses ranging from 5 to 500 ng/ml but depends on the duration of treatment; when cells are treated for 168 h with TNF, the maximal index of proliferation is observed between 120 and 144 h of treatment. The two neuroblastoma cell lines express type A and type B TNF receptors and contain TNF protein; however, TNF is undetectable in culture supernatants. Treatment of the two neuroblastoma cell lines with a rabbit polyclonal antibody to TNF for 96 h fully inhibits [3H]thymidine incorporation; less than 5% viable cells are left in the samples after treatment. A combination of two monoclonal antibodies against type A and type B TNF receptors also inhibits over 85% of the [3H]thymidine incorporation by the two cell lines after 96 h of treatment; the use of a single antibody has a partial effect, suggesting that both receptors are functional on the neuroblastoma cell lines. Taken together, these results show that TNF is an autocrine growth factor for the two neuroblastoma cell lines SKNFI and SKNBE. The results described above have been confirmed on two other neuroblastoma cell lines, IRM32 and CLB-PE.

Cell Division↗
PubMed1992

Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Correlation of metal decoration and topochemistry on protein surfaces.

On the surface of protein molecules the formation of metal clusters during vacuum condensation is controlled by topochemical features of the substrate and by specific properties of the decorating material. The resulting metal distribution (decoration pattern) can be mapped by electron microscopy in conjunction with image processing. We have applied this technique to freeze-etched crystals of the lumazine synthase-riboflavin synthase complex and its derivative obtained by binding of the heteropolytungstate (NaP5W30O110)(NH4)14.31 H2O. The decoration pattern of the free protein and its heteropolytungstate derivative showed marked differences. The correlation of these data with the X-ray structure of the protein showed an increased affinity of both gold and silver to the location of heteropolytungstate. Decoration sites can, but do not need to, be close to the protein surface. Actually, two of the observed decoration sites are located on a layer of ice as thick as 20 A, which apparently transmits underlying topochemical features. Preferential affinity of a site to a given metal must be seen as a property that depends on specific interaction with the decorating material but also on the differential affinities in adjacent areas.

Bacillus subtilis↗
PubMed1991

Class pi glutathione S-transferase from pig lung. Purification, biochemical characterization, primary structure and crystallization.

A cytosolic glutathione S-transferase from pig lung was purified 210-fold to apparent homogeneity. The enzyme was classified as a class pi isoenzyme on the basis of its physical and chemical properties. It is homodimeric with a subunit Mr of 23,500, has a pI of 7.2, and shows a high specific activity towards ethacrynic acid. The glutathione analogues, S-hexylglutathione and glutathione sulfonate, were strong reversible inhibitors. The enzyme's primary structure, established entirely by protein chemical methods, consists of 203 amino acids and is highly similar (82-84% residue identity) to the rat and human class pi isoenzymes. Furthermore, there was no evidence of microheterogeneity or post-translational modifications. Each subunit contains a highly reactive cysteine residue, the modification of which leads to enzyme inactivation. None of the cysteine residues in the pig enzyme appear to form intramolecular disulfide bonds. Singel crystals of the glutathione-S-transferase-glutathione-sulfonate complex were obtained by the hanging-drop method of vapour diffusion from poly(ethylene glycol) 4000 solutions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 10.125 nm, b = 8.253 nm and c = 5.428 nm and diffract to better than 0.22 nm.

Amino Acid Sequence↗
PubMed1991

The three-dimensional structure of class pi glutathione S-transferase in complex with glutathione sulfonate at 2.3 A resolution.

The three-dimensional structure of class pi glutathione S-transferase from pig lung, a homodimeric enzyme, has been solved by multiple isomorphous replacement at 3 A resolution and preliminarily refined at 2.3 A resolution (R = 0.24). Each subunit (207 residues) is folded into two domains of different structure. Domain I (residues 1-74) consists of a central four-stranded beta-sheet flanked on one side by two alpha-helices and on the other side, facing the solvent, by a bent, irregular helix structure. The topological pattern resembles the bacteriophage T4 thioredoxin fold, in spite of their dissimilar sequences. Domain II (residues 81-207) contains five alpha-helices. The dimeric molecule is globular with dimensions of about 55 A x 52 A x 45 A. Between the subunits and along the local diad, is a large cavity which could possibly be involved in the transport of nonsubstrate ligands. The binding site of the competitive inhibitor, glutathione sulfonate, is located on domain I, and is part of a cleft formed between intrasubunit domains. Glutathione sulfonate is bound in an extended conformation through multiple interactions. Only three contact residues, namely Tyr7, Gln62 and Asp96 are conserved within the family of cytosolic glutathione S-transferases. The exact location of the binding site(s) of the electrophilic substrate is not clear. Catalytic models are discussed on the basis of the molecular structure.

Amino Acid Sequence↗
PubMed1990

The lumazine synthase-riboflavin synthase complex of Bacillus subtilis. Crystallization of reconstituted icosahedral beta-subunit capsids.

The lumazine synthase-riboflavin synthase complex (heavy riboflavin synthase) of Bacillus subtilis consists of an icosahedral capsid of 60 beta-subunits containing a core of three alpha-subunits. The enzyme has been purified from the derepressed mutant H 94 of B. subtilis by a novel efficient procedure using column chromatography and preparative crystallization. Beta-Subunits were isolated after dissociation of the enzyme at pH 8.0. Ligand-driven renaturation of beta-subunits yields hollow icosahedral beta 60 capsids which could be crystallized in 1.55 M phosphate, pH 8.7, in three different modifications. A monoclinic modification belongs to space group C2 with unit cell dimensions of a = 235.5, b = 191.2, and c = 165.4 A and alpha = gamma = 90 degrees and beta = 134.4 degrees. The crystals contain two hollow beta 60 particles/unit cell and diffract to approximately 2.8-A resolution. A hexagonal modification has the space group P6(3)22 with unit cell dimensions of a = b = 157.2 and c = 300.8 A and alpha = beta = 90 degrees and gamma = 120 degrees. These cell parameters are similar to the dimensions of hexagonal crystals of native heavy riboflavin synthase (alpha 3 beta 60). A second hexagonal modification shows unit cell parameters of a = b = 156.3 and c = 622.6 A and alpha = beta = 90 degrees and gamma = 120 degrees. The space group of this modification could not be determined unambiguously.

Bacillus subtilis↗
PubMed1989

X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands.

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.

Ascorbate Oxidase↗
PubMed1989

Electrostatic effects in a large enzyme complex: subunit interactions and electrostatic potential field of the icosahedral beta 60 capsid of heavy riboflavin synthase.

The role of the electrostatic interactions in the stability of the icosahedral beta 60 capsid of heavy riboflavin synthase from Bacillus subtilis has been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interactions agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand-free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between beta-subunits forming dimers, which connect the relatively stable pentamers in the beta-60 capsid. The release of the ligand causes a reduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due to the titratable groups and alpha-helix macrodipoles has been calculated on the basis of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polar distribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of positive potential "channels" that coincide with the channels constituted by the pentameric and trimeric beta-subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme-substrate recognition.

Bacillus subtilis↗