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Europaische Rahmenbedingungen Mit Inkrafttreten der „Paediatric Regulation“ [Verordnung (EG) Nr. 1901/2006] im Januar 2007 wurde auf europaischer Ebene und in der Folge von den Mitgliedsstaaten eindeutig politischer Wille gezeigt, das Dilemma der Kindermedikation trotz des ethischen Spannungsfelds im Umfeld von Studien einer Losung zuzufuhren. Seit Juli 2008 besteht in ganz Europa die Verpflichtung zur Durchfuhrung von Kinderstudien.

Das Neuroblastom ist der haufigste extrakraniale solide Tumor des Kindesalters. Je nach seiner Lokalisation kommt es zu den fur Neuroblastome charakteristischen, aber lageabhangigen Symptomen. Ein 123I-MIBG-Scan ist das Standarddiagnoseverfahren der Wahl fur alle Tumorlokalisationen. Ebenso sind beidseitig gewonnene Markaspirate der hinteren Darmbeinschaufelleiste und Biopsien des Knochenmarkkerns unverzichtbar. Entscheidender Marker fur die Beurteilung der Dignitat von neuroblastischen Tumoren ist die MYCN-Onkogen-Amplifikation. Das Neuroblastom muss risikoadaptiert behandelt werden. Aus diesem Grund sind eine entsprechend exakte Diagnostik und Klassifikation unverzichtbar, und die Therapie soll an dafur spezialisierten Zentren und im Rahmen von qualitatssichernden Therapieoptimierungsstudien durchgefuhrt werden.

Neuroblastoma (NB) is the most frequent solid tumour in early childhood. NBs are extremely heterogeneous in terms of their biological characteristics and the clinical behaviour which ranges from lethal disease to spontaneous regression and/or differentiation. Genetically, the heterogeneity of these tumours is reflected mainly by differences in the DNA content, presence of chromosome lp deletions and amplification of the proto-oncogene N-myc. Up to now, only the prognostic impact of N-myc amplification, based on a large series of analysed tumour samples, was established and is now used as the discriminating factor for treatment stratification in a newly developed European protocol. The influence of other genetic aberrations, i.e. dellp36, t(l;17), ploidy and expression of certain proteins, such as nerve growth factor (NGF) receptor proteins and CD44, on the biological behaviour were studied only in a limited number of cases. We will review the literature and outline our own observations dealing with the peculiar phenomena observed in some NBs, i. e. spontaneous regression and/or maturation.

The electrostatic interactions in the channels of OmpF and PhoE porins from Escherichia coli were analysed on the basis of a macroscopic multi-dielectric model of the protein-membrane complex derived from the respective porin X-ray structures. The membrane was represented as layers of distinct dielectric constants corresponding to the aliphatic core and the polar head groups of the lipids. The pKa values of the titratable groups and the electrostatic field in the region of the channel were calculated by the finite difference technique. In spite of the differences in sequences and charge constellations, the calculated electrostatic properties of the two porins are similar in several aspects: (1) unusual titration behaviour (pKa below 7) was found for some groups of the cluster of basic residues at the constriction of the pore; (2) a number of acidic groups buried between the internal loop and the barrel wall are stabilized in their protonated forms at neutral pH; (3) there is a strong transverse electrostatic field in the channel characterized by a screw-like form. The strength of the field is greatest at the region of the constriction zone. This would facilitate the diffusion of solutes with a large dipole moment such as free amino acids. Differences between the electrostatic fields of OmpF and PhoE are mainly confined to that end of the pore that faces the cell exterior in vivo. In OmpF the electrostatic potential is close to zero in this region of the channel, whereas a positive potential was found in PhoE. It was shown that the experimentally observed difference in ion selectivity of the two porins can largely be attributed to this distinct electrostatic property.

Overcoming childhood cancers is critically dependent on the state of research. Understanding how, with whom and what the research community is doing with childhood cancers is essential for ensuring the evidence-based policies at national and European level to support children, their families and researchers. As part of the European Union funded EUROCANCERCOMS project to study and integrate cancer communications across Europe, we have carried out new research into the state of research in childhood cancers. We are very grateful for all the support we have received from colleagues in the European paediatric oncology community, and in particular from Edel Fitzgerald and Samira Essiaf from the SIOP Europe office. This report and the evidence-based policies that arise from it come at a important junction for Europe and its Member States. They provide a timely reminder that research into childhood cancers is critical and needs sustainable long-term support., peer-reviewed

Das Neuroblastom ist der haufigste extrakranielle solide Tumor des Kindesalters. Der Tumor geht von embryonalen sympathischen Neuroblasten des Grenzstranges, der Nebenniere oder den Sympathikusganglien des vegetativen Nervensystems aus. Die Morphologie entspricht den Entwicklungsstadien der sympathischen Ganglien. Etwa 2,6% sind zervikal, 14,7% thorakal 79% im Abdomen (51% in der Nebenniere und 28% extraadrenal), der Rest im Becken oder unentdeckt. Der Tumor ist heterogen mit einem weiten Spektrum in seiner klinischen Manifestation und in seinem biologischen Verhalten. Der Verlauf der Erkrankung kann sehr variabel sein: im Sauglingsalter konnen Spontanremissionen sogar bei metastasierten Erkrankungen auftreten, wahrend das 5-Jahres-Uberleben alterer Kinder mit metastaslerter Erkrankung nur bei etwa 40% liegt. Wichtige prognostische Faktoren sind das Alter des Patienten bei Diagnosestellung, das Erkrankungsstadium sowie molekulargenetische Marker.

The structure-function relationships of alcohol dehydrogenases from the large family of short-chain dehydrogenase/reductase (SDR) enzymes are described. It seems that while mammals evolved with a medium-chain alcohol dehydrogenase family (MDR), fruit flies utilized an ancestral SDR enzyme. They have modified its function into an efficient alcohol dehydrogenase to aid them in colonizing the emerging ecological niches that appeared around 65 million years ago. To the scientific community, Drosophila has now served as a model organism for quite some time, and Drosophila alcohol dehydrogenase is one of the best-studied members of the SDR family. The availability of a number of high-resolution structures, accurate and thorough kinetic work, and careful theoretical calculations have enabled an understanding of the structure-function relationships of this metal-free alcohol dehydrogenase. In addition, these studies have given rise to various hypotheses about the mechanism of action of this enzyme and contribute to the detailed knowledge of the large superfamily of SDR enzymes.

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.

At least three genetic hallmarks identify aggressive tumour behaviour in neuroblastomas; amplification of the oncogene MYCN; deletion (loss of heterozygosity [LOH]) at the short arm of chromosome 1 (del1p36), seen in approximately 28% of the cases; and di-tetraploidy. The MYCN oncogene is amplified in approximately 23% of all neuroblastomas and becomes important for the stratification of therapy in localised and 4s tumours. Up to now, it has been believed that the genetic constellation of neuroblastic tumours is stable and does not alter during tumour evolution or during tumour progression.Using fluorescence in situ hybridisation techniques (FISH) to investigate different tumour areas on touch preparations and histological sections, we show that genetic heterogeneity can be detected in neuroblastomas, especially in tumours detected by urinary mass screening.The identification of such cell clones is important, because the MYCN amplification and/or the deletion at 1p36 appear to be responsible for aggressive local growth and development of metastases.

An update of results from the High Risk Protocol of the Meta-EICESS Study, conducted at the Pediatric Stem-Cell Transplant Centers of Düsseldorf and Vienna. In order to evaluate a possible therapeutic benefit after allogeneic SCT in patients with advanced Ewing tumors (AET), we compared outcome after autologous and allogeneic stem-cell transplantation (SCT).We analyzed 36 patients treated with the myeloablative Hyper-ME protocol (hyperfractionated total body irradiation, melphalan, etoposide +/- carboplatin) between November 1986 and December 1994. Minimal follow-up for all patients was five years. All patients underwent remission induction chemotherapy and local treatment before myeloablative therapy. Seventeen of thirty-six patients had multifocal primary Ewing's tumor, eighteen of thirty-six had early, multiple or multifocal relapse, one of thirty-six patients had unifocal late relapse. Twenty-six of thirty-six were treated with autologous and ten of thirty-six with allogeneic hematopoietic stem cells. We analyzed the following risk factors, that could possibly influence the event-free survival (EFS): number of involved bones, degree of remission at time of SCT, type of graft, indication for SCT, bone marrow infiltration, bone with concomitant lung disease, age at time of diagnosis, pelvic involvement, involved compartment radiation, histopathological diagnosis.EFS for the 36 patients was 0.24 (0.21) +/- 0.07. Eighteen of thirty-six patients suffered relapse or died of disease, nine of thirty-six died of treatment related toxicity (DOC). Nine of thirty-six patients are alive in CR. Ageor = 17 years at initial diagnosis (P0.005) significantly deteriorated outcome. According to the type of graft, EFS was 0.25 +/- 0.08 after autologous and 0.20 +/- 0.13 after allogeneic SCT. Incidence of DOC was more than twice as high after allogeneic (40%) compared to autologous (19%) SCT, even though the difference did not reach significance (P = 0.08, Fisher's exact test).Because of the rather short observation period. secondary malignant neoplasms (SMN) may complicate the future clinical course of some of our patients who are currently viewed as event-free survivors. EFS in AET is not improved by allogeneic SCT due to a higher complication rate. The patient group was to small to analyze for a possible graft-versus-tumor effect.

Lumazine synthase of Saccharomyces cerevisiae is a homopentamer with a molecular weight of 90 kDa. Crystals of the recombinant enzyme with a size of up to 1.6 mm were obtained. The space group is P4(1)2(1)2 with lattice dimensions 82.9 A x 82.9 A x 300.2 A. X-ray diffraction data collected under cryogenic conditions were complete to 1.85 A resolution. The structure of the enzyme in complex with the intermediate analogue, 5-(6-D-ribitylamino-2,4-dihydroxypyrimidine-5-yl)-1-pentyl-p hosphonic acid was solved via molecular replacement using the structure of the Bacillus subtilis enzyme as search model and was refined to a final R-factor of 19.8% (Rfree: 22.5%). The conformation of the active site ligand of the enzyme mimicks that of the Schiff base intermediate of the enzyme-catalyzed reaction. The data enable the reconstruction of the reactant topology during the early steps of the catalytic reaction. Structural determinants, which are likely to be responsible for the inability of the S. cerevisiae enzyme to form icosahedral capsids, will be discussed.

Drosophila melanogaster alcohol dehydrogenase (ADH), a paradigm for gene-enzyme molecular evolution and natural selection studies, presents three main alleloforms (ADHS, ADHF and ADHUF) differing by one or two substitutions that render different biochemical properties to the allelozymes. A three-dimensional molecular model of the three allozymes was built by homology modeling using as a template the available crystal structure of the orthologous D. lebanonensis ADH, which shares a sequence identity of 82.2%. Comparison between D. lebanonensis and D. melanogaster structures showed that there is almost no amino-acid change near the substrate or coenzyme binding sites and that the hydrophobic active site cavity is strictly conserved. Nevertheless, substitutions are not distributed at random in nonconstricted positions, or located in external loops, but they appear clustered mainly in secondary structure elements. From comparisons between D. melanogaster allozymes and with D. simulans, a very closely related species, a model based on changes in the electrostatic potential distribution is presented to explain their differential behavior. The depth of knowledge on Drosophila ADH genetics and kinetics, together with the recently obtained structural information, could provide a better understanding of the mechanisms underlying molecular evolution and population genetics.

The extremely thermostable superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius was crystallized and the three-dimensional structure was determined by X-ray diffraction methods. The enzyme crystallized in the monoclinic spacegroup C2 with the cell dimensions a=168.1 A, b=91.3 A, c=85.7 A, beta=91.4 degrees. The diffraction limit of these crystals was 2.2 A. The crystals were very stable in the X-ray beam and measured diffraction data of a single crystal had a completeness of 99.5 % up to a resolution of 2.2 A. The crystal structure of S. acidocaldarius superoxide dismutase was solved by Patterson search methods using a dimer of Thermus thermophilus superoxide dismutase as a search model. The asymmetric unit accommodates three dimers. Two dimers form a tetramer by using only local symmetries; the third dimer forms a tetramer as well, however, by using the crystallographic 2-fold symmetry. The three-dimensional structure of the S. acidocaldarius dismutase has typical features of tetrameric dismutases. Secondary structure elements as well as residues important for the catalytic activity of the enzyme were found to be highly conserved. The model was refined at a resolution of 2.2 A and yielded a crystallographic R-value of 17.4 % (Rfree=22.3 %). A structural comparison of the two extremely stable tetrameric dismutases from S. acidocaldarius and Aquifex pyrophilus with the less stable enzyme from T. thermophilus and Mycoplasma tuberculosis revealed the structural determinants which are probably responsible for the high intrinsic stability of S. acidocaldarius dismutase. The most obvious factor which may give rise to the extraordinary thermal stability of S. acidocaldarius dismutase (melting temperature of about 125 degreesC) is the increase in intersubunit ion pairs and hydrogen bonds and, more importantly, the significant reduction of solvent-accessible hydrophobic surfaces, as well as an increase in the percentage of buried hydrophobic residues.

The aim of this study was to investigate genetic changes occurring in Ewing Tumors (ETs) and Neuroblastomas (NBs). We analyzed whether the same genetic alteration, i.e. the deletion of genetic material at chromosomal region 1p36, occurs in both tumor types at a comparable rate and whether these aberrations are acquired during tumor progression. Consecutive tumor samples from the same patients at different stages of the disease and topographically different samples from single tumors were studied. Furthermore, we focused on the detection of the status of the MYCN oncogene in NBs. Deletions at 1p36 are found in ETs and NBs at a comparable frequency (approx. 20%) and account for an unfavorable prognosis in localized disease.1,2,3 In ETs, the loss of 1p36 material was frequently observable in a sub-population of cells and is therefore regarded as a later event in the tumor development. In NBs, it seemed more likely that the aberrations found in a subgroup of tumors (i.e. dellp, MYCN amplification, and gain of 17q) are very early on initial events as virtually all tumor cells within an individual tumor were thought to bear the respective genetic aberra- tion. This view is supported by the results obtained so far.4 However, recent data indicate that deletions at 1p36 and also MYCN amplifications can be acquired during tumor progression (Ambros et al. in press), because sub-populations of cells showing these aberrations interspersed between cells not showing these aberrations exist in evolutionary early tumors.

Drosophila alcohol dehydrogenase (DADH; EC 1.1.1.1) is a NAD(H)-dependent oxidoreductase belonging to the short-chain dehydrogenases/reductases (SDR) family. This homodimeric enzyme catalyzes the dehydrogenation of alcohols to their respective ketones or aldehydes in the fruit-fly Drosophila, both for metabolic assimilation and detoxification purposes. The crystal structure of the apo form of DADH, one of the first biochemically characterized member of the SDR family, was solved at 1.9 A resolution by Patterson methods. The initial model was improved by crystallographic refinement accompanied by electron density averaging, R-factor=20.5%, R-free=23.8%.DADH subunits show an alpha/beta single domain structure with a characteristic NAD(H) binding motif (Rossmann fold). The peptide chain of a subunit is folded into a central eight-stranded beta-sheet flanked on each side by three alpha-helices. The dimers have local 2-fold symmetry. Dimer association is dominated by a four-helix bundle motif as well as two C-terminal loops from each subunit, which represent a unique structural feature in SDR enzymes with known structure. Three structural features are characteristic for the active site architecture. (1) A deep cavity which is covered by a flexible loop (33 residues) and the C-terminal tail (11 residues) from the neighboring subunit. The hydrophobic surface of the cavity is likely to increase the specificity of this enzyme towards secondary aliphatic alcohols. (2) The residues of the catalytic triad (Ser138, Tyr151, Lys155) are known to be involved in enzymatic catalysis in the first line. The Tyr151 OH group is involved in an ionic bond with the Lys155 side-chain. Preliminary electrostatic calculations have provided evidence that the active form of Tyr151 is a tyrosinate ion at physiological pH. (3) Three well-ordered water molecules in hydrogen bond distance to side-chains of the catalytic triad may be significant for the proton release steps in DADH catalysis.A ternary structure-based sequence alignment with ten members of the SDR family with known three-dimensional structure has suggested to define a model consisting of four groups of residues, which relates the observed low degree of sequence identity to quite similar folding patterns and nearly identical distributions of residues involved in catalysis.

To determine the current approach to stem cell transplantation (SCT) in centres which treat predominantly paediatric patients, a questionnaire was sent to 67 centres known by the EBMT registry to perform SCT mainly in children. Fifty-five centres from 19 countries responded. Forty centres (75%) started their transplantation activities between 1980 and 1992. Median number of transplants/centre was 95 (range 8-400). Median number of transplants/centre/year was 18 (range 5-85). On average, there was one physician responsible for seven SCT/year while one nurse was involved for a median of 1.7 SCT/year. Median four rooms/centre (range 1-17) were available for paediatric SCT. The most common isolation facilities were rooms with high efficiency particulate air filtration (HEPA). Eighty-two percent (45/55) of the centres performed allogeneic as well as autologous SCT, while 5% (three centres) offered exclusively allogeneic SCT and 13% (seven centres) used only autologous stem cell rescue. Stem cell source for allogeneic SCT was bone marrow in 87%, peripheral blood (PB) in 10% and umbilical cord blood in 3%. Donors were HLA matched related in 57%, mismatched related in 13%, and matched unrelated in 30% of allogeneic SCT. PB was the most commonly used stem cell source for autologous SCT (48%), followed by BM (41%) and the two together (11%). Data analysis revealed substantial differences in protective care, stem cell processing and transplantation procedures within the centres, irrespective of the country, centre size and transplant type.

It has become clear since about a decade ago, that the biosphere contains a variety of microorganisms that can live and grow in extreme environments. Hyperthermophilic microorganisms, present among Archaea and Bacteria, proliferate at temperatures of around 80-100 degrees C. The majority of the genera known to date are of marine origin, however, some of them have been found in continental hot springs and solfataric fields. Metabolic processes and specific biological functions of these organisms are mediated by enzymes and proteins that function optimally under these extreme conditions. We are now only starting to understand the structural, thermodynamic and kinetic basis for function and stability under conditions of high temperature, salt and extremes of pH. Insights gained from the study of such macromolecules help to extend our understanding of protein biochemistry and -biophysics and are becoming increasingly important for the investigation of fundamental problems in structure biology such as protein stability and protein folding. Extreme conditions in the biosphere require either the adaptation of the amino acid sequence of a protein by mutations, the optimization of weak interactions within the protein and at the protein-solvent boundary, the influence of extrinsic factors such as metabolites, cofactors, compatible solutes. Furthermore folding catalysts, known as chaperones, that assist the folding of proteins may be involved or increased protein protein synthesis in order to compensate for destruction by extreme conditions. The comparison of structure and stability of homologous proteins from mesophiles and hyperthermophiles has revealed important determinants of thermal stability of proteins. Rather than being the consequence of one dominant type of interactions or of a general stabilization strategy, it appears that the adaptation to high temperatures reflects a number of subtle interactions, often characteristic for each protein species, that minimize the surface energy and the hydration of apolar surface groups while burying hydrophobic residues and maximizing packing of the core as well as the energy due to charge-charge interactions and hydrogen bonds. In this article, mechanisms of intrinsic stabilization of proteins are reviewed. These mechanisms are found on different levels of structural organization. Among the extrinsic stabilization factors, emphasis is put on archaea chaperonins and their still strongly debated function. It will be shown, that optimization of weak protein-protein and protein-solvent interactions plays a key role in gaining thermostability. The difficulties in correlating suitable optimization criteria with real thermodynamic stability measures are due to experimental difficulties in measuring stabilization energies in large proteins or protein oligomers and will be discussed. Thus small single domain proteins or isolated domains of larger proteins may serve as model systems for large or multidomain proteins which due to the complexity of their thermal unfolding transitions cannot be analyzed by equilibrium thermodynamics. The analysis of the energetics of the thermal unfolding of a small, hyperthermostable DNA binding protein from Sulfolobus has revealed that a high melting temperature is not synonymous with a larger maximum thermodynamic stability. Finally, it is now well documented, that many thermophilic and hyperthermophilic proteins show a statistically increased number of salt bridges and salt bridge networks. However their contribution to thermodynamic and functional stability is still obscure.

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5pH7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities.

1070 myeloablative procedures followed by stem cell rescue for neuroblastoma are reviewed. These 1070 procedures are part of the European Group for Blood and Marrow Transplant (EBMTG) registry from the last 17 years (in 4536 patients). In 1070 neuroblastoma patients, survival at 2 years was 49%, at 5 years, 33% and relapses were observed as late as 7 years post-BMT (bone marrow transplant). However, 5-year survivors after megatherapy with BMT for stage 4 disease do have an 80% chance of becoming a long-term survivor. When BMT had been used in first complete response (CR1) no salvage was possible, whereas 15% survivors may be seen if BMT is used for the first time at relapse. Infants with stage 4 neuroblastoma had a 17% toxic death rate and indication in this group is exceptional and not recommended. In a matched cohort (17 allogeneic and 34 autologous), autologous stem cell rescue (SCR) was shown to be at least equal to allogeneic SCR. Multivariate analysis of clinical prognostic factors in children with stage 4 disease over 1 year showed that event-free survival was mainly influenced by two adverse factors before the megatherapy procedure: persisting skeleton lesions (99Tc and/or mIBG scan positive) as well as persisting bone marrow (BM) involvement.

The cross vectors of the native Patterson map are shown to exhibit non-crystallographic symmetry in the case of local axes parallel to one another. This information can be used to determine the translation component of such axes. A program is described to search for this cross vector, and is tested on low-resolution data from crystals of the tetradecameric GroEL molecule, the decameric GTP cyclohydrolase I and the tetradecameric proteosome. For GroEL, the function produces a packing arrangement optimal for sevenfold symmetry, and is in agreement with the dimensions of the molecule as given by electron microscopy data and the recently determined crystal structure. Positioning of local axes is confirmed by two high-resolution crystal structure analyses: the fivefold axis in cyclohydrolase I and the sevenfold axis in the proteosome. Implications for the location of heavy-atom positions are discussed for these two cases.

The European BMT Solid Tumour Registry (EBMT-STR) received reports from 21 European transplant centers on 63 patients (50 Ewing's sarcomas and 13 peripheral neuroectodermal tumours) in first (n = 32) or second CR (n = 31) consolidated with megatherapy and BM and/or PSC rescue between December 1982 and November 1992. There were 31 males and 32 females with a median age of 12 years (range 1-30 years) at megatherapy. The median follow-up time since megatherapy is 4 years (range 1 month to 10 years), Thirty-two patients with metastatic disease at diagnosis (22 had metastases to the bone and/or bone marrow) and consolidated in CR1 reached an actuarial event-free survival (EFS) of 21% at 5 years. Thirty one patients in CR2 achieved an actuarial EFS of 32% at 5 years. Favourable outcome was limited to relapse patients with localised disease at initial diagnosis. Distant relapse had a more favourable prognosis than local failure. Analysis of the different megatherapy strategies could not identify a significantly superior approach, nor is there convincing evidence in favour of double graft procedures. From the above results it appears that consolidation treatment by megatherapy contributes to improved EFS rates in high-risk patients compared with historical experience. Major questions for the future to be addressed prior to randomised studies include agreement on the definition of high-risk patients and the most efficient megatherapy procedure.

The purification and characterization of a new type of thermostable chaperonin from the archaebacterium Sulfolobus solfataricus is described. The chaperonin forms a hetero-oligomeric complex of two different, but closely related, subunits, which we have assigned TF55-alpha and TF55-beta. Their N-terminal sequences and amino acid residue compositions are reported. Two-dimensional projections of the chaperonin have been reconstructed from electron microscopy images, showing a 9-fold symmetrical complex, about 17.5 nm in height and 16 nm in diameter, with a central cavity of 4.5 nm. The complex is resistant to denaturing agents at room temperature and only pH values lower than 2 lead to dissociation. The separated subunits do not reassemble spontaneously but require Mg2+ and ATP for complex formation. Both subunits are necessary for formation of the TF55 oligomer. Significant structural changes have been observed after phosphorylation, thus providing evidence for a structural mobility during the chaperonin-assisted folding process of a protein. The phosphorylation reaction is modulated by potassium and magnesium ions. Magnesium seems to have an inhibitory effect, whereas potassium enhances this reaction.

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.

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.

Glutathione peroxidase showed an X-ray photoelectron spectroscopy signal of the Se 3d (3/2, 5/2) electrons at 54.4 eV. After the addition of the acceptor substrate H2O2, a marked shift of this signal to a value of 58.0 eV was observed. Upon subsequent treatment with the donor substrate glutathione, this chemical shift was reversed and the original signal was obtained. These data demonstrate that the enzyme-bound selenium moiety participates in the catalytic process. From the chemical shift obtained it is concluded that the enzyme shuttles between a selenol or selenol derivative in its reduced form and a seleninyl or selenonyl compound in its oxidized form.

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.

A method was developed to purify glutathione transferase from bovine placenta by affinity chromatography and fast protein liquid chromatography. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. The dimeric enzyme is composed of identical subunits with a molecular weight of 23,000; its isoelectric point is 6.9. In contrast to previously described isoenzymes of glutathione transferase, the protein we have purified exists in two forms, an active reduced form and a less active oxidized form. These can be reversibly transformed into each other but behave differently in sedimentation analysis, gel chromatography, and gel electrophoresis. These differences may reflect a change in the molecular shape of glutathione transferase. Chemical modification with iodoacetate, iodoacetamide (presumably of thiol groups), phenylglyoxal, and butadione (presumably of arginyl groups), and their inhibitory effects on the activity were investigated. From substrate specificity studies and N-terminal sequence analysis it is obvious that this glutathione transferase must belong to the isoenzyme class pi. The purified enzyme could be crystallized from 1.4 M ammonium sulfate solution, pH 8.0, in the presence of S-hexyl-glutathione. The crystals are tetragonal, with space group P4(1)2(1)2 or P4(3)2(1)2. The cell constants are a = b = 6.1 nm, c = 23.7 nm, alpha = beta = gamma = 90 degrees. The crystals diffract to 0.26-nm resolution and are suitable for x-ray structure analysis.

Geometric features as well as possible functional properties of the substrate binding sites at the pentamer interfaces are described. Ligand binding at the pentamer interface regions increases the stability of the beta 60 capsid considerably and influences the reassembly of isolated beta-subunits.

Heavy riboflavin synthase from Bacillus subtilis is an enzyme complex consisting of approximately three alpha-subunits (Mr 23.5 X 10(3)) and 60 beta-subunits (Mr 16 X 10(3)). The enzyme has been crystallized from phosphate buffer in a hexagonal crystal modification that belongs to space group P6(3)22. The asymmetric unit of the crystal cell contains ten beta-subunits. The structure of this unusual 10(6) Mr protein has been studied by small-angle X-ray scattering, electron microscopy of three-dimensional crystals, and crystallographic methods. The scattering curves can be interpreted in terms of a hollow sphere model with a ratio of inner and outer radius of 0.3:1. A diameter of 168 A was estimated from the scattering curves, in close agreement with electron microscopic studies. An aggregate with the stoichiometry beta 60, which was obtained by ligand-driven reaggregation of isolated beta-subunits, showed similar shape and dimensions, but a larger value for the ratio Ri/Ra. Electron micrographs of freeze-etched enzyme crystals showed approximately spherical molecules, which were arranged in hexagonal layers. The lattice constants found from the micrographs are in good agreement with the values derived from X-ray diffraction data. Rotation function calculations in Patterson space showed a set of peaks for 2-fold, 3-fold and 5-fold local rotation axes, accurately consistent with icosahedral symmetry and with the particle orientation A shown in the Appendix. The crystal packing can be described as follows: enzyme particles with icosahedral symmetry (point group 532) are located at points 32 of the hexagonal cell, corresponding to positions (0, 0, 0) and (0, 0, 1/2) on the 6-fold screw axes. From the data reported, it may be concluded that the enzyme structure can be described as an icosahedral capsid of 60 beta-subunits with the triangulation number T = 1. The alpha-subunits are located in the central core space of the capsid, but their spatial orientation is incompletely understood.