[Requirements and performance profile of the Paediatric Radiation Oncology Working Group (APRO): evaluation of the present situation and description of future developments]. / Anforderungen und Leistungsprofile der Arbeitsgemeinschaft Pädiatrische Radioonkologie (APRO): Eine Bestandsaufnahme und Darstellung zukünftiger Entwicklungen.

BACKGROUND: Radiation therapy is an integral component in the management of childhood malignancies and undergoes a continuous process of optimization within the prospective trials of the GPOH. At present there are approximately 20 active protocols, some specifying radio-oncological study questions, in which about 500 to 600 children annually are given radiotherapy. MATERIALS/METHODS: The Pediatric Radiation Oncology Working Group (APRO) of the German Society for Radiation Oncology (DEGRO) represents the organizational link between GPOH and DEGRO. Their activities range from phrasing guidelines of radio-oncological therapy, through writing a protocol for a prospective study on radiation-induced late effects (RISK--in co-operation with GPOH, 695 patients registered so far) and organizing meetings for information transfer, to implementing radio-oncology within the prospective studies of the GPOH by establishing study chairs for radio-oncology when radio-oncological questions are a primary focus and/or to function as a reference institution for quality assurance. These activities also include individual case consultations outside the study proper. Twice annually the members of the APRO meet for an update on current knowledge and future directions where a representative of the GPOH is invited to contribute special aspects of pediatric oncology. CONCLUSIONS: In the future, modern technology (intensity modulated radiotherapy, proton therapy, inclusion of imaging in treatment planning) will be part of disease management in pediatric oncology. A working group for modern radiotherapy technology was established to enhance this development. Prospective studies of the GPOH with primary or secondary radio-oncological questions require the implementation of corresponding tasks (documentation, monitoring, etc.) in order to meet future demands on clinical trials and to achieve the aims of the protocol. Consequently adequate financial support is indispensable.

Results of treatment for soft tissue sarcoma in childhood and adolescence: a final report of the German Cooperative Soft Tissue Sarcoma Study CWS-86.

PURPOSE: The goal of the second German Soft Tissue Sarcoma Study CWS-86 (1985 to 1990) was to improve the prognosis in children and adolescents with soft tissue sarcoma by means of a clinical trial comprising intensive chemotherapy and risk-adapted local therapy. PATIENTS AND METHODS: There were 372 eligible patients. A staging system based on the postsurgical extent of disease was used. Chemotherapy consisted of vincristine, dactinomycin, doxorubicin, and ifosfamide. Radiotherapy was administered early at 10 to 13 weeks simultaneously with the second chemotherapy cycle (32 Gy or 54. 4 Gy). The single dose was reduced to 1.6 Gy and given twice daily (accelerated hyperfractionation). RESULTS: The event-free survival (EFS) and overall survival rates at 5 years were 59% +/- 3% and 69% +/- 3%, respectively. The 5-year EFS rate according to stage was as follows: stage I, 83% +/- 5%; stage II, 69% +/- 6%; stage III, 57% +/- 4%; and stage IV, 19% +/- 6%. The outcome for patients with stage III disease who required radiotherapy was much better in the CWS-86 study compared with the CWS-81 study (5-year EFS, 60% +/- 5% v 44% +/- 6%; P =.053). The most common treatment failure was isolated local relapse, with 14% of patients relapsing at the primary tumor site. CONCLUSION: The improved design of the study incorporating risk-adapted radiotherapy allowed treatment to be reduced for selected groups of patients without compromising survival.

[Radiation therapy in juvenile aggressive fibromatosis]. / Strahlentherapie bei juvenilen aggressiven Fibromatosen.

BACKGROUND: In about one third of patients suffering from a desmoid tumor primary complete resection is not feasible. Furthermore in locally relapsing tumors reoperation alone does not result in cure in many cases. Radiotherapy can be applied in both groups of patients with curative intention. But the indication of radiotherapy is challenging particularly in children and adolescents due to the impending late radiation sequelae such as growth delay, fibrosis and radiation induced secondary malignancy. PATIENTS AND METHOD: The follow up and outcome of five irradiated children/adolescents with desmoid tumors, registered in the German-Cooperative-Soft-Tissue-Sarcoma Study (CWS) was looked at, and the corresponding literature was reviewed. RESULTS: Radiotherapy of gross residual or relapsing tumors resulted in long lasting event free survival in two cases (3/8 years), but in one patient local progression occurred despite irradiation. Postoperative radiotherapy in patients with microscopic residual disease resulted in both, long lasting event free survival (14 years, 1 patient) and in early local relapse (1.5 years, 1 patient). The role of radiotherapy could not be evaluated clearly by the CWS-experience due to the fact that the irradiated patients were treated individually also by chemotherapy and/or tamoxifen. But despite sparse and retrospective data there is evidence in the literature, that radiotherapy is able to control 65-90% of the unresectable desmoid tumors and that the local relapse rate can be reduced by radiotherapy by 10-20% in patients with microscopic residual disease following resection. CONCLUSIONS: Radiotherapy as primary treatment should be given if complete tumor resection is not feasible without mutilation. Radiotherapy can be applied postoperatively if the risk of local relapse seems to be highly life- or function threatening.

Heterologous expression of human granzyme K in Bacillus subtilis and characterization of its hydrolytic activity in vitro.

Human granzyme K, a serine protease found in secretory granules of cytotoxic T-lymphocytes, was produced in its catalytically active form by recombinant technology using Bacillus subtilis as host. The enzyme displays 40-45% identity to other members of the human granzyme group, and its closest homologue (75% identity) is the rat tryptase RNK-tryp2. The recombinant protein can be recovered in its mature form from the bacterial culture supernatant and purified by cation exchange chromatography. Initial characterization reveals a protein of approximately 28 kDa that is specifically labelled by [3H]di-isopropyl fluorophosphate. Measurements of Kcat/K(m) for single-residue thioester substrates show approximately a two-fold preference for a Lys versus Arg residue at Pl. No activity was observed on ester substrates with various other residues at the Pl position. Using oligopeptide substrates, the enzyme displays peptidolytic activity C-terminal to both Lys and Arg residues with comparable rates of hydrolysis. Likewise, substrate hydrolysis is blocked most efficiently by inhibitors that contain Lys or Arg at position Pl. The availability of the cloned enzyme will facilitate the analysis of biological roles for this novel granzyme, and differentiate its activity from that of other granzymes.

Crystal structure of phenylmethanesulfonyl fluoride-treated human chymase at 1.9 A.

The X-ray crystal structure of human chymase has been determined to 1.9 A resolution using molecular replacement methods. This first structure of human chymase is present as the Ser 195 ester of alpha-toluenesulfonic acid. The refined structure (Rcryst = 0.183) shows that the inhibitor phenyl moiety lies at the top of the major specificity pocket, S1, while the sulfur is covalently linked to Ser 195-O gamma. The sulfonyl oxygens interact with the oxyanion hole and with His 57-N delta 1. The presence of the inhibitor disturbs the usual gauche position of His 57 and forces it to the trans conformer. Though the primary binding pockets are similarly specific in chymase and chymotrypsin, examination of the extended substrate binding sites reveals the structural basis for chymase's greater discrimination in choosing substrates. The larger 30s loop and its proximity to the active site indicates that it contacts substrate residues C-terminal to the scissile bond. Modeling of substrate at the chymase active site suggests that binding energy may be gained by three main-chain hydrogen bonds provided by substrate residues P2' and P4' and that discriminating interactions with substrate side chains are also likely. The presence of Lys 40 in S1' of human chymase explains its preference for Asp/Glu at P1'. Moreover, the cationic nature of S1' provides a structural basis for human chymase's poor catalytic efficiency when angiotensin II is the substrate.

Production of crystallizable human chymase from a Bacillus subtilis system.

A Bacillus subtilis strain deficient in seven extracellular proteases was used to produce human mast cell chymase and is a viable expression system for serine proteases and other classes of proteins. Chymase is produced at 0.3-0.5 mg/l and is purified by three chromatography steps. Two crystal forms of PMSF-treated chymase were optimized. The first is C2 with a=47.94 A, b=85.23 A, c=174.18 A, beta=96.74 degrees, and diffracts to at least 2.1 A, while the second is P212121, with cell dimensions a=43.93 A, b=58.16 A, and c=86.09 A, and a diffraction limit of approximately 1.9 A. The first crystal form has either three or four molecules/asymmetric unit, while the second has one molecule/asymmetric unit.

Signal amplification in the detection of single-copy DNA and RNA by enzyme-catalyzed deposition (CARD) of the novel fluorescent reporter substrate Cy3.29-tyramide.

We demonstrate that the CAtalyzed Reporter Deposition method (CARD), utilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the Fluorescent in Situ Hybridization (FISH) detection of RNA and single-copy DNA. Histone 4 expression is detected in RNA extracts of 5-phase, synchronized HeLa cells by dot-blot analysis. Gene expression of histone 4 in HeLa cells is demonstrated by FISH via CARD, utilizing oligonucleotide probes. Fluorescence intensity measurements on CARD-amplified histone 4 RNA detection showed (a) a 25-fold amplification of the signal brightness by biotinylated oligonucleotide probes and (b) a sixfold amplification of the signal brightness by horseradish peroxidase (HRP)-labeled histone 4 probes vs the directly stained control. The sensitivity of the CARD method is demonstrated by the FISH detection of single-copy DNA on human corneal fibroblast and HeLa S5 interphase nuclei. Chromosomal localization of the single copy DNA is demonstrated on HeLa S3 metaphase chromosome spreads.

Specificity of DNA alkylation by 1-(2-chloroethyl)-3-alkyl-3-acyltriazenes depends on the structure of the acyl group: kinetic and product studies.

The reactions of calf thymus DNA with ten 1-(2-chloroethyl)-3-alkyl-3-acyltriazenes of varying acyl side chain structure were studied alone, or in the presence of porcine liver esterase in pH 7.0 phosphate buffer. In several of the key triazenes, the acyl substituent contained a free carboxylic acid group. With esterase present in the reaction mixture, the resultant levels of DNA alkylation could be correlated with the kinetic rates of decomposition of the triazenes. Under these conditions, the predominant pathway of decomposition involved deacylation of the parent triazene and eventual production of an alkanediazonium ion. This intermediate subsequently alkylated DNA--guanine to give 7-alkylguanine as the principal reaction product. In the absence of esterase, the order of DNA alkylation for all of the acyltriazenes did not correlate with their respective rates of decomposition, leading to the conclusion that the triazenes did not decompose by the expected mode of uncatalyzed N(2)-N(3) heterolyic cleavage. The major DNA alkylation product from the N(3)-methyltriazenes was 7-methylguanine, instead of the expected 7-(chloroethyl)- and 7-(hydroxyethyl)guanine products, which suggested that the acyl group was being hydrolyzed. However, acyltriazenes with an N(3)-benzyl group rather than a methyl in this position produced very little 7-benzylguanine product, contrary to prediction. An alternative mechanism involving internally assisted hydrolysis of the side chain ester is proposed to explain these results. NMR product analysis and computational studies were carried out to lend support to the postulated mechanism.

Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD).

Progress has been made in improving the immunohistochemical detection of antigens for imaging and flow cytometry. We report the synthesis of a novel fluorescent horseradish peroxidase substrate, Cy3.29-tyramide, and its application in an enzyme-based signal amplification system, catalyzed reporter deposition (CARD). The catalyzed deposition of Cy3.29-tyramide was used to detect cell surface markers such as CD8 and CD25 on tonsil tissue and human lymphocytes. We compared the fluorescence CARD method to standard indirect immunofluorescence detection methods and found that an amplification of up to 15-fold was possible with CARD. The detection of the intracellular protein myosin II in fibroblastic cells and rabbit serum proteins blotted onto nitrocellulose was also improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance significantly the detection of antigens.

Alkalophilic Bacillus sp. strain LG12 has a series of serine protease genes.

Four tandem subtilisin-like protease genes were found on a 6,854-bp DNA fragment cloned from the alkalophilic Bacillus sp. strain LG12. The two downstream genes (sprC and sprD) appear to be transcribed independently, while the two upstream genes (sprA and sprB) seem to be part of the same transcript.

Cloning of cDNA for human granzyme 3.

A serine protease gene was cloned from cDNA prepared from tissue isolated from human ascites. The gene codes for a protein of 264 amino acids, identified as human granzyme 3 by the N-terminal amino acid sequence. Granzyme 3 has the expected features of the chymotrypsin family of serine proteases, and is closely related to other human granzymes (40-45% identity). However, the closest granzyme 3 homologue is the recently characterized rat tryptase, RNK-Tryp-2 (75% identity). From Northern blots, granzyme 3 appears to be highly expressed in peripheral blood leukocytes, spleen, thymus, and lung tissues.

Peptide-linked 1,3-dialkyl-3-acyltriazenes: gastrin receptor directed antineoplastic alkylating agents.

The gastrin receptor is expressed in various human cancers, such as the adenocarcinoma of the colon. The peptide hormone gastrin and the C-terminal peptides derived from it act as growth factors for these cancers. The hypothesis for the present work was to use the gastrin receptor as a target for appropriately constructed cytotoxic agents. We developed methods to link tetragastrin and pentagastrin by their N-termini to cytotoxic 1-(2-chloroethyl)-3-benzyl-3-succinoyltriazene. These compounds, CBS-4 and CBS-5, respectively, whose complete structures were determined by multinuclear NMR and mass spectrometry, competed effectively with gastrin in an assay using either guinea pig stomach fundus or the rat acinar tumor cell line AR42J as the source of the receptor. CBS-5 was cytotoxic to AR42J cells but was not toxic to A549 human lung cancer cells, which do not express the receptor.

Two histidines are essential for the activity of the beta-galactosidase from Lactobacillus delbrückii subsp. bulgaricus.

Twelve tyrosines, ten glutamates, and five histidines were individually substituted for phenylalanine, glutamine, and asparagine, respectively, in the beta-galactosidase from Lactobacillus delbrückii subsp. bulgaricus. Only Y509, E464, H351, and H534 appear to be essential for the activity of the enzyme. The residues Y509 and E464 are homologous to Y503 and E461, respectively, of the Escherichia coli lacZ beta-galactosidase which have been shown to be necessary for its activity. Surprisingly, a number of amino acids that are highly conserved in the sequence alignments of nine beta-galactosidases and four beta-glucuronidases are not essential for enzyme activity.

Identification of two aspartates and a glutamate essential for the activity of endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus.

In order to identify groups essential for the activity of endo-beta-N-acetylglucosaminidase H (Endo H), all 8 glutamate residues, all 19 aspartates, and both tryptophans were individually substituted with glutamines, asparagines, and phenylalanines, respectively, by oligonucleotide site-directed mutagenesis. Only variants D170N, D172N, and E174Q were found to have specific activities significantly less than wild-type Endo H. Another variant, D173N, did not produce detectable amounts of protein. Wild-type enzyme was found to have a bell-shaped pH activity profile, which was retained in the essential aspartate mutants, but E174Q lost the basic pH limb of the curve, indicating that E174 is good candidate for the proton donating group necessary for catalysis. The general base needed for activity could not be unambiguously identified; although, of the essential aspartates, D172 is the only one conserved in other related glucosidases.

Generation and Characterization of Environmentally Sensitive Variants of the beta-Galactosidase from Lactobacillus delbrueckii subsp. bulgaricus.

A method is described for generating and screening variants of the beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus sensitive to several environmental stresses, with potential application in the food industry. Chemical mutagenesis with hydroxylamine or methoxylamine was performed on the beta-galactosidase gene carried on an Escherichia coli expression vector. Mutants sensitive to cold, heat, low pH, low magnesium concentration, and the presence of urea were isolated by screening for reduced color development on beta-galactosidase indicator plates. The mutations responsible for three variant beta-galactosidases were localized, and the base substitutions were determined by DNA sequencing. The amino acid alterations associated with one low-pH-sensitive (pHs) and two urea-sensitive (Us) variants correspond to P584L (pHs1), G400S/R479Q (Us26), and G167E/E168K/E363K/V492M (Us17), respectively. Mutant pHs1 is also heat, cold, low magnesium, and urea sensitive; Us26 is also cold sensitive; and Us17 is also low-pH sensitive.

Characterization of two cold-sensitive mutants of the beta-galactosidase from Lactobacillus delbruckii subsp. bulgaricus.

Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobacillus delbrückii subsp. bulgaricus was used to generate cold-sensitive variants. Two variants, P429S and L317F, were characterized kinetically in order to determine the enzymatic consequences of these mutations. The kinetic parameters Km and Vmax on the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside have been determined over a temperature range of 11-45 degrees C. Only the Vmax of the two variants was significantly different than the wild-type enzyme over the temperature range studied. The Vmax of the L317F variant is reduced proportionately at all temperatures compared to the wild-type enzyme while the value of Vmax for the P429S mutant deviates from wild-type only at lower temperatures (in 2 mM Mg2+). This temperature-dependent effect on the Vmax of P429S can be suppressed by increasing the Mg2+ concentration. The results suggest that the binding of this essential metal ion is altered in the P429S variant such that its dissociation is increased by lowering the temperature.

[The value of NMR tomography in the planning of HDR-afterloading brachytherapy in cervical carcinomas. The experiences with 41 patients]. / Wert der Kernspintomographie zur Planung der HDR-Afterloading-Brachytherapie bei Zervixkarzinomen. Erfahrungen bei 41 Patientinnen.

In 41 patients with inoperable cervical carcinomas, afterloading brachytherapy was used in combination with radiotherapy after planning with MRI (1.5 T spin echo multislice technique). In 27 women, phantoms neutral to MRI were introduced into the uterus to create a topographic situation comparable to that during brachytherapy; at the same time, the position of the applicators was marked. Under the circumstances, MRI demonstrated the position of the uterus and the tumour, the vagina, the bladder, the rectosigmoid and the applicator phantoms in every case. In 88% it was possible to demonstrate the margin of the tumour and normal myometrium. In 18% of the patients, the technique lead to a significant and, in a further 30%, to a marginal improvement in brachytherapy technique.