In Berlin wurde 2003 die politische Entscheidung zur Einführung der Soziakaumorientierung in der Jugendhilfe getroffen. „Optimierung der Entscheidungsprozesse, der Organisation und der Finanzierung der Berliner Jugendhilfe“ waren die mit der Einführung verbundenen Ziele. Sie wurden durch ein Programm von Unterstützungs-und Qualifizierungsmodulen flankiert.

Wiskott-Aldrich syndrome (WAS) predisposes patients to leukemia and lymphoma. WAS is caused by mutations in the protein WASP which impair its interaction with the WIPF1 protein. Here, we aim to identify a module of WIPF1-coexpressed genes and to assess its use as a prognostic signature for colorectal cancer, glioma, and breast cancer patients. Two public colorectal cancer microarray data sets were used for discovery and validation of the WIPF1 co-expression module. Based on expression of the WIPF1 signature, we classified more than 400 additional tumors with microarray data from our own experiments or from publicly available data sets according to their WIPF1 signature expression. This allowed us to separate patient populations for colorectal cancers, breast cancers, and gliomas for which clinical characteristics like survival times and times to relapse were analyzed. Groups of colorectal cancer, breast cancer, and glioma patients with low expression of the WIPF1 co-expression module generally had a favorable prognosis. In addition, the majority of WIPF1 signature genes are individually correlated with disease outcome in different studies. Literature gene network analysis revealed that among WIPF1 co-expressed genes known direct transcriptional targets of c-myc, ESR1 and p53 are enriched. The mean expression profile of WIPF1 signature genes is correlated with the profile of a proliferation signature. The WIPF1 signature is the first microarray-based prognostic expression signature primarily developed for colorectal cancer that is instrumental in other tumor types: low expression of the WIPF1 module is associated with better prognosis.

The six-coordinate dihydridoosmium(IV) complex [OsH2Cl2(PiPr(3))(2)] (1) reacted with the hemilabile chelating phosphanes iPr(2)PCH(2)CH(2)OMe and iPr(2)PCH(2)CO(2)R (R = Me, Et) at room temperature to give the substitution products [OsH2Cl2(iPr(2)PCH(2)CH(2)OMe)(2)] (2) and [OsH2Cl2{kappa(2)(P,O)- iPr(2)PCH(2)C(=O)OR}{kappa(P)-iPr(2)PCH(2)CO(2)R}] (3,4) in good to excellent yields. Treatment of 1 with iPr(2)PCH(2)CH(2)NMe(2) led to the displacement of only one PiPr(3) ligand and gave [OsH2Cl2(PiPr(3))(iPr(2)PCH(2)CH(2)NMe(2))] (5). The reaction of 1 with iPr(2)PCH(2)CH(2)OMe and iPr(2)PCH(2)CO(2)R (R = Me, Et) at elevated temperature afforded the osmium(11) complexes [OsCl2{kappa(2) (P, O)-iPr(2)PCH(2)CH(2)OMe}(2)] (6) and [OsCl2{kappa(2)(P,O)- iPr(2)PCH(2)C(=O)OR}(2)] (7, 8), which were also obtained on heating the osmium(IV) precursor 3 or the labile 1: 1 adducts of 3 and 4 with ethene in benzene under reflux. The dihydrido compounds 2 and 3 reacted with CO at room temperature to give initially the octahedral all-cis-configured complexes [OsCl2(CO)(2)(kappa(P)-iPr(2)PCH(2)CH(2)OMe}(2)] (11a) and [OsCl2(CO)(2)(kappa(P)-iPr(2)PCH(2)CO(2)Me}(2)] (12a), which rearrange to the more stable cis,cis,trans isomers 11b and 12b in benzene under reflux. The all-trans isomer 11c and the monocarbonyl complexes trans, trans- [OsCl2(CO){kappa(2)(P,O)-iPr(2)PCH(2)C(=O)OR}{kappa(P)-iPr(2)PCH(2)CO(2)R}] (13, 14) were prepared by passing CO through a solution of 6-8 in benzene or toluene/dichloromethane at room temperature. While the osmium(IV) compounds 3, 4 as well as the osmium(II) complexes 7, 8 reacted with CNtBu to give the neutral complexes cis,cis,trans- [OsCl2(CNtBu)(2)(kappa(P)-iPr(2)PCH(2)CO(2)R}(2)] (15, 16), treatment of 5 with CNtBu afforded the ionic product [OsHCl(CNtBu)(2)(PiPr(3))(iPr(2)PCH(2)CH(2)NHMe(2))]Cl (17). The reaction of 7 with Ph2CN2 did not lead to the formation of a (carbene)osmium(II) compound but gave the dinitrogen complex trans, trans- [OsCl2(N-2) {kappa(2)(P,O)-iPr(2)PCH(2)C(=O)OMe}(kappa(P)-iPr(2)PCH(2)CO(2)Me)] (18) instead. The structurally related vinylidene complexes trans, trans- [OsCl2(=C=CHPh) {kappa(2)(P, O)- iPr(2)PCH(2)CH(2)OMe}{kappa(P)-iPr(2)PCH(2)CH(2)OMe}] (19a) and trans, trans- [OsCl2(=C=CHPh) {kappa(2) (P, O)-iPr(2)PCH(2)C(=O)OR){kappa(P)-iPr(2)PCH(2)CO(2)R}] (20, 21) were prepared from 6-8 and PhC=CH as starting materials.The cis, cis-configured compound [OsCl2(=C=CHPh)(PiPr(3)){kappa(2) (P,N)-iPr(2)PCH(2)CH(2)NMe(2)}] (22) was obtained analogously from 5 and phenylacetylene. (c) Wiley-VCH Verlag GmbH & Co.

Colorectal tumors have characteristic genome-wide expression patterns that allow their distinction from normal colon epithelia and facilitate clinical prognosis. The expression heterogeneity within a primary colorectal tumor has not been studied on a genome scale yet. Here we investigated three compartments of colorectal tumors, the invasion front, the inner tumor mass, and surrounding normal epithelial tissue by microdissection and microarray-based expression profiling. In both tumor compartments many genes were differentially expressed when compared to normal epithelium. The sets of significantly deregulated genes in both compartments overlapped to a large extent and revealed various interesting known and novel pathways that could have contributed to tumorigenesis. Cells from the invasion front and inner tumor mass, however, did not show significant differences in their expression profile, neither on the single gene level nor on the pathway level. Instead, gene expression differences between individuals are more pronounced as all patient-matched tumor samples clustered in close proximity to each other. With respect to invasion front and inner tumor mass we conclude that the specific tumor cell micro-environment does not have a strong influence on expression patterns: largely similar genome-wide expression programs operate in the invasion front and interior compartment of a colorectal tumor.

BACKGROUND:Cancer development is accompanied by genetic phenomena like deletion and amplification of chromosome parts or alterations of chromatin structure. It is expected that these mechanisms have a strong effect on regional gene expression.RESULTS:We investigated genome-wide gene expression in colorectal carcinoma (CRC) and normal epithelial tissues from 25 patients using oligonucleotide arrays. This allowed us to identify 81 distinct chromosomal islands with aberrant gene expression. Of these, 38 islands show a gain in expression and 43 a loss of expression. In total, 7.892 genes (25.3% of all human genes) are located in aberrantly expressed islands. Many chromosomal regions that are linked to hereditary colorectal cancer show deregulated expression. Also, many known tumor genes localize to chromosomal islands of misregulated expression in CRC.CONCLUSION:An extensive comparison with published CGH data suggests that chromosomal regions known for frequent deletions in colon cancer tend to show reduced expression. In contrast, regions that are often amplified in colorectal tumors exhibit heterogeneous expression patterns: even show a decrease of mRNA expression. Because for several islands of deregulated expression chromosomal aberrations have never been observed, we speculate that additional mechanisms (like abnormal states of regional chromatin) also have a substantial impact on the formation of co-expression islands in colorectal carcinoma.

Differential gene expression as a result of molocular alterations in cancer play an important role for defining new diagnostic biomarkers and treatment targets. Chiparray technology enabling expression analysis of thousands of genes simultaneously has already been successfully applied for the identification of differentially expressed genes (DEGs) in several tumor entities. The aim of this study was to identify DEGs in colorectal cancer by comparing chiparray-based expressiondata from normal and corresponding cancerous epithelium after laser microdissection. We used Affymetrix GeneChips to monitor the gene expression of about 33.000 known genes in 25 patients with colorectal cancer. By generating a rank for all detected genes based on statistical tests 2352 genes and expressed sequence tags (ESTs) were identified to be differentially expressed. Besides several DEGs which were already known to be associated with colorectal cancer or other tumor entities, genes like carbonic anhydrase IV, fatty acid binding protein 1 and hevin were found to be differentially downregulated whereas genes like melanoma growth stimulating activity (Onkogen), S-adenosylcystein-hydrolase, general transcription factor IIIa and transforming growth factor showed a significant upregulated expression in tumor. Validation of selected genes on RNA and protein-level showed good reproducibility. Whether the identified DEGs and ESTs presented in this study encode new potential tumor markers or potential novel therapeutic targets in colorectal cancer has to be further evaluated.

Trotz etablierter Klassifikationen (TNM/UICC) ist die Prognosevorhersage für den individuellen Patienten mit einem kolorektalen Karzinom bislang nur unzuverlässig möglich. Genexpressionsprofilanalysen auf mRNA-Ebene ermöglichen, wie für andere Tumorentitäten (z. B. Mamma- und Ösophagus-Ca.) gezeigt, eine über die UICC-Stadien hinaus gehende Aussage über die Prognose. Ziel dieser Studie ist die Identifikation von charakteristischen Genexpressionsprofilen für Patienten unterschiedlicher UICC-Stadien (I–IV), insbesondere Patienten ohne versus mit Lymphknotenbefall (N0 vs. N +) und Patienten ohne versus mit Fernmetastasen (M0 vs. M +). Um der inhomogenen Genexpression innerhalb des Tumors Rechnung zu tragen, sollen definierte Tumorareale, die Invasionsfront und zentrale Tumoranteile, durch Mikrodissektion getrennt werden.

Sanfilippo syndrome (mucopolysaccharidosis type III, MPS III) is a progressive disorder in which patients are characterized by severe central nervous system degeneration together with mild somatic disease. MPS III results from a deficiency in one of the four enzymes involved in the degradation of heparan sulfate, with sulfamidase (SGSH) being deficient in MPS IIIA and a-N-acetylglucosaminidase (NAGLU) deficient in MPS IIIB. Mutation screening using SSCP/heteroduplex analysis on genomic DNA fragments was performed in five Turkish MPS IIIA and eight Turkish MPS IIIB patients. In this study two mutations of SGSH were identified in MPS IIIA patients: R74C and the novel mutation P288S, and one polymorphism (IVS1+23 C>G). Five different mutations of NAGLU were identified in MPS IIIB patients: L682R, H248R, E153K, g.17703 A>G (novel), and T437I (novel). The clinical data of all patients are reported in detail. A high degree of genetic heterogeneity was observed in the Turkish MPS IIIA and MPS IIIB patients.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of heparan-N-sulfamidase (NS; EC 3.10.1.1), resulting in defective degradation and subsequent storage of heparan sulfate and leading to a clinical phenotype known as Sanfilippo syndrome. A sensitive and specific monoclonal/polyclonal-based immunoquantification assay has enabled the determination of NS protein, down to ∼3 pg NS protein, in cultured fibroblasts from control and MPS-IIIA patients. Cultured skin fibroblasts from 15 normal controls contained 11.9 to 105 ng of NS protein/mg extracted cell protein, whereas NS protein ranged from “none detected” to 11 ng/mg in fibroblasts from 35 MPS-IIIA patients. A relationship between genotype/phenotype and amount of NS protein present in these MPS-IIIA fibroblasts was established. Immunoquantification, in combination with a specific and highly sensitive tetrasaccharide-based assay of NS activity, enabled the determination of residual specific NS activity in these fibroblasts. Specific NS activity ranged from 28 to 1289 nmol/min/mg NS protein for MPS-IIIA patients, compared to 870 nmol/min/mg of recombinant human NS. It is proposed that this immunoquantification method, in conjunction with the specific NS activity assay, may be used to predict clinical severity in MPS-IIIA patients, allowing for the selection of individuals best suited for gene- and enzyme-replacement therapy when these methods become available. Also proposed is that an enzyme-replacement therapy achieving a correction of approximately 10% of normal NS activity is required to avoid the onset of a Sanfilippo clinical phenotype.

Mucopolysaccharidosis type IIIB (MPS-IIIB, Sanfilippo type B Syndrome) is a heterosomal, recessive lysosomal storage disorder resulting from a deficiency of [alpha]-N-acetylglucosaminidase (NAGLU). To characterize this enzyme further and evaluate its potential for enzyme replacement studies we expressed the NAGLU-encoding cDNA in Chinese hamster ovary cells (CHO-K1 cells) and purified the recombinant enzyme from the medium of stably transfected cells by a two-step affinity chromatography. Two isoforms of recombinant NAGLU with apparent molecular weights of 89 and 79 kDa were purified and shown to differ in their glycosylation pattern. The catalytic parameters of both forms of the recombinant enzyme were indistinguishable from each other and similar to those of NAGLU purified from various tissues. However, compared to other recombinant lysosomal enzymes expressed from CHO-K1 cells, the mannose-6-phosphate receptor mediated uptake of the secreted form of recombinant NAGLU into cultured skin fibroblasts was considerably reduced. A small amount of phosphorylated NAGLU present in purified enzyme preparations was shown to be endocytosed by MPS-IIIB fibroblasts via the mannose-6-phosphate receptor-mediated pathway and transported to the lysosomes, where they corrected the storage phenotype. Direct metabolic labeling experiments with Na(2) (32)PO(4) confirmed that the specific phosphorylation of recombinant NAGLU secreted from transfected CHO cells is significantly lower when compared with a control lysosomal enzyme. These results suggest that the use of secreted NAGLU in future enzyme and gene replacement therapy protocols will be severely limited due to its small degree of mannose-6-phosphorylation.

Mucopolysaccharidosis type IIIB (MPS-IIB) is a lysosomal storage disorder characterised by the defective degradation of heparan sulfate due to a deficiency of α-N-acetylglucosaminidase (NAG). The clinical severity of MPS-IIIB ranges from an attenuated to severely affected Sanfilippo phenotype. This paper describes the expression and characterisation of wild-type recombinant NAG and the molecular characterisation of a previously identified R297X/F48L compound heterozygous MPS-IIIB patient with attenuated Sanfilippo syndrome. We have previously shown R297X to be the most common mutation in a cohort of Dutch and Australian patients, occurring at a frequency of approximately 12.5%. To date F48L has only been described in the proband. To determine the contribution of each mutation to the overall clinical phenotype of the patient, both mutant alleles were engineered into the wild-type NAG cDNA and expressed in Chinese hamster ovary cells. The wild-type NAG and F48L mutant alleles were also retrovirally expressed in MPS-IIIB skin fibroblasts. Residual NAG activity and the stability and maturation of immunoprecipitated NAG were determined for wild-type NAG and mutant NAG. The combined biochemical phenotypes of the two NAG mutant alleles demonstrated a good correspondence with the observed attenuated Sanfilippo phenotype of the patient.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), required for the degradation of heparan sulfate. Molecular defects of 24 Italian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutagenized to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes.

Mucopolysaccharidosis IIIA (MPS-IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of sulfamidase (NS; EC 3.10.1.1), resulting in defective degradation and storage of heparan sulfate. This paper reports the production and characterization of monoclonal and polyclonal antibodies against recombinant human sulfamidase (rhNS) to quantitate and characterize normal and mutant sulfamidase produced from the wild type NS expression vector. Glycosylation and phosphorylation studies of immunoprecipitated rhNS show that all five potential glycosylation sites are utilized, with three high mannose/hybrid oligosaccharides and two simpler chains, with at least one functional mannose 6-phosphate group. An NS quantification system was developed to determine the effect of the three most common and severe patient mutations: S66W (Italy), R74C (Poland), and R245H (The Netherlands). The quantity and specific activity of expressed mutant rhNS was significantly lower than expressed normal rhNS, with 0.3, 0.2, and 0.05% of normal rhNS produced and 15, 17, and 83% of normal specific activity for S66W, R74C, and R245H observed, respectively. The recent structural elucidation of N-acetylgalactosamine-4-sulfatase was utilized to postulate the effect on the structure-function relationship of NS. The characterization of normal and mutated rhNS has relevance for efficient diagnosis and therapeutic developments for MPS-IIIA patients.

Sanfilippo B syndrome (mucopolysaccharidosis IIIB, MPS IIIB) is caused by a deficiency of α-N-acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes leads to degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. Age of onset and rate of progression vary considerably, whilst diagnosis is often delayed due to the absence of the pronounced skeletal changes observed in other mucopolysaccharidoses. Cloning of the gene and cDNA encoding α-N-acetylglucosaminidase enabled a study of the molecular basis of this syndrome. We were able to identify 31 mutations, 25 of them novel, and two polymorphisms in the 40 patients mostly of Australasian and Dutch origin included in this study. The observed allellic heterogeneity reflects the wide spectrum of clinical phenotypes reported for MPS IIIB patients. The majority of changes are missense mutations; also four nonsense and nine frameshift mutations caused by insertions or deletions were identified. Only five mutations were found in more than one patient and the observed frequencies are well below those observed for the common mutations in MPS IIIA. R643C and R297X each account for around 20% of MPS IIIB alleles in the Dutch patient group, whilst R297X, P521L, R565W and R626X each have a frequency of about 6% in Australasian patients. R643C seems to be a Dutch MPS IIIB allele and clearly confers the attenuated phenotype. One region of the gene shows a higher concentration of mutations, probably reflecting the instability of this area which contains a direct repeat. Several arginine residues seem to be ‘hot-spots’ for mutations, being affected by two or three individual base pair exchanges.

We have identified a common mutation (R245H) in the sulphamidase gene of Sanfilippo syndrome type A (mucopolysaccharidosis type IIIA, MPS IIIA) patients from The Netherlands. Allele-specific oligonucleotide hybridization was used to determine the incidence of this mutation in 45 unrelated MPS IIIA patients from different regions of The Netherlands. R245H was present in 51 alleles, representing 56.7% of the total allelic population. Of 39 patients, for whom we have uniform clinical details, 13 MPS IIIA patients who were homozygous for this common mutation had a more uniform but severe clinical phenotype than the remaining 21 or 5 patients, containing respectively one or no R245H alleles. The R245H allele had a higher prevalence in western rather than eastern regions of The Netherlands.