Search

Die Kinder- und Jugendarbeit der Stadt Nurnberg hat eine ausgepragte Tradition und eine ausgepragte Struktur. Die historischen Wurzeln reichen bis in die reformpadagogischen und sozialreformerisch orientierten, jugendbewegten Ansatze der Weimarer Republik zuruck. Seit Ende der 1960er Jahre wird das Handlungsfeld Kinder- und Jugendarbeit als eine der zentralen kommunalpolitischen Aufgaben verstanden, die auch mit hohen Anteilen in eigener Tragerverantwortung realisiert wird. Der konzeptionell gesteuerte, sukzessive Auf- und Ausbau erfolgte spatestens seit den 1990er Jahren in der Regel parteiubergreifend im weitgehenden Konsens. Kinder- und Jugendarbeit wird dabei von einem offensiven Grundverstandnis der Kinder- und Jugendhilfe gepragt, was dazu gefuhrt hat, dass auch in Zeiten hoher finanzieller Haushaltsbelastungen nicht auf den weiter notwendigen Ausbau von Einrichtungen verzichtet wurde. Kinder- und Jugendarbeit ist heute als Bereich im Amt fur Kinder, Jugendliche und Familien organisatorisch verortet und mit einem Gesamtansatz von 18,84 Mio. € (das sind 9,1 % der gesamten Jugendhilfeaufwendungen der Stadt Nurnberg; Bezugsjahr 2009), 120 Planstellen und 45 kommunalen Einrichtungen und Projekten versehen.

The development of reliable analytic methods, capable of separating mixtures of secondary metabolites as well as providing structural information, is essential for the investigation of secondary metabolites, e.g. from Streptomyces. Here we report a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using a triple quadrupole mass analyzer for the structural elucidation of caprazamycins and liposidomycins from culture extracts of the wild-type producer strains. Comparison of the fragmentation patterns in positive as well as in negative ionization mode revealed several characteristic product ions used for identification of six new caprazamycins. Furthermore, a chromatographic method for the purification of nucleosides from cell cultures using a boronic acid gel was adapted for the partial purification of the culture extracts.

Caprazamycins are potent antimycobacterials isolated from Streptomyces sp. MK730F-62F2 (23). In a pulmonary tuberculosis mouse model, they showed a therapeutic effect but no significant toxicity (24). The caprazamycins are assigned to the translocase I inhibitors (27) due to their structural similarity to the liposidomycins (34, 43), which have been studied in more detail. Translocase I catalyzes the first step in the membrane-linked reaction cycle of bacterial cell wall formation (52): the transfer of phospho-N-acetylmuramic acid-l-Ala-γ-d-Glu-m-diaminopimelic acid-d-Ala-d-Ala from UMP to the lipid carrier undecaprenyl phosphate. Structurally unique in nature, the caprazamycins and liposidomycins share a 5′-β-O-aminoribosyl-glycyluridine and a rare N-methylated diazepanone as their characteristic feature (Fig. ​(Fig.1)1) (25, 53). Attached at the 3″ position are β-hydroxylated fatty acid groups of different chain lengths, carrying a 3-methylglutarate. While the liposidomycins are sulfated, the caprazamycins lack this group. Instead, they are glycosylated with a 2,3,4-O-methyl-l-rhamnose and therefore belong to the large number of bioactive compounds containing 6-deoxyhexoses. Usually, these moieties contribute significantly to the compounds' properties, influencing, e.g., molecule-target interactions, cell import and export, pharmacokinetics, and solubility (56). The biosynthesis of deoxysugars has been studied in detail and generally starts from NDP-activated hexoses via 4-keto-6-deoxy intermediates (36). The formation of l-rhamnose involves four enzymes, a dTDP-d-glucose synthase, a dTDP-d-glucose 4,6-dehydratase, a dTDP-3,5-deoxyglucose epimerase, and a 4-ketoreductase. Recent advances in combinatorial biosynthesis have led to a variety of novel natural products with an engineered glycosylation pattern and altered bioactivity (37, 44). The key step in this approach is the attachment of different deoxysugars to the aglycones, which demands substrate-flexible glycosyltransferases. FIG. 1. Structure of caprazamycins and organization of the caprazamycin gene cluster (cpz9 to cpz31) lacking the genes for deoxysugar formation. Assignments of genes to different steps in the biosynthesis are indicated. We recently reported cloning and heterologous expression of cosmid cpzLK09 containing the first identified gene cluster of a translocase I inhibitor, the caprazamycins. However, genes for the formation of the dTDP-l-rhamnose could not be identified on this cosmid (Fig. ​(Fig.1)1) and therefore only caprazamycin aglycones were produced by the heterologous host, Streptomyces coelicolor M512/cpzLK09 (31). The absence of genes for the formation of the l-rhamnose moiety within the corresponding biosynthetic gene cluster has previously been reported for aranciamycin (38), steffimycin (16), spinosyn (55), and elloramycin (9). Since potential genes for O methylation (cpz28 to cpz30) and a glycosyltransferase (cpz31) are encoded in the caprazamycin gene cluster, we speculated that only four genes are missing for successful heterologous production of intact caprazamycins. Here we report the identification of the genes required for the biosynthesis of the caprazamycin deoxysugar moiety elsewhere on the genome of Streptomyces sp. MK730-62F2. A new strategy was developed, based on Red/ET-mediated recombination, to assemble the identified subcluster into cosmid cpzLK09. Expression of the assembled cluster readily resulted in the production of intact caprazamycins in the heterologous producer strain. Moreover, in vitro studies demonstrated that Cpz31 is the glycosyltransferase in caprazamycin biosynthesis.

Background Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer. Results Besides the determination of ribonucleosides, additional compounds with cis-diol structure, deriving from cross-linked biochemical pathways, like purine-, histidine- and polyamine metabolism were detected. In total, 36 metabolites were identified by comparison of fragmentation patterns and retention time. Relation to the internal standard isoguanosine yielded normalized area ratios for each identified compound and enabled a semi-quantitative metabolic signature of both analyzed cell lines. 13 of the identified 26 modified ribonucleosides were elevated in the cell culture supernatants of MCF-7 cells, with 5-methyluridine, N 2,N 2,7-trimethylguanosine, N 6-methyl-N 6-threonylcarbamoyladenosine and 3-(3-aminocarboxypropyl)-uridine showing the most significant differences. 1-ribosylimidazole-4-acetic acid, a histamine metabolite, was solely found in the supernatants of MCF-10A cells, whereas 1-ribosyl-4-carboxamido-5-aminoimidazole and S-adenosylmethionine occurred only in supernatants of MCF-7 cells. Conclusion The obtained results are discussed against the background of pathological changes in cell metabolism, resulting in new perspectives for modified nucleosides and related metabolites as possible biomedical markers for breast carcinoma in vivo.

Local application of dexamethasone-21-dihydrogen-phosphate (Dex-P) to the round window (RW) membrane of guinea pigs produces a substantial basal-apical concentration gradient in scala tympani (ST) perilymph.In recent years, intratympanically applied glucocorticoids are increasingly being used for the treatment of inner ear disease. Although measurements of intracochlear concentrations after RW application exist, there is limited information on the distribution of these drugs in the inner ear fluids. It has been predicted from computer simulations that substantial concentration gradients will occur after RW application, with lower concentrations expected in apical turns. Concentration gradients of other substances along the cochlea have recently been confirmed using a sequential apical sampling method to obtain perilymph.Dexamethasone-21-dihydrogen-phosphate (10 mg/ml) was administered to the RW membrane of guinea pigs (n = 9) in vivo for 2 to 3 hours. Perilymph was then collected using a protocol in which 10 samples, each of approximately 1 mul, were taken sequentially from the cochlear apex into capillary tubes. Dexamethasone-21-dihydrogen-phosphate concentration of the samples was analyzed by high-performance liquid chromatography. Interpretation of sample data using a finite element model allowed the longitudinal gradients of Dex-P in ST to be quantified.The Dex-P content of the first sample in each experiment (dominated by perilymph from apical regions) was substantially lower than that of the third and fourth sample (dominated by basal turn perilymph). These findings qualitatively demonstrated the existence of a concentration gradient along ST. After detailed analysis of the measured sample concentrations using an established finite element computer model, the mean basal-apical concentration gradient was estimated to be 17,000. Both absolute concentrations of Dex-P in ST and the basal-apical gradients were found to vary substantially.The existence of substantial basal-apical concentration gradients of Dex-P in ST perilymph were demonstrated experimentally. If the variability in peak concentration and gradient is also present under clinical conditions, this may contribute to the heterogeneity of outcome that is observed after intratympanic application of glucocorticoids for various inner ear diseases.

The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS(3) screening runs and MS(n(n=4-6)) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N(omega)-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N(6)-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N(6)-methyl-N(6)-threonylcarbamoyladenosine, and 2-methylthio-N(6)-threonylcarbamoyladenosine in human urine is verified for the first time.

Three new aminocoumarin antibiotics, termed ferulobiocin, 3-chlorocoumarobiocin and 8'-dechloro-3-chlorocoumarobiocin, were isolated from the culture broth of a Streptomyces coelicolor M512 strain expressing a modified clorobiocin biosynthetic gene cluster. Structural analysis showed that these new aminocoumarins were very similar to clorobiocin, with a substituted 4-hydroxycinnamoyl moieties instead of the prenylated 4-hydroxybenzoyl moiety of clorobiocin. The possible biosynthetic origin of these moieties is discussed.

Three new aminocoumarin antibiotics, termed ferulobiocin, 3-chlorocoumarobiocin and 8'-dechloro-3-chlorocoumarobiocin, were isolated from the culture broth of a Streptomyces coelicolor M512 strain expressing a modified clorobiocin biosynthetic gene cluster. Structural analysis showed that these new aminocoumarins were very similar to clorobiocin, with a substituted 4-hydroxycinnamoyl moieties instead of the prenylated 4-hydroxybenzoyl moiety of clorobiocin. The possible biosynthetic origin of these moieties is discussed.

Prediction of the metabolic profile of a potential new drug is recommended at an early stage in industrial drug discovery process to determine whether or not any potentially reactive or toxic metabolites are formed. In the present study, we investigated the in vitro metabolism of ML3403 ({4- [5-(4-Fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl -(1-phenylethyl)-amine), a potent and selective p38 MAP kinase inhibitor using mouse liver microsomes. The combination of LC-ESI-Qq-TOF (tandem quadrupole time-of-flight)-MS (mass spectrometer) and LC-SPE (solid phase extraction)-cryo-NMR (nuclear magnetic resonance)/MS at 600 MHz has been applied for comprehensive and straightforward structural elucidation of ML3403 metabolites. It was possible to determine the metabolic profile of ML3403, revealing eight different metabolites formed by N-desalkylation, S-mono- and di-oxidation, aliphatic hydroxylation and pyridine-N-oxidation. The ESI-Qq-TOF-MS data yielded elemental compositions of all metabolites and their fragments by evaluation of the accurate mass and isotopic pattern information using the sigma-fit algorithm. Evaluation of 2D NMR spectra obtained from pure ML3403 an its major metabolite ML3603 allowed the unequivocal assignment of the resonances in 1D NMR spectra obtained directly from the microsomal incubation by LC-SPE-cryo-NMR/MS. The presented method significantly decreases the time required for a complete structural assignment of metabolites from microsomal in vitro assays.

Based on the purine scaffold of ATP, derivatives of 6,9-diarylpurine-8-one were prepared and tested for their ability to inhibit p38 MAP kinase, a key enzyme in the cellular regulation of proinflammatory cytokines. The inhibitor design combines the purine system of the authentic cosubstrate ATP with various phenyl moieties to explore the selectivity for the two hydrophobic regions of the kinase's ATP-binding cleft. The present study indicates a new binding mode of our scaffold to p38 MAP kinase, which comprises the desired structural features of ATP and the N-phenyl-N-purin-6-yl ureas previously published by Wan et al. Combinations of Autodock and FlexX docking with different scoring functions were used to assess the postulated binding mode. The predictive power of different docking-scoring combinations was determined. The presented results may form a solid basis for further optimization cycles since our theoretical findings are consistent with our experimental binding data and supported by the literature.

The p38 mitogen-activated protein kinase (MAPK) is a key mediator in cytokine-induced signaling events that are activated in response to a variety of extracellular stimuli such as stress factors, apoptosis, and proliferation. Therefore, the MAPK family plays an integral role in disease states including oncogenesis, autoimmune diseases, and inflammatory processes. Inhibition of these protein kinases represents an attractive strategy for therapeutic intervention. In particular, one class of p38 MAP kinase inhibitors, the pyridinyl imidazole derivatives, is intensely investigated by several industrial groups, but so far no studies concerning the metabolism of these structurally related substances seem to be available. The objective of our examinations was the preclinical characterization of ML3403, {4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine, a potent inhibitor of p38 MAP kinase, comprising the basic pyridinyl imidazole structure. In human hepatic microsomal incubations, the sulfoxidation to ML3603 ({4-[5-(4-fluorophenyl)-2-methylsulfinyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine) and M-sulfone ({4-[5-(4-fluorophenyl)-2-methylsulfonyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine) was found to be the predominant metabolic transformation. In addition, oxidative removal of the phenylethyl moiety, pyridine N-oxidation, and hydroxylation reactions were observed. Incubations were carried out with hepatic microsomes from various species and with recombinant human cytochrome P450 isoenzymes, showing that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 are the prominent enzymes in the metabolism of ML3403. Michaelis-Menten kinetics of ML3603 formation by these recombinant isoenzymes showed that CYP3A4 plays a pivotal role in the sulfoxidation reaction. In addition, pharmacokinetics of ML3403 were evaluated in male and female Wistar rats after oral gavage, showing a fast and high conversion to its active sulfoxide metabolite ML3603. A remarkable gender-specific difference in the systemic exposure to ML3403 and ML3603 was found in rats. No gender-specific difference was detected in incubations with human liver microsomes.

Drought is a major environmental stress affecting growth and vitality of forest ecosystems. In the present study, foliar nitrogen (N) and carbon (C) metabolism of two Douglas fir (Pseudotsuga menziesii) provenances with assumed different drought tolerance were investigated. We worked with 1-year-old seedlings of the interior provenance Fehr Lake (FEHR) originating from a dry environment and the coastal provenance Snoqualmie (SNO) from a more humid origin. Total C and N, structural N and the concentrations of soluble protein, total amino acids (TAAs) and individual amino acids as well as the relative abundance of polar, low-molecular-weight metabolites including antioxidants were determined in current-year needles exposed either to 42 days of drought or to 42 days drought plus 14 days of rewatering. The seedlings reacted in a provenance-specific manner to drought stress. Coastal provenance SNO showed considerably increased contents of TAAs, which were caused by increased abundance of the quantitatively most important amino acids arginine, ornithine and lysine. Additionally, the polyamine putrescine accumulated exclusively in drought-stressed trees of this provenance. In contrast, the interior provenance FEHR showed the opposite response, i.e., drastically reduced concentrations of these amino acids. However, FEHR showed considerably increased contents of pyruvate-derived and aromatic amino acids, and also higher drought-induced levels of the antioxidants ascorbate and α-tocopherol. In response to drought, both provenances produced large amounts of carbohydrates, such as glucose and fructose, most likely as osmolytes that can readily be metabolized for protection against osmotic stress. We conclude that FEHR and SNO cope with drought stress in a provenance-specific manner: the coastal provenance SNO was mainly synthesizing N-based osmolytes, a reaction not observed in the interior provenance FEHR; instead, the latter increased the levels of scavengers of reactive oxygen species. Our results underline the importance of provenance-specific reactions to abiotic stress.

Streptomyces cinnamonensis DSM 1042 produces two classes of secondary metabolites of mixed isoprenoid/nonisoprenoid origin: the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and several prenylated phenazines, predominantly endophenazine A. We now report the cloning and sequence analysis of a 55 kb gene cluster required for the biosynthesis of these compounds. Several inactivation experiments confirmed the involvement of this gene cluster in the biosynthesis of FNQ I and endophenazine A. The six identified genes for endophenazine biosynthesis showed close similarity to phenazine biosynthetic genes from Pseudomonas. Of the 28 open reading frames identified in the adjacent FNQ I cluster, 13 showed close similarity to genes contained in the cluster for furaquinocin-a structurally similar metabolite from another Streptomyces strain. These genes included a type III polyketide synthase sequence, a momA-like monooxygenase gene, and two cloQ-like prenyltransferase genes designated fnq26 and fnq28. Inactivation experiments confirmed the involvement of fnq26 in FNQ I biosynthesis, whereas no change in secondary-metabolite formation was observed after fnq28 inactivation. The FNQ I cluster contains a contiguous group of five genes, which together encode all the enzymatic functions required for the recycling of S-adenosylhomocysteine (SAH) to S-adenosylmethionine (SAM). Two SAM-dependent methyltransferases are encoded within the cluster. Inactivation experiments showed that fnq9 is responsible for the 7-O-methylation and fnq27 for the 6-C-methylation reaction in FNQ I biosynthesis.

Before new drugs for the treatment of inner ear disorders can be studied in controlled clinical trials, it is important that their pharmacokinetics be established in inner ear fluids. Microdialysis allows drug levels to be measured in perilymph without the volume disturbances and potential cerebrospinal fluid contamination associated with fluid sampling. The aims of this study were to show: (i) that despite low recovery rates from miniature dialysis probes, significant amounts of drug are removed from small fluid compartments, (ii) that dialysis sampling artifacts can be accounted for using computer simulations and (iii) that microdialysis allows quantification of the entry rates through the round window membrane (RWM) into scala tympani (ST). Initial experiments used microdialysis probes in small compartments in vitro containing sodium fluorescein. Stable concentrations were observed in large compartments (1000 microl) but significant concentration declines were observed in smaller compartments (100, 10 and 5.6 microl) comparable to the size of the inner ear. Computer simulations of these experiments closely approximated the experimental data. In in vivo experiments, sodium fluorescein 10 mg/ml and dexamethasone-dihydrogen-phosphate disodium salt 8 mg/ml were simultaneously applied to the RWM of guinea pigs. Perilymph concentration in the basal turn of ST was monitored using microdialysis. The fluorescein concentration reached after 200 min application (585+/-527 microg/ml) was approximately twice that of dexamethasone phosphate (291+/-369 microg/ml). Substantial variation in concentrations was found between animals by approximately a factor of 34 for fluorescein and at least 41 for dexamethasone phosphate. This is, to a large extent, thought to be the result of the RWM permeability varying in different animals. It was not caused by substance analysis variations, because two different analytic methods were used and the concentration ratio between the two substances remained nearly constant across the experiments and because differences were apparent for the repeated samples obtained in each animal. Interpretation of the results using computer simulations allowed RWM permeability to be quantified. It also demonstrated, however, that cochlear clearance values could not be reliably obtained with microdialysis because of the significant contribution of dialysis to clearance. The observed interanimal variation, e.g., in RWM permeability, is likely to be clinically relevant to the local application of drugs in patients.

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard. For the off-line SPE plasma method mixed cation-exchange SPE cartridges and a 150 x 2 mm C18 column with isocratic elution were used. For the on-line SPE method, a Waters Oasis HLB extraction column and the same C18 analytical column in a column-switching set-up with gradient elution were utilized. All assays were linear within a range of 0.5-100 ng/mL with a limit of detection of 0.05 ng/mL. The intra- and interday coefficients of variance ranged from 1.3 to 6.1% for plasma and 0.5 to 6.4% for urine, respectively. The extraction recovery for the off-line plasma assay was between 90.7 and 100.0%. Influence of matrix effects, and freeze/thaw and long-term stability were validated for both approaches; influence of urine pH additionally for quantification in urine.

Preparations containing willow bark extract are popular herbal remedies, but they are mostly standardised with respect to only one compound (usually salicin). RP-HPLC using a C18-column eluted with water:methanol: tetrahydrofuran and coupled to electrospray triple-quadrupole MS and MS/MS was used for the characterisation of dried extracts of Salix spp. and for the identification of their constituents. Comparison with reference substances led to the identification of 13 compounds (saligenin, salicylic acid, salicin, isosalicin, picein, salidroside, triandrin, salicoylsalicin, salicortin, isosalipurposide, salipurposide, naringenin-7-O-glucoside and tremulacin). Two pharmaceutical preparations containing willow bark extract, used in clinical trials and labelled Salix daphnoides and S. purpurea × daphnoides extracts, were compared using the described method and exhibited several clear differences, the most prominent of which was the possible presence of picein in the former preparation. The described method may be utilised for the characterisation of herbal medicines in order to ensure comparability of medication in further clinical trials. Copyright © 2005 John Wiley & Sons, Ltd.

BackgroundThe anticancer drug Ukrain (NSC-631570) which has been specified by the manufacturer as semisynthetic derivative of theChelidonium majusL. alkaloid chelidonine and the alkylans thiotepa was reported to exert selective cytotoxic effects on human tumour cell linesin vitro. Few clinical trials suggest beneficial effects in the treatment of human cancer. Aim of the present study was to elucidate the importance of apoptosis induction for the antineoplastic activity of Ukrain, to define the molecular mechanism of its cytotoxic effects and to identify its active constituents by mass spectrometry.MethodsApoptosis induction was analysed in a Jurkat T-lymphoma cell model by fluorescence microscopy (chromatin condensation and nuclear fragmentation), flow cytometry (cellular shrinkage, depolarisation of the mitochondrial membrane potential, caspase-activation) and Western blot analysis (caspase-activation). Composition of Ukrain was analysed by mass spectrometry and LC-MS coupling.ResultsUkrain turned out to be a potent inducer of apoptosis. Mechanistic analyses revealed that Ukrain induced depolarisation of the mitochondrial membrane potential and activation of caspases. Lack of caspase-8, expression of cFLIP-L and resistance to death receptor ligand-induced apoptosis failed to inhibit Ukrain-induced apoptosis while lack of FADD caused a delay but not abrogation of Ukrain-induced apoptosis pointing to a death receptor independent signalling pathway. In contrast, the broad spectrum caspase-inhibitor zVAD-fmk blocked Ukrain-induced cell death. Moreover, over-expression of Bcl-2 or Bcl-xLand expression of dominant negative caspase-9 partially reduced Ukrain-induced apoptosis pointing to Bcl-2 controlled mitochondrial signalling events.However, mass spectrometric analysis of Ukrain failed to detect the suggested trimeric chelidonine thiophosphortriamide or putative dimeric or monomeric chelidonine thiophosphortriamide intermediates from chemical synthesis. Instead, theChelidonium majusL. alkaloids chelidonine, sanguinarine, chelerythrine, protopine and allocryptopine were identified as major components of Ukrain.Apart from sanguinarine and chelerythrine, chelidonine turned out to be a potent inducer of apoptosis triggering cell death at concentrations of 0.001 mM, while protopine and allocryptopine were less effective. Similar to Ukrain, apoptosis signalling of chelidonine involved Bcl-2 controlled mitochondrial alterations and caspase-activation.ConclusionThe potent proapoptotic effects of Ukrain are not due to the suggested "Ukrain-molecule" but to the cytotoxic efficacy ofChelidonium majusL. alkaloids including chelidonine.

Direct 1N-amination of the triazole-fused zinc phthalocyanine 2 and oxidation of the formed amino derivative 3 resulted in the generation of the very reactive intermediate, the dehydrometallophthalocyanine 4, which was not known previously. The latter was trapped in situ with different dienes, for example, furan, tetraphenylcyclopentadienone, and anthracene to form the corresponding Diels-Alder adducts. The products were characterized by 1 H and 1 3 C-dept135 NMR, and UV/Vis spectroscopy. MALDI-TOF mass spectrometry, and elemental analysis, which are fully in agreement with their structure. The developed synthetic procedure opens a simple and versatile pathway towards unsymmetrical peripheral modification of phthalocyanines, which is readily applicable to the micromol scale and is important for the design of new interesting Pc-based systems.

The total flavonoids and saponins of the seeds of Glinus lotoides in the crude extracts and tablet formulation thereof were quantified by reversed-phase high-performance liquid chromatographic (RP-HPLC) methods with UV detection. The saponins were analyzed after acid hydrolysis in 3 M HCI at 100 degrees C for 1 h. Vicenin-2 and mollugogenol B were isolated and used as reference substances for the quantification of total flavonoids and saponins, respectively. The identity and purity (> 97%) of the standards were confirmed by spectroscopic (UV, MS, and NMR) and chromatographic (HPLC) methods. The flavonoids and saponins of the crude extract of the seeds and tablet formulation were separated by RP-HPLC (Nucleosil RP-18 column, 250 mm x 4.6 mm) using linear gradient elution systems of acetonitrile-water-0.1 M H3PO4 for flavonoids and methanol-water for saponins. Satisfactory separation of the compounds was obtained in less than 30 and 25 min, for the flavonoids and saponins, respectively. The methods were validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). Repeatability (inter- and intra-day, n = 6 and 9, respectively) showed less than 2% relative standard deviation (RSD). The LOD and LOQ were found to be 0.075 and 0.225 mg/mL, respectively, for vicenin-2 and 0.027 and 0.082 mg/100 mL, respectively, for mollugogenol B. The content of flavonoids and saponins of six single tablets was between 95 and 103% for flavonoids and 94-98% for saponins. The validated HPLC methods were employed to standardize a fingerprint of a laboratory produced purified extract, which could be used as a secondary standard for the routine quality control. Accordingly, the purified extract was found to contain 21.3% flavonoids (vicenin-2, 10%) and 25.4% saponins (glinuside G, 14.2%).

The effect of cytochrome P450 (CYP) 2C9 polymorphisms on the stereoselective biotransformation of the oral anticoagulant phenprocoumon (PPC) to inactive, monohydroxylated metabolites was studied in vitro and in vivo. In human liver microsomes, the (S)-7-hydroxylation--being the major metabolic pathway--was significantly compromised in a gene-dose-dependent manner in samples expressing the CYP2C9*2 or CYP2C9*3 allele. The CYP2C9*3/*3 genotype corresponded to an almost fourfold lower (S)-7-hydroxylation rate than CYP2C9*1/*1 (wild-type). The intrinsic clearance of human recombinant CYP2C9*2 and CYP2C9*3 for the (S)-7-hydroxylation was 28.9 and 50.9% lower than of CYP2C9*1, respectively. The area under the plasma concentration-time curve (AUC) of PPC metabolites after oral intake of 12 mg racemic PPC was significantly lower in volunteers expressing the CYP2C9*2 or CYP2C9*3 allele. Increasing plasma AUC metabolic ratios (parent compound/metabolite) in CYP2C9*2 and CYP2C9*3 variant allele carriers were found for each hydroxylation reaction and the CYP2C9*3/*3 genotype corresponded to an about 10-fold higher metabolic ratio of PPC (S)-7-hydroxylation relative to CYP2C9*1/*1. CYP2C9 polymorphisms cause a markedly compromised PPC (S)-7-hydroxylation. However, PPC metabolism appears overall less influenced by CYP2C9 genotype compared with other oral anticoagulants and it may thus be a valuable alternative for therapeutic anticoagulation of patients expressing CYP2C9 variant alleles.

In diseases accompanied by strong metabolic disorders, like cancer and AIDS, modifying enzymes are up- or down-regulated. As a result, many different types of metabolic end-products, including abnormal amounts of modified nucleosides, are found in urine. These nucleosides are degradation products of an impaired ribonucleic acid (RNA) metabolism, which affects the nucleoside pattern in urine. In several basic experiments we elucidated the fragmentation pathways of 16 characteristic nucleosides and six corresponding nucleic bases that occur in urine using electrospray ionization ion trap MS(5) (ESI-ITMS) experiments operated in positive ionization mode. For urinary nucleoside analysis, we developed an auto-LC-MS3 method based on prepurification via boronate gel affinity chromatography followed by reversed phase chromatography. For this purpose, an endcapped LiChroCART Superspher RP 18 column with a gradient of ammonium formate and a methanol-water mixture was used. This method gives a limit of detection of between 0.1 and 9.6 pmol for 15 standard nucleosides, depending on the basicity of the nucleoside. Overall, the detection of 36 nucleosides from urine was feasible. It was shown that this auto-LC-MS3 method is a valuable tool for assigning nucleosides from complex biological matrices, and it may be utilized in the diagnosis of diseases associated with disorders in RNA metabolism.

As RNA turnover seems to be impaired in cancer patients, modified nucleosides have been evaluated as potential tumor markers. Modified nucleosides are mainly formed post-transcriptionally in tRNA, set free during RNA metabolism, and excreted in urine. Especially methylated nucleosides play an important role, as their levels are higher in urine from cancer patients. For structural elucidation of known and unknown nucleosides from urine samples of cancer patients, MALDI-TOF MS and MALDI-PSD were used for the first time. This technique generally ensures high sensitivity, mass resolution, and accuracy. In our analytical approach we prepurified nucleosides from urine by affinity chromatography and subsequently separated them by semipreparative high performance liquid chromatography. The different fractions were collected separately and analyzed by MALDI-TOF MS and PSD-MALDI using a mixture of six low molecular weight calibrants for internal or external calibration. The molecular totals formulas based on a mass accuracy of 10 ppm and below were calculated and a systematic data base search was performed. The inherent problem of the MALDI-technique, the reduced sensitivity for low molecular weight substances caused by matrix suppression effects, was reduced by our technique. We identified several nucleosides in urine, which were previously identified via retention times and UV spectra of standards after HPLC analysis. Eight further nucleosides were observed. This work demonstrates for the first time the potential of MALDI-TOF and PSD-MALDI in combination with semipreparative HPLC for assignment of nucleosides in urine. The particularly high mass accuracy of this mass spectrometric method provides opportunities for identifying unknown compounds.

CYP2C9 catalyses the biotransformation of the oral anticoagulants S-warfarin and R- and S-acenocoumarol. According to data obtained in vitro, phenprocoumon is also metabolized by CYP2C9 but the impact of the CYP2C9 polymorphism on phenprocoumon pharmacokinetics has not been studied. Twenty-six healthy heterozygous and homozygous carriers of the CYP2C9 alleles *1 (wild-type), *2 (Arg144Cys), and *3 (Ile359Leu) received a single oral dose of 12 mg of racemic phenprocoumon. Plasma and 12 h urine concentrations of both enantiomers and their monohydroxylated metabolites were measured by high-performance liquid chromatography with mass spectrometry detection. No significant effect of the CYP2C9 variants *2 and *3 on R-phenprocoumon pharmacokinetic parameters was detected, but S-phenprocoumon clearance tended to decrease with increasing number of CYP2C9*2 and *3 alleles. The ratios of S- to R-phenprocoumon plasma clearances were higher with a median of 0.95 in carriers of *1/*1 versus 0.65 in *3/*3 (P0.001 for trend). Plasma and urine concentrations of 4'-, 6- and 7-hydroxyphenprocoumon were significantly lower in homozygous carriers of the CYP2C9*2 and *3 variants compared to CYP2C9*1/*1. Carriers of CYP2C9*3/*3 had a median AUC of (R,S) 7-OH-phenprocoumon of only approximately 25% compared to the wild-type genotype. The AUC of (R,S) 6-OH-phenprocoumon was only approximately 50% in CYP2C9*3/*3 compared to the homozygous wild-type genotype. In conclusion, carriers of CYP2C9*2 and *3 alleles had a lower metabolic capacity regarding phenprocoumon hydroxylation than those with CYP2C9*1/*1. However, regarding phenprocoumon hydroxylation CYP2C9 genotypes had only marginal effects on S- and R-phenprocoumon total clearance in healthy volunteers.

Phenprocoumon is a commonly used oral anticoagulant of the coumarin type, and has found extensive clinical use in the treatment of thrombophlebitis, pulmonary embolism and atrial fibrillation. In the course of a clinical study to investigate the influence of genetic polymorphisms of the CYP2C9 enzyme on phenprocoumon metabolism, we developed a new enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/MS/MS) method to quantify (R)- and (S)-phenprocoumon in human plasma. HPLC separation of the enantiomers was achieved on a Chira-Grom-2 column under isocratic conditions using a water/acetonitrile/formic acid eluent. For detection and quantification a triple-quadrupole MS system was used in the selected reaction monitoring (SRM) mode. As an internal standard the structurally homologous compound warfarin was chosen. The detector response was linear with a correlation coefficient of 0.988-0.999 for (R)-phenprocoumon and 0.989-0.999 for (S)-phenprocoumon in the investigated concentration range between 62.5 and 1000 ng/mL (per enantiomer). The limit of detection (LOD) was 12.5 ng/mL.

Ring A (3-dimethylallyl-4-hydroxybenzoic acid) is a structural moiety of the aminocoumarin antibiotics novobiocin and clorobiocin. In the present study, the prenyltransferase involved in the biosynthesis of this moiety was identified from the clorobiocin producer ( Streptomyces roseochromogenes ), overexpressed, and purified. It is a soluble, monomeric 35-kDa protein, encoded by the structural gene cloQ . 4-Hydroxyphenylpyruvate and dimethylallyl diphosphate were identified as the substrates of this enzyme, with K m values determined as 25 and 35 μM, respectively. A gene inactivation experiment confirmed that cloQ is essential for ring A biosynthesis. Database searches did not reveal any similarity of CloQ to known prenyltransferases, and the enzyme did not contain the typical prenyl diphosphate binding site (N/D)DXXD. In contrast to most of the known prenyltransferases, the enzymatic activity was not dependent on the presence of magnesium, and in contrast to the membrane-bound polyprenyltransferases involved in ubiquinone biosynthesis, CloQ did not accept 4-hydroxybenzoic acid as substrate. CloQ and the similar NovQ from the novobiocin producer seem to belong to a new class of prenyltransferases.

KIDS ist ein gemeinsames Projekt von Kreisjugendring Nurnberg-Stadt und Jugendamt der Stadt Nurnberg. KIDS ist ein Instrument der Jugendhilfeplanung. Ziel ist es, die Offene Kinder- und Jugendarbeit in Nurnberg stadtteil-orientiert zu planen, weiterzuentwickeln und die Rahmenbedingungen fur Kinder- und Jugendarbeit zu verbessern. Dies umfasst eine Bestandsaufnahme uber die gesamte Kinder- und Jugendarbeit, die Koordination und Kooperation in den Stadtteilen, die bessere Nutzung von Ressourcen, die starkere Partizipation von Kindern und Jugendlichen und die Initiierung von neuen Projekten. Hierbei geht es um die Verbesserung der Kinder- und Jugendarbeit in Nurnberg insgesamt. Um Missverstandnisse zu vermeiden, soll betont werden, dass die Initiative dazu aus den Reihen der Kinder-und Jugendarbeit selbst kam. Es ging und geht um die fachliche Weiterentwicklung, nicht um Sparzwange. Die Offene Kinder- und Jugendarbeit soll sich verstarkt stadtteilorientiert und vernetzt ausrichten. Die Kenntnis uber Schwerpunkte, Umfang und Zielgruppen der jeweils in der gleichen Region tatigen Jugendeinrichtungen bzw. Initiativen und Projekten ist ein erster Schritt. Zugleich ist es von Vorteil, wenn durch Abstimmungen und Absprachen Reibungsverluste vermindert werden. Durch Zusammenarbeit kann viel Neues angestosen und umgesetzt werden.

Four tetrahydropyridocarbazole derivatives were analysed by different mass spectrometry techniques: electrospray ionization, fast atom bombardment and by low and high resolution 70 eV electron ionization. Retro Diels Alder is the main fragmentation pathway, whereas other pathways leading to [M—1]+, [M—CH3]+and double charge ions also occur to considerable extents. Semi-empirical calculation provided some evidence on the nature of tropylium ions [M—1]+. Calculation of ΔHf0indicated that [M+—1] could be formed preferentially when a hydrogen atom is lost from the methyl substituent of the homoaromatic ring.

Offene Kinder- und Jugendarbeit ist ein elementarer Bestandteil der Infrastruktur der Stadt Nurnberg im Sinne des gesetzlichen Auftrags des Kinder- und Jugendhilfegesetzes (§ 1 KJHG): „Jeder junge Mensch hat ein Recht auf Forderung seiner Entwicklung und auf Erziehung zu einer eigenverantwortlichen und gemeinschaftsfahigen Personlichkeit“.

SummaryBecause the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity of the glycosidic linkage limits their therapeutic application. One potential solution to this problem is to replace the labile O-glycosidic bond with an enzymatically and chemically stable C-glycosidic bond. In this study, computational analysis of the O-glycosyltransferase LanGT2 and the C-glycosyltransferase UrdGT2 was used to predict the changes necessary to switch the O-glycosylating enzyme to a C-glycosyltransferase. By screening rationally designed LanGT2 mutants a number of LanGT2 variants with C-glycosyltransferase activity were identified. One variant, having 10 amino acid substitutions, revealed the primary region that determines O- versus C-glycosylation. By modeling the active site of this mutant and probing the role of active site residues with alanine substitutions, this work also illuminates the mechanistic features of O- and C-glycosylation.

Background Breast cancer belongs to the most frequent and severe cancer types in human. Since excretion of modified nucleosides from increased RNA metabolism has been proposed as a potential target in pathogenesis of breast cancer, the aim of the present study was to elucidate the predictability of breast cancer by means of urinary excreted nucleosides. Methods We analyzed urine samples from 85 breast cancer women and respective healthy controls to assess the metabolic profiles of nucleosides by a comprehensive bioinformatic approach. All included nucleosides/ribosylated metabolites were isolated by cis-diol specific affinity chromatography and measured with liquid chromatography ion trap mass spectrometry (LC-ITMS). A valid set of urinary metabolites was selected by exclusion of all candidates with poor linearity and/or reproducibility in the analytical setting. The bioinformatic tool of Oscillating Search Algorithm for Feature Selection (OSAF) was applied to iteratively improve features for training of Support Vector Machines (SVM) to better predict breast cancer. Results After identification of 51 nucleosides/ribosylated metabolites in the urine of breast cancer women and/or controls by LC- ITMS coupling, a valid set of 35 candidates was selected for subsequent computational analyses. OSAF resulted in 44 pairwise ratios of metabolite features by iterative optimization. Based on this approach ultimately estimates for sensitivity and specificity of 83.5% and 90.6% were obtained for best prediction of breast cancer. The classification performance was dominated by metabolite pairs with SAH which highlights its importance for RNA methylation in cancer pathogenesis. Conclusion Extensive RNA-pathway analysis based on mass spectrometric analysis of metabolites and subsequent bioinformatic feature selection allowed for the identification of significant metabolic features related to breast cancer pathogenesis. The combination of mass spectrometric analysis and subsequent SVM-based feature selection represents a promising tool for the development of a non-invasive prediction system.

Background Bioinformatics data analysis often deals with additive mixtures of signals for which only class labels are known. Then, the overall goal is to estimate class related signals for data mining purposes. A convenient application is metabolic monitoring of patients using infrared spectroscopy. Within an infrared spectrum each single compound contributes quantitatively to the measurement. Results In this work, we propose a novel factorization technique for additive signal factorization that allows learning from classified samples. We define a composed loss function for this task and analytically derive a closed form equation such that training a model reduces to searching for an optimal threshold vector. Our experiments, carried out on synthetic and clinical data, show a sensitivity of up to 0.958 and specificity of up to 0.841 for a 15-class problem of disease classification. Using class and regression information in parallel, our algorithm outperforms linear SVM for training cases having many classes and few data. Conclusions The presented factorization method provides a simple and generative model and, therefore, represents a first step towards predictive factorization methods.