Andreas Pich

Publications of Andreas Pich

• Secretome analysis of Clostridium difficile strains.

  Authors: Alexander Boetzkes, Katharina Wiebke Felkel, Johannes Zeiser, Nelli Jochim, Ingo Just, Andreas Pich

  Archives of microbiology. 03/2012;

  Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027)Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.

• Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene.

  Authors: Alexandra Olling, Sophie Seehase, Nigel P Minton, Helma Tatge, Saskia Schröter, Saskia Kohlscheen, Andreas Pich, Ingo Just, Ralf Gerhard

  Microbial pathogenesis. 11/2011; 52(1):92-100.

  The small open reading frame tcdE is located between the genes tcdA and tcdB which encode toxin A (TcdA) and B (TcdB), respectively, within the pathogenicity locus of Clostridium difficile. SequenceThe small open reading frame tcdE is located between the genes tcdA and tcdB which encode toxin A (TcdA) and B (TcdB), respectively, within the pathogenicity locus of Clostridium difficile. Sequence and structure similarities to bacteriophage-encoded holins have led to the assumption that TcdE mediates the release of the toxins from C. difficile into the extracellular environment. A TcdE-deficient C. difficile 630 strain was generated by insertional inactivation of the tcdE gene. Data revealed that TcdE does not regulate or affect growth or sporogenesis. TcdE-deficiency was accompanied by a moderately increased accumulation of TcdA and TcdB prior to sporulation in this microorganism. Interestingly, this observation did not correlate with a delayed or inhibited toxin release: inactivation of TcdE neither significantly altered kinetics of release nor the absolute level of secreted TcdA and TcdB, indicating that TcdE does not account for the pathogenicity of C. difficile strain 630. Furthermore, mass spectrometry analysis could not reveal differences in the secretome of wild type and TcdE-deficient C. difficile, indicating that TcdE did not function as a secretion system for protein release. TcdE was expressed as a 19 kDa protein in C. difficile, whereas TcdE expressed in Escherichia coli appeared as a 19 and 16 kDa protein. Expression of the short 16 kDa TcdE correlated with bacterial cell death. We conclude that TcdE does not exhibit pore-forming function in C. difficile since in these cells only the non-lytic full length 19 kDa protein is expressed.

• Effects of Clostridium difficile Toxin A on the proteome of colonocytes studied by differential 2D electrophoresis.

  Authors: Johannes J Zeiser, Jennifer Klodmann, Hans-Peter Braun, Ralf Gerhard, Ingo Just, Andreas Pich

  Journal of proteomics. 08/2011; 75(2):469-79.

  Clostridium difficile is a spore-forming anaerobic pathogen, commonly associated with severe diarrhea or life-threatening pseudomembraneous colitis. Its main virulence factors are the single-chain,Clostridium difficile is a spore-forming anaerobic pathogen, commonly associated with severe diarrhea or life-threatening pseudomembraneous colitis. Its main virulence factors are the single-chain, multi-domain toxin A (TcdA) and B (TcdB). Their glucosyltransferase domain selectively inactivates Rho proteins leading to a reorganization of the cytoskeleton. To study exclusively glucosyltransferase-dependent molecular effects of TcdA, human colonic cells (Caco-2) were treated with recombinant wild type TcdA and the glucosyltransferase deficient variant of the toxin, TcdA(gd) for 24h. Changes in the protein pattern of the colonic cells were investigated by 2-D DIGE and LCMS/MS methodology combined with detailed proteome mapping. gdTcdA did not induce any detectable significant changes in the protein pattern. Comparing TcdA-treated cells with a control group revealed seven spots of higher and two of lower intensity (p<0.05). Three proteins are involved in the assembly of the cytoskeleton (β-actin, ezrin, and DPYL2) and four are involved in metabolism and/or oxidative stress response (ubiquitin, DHE3, MCCB, FABPL) and two in regulatory processes (FUBP1, AL1A1). These findings correlate well to known effects of TcdA like the reorganization of the cytoskeleton and stress the importance of Rho protein glucosylation for the pathogenic effects of TcdA.

• Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI.

  Authors: Nelli Jochim, Ralf Gerhard, Ingo Just, Andreas Pich

  Proteome science. 08/2011; 9:48.

  ABSTRACT: The anaerobe Clostridium difficile produces two major virulence factors toxin A and B that inactivate Rho proteins by glucosylation of a pivotal threonine residue. Purified toxins induceABSTRACT: The anaerobe Clostridium difficile produces two major virulence factors toxin A and B that inactivate Rho proteins by glucosylation of a pivotal threonine residue. Purified toxins induce reorganization of the cytoskeleton and cell death in colonic cells. Whether all toxin effects on target cells depend on catalytic glucosyltransferase activity is unclear at present. Thus, we conducted a proteome approach to compare the protein profile of target cells treated either with wild type toxin A (rTcdA wt) or with a catalytically inactive mutant toxin A (mutant rTcdA). Relative protein quantification was feasible using isotope-coded protein labeling techniques (ICPL) and mass spectrometry (LC-MALDI). Altogether we found a significant differential expression of thirty proteins after treatment with rTcdA wt or mutant rTcdA. Mutant rTcdA caused up-regulation of seven proteins and sixteen proteins were responsive to rTcdA wt after 5 h. Long-term effect of rTcdA wt on protein expression was the down-regulation of eleven proteins. Up- or down-regulation of several proteins was verified by western blot analysis confirming the MS results. Our results indicate incubation time-dependent effects of the clostridial glucosylating toxin A on colonic cells. The rTcdA wt impact more cellular functions than actin cytoskeleton reorganization and apoptosis. Furthermore, these data give insight into glucosyltransferase independent effects of clostridial glucosylating toxins on target cells after short incubation time. Additionally, our data reveal pro-inflammatory and proliferative effects of mutant rTcdA after short-term incubation.

• The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication.

  Authors: Anja Puklowski, Yahya Homsi, Debora Keller, Martin May, Sangeeta Chauhan, Uta Kossatz, Viktor Grünwald, Stefan Kubicka, Andreas Pich, Michael P Manns, Ingrid Hoffmann, Pierre Gönczy, Nisar P Malek

  Nature cell biology. 07/2011; 13(8):1004-9.

  Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligaseDeregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.

• Proteomic bronchiolitis obliterans syndrome risk monitoring in lung transplant recipients.

  Authors: Thomas Wolf, Tonio Oumeraci, Jens Gottlieb, Andreas Pich, Benedikt Brors, Roland Eils, Axel Haverich, Brigitte Schlegelberger, Tobias Welte, Marc Zapatka, Nils von Neuhoff

  Transplantation. 06/2011; 92(4):477-85.

  Obliterative bronchiolitis poses a primary obstacle for long-term survival of lung transplant recipients and manifests clinically as bronchiolitis obliterans syndrome (BOS). Establishing a molecularObliterative bronchiolitis poses a primary obstacle for long-term survival of lung transplant recipients and manifests clinically as bronchiolitis obliterans syndrome (BOS). Establishing a molecular level screening method to detect BOS-related proteome changes before its diagnosis by forced expiratory volume surrogate marker criteria was the main objective of this study. Bronchoalveolar lavage was performed in 82 lung transplant recipients (48/34 with/without known BOS development) at different time points between 12 and 48 months after lung transplantation. A mass spectrometry-based method was devised to generate bronchoalveolar lavage fluid proteome profiles that were screened for BOS-specific alterations. Statistically significant marker peptides and proteins were identified and validated by in-gel digestion, tandem mass spectrometric sequencing, and quantitative immunoassays. Among the panel of statistically significant markers were Clara cell protein, calgranulin A, human neutrophil peptides, and the antimicrobial agent histatin. To assess their clinical relevance, a highly sensitive and specific classifier model was developed. Positive BOS classification by monitoring of seven polypeptides correlated strongly with a significant decrease in BOS-free time. Thus, it was possible to detect high-risk patients early on in the pathogenetic process. Monitoring the bronchoalveolar lavage fluid levels of seven polypeptides detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allows a reliable prediction of early BOS using a Random Forest decision tree-based classifier model. The high accuracy of this robust model and its synergistic potential in combination with established forced expiratory volume-based diagnostics could make it an effective tool to supplement the current diagnostic regime after multicentric validation.

• Bronchoalveolar lavage fluid of lung cancer patients: mapping the uncharted waters using proteomics technology.

  Authors: Tonio Oumeraci, Bernd Schmidt, Thomas Wolf, Marc Zapatka, Andreas Pich, Benedikt Brors, Roland Eils, Michael Fleischhacker, Brigitte Schlegelberger, Nils von Neuhoff

  Lung cancer (Amsterdam, Netherlands). 02/2011; 72(1):136-8.

  The search for proteome-level markers of non-small cell lung cancer (NSCLC) has been mainly limited to serum or cell line screening approaches up to this point. We would like to demonstrate by thisThe search for proteome-level markers of non-small cell lung cancer (NSCLC) has been mainly limited to serum or cell line screening approaches up to this point. We would like to demonstrate by this proof-of-principle study investigating bronchoalveolar lavage fluid samples from a cohort of NSCLC and control patients, that this readily available biofluid might be a more suitable source for discovering clinically usable NSCLC biomarkers.

• Endo- and exopolygalacturonases of Ralstonia solanacearum are inhibited by polygalacturonase-inhibiting protein (PGIP) activity in tomato stem extracts.

  Authors: Tanja Schacht, Christoph Unger, Andreas Pich, Kerstin Wydra

  Plant physiology and biochemistry : PPB / Société française de physiologie végétale. 02/2011; 49(4):377-87.

  Polygalacturonases (PGs) of wild-type and non-virulent phenotype conversion mutant (PC) strains of Ralstonia solanacearum were compared by investigating their activities and their inhibition byPolygalacturonases (PGs) of wild-type and non-virulent phenotype conversion mutant (PC) strains of Ralstonia solanacearum were compared by investigating their activities and their inhibition by polygalacturonase-inhibiting proteins (PGIPs) from tomato stems. In cultures of wild-type strain ToUdk2, slimy (s), retarded slimy (rs) and non-slimy (ns) colonies appeared. The conversion of the 's' into the 'rs' colony form coincided with the beginning of PG production. PG activity of the PC strain increased about 5 h earlier (at 6 hpi), and was up to 35 times higher in media supplemented with two different tomato stem extracts or polygalacturonic acid, compared to the wild-type at 6 hpi, and generally 4-8 times higher across test media and time. By hydrophobic interaction chromatography (HIC), fluorophor-assisted carbohydrate-polyacrylamid-gel electrophoresis (FACE-PAGE) and mass spectrometry analyses, endo-PG PehA, exo-PGs PehB and PehC were identified. PGs of the PC mutant consisted mainly of endo-PG. The increased PG production after supplementing the medium with tomato cell wall extract was reflected by a higher activity of exo-PGs for both strains. Total PGs (endo-PG and exo-PGs) activities were inhibited by PGIPs of tomato stem extracts. PGIP activity was concentration dependent, constitutively present, and not related to resistance nor susceptibility of tomato recombinant inbred lines to R. solanacearum. The proteinaceous character of the inhibiting component was inferred from ammonium sulphate precipitation. For the first time a plant PGIP activity against a bacterial pathogen is reported. Observations support that endo- and exo-PG synthesis is governed by a sensitive regulatory network, which, in interaction with PGIP and cell wall degradation products, leads to generation or avoidance of elicitor-active oligomers, and, thus, may contribute to the development of the compatible or incompatible interaction.

• A universal matrix-assisted laser desorption/ionization cleavable cross-linker for protein structure analysis.

  Authors: Mathias Q Müller, Johannes J Zeiser, Frank Dreiocker, Andreas Pich, Mathias Schäfer, Andrea Sinz

  Rapid communications in mass spectrometry : RCM. 01/2011; 25(1):155-61.

  The concept of protein cross-linking in combination with mass spectrometry holds great promise to derive structural information on protein conformation and protein-protein interactions. We recentlyThe concept of protein cross-linking in combination with mass spectrometry holds great promise to derive structural information on protein conformation and protein-protein interactions. We recently presented a dissociative amine-reactive cross-linker (NHS-BuUrBu-NHS) that is shown herein to be universally applicable to protein structure analysis under matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) conditions, based on the examples of the peptides substance P, luteinizing hormone releasing hormone (LHRH), and the 32-kDa ligand-binding domain of peroxisome proliferator-activated receptor alpha (PPARα). The characteristic fragment ion patterns and constant neutral losses of the cross-linker greatly simplify the identification of different cross-linked species from complex mixtures and drastically reduce the potential of identifying false-positive cross-links. Therefore, this cross-linker holds an enormous potential for deriving structural information of proteins and protein complexes in a highly automated fashion.

• Autoproteolytic cleavage mediates cytotoxicity of Clostridium difficile toxin A.

  Authors: Isa Kreimeyer, Friederike Euler, Alexander Marckscheffel, Helma Tatge, Andreas Pich, Alexandra Olling, Janett Schwarz, Ingo Just, Ralf Gerhard

  Naunyn-Schmiedeberg's archives of pharmacology. 11/2010; 383(3):253-62.

  Toxin A and toxin B from Clostridium difficile are the causative agents of the antibiotic-associated pseudomembranous colitis. They are of an A/B structure type and possess inositolToxin A and toxin B from Clostridium difficile are the causative agents of the antibiotic-associated pseudomembranous colitis. They are of an A/B structure type and possess inositol hexakisphosphate-inducible autoproteolytic activity to release their glucosyltransferase domain to the cytoplasm of target cells. In this study, we investigated the effect of extracellular and intracellular autoproteolytic cleavage on the function of TcdA. Extracellular cleavage led to functional inactivation albeit TcdA was less susceptible to inositol hexakisphosphate-induced autoproteolysis than TcdB. A non-cleavable TcdA mutant (TcdA A541 G542 A543) was generated to investigate whether autoproteolysis is a prerequisite for intracellular function of TcdA. Although the EC(50) regarding cell rounding was about 75-fold reduced in short-term assay, non-cleavable TcdA was able to induce complete cell rounding and apoptosis after 36 h comparable to wildtype TcdA when continuously present. Studies with limited uptake of toxins revealed progressive Rac1 glucosylation and complete cell rounding for TcdA, whereas the effect induced by non-cleavable TcdA was reversible. These findings argue for cytosolic accumulation of the released glucosyltransferase domain of wild-type TcdA and rapid degradation of the non-cleavable TcdA. In summary, extracellular cleavage functionally inactivates TcdA (and TcdB), whereas intracellular autoproteolytic cleavage is not essential for function of TcdA but defines its potency.

• Site-directed mutagenesis of the χ subunit of DNA polymerase III and single-stranded DNA-binding protein of E. coli reveals key residues for their interaction.

  Authors: Natalie Naue, Roman Fedorov, Andreas Pich, Dietmar J Manstein, Ute Curth

  Nucleic acids research. 10/2010; 39(4):1398-407.

  During DNA replication in Escherichia coli, single-stranded DNA-binding protein (SSB) protects single-stranded DNA from nuclease action and hairpin formation. It is known that the highly conservedDuring DNA replication in Escherichia coli, single-stranded DNA-binding protein (SSB) protects single-stranded DNA from nuclease action and hairpin formation. It is known that the highly conserved C-terminus of SSB contacts the χ subunit of DNA polymerase III. However, there only exists a theoretical model in which the 11 C-terminal amino acids of SSB have been docked onto the surface of χ. In order to refine this model of SSB/χ interaction, we exchanged amino acids in χ and SSB by site-directed mutagenesis that are predicted to be of key importance. Detailed characterization of the interaction of these mutants by analytical ultracentrifugation shows that the interaction area is correctly predicted by the model; however, the SSB C-terminus binds in a different orientation to the χ surface. We show that evolutionary conserved residues of χ form a hydrophobic pocket to accommodate the ultimate two amino acids of SSB, P176 and F177. This pocket is surrounded by conserved basic residues, important for the SSB/χ interaction. Mass spectrometric analysis of χ protein cross-linked to a C-terminal peptide of SSB reveals that K132 of χ and D172 of SSB are in close contact. The proposed SSB-binding site resembles those described for RecQ and exonuclease I.

• Analysis of cell wall proteins regulated in stem of susceptible and resistant tomato species after inoculation with Ralstonia solanacearum: a proteomic approach.

  Authors: Diwakar Dahal, Andreas Pich, Hans Peter Braun, Kerstin Wydra

  Plant molecular biology. 05/2010; 73(6):643-58.

  Proteomics approach was used to elucidate the molecular interactions taking place at the stem cell wall level when tomato species were inoculated with Ralstonia solanacearum, a causative agent ofProteomics approach was used to elucidate the molecular interactions taking place at the stem cell wall level when tomato species were inoculated with Ralstonia solanacearum, a causative agent of bacterial wilt. Cell wall proteins from both resistant and susceptible plants before and after the bacterial inoculation were extracted from purified cell wall with salt buffers and separated with 2-D IEF/SDS-PAGE and with 3-D IEF/SDS/SDS-PAGE for basic proteins. The gels stained with colloidal Coomassie revealed varied abundance of protein spots between two species (eight proteins in higher abundance in resistant and six other in susceptible). Moreover, proteins were regulated differentially in response to bacterial inoculation in resistant (seven proteins increased and eight other decreased) as well as in susceptible plants (five proteins elevated and eight other suppressed). Combination of MALDI-TOF/TOF MS and LC-ESI-IonTrap MS/MS lead to the identification of those proteins. Plants responded to pathogen inoculation by elevating the expression of pathogenesis related, other defense related and glycolytic proteins in both species. However, cell wall metabolic proteins in susceptible, and antioxidant, stress related as well as energy metabolism proteins in resistant lines were suppressed. Most of the proteins of the comparative analysis and other randomly picked spots were predicted to have secretion signals except some classical cytosolic proteins.

• Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix.

  Authors: Nina Luecke, Christian Templin, Marika Victoria Muetzelburg, Detlef Neumann, Ingo Just, Andreas Pich

  Rapid communications in mass spectrometry : RCM. 03/2010; 24(5):561-70.

  Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrineAdministration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1alpha (MIP-1alpha) and monocyte-chemoattractant protein-1beta (MIP-1beta) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration.

• Characterization of the acidic N-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach.

  Authors: Dorothee von Witzendorff, Kai Maass, Andreas Pich, Silja Ebeling, Sabine Kölle, Christine Kochel, Mahnaz Ekhlasi-Hundrieser, Hildegard Geyer, Rudolf Geyer, Edda Töpfer-Petersen

  Carbohydrate research. 06/2009;

  Oocyte maturation is a prerequisite for successful fertilization. Growing evidence suggests that not only the oocyte but also the surrounding zona pellucida has to undergo maturational changes. InOocyte maturation is a prerequisite for successful fertilization. Growing evidence suggests that not only the oocyte but also the surrounding zona pellucida has to undergo maturational changes. In the pig, two-dimensional electrophoretic analysis demonstrated an acidic shift of the zona pellucida glycoproteins of about 1.5-2.0 pH units during the maturation process. These findings were corroborated by histological studies that indicated the synthesis of acidic glycoconjugates in the cumulus cells and an increased occurrence of acidic glycans in the zona pellucida after oocyte maturation. In order to provide structural data on prepuberal zona pellucida N-glycosylation, N-glycans were released from prepuberal zona pellucida glycoproteins by N-glycosidase F and studied by mass spectrometry before and after desialylation and treatment with endo-beta-galactosidase. Our results verified the presence of high-mannose-type Man(5)GlcNAc(2) compounds as well as diantennary N-glycans as major neutral species, whereas sialylated diantennary and triantennary species constituted the dominant non-sulfated acidic sugar chains. The major acidic N-glycans of prepuberal animals, however, represented mono-sulfated diantennary, triantennary and tetraantennary oligosaccharides carrying, in part, N-acetyllactosamine repeating units as well as additional Neu5Ac or Neu5Gc residues. Glycans comprising more than one sulfate residue were not detected. In contrast to the literature data on zona pellucida glycoprotein-N-glycans of cyclic animals, our data thus reveal a lower degree in glycan sulfation of the prepuberal zona pellucida.

• Threonine 66 in the death domain of IRAK-1 is critical for interaction with signaling molecules but is not a target site for autophosphorylation.

  Authors: Detlef Neumann, Christian Kollewe, Andreas Pich, Ping Cao, Klaus Resch, Michael U Martin

  Journal of leukocyte biology. 09/2008; 84(3):807-13.

  Ligand binding in the TLR/IL-1R family results in the transient formation of an intracellular signaling complex, which contains, amongst others, the serine/threonine-specific kinase IL-1R-associatedLigand binding in the TLR/IL-1R family results in the transient formation of an intracellular signaling complex, which contains, amongst others, the serine/threonine-specific kinase IL-1R-associated kinase 1 (IRAK-1). Concomitantly, the kinase function of IRAK-1 becomes activated, resulting in massive autophosphorylation and finally in the dissociation of the initially constituted signaling complex. The death domain (DD) of IRAK-1 mediates the interaction with other molecules of the signaling complex, e.g., the adaptor MyD88, the silencer Tollip, and the activator kinase IRAK-4. The conserved threonine at position 66 (T66), located within the DD, is a putative autophosphorylation target site. Here, we provide evidence that T66 critically impacts the secondary structure of the IRAK-1 DD. Thereby, it ensures the transient manner of interactions between IRAK-1 and the other signaling molecules. This essential role, however, is not regulated by phosphorylation of T66 itself.

• Experimental bronchial asthma - the strength of the species rat.

  Authors: Thomas Tschernig, Detlef Neumann, Andreas Pich, Martina Dorsch, Reinhard Pabst

  Current drug targets. 07/2008; 9(6):466-9.

  No single animal species reflects the complete range of human respiratory anatomy, physiology and - often not mentioned - age-related changes. The rat was the first experimental asthma model, but wasNo single animal species reflects the complete range of human respiratory anatomy, physiology and - often not mentioned - age-related changes. The rat was the first experimental asthma model, but was overtaken in numbers by murine models many years ago. Data will be compiled to document that the rat model still has an important role in the research of bronchial asthma and other lung diseases. In pharmaceutical research for new drugs the rat model is still indispensable. Here specific aspects will be highlighted where the rat offers advantages in comparison to other species: the anatomical feature of the proprietary bronchial circulation, genetics and proteomics, lung function and finally economical considerations.

• Distinct contributions of beta 4GalNAcTA and beta 4GalNAcTB to Drosophila glycosphingolipid biosynthesis.

  Authors: Anita Stolz, Nicola Haines, Andreas Pich, Kenneth D Irvine, Cornelis H Hokke, André M Deelder, Rita Gerardy-Schahn, Manfred Wuhrer, Hans Bakker

  Glycoconjugate journal. 03/2008; 25(2):167-75.

  Drosophila melanogaster has two beta4-N-acetylgalactosaminyltransferases, beta4GalNAcTA and beta4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcbeta,4GlcNAc). LacdiNAc is foundDrosophila melanogaster has two beta4-N-acetylgalactosaminyltransferases, beta4GalNAcTA and beta4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcbeta,4GlcNAc). LacdiNAc is found as a structural element of Drosophila glycosphingolipids (GSLs) suggesting that beta4GalNAcTs contribute to the generation of GSL structures in vivo. Mutations in Egghead and Brainaic, enzymes that generate the beta4GalNAcT trisaccharide acceptor structure GlcNAcbeta,3Manbeta,4GlcbetaCer, are lethal. In contrast, flies doubly mutant for the beta4GalNAcTs are viable and fertile. Here, we describe the structural analysis of the GSLs in beta4GalNAcT mutants and find that in double mutant flies no lacdiNAc structure is generated and the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer accumulates. We also find that phosphoethanolamine transfer to GlcNAc in the trisaccharide does not occur, demonstrating that this step is dependent on prior or simultaneous transfer of GalNAc. By comparing GSL structures generated in the beta4GalNAcT single mutants we show that beta4GalNAcTB is the major enzyme for the overall GSL biosynthesis in adult flies. In beta4GalNAcTA mutants, composition of GSL structures is indistinguishable from wild-type animals. However, in beta4GalNAcTB mutants precursor structures are accumulating in different steps of GSL biosynthesis, without the complete loss of lacdiNAc, indicating that beta4GalNAcTA plays a minor role in generating GSL structures. Together our results demonstrate that both beta4GalNAcTs are able to generate lacdiNAc structures in Drosophila GSL, although with different contributions in vivo, and that the trisaccharide GlcNAcbeta,3Manbeta,4GlcbetaCer is sufficient to avoid the major phenotypic consequences associated with the GSL biosynthetic defects in Brainiac or Egghead.

• A tetraantennary glycan with bisecting N-acetylglucosamine and the Sd(a) antigen is the predominant N-glycan on bovine pregnancy-associated glycoproteins.

  Authors: Karl Klisch, Evelyne Jeanrond, Poh-Choo Pang, Andreas Pich, Gerhard Schuler, Vibeke Dantzer, Mariusz P Kowalewski, Anne Dell

  Glycobiology. 02/2008; 18(1):42-52.

  Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ruminants. Binucleate trophoblast giant cells (BNCs) store these proteins in secretory granules andPregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ruminants. Binucleate trophoblast giant cells (BNCs) store these proteins in secretory granules and release them into the maternal organism after fusion with maternal uterine epithelial cells. By matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) analysis and linkage analysis, we show that by far, the most abundant N-glycan of PAGs in midpregnancy is a tetraantennary core-fucosylated structure with a bisecting N-acetylglucosamine (GlcNAc). All four antennae consist of the Sd(a)-antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc-). Immunohistochemistry with the mono- clonal antibody CT1, which recognizes the Sd(a)-antigen, shows that BNC granules contain the Sd(a)-antigen from gestation day (gd) 32 until a few days before parturition. Lectin histochemistry with Maackia amurensis lectin (MAL), which binds to alpha2-3sialylated lactosamine, shows that BNC granules are MAL-positive prior to gd 32 and also at parturition. The observed tetraantennary glycan is a highly unusual structure, since during the synthesis of N-glycans, the insertion of a bisecting GlcNAc inhibits the activity of the GlcNAc-transferases that leads to tri- and tetraantennary glycans. The study defines the substantial changes of PAG N-glycosylation in the course of pregnancy. This promotes the hypothesis that PAGs may have different carbohydrate-mediated functions at different stages of pregnancy.

• Peptidomic analysis of breast cancer reveals a putative surrogate marker for estrogen receptor-negative carcinomas.

  Authors: Frank Traub, Marco Jost, Rüdiger Hess, Karl Schorn, Christoph Menzel, Petra Budde, Peter Schulz-Knappe, Norbert Lamping, Andreas Pich, Hans Kreipe, Harald Tammen

  Laboratory investigation; a journal of technical methods and pathology. 04/2006; 86(3):246-53.

  Estrogen-receptor status provides a major biomarker in breast cancer classification and has an important impact on prognosis and treatment options. The aim of this study was to investigate peptideEstrogen-receptor status provides a major biomarker in breast cancer classification and has an important impact on prognosis and treatment options. The aim of this study was to investigate peptide profiles of invasive breast cancer with positive (n=39) and negative receptor status (n=41). Peptide profiles were generated by 'Differential Peptide Display', which is an offline-coupled combination of reversed-phase-HPLC and MALDI mass spectrometry. Mass spectrometric data were correlated with the immunohistochemically determined receptor state. Identification of peptides of interest was carried out by additional mass spectrometric methods (eg MALDI-TOF-TOF-MS-MS). Approximately 3000-7000 signals were detected per sample and thymosin alpha-1, an asparaginyl endopeptidase generated cleavage product of the ubiquitous acidic protein prothymosin-alpha, was found to differentiate the tumor samples according to their receptor status with the highest specificity. The concentration of Thymosin alpha-1 was found to be upregulated (n=37) in estrogen-negative cancer samples and downregulated (n=32) in estrogen-positive breast cancer samples. The expression of the precursor protein (Prothymosin-alpha) has been discussed previously as a prognostic factor in breast cancer. It is involved in the ER signal transduction pathway as an anti-coactivator-inhibitor. From our findings we conclude that Thymosin alpha-1 could serve as a surrogate marker in breast cancers and may indicate ER functionality.

• High-resolution genomic profiling reveals association of chromosomal aberrations on 1q and 16p with histologic and genetic subgroups of invasive breast cancer.

  Authors: Daniel E Stange, Bernhard Radlwimmer, Falk Schubert, Frank Traub, Andreas Pich, Grischa Toedt, Frank Mendrzyk, Ulrich Lehmann, Roland Eils, Hans Kreipe, Peter Lichter

  Clinical cancer research : an official journal of the American Association for Cancer Research. 02/2006; 12(2):345-52.

  PURPOSE: Invasive ductal carcinoma and invasive lobular carcinoma (ILC) represent the major histologic subtypes of invasive breast cancer. They differ with regard to presentation, metastatic spread,PURPOSE: Invasive ductal carcinoma and invasive lobular carcinoma (ILC) represent the major histologic subtypes of invasive breast cancer. They differ with regard to presentation, metastatic spread, and epidemiologic features. To elucidate the genetic basis of these differences, we analyzed copy number imbalances that differentiate the histologic subtypes.EXPERIMENTAL DESIGN: High-resolution genomic profiling of 40 invasive breast cancers using matrix-comparative genomic hybridization with an average resolution of 0.5 Mb was conducted on bacterial artificial chromosome microarrays. The data were subjected to classification and unsupervised hierarchical cluster analyses. Expression of candidate genes was analyzed in tumor samples.RESULTS: The highest discriminating power was achieved when combining the aberration patterns of chromosome arms 1q and 16p, which were significantly more often gained in ILC. These regions were further narrowed down to subregions 1q24.2-25.1, 1q25.3-q31.3, and 16p11.2. Located within the candidate gains on 1q are two genes, FMO2 and PTGS2, known to be overexpressed in ILC relative to invasive ductal carcinoma. Assessment of four candidate genes on 16p11.2 by real-time quantitative PCR revealed significant overexpression of FUS and ITGAX in ILC with 16p copy number gain. Unsupervised hierarchical cluster analysis identified three molecular subgroups that are characterized by different aberration patterns, in particular concerning gain of MYC (8q24) and the identified candidate regions on 1q24.2-25.1, 1q25.3-q31.3, and 16p11.2. These genetic subgroups differed with regard to histology, tumor grading, frequency of alterations, and estrogen receptor expression.CONCLUSIONS: Molecular profiling using bacterial artificial chromosome arrays identified DNA copy number imbalances on 1q and 16p as significant classifiers of histologic and molecular subgroups.