@phdthesis{Duraphe2010,
  author    = {Duraphe, Prashant},
  title     = {Identification and characterization of AUM, a novel human tyrosine phosphatase},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-44256},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Protein Phosphatasen werden aufgrund der Aminos{\"a}uresequenzen ihrer aktiven Zentren in drei große Familien unterteilt. In einer neu entdeckten Familie von Phosphatasen ist das aktive Zentrum durch die Sequenz DXDX(T/V) charakterisiert. Diese Aspartat-abh{\"a}ngigen Phosphatasen geh{\"o}ren zu der Superfamilie der Hydrolasen vom Haloazid Dehalogenase(HAD)-Typ, einer evolution{\"a}r konservierten und ubiquit{\"a}r verbreiteten Enzymfamilie. Bislang konnten 58 menschliche HAD Enzyme durch Datenbankanalysen identifiziert werden. Ihre Funktionen sind jedoch nach wie vor nur rudiment{\"a}r verstanden. Im Rahmen dieser Arbeit wurde zun{\"a}chst das Komplement aller menschlichen HAD Phosphatasen durch Datenbank-Recherchen erfasst. Zusammen mit phylogenetischen Analysen gelang es, eine zum damaligen Zeitpunkt unbekannte, putative Phosphatase zu identifizieren, die eine vergleichsweise hohe Sequenz-Homologie zu der Zytoskelettregulierenden HAD Phosphatase Chronophin aufweist. Dieses neuartige Enzym wurde kloniert und mit biochemischen und zellbiologischen Methoden charakterisiert. Auf der Basis dieser Befunde bezeichnen wir dieses neuartige Protein als AUM (actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase).Mittels Northern blot, real-time PCR und Western blot Analysen konnte gezeigt werden, dass AUM in allen untersuchten menschlichen und murinen Geweben exprimiert wird. Die h{\"o}chste Expression konnte in Hodengewebe nachgewiesen werden. Durch immunohistochemische Untersuchungen konnte gezeigt werden, dass AUM spezifisch in reifenden Keimzellen mit einem Expressionsmaximum zum Zeitpunkt der Spermiogenese exprimiert wird. Um die Substratpr{\"a}ferenz von AUM zu charakterisieren, wurde zun{\"a}chst ein peptidbasierter in vitro Phosphatase-Substrat-Screen durchgef{\"u}hrt. Hierbei wurden 720 aus menschlichen Phosphoproteinen abgeleitete Phosphopeptide untersucht. Interessanterweise dephosphorylierte AUM ausschließlich Phosphotyrosin (pTyr)-enthaltende Peptide. Nur 17 pTyr-Peptide (~2\% aller untersuchten Peptide) fungierten als AUM-Substrate. Diese Daten legen eine hohe Substratspezifit{\"a}t von AUM nahe. Zu den putativen AUM Substraten geh{\"o}ren Proteine, die in die Dynamik der Zytoskelett-Reorganisation sowie in Tyrosin Kinasevermittelte Signalwege eingebunden sind. In {\"U}bereinstimmung mit den Ergebnissen dieses Phosphopeptid-Screens konnte mittels Phosphatase overlay assays sowie in Zellextrakten aus Pervanadat-behandelten HeLa Zellen demonstriert werden, dass AUM eine begrenzte Anzahl Tyrosin-phosphorylierter Proteinen dephosphorylieren kann.In zellul{\"a}ren Untersuchungen wurde die m{\"o}gliche Rolle von AUM im Rahmen der durch den epidermalen Wachstumsfaktor (EGF) ausgel{\"o}sten Tyrosin-Phosphorylierung in einer Spermatogonien Zelllinie (GC-1 spg-Zellen) analysiert. So konnte nachgewiesen werden, dass die {\"U}berexpression von AUM zu einer moderaten Abnahme Tyrosin phosphorylierter Proteine nach EGF-Stimulation f{\"u}hrte. Im Gegensatz dazu l{\"o}ste jedoch die durch RNAInterferenz vermittelte Depletion von endogenem AUM einen robusten Anstieg Tyrosinphosphorylierter Proteine aus, zu denen auch der EGF-Rezeptor selbst z{\"a}hlt. Zus{\"a}tzlich zu dem EGF-Rezeptor wurde die Src-Kinase im Zuge des Phosphopeptid- Screens als m{\"o}gliches AUM Substrat identifiziert. Daher wurden in vitro Kinase/Phosphatase-Assays mit gereinigtem Src und AUM durchgef{\"u}hrt. Mit diesem Ansatz konnte erstmals gezeigt werden, dass AUM in der Lage ist, die Src-Kinase zu aktivieren, w{\"a}hrend Src AUM phosphoryliert und die AUM Phosphatase-Aktivit{\"a}t blockiert. Diese Ergebnisse deuten auf eine gekoppelte, wechselseitige Regulation von AUM und Src hin. Obwohl die Details dieser Regulation derzeit noch unklar sind, zeigen unsere initialen Ergebnisse, dass AUM die Src-Aktivit{\"a}t unabh{\"a}ngig von seiner Phosphatase Aktivit{\"a}t steigert, w{\"a}hrend Src die AUM Phosphatase-Aktivit{\"a}t Kinase-abh{\"a}ngig vermindert. Auf zellul{\"a}rer Ebene sind AUM-depletierte Zellen durch Ver{\"a}nderungen der Aktin- Zytoskelett-Dynamik und der Zelladh{\"a}sion charakterisiert. So weisen AUM-defiziente Zellen stabilisierte Aktin Streßfasern und vergr{\"o}ßerte fokale Adh{\"a}sionen auf. Weiterhin sind AUMdepletierte Zellen durch ein beschleunigtes spreading auf Fibronektin gekennzeichnet. Wir haben mit AUM ein bisher nicht beschriebenes Mitglied der Familie Aspartat-abh{\"a}ngiger Phosphatasen entdeckt. In dieser Arbeit ist es gelungen, AUM phylogenetisch, biochemisch und zellbiologisch zu charakterisieren. Unsere Ergebnisse legen nahe, dass AUM einen wichtigen, neuartigen Regulator der Src-vermittelten Zytoskelett-Dynamik im Rahmen der Zelladh{\"a}sion und Migration darstellt.},
  subject      = {Tyrosin},
  language  = {en}
}
@article{ShephardSengstagLutzetal.1993,
  author    = {Shephard, S. E. and Sengstag, C. and Lutz, Werner K. and Schlatter, C.},
  title     = {Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60683},
  year      = {1993},
  abstract  = {2-Acetylaminofluorene (2-AAF) was administered at Ievels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic micc bearing the lacl genein a Iambda vector (Big Blue® mice). The Iambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10\(^5\) plaques wcre screened per animal for the appearance of a bluc colour, indicative of mutations in the lac/ gcnc which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10\(^{-5}\) (pooled results of two animals, 8 mutant plaques/289 530 plaques). At 300 ppm in the diet, the rate of 3.5 X 10\(^{-5}\)(8/236 300) was not significantly increased over background. At 600 ppm in the dict, the rate increased approximately 3 fold to 7.7 x 10\(^{-5}\) (17 /221240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose Ievels 00-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2·AAF.},
  subject      = {Toxikologie},
  language  = {en}
}
@article{FischerBelandLutz1993,
  author    = {Fischer, W. H. and Beland, P. E. and Lutz, Werner K.},
  title     = {DNA adducts, cell proliferation and papilloma latency time in mouse skin after repeated dermal application of DMBA and TPA},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60673},
  year      = {1993},
  abstract  = {'lbe mouse skin tumor model was used to investigate whether the Ievel of DNA 8dducts and/or the rate of cell division in the epidermis are indicators of the risk of cancer formation for an individual in an outbred animal popul8tion. A high risk was considered to be reftected by 8 short latency period for the 8ppearance of 8 papilloma. Fernale NMRI mice were treated twice weekly with 2.5 nmol 7 ,12-dimethylbenz[a]antbracene (DMBA) and 3 nmoi12-0-tetradecanoylphorbol-13- 8cetate (TPA) and the appearance of papillomas was registered. The first papilloma 8ppeared after 7.5 weeks. After 17 weeks, when 12 of 14 mice bad 8t least one papilloma, an osmotic minipump deliverlog 5-bromo-2'deoxyuridine (BrdU) was implanted into eacb mouse for 24 h. The mice were killed after 24 h ~d the epidermis was analyzed for D:MBA-nucleotide 8dducts by 32p.postlabeling, for the cell number per unit skin length, and for the labeling index for DNA synthesls. Unexpectedly, D:MBA-nucleotide 8dduct Ievels were highest in those anima1s wbich showed the Iongest latency periods. Adduct Ievels were negatively correlated with the 18beling index, indicating that dilution of adducts by cell division was a predominant factor in determining average adduct concentrations. Individual tumor-latency time was not corTelated with either cell ntunber or labeling index. This could be due to the fact that the measurements only provided 8veraged data and gave no infonnation on the specific situation in clones of premalignant cells. Under the conditions of tbis assay, therefore, neither DNA adduct Ievels nor information on the average kinetics of cell division bad a predidive value for the individual amcer risk withln a group of outbred animals receiving the same treatment},
  subject      = {Toxikologie},
  language  = {en}
}
@article{CantoreggiDietrichLutz1993,
  author    = {Cantoreggi, S. and Dietrich, D. R. and Lutz, Werner K.},
  title     = {Induction of cell proliferation in the forestomach of F344 rats following subchronic administration of styrene 7,8-oxide and butylated hydroxyanisole},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60669},
  year      = {1993},
  abstract  = {The question addressed was whether Stimulation of cell proliferation could be responsible for tumor induction in the torestornach by styrene 7,8-oxide (SO). Male F344 rats were treated for 4 weeks with 0, 137,275, and 550 mglkg SO by p.o. gavage 3 times/week. Positive controls received 0, 0.5, I, and 2\% butylated hydroxyanisole (BHA) in the diet for 4 weeks. Twenty-four h before termination of the experlment, the rats were implanted s.c. with an osmotic minipump deliverlog S-bromo-2'-deoxyuri· dine (BrdU). Cell proliferation in the forestomach was assessed by immunohistochemistry for BrdU incorporated into DNA. Cell number/mm section length and fraction of replicating cells (labeling Index) were determined in 3 domains of the forestomach, the saccus caecus, the midregion, and the prefundic region. With the exception of the prefundic reglon of the low-dose SO group, a significant increase of the labeling index was found in all regions both with SO and BHA. Rats treated with BHA showed, in addition, a dose-dependent increase in number and size of hyperplastic lesions. This was most pronounced in the prefundic region where carcinomas were reported to be localized. In this region, the number of dividing cells/mm section length was increased up to 17-fold. With SO, only marginal morphological changes were occasionally observed, despite the fact that the respective long-term treatment bad been reported to result in a higher carcinoma incidence than treatment with BHA. It ls concluded that the rate of replicating cells alone, numerically expressed by the labeling Index, is an lnsufficient tool for interpretlog the role of cell division in carcinogenesis. It is postulated that SO and BHA induce forestomach tumors via different mechanisms. While hyperplasia in the prefundic region most likely dominates the carcinogenicity of BHA, a mechanism combining marginal genotoxicity with strong promotion by increased cell proliferation appears to be involved in the tumorigenic action of SO.},
  subject      = {Toxikologie},
  language  = {en}
}
@article{GrunickePyerinEisenbrandetal.1994,
  author    = {Grunicke, H. and Pyerin, W. and Eisenbrand, G. and Havemann, K. and Rabes, H. M. and Molling, K. and Schwab, M. and Lutz, Werner K. and Wahrendorf, J. and Schirrmacher, V.},
  title     = {7th International Symposium of the Division of Experimental Cancer Research (AEK) of the German Cancer Society : [Meeting report]},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60651},
  year      = {1994},
  abstract  = {No abstract available},
  subject      = {Toxikologie},
  language  = {en}
}
@article{FischerLutz1994,
  author    = {Fischer, W. H. and Lutz, Werner K.},
  title     = {Short communication : Mouse skin papilloma formation by chronic dermal application of 7,12-dimethylbenz[a]anthracene is not reduced by diet restriction},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60644},
  year      = {1994},
  abstract  = {No abstract available},
  subject      = {Toxikologie},
  language  = {en}
}
@article{ShephardLutzSchlatter1994,
  author    = {Shephard, S. E. and Lutz, Werner K. and Schlatter, C.},
  title     = {The lacI transgenic mouse mutagenicity assay: quantitative evaluation in comparison to tests for carcinogenicity and cytogenetic damage in vivo},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60638},
  year      = {1994},
  abstract  = {The detection Iimit of the lacl transgenic mouse mutagenicity assay lies, in practice, at approximately a 50-100\% increase in mutant frequency in treated animals over controls. The sensitivity of this assay in detecting genotoxins can be markedly improved by subchronic rather than acute application of the test compound. The lac/ transgenic mouse mutagenicity assay was compared quantitatively to rodent carcinogenicity tests and to presently used in vivo mutagenicity assays. With the genotoxic carcinogens tested thus far, a rough correlation between mutagenic potency and carcinogenic potency was observed: on average, to obtain a doubling in lacl mutant frequency the mice bad to be treated with a total dose equal to 50 times the TD50 daily dose Ievel. This total dose could be administered eilher at a high dose rate within a few days or, preferably, at a low dose rate over several weeks. This analysis also indicated that a lacl experiment using a 250-day exposure period would give a detection Iimit approximately equal to that of a long-term carcinogenicity study. In comparison to the micronucleus test or the chromosome aberration assay, acute sturlies with the presently available lacl system offered no increase in sensitivity. However, subchronic lacl sturlies (3-4-month exposure) resulted in an increase in sensitivity over the established tests by 1-2 orders of magnitude (shown with 2-acetylaminofluorene, N-nitrosomethylamine, N-nitrosomethylurea and urethane). 1t is concluded that a positive result in the lacl test can be highly predictive of carcinogenicity butthat a negative result does not provide a large margin of safety.},
  subject      = {Toxikologie},
  language  = {en}
}
@article{KlotzJesaitis1994,
  author    = {Klotz, Karl-Norbert and Jesaitis, A. J.},
  title     = {The interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton is energy-dependent},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60499},
  year      = {1994},
  abstract  = {Desensitization of N-fonnyl peptide chemoattractant receptors (FPR) in human neutrophils is thought to be achieved by lateral segregation of receptors and G proteins within the plane of the plasma membrane resulting in an interruption of the signalling cascade. Direct coupling of FPR to membrane skeletal actin appears to be the basis of this process~ however, the molecular mechanism is unknown. In this study we investigated the effect of energy depletion on formation of FPR-membrane skeleton complexes. In addition the effect of the protein kinase C inhibitor stauroporine and the phosphatase inhibitor okadaic acid on coupling of FPR to the membrane skeletonwas studied. Human neutrophils were desensitized using the photoreactive agonist N-formy1-met-leu-phe-1ys-N'[\(^{125}\)I]2(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (fMLFK-[\(^{125}\)I]ASD) after ATP depletion with NaF or after incubation with the respective inhibitors. The interaction of FPR with the membrane skeleton was studied by Sedimentation of the membrane skeleton-associated receptors in sucrose density gradients. Energy depletion of the cells markedly inhibited the formation of FPR-membrane skeleton complexes. This does not appear tobe related to inhibition of protein phosphorylation due to ATP depletion because inhibition of protein kinases and phosphatases bad no significant effect on coupling of FPR to the membrane skeleton. We conclude, therefore, that coupling of FPR to the membrane skeleton is an energy,dependent process which does not appear to require modification of the receptor protein by phosphorylation.},
  subject      = {Toxikologie},
  language  = {en}
}
@article{KlotzJesaitis1994,
  author    = {Klotz, Karl-Norbert and Jesaitis, A. J.},
  title     = {Physical coupling of N-formyl peptide chemoattractant receptors to G protein is not affected by desensitization},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60483},
  year      = {1994},
  abstract  = {Desensitization of N-formyl peptide chemoattractant receptors (FPR) in human neutrophils results in association of these receptors to the membrane skeleton. This is thought to be the critical event in the lateral segregation of receptors and guanyl nucleotide-binding proteins (G proteins) within the plane of the plasma membrane resulting in an interruption of the signaling cascade. In this study we probed the interaction of FPR with G protein in human neutrophils that were desensitized to various degrees. Human neutrophils were desensitized using the photoreactive agonist N-formyl-met-leu-phelys- N\(^\epsilon\)-[\(^{125}\)I]2(p-azidosalicylamido )ethyl-1 ,3 '-dithiopropionate (/MLFK-[\(^{125}\)I]ASD). The interaction if FPR with G protein was studied via a reconstitution assay and subsequent analysis of FPR-G protein complexes in sucrose density gradients. FPR-G protein complexes were reconstituted with solubilized FPR from partially and fully desensitized neutrophils with increasing concentrations of Gi purified from bovine brain. The respective EC\(_{50}\) values for reconstitution were similar to that determined for FPR from unstimulated neutrophils (Bommakanti RK et al., J Bio[ Chem 267: 757~7581, 1992). We conclude, therefore, that the affinity of the interaction of FPR with G protein is not affected by desensitization, consistent with the model of lateral segregation of FPR and G protein as a mechanism of desensitization.},
  subject      = {Toxikologie},
  language  = {en}
}
@article{KlotzJesaitis1994,
  author    = {Klotz, Karl-Norbert and Jesaitis, A. J.},
  title     = {Neutrophil chemoattractant receptors and the membrane skeleton},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-60471},
  year      = {1994},
  abstract  = {Signal transduction via receptors for N-formylmethionyl peptide chemoattractants (FPR) on human neutrophils is a highly regulated process which involves participation of cytoskeletal elements. Evidence exists suggesting that the cytoskeleton and/or the membrane skeleton controls the distributJon of FPR in the plane of the plasma membrane, thus controlling the accessibility of FPR to different proteins in functionally distinct domains. In desensitized cells, FPR are restricted todomains which are depleted of G proteins but enriched in cytoskeletal proteins such as actin and fodrin. Thus, the G protein signal transduction partners of FPR become inaccessible to the agonist-occupied receptor, preventing cell activation. The mechanism of interaction of FPR with the membrane skeleton is poorly understood but evidence is accumulating that suggests a direct binding of FPR (and other receptors) to cytoskeletal proteins such as actin.},
  subject      = {Toxikologie},
  language  = {en}
}