The cysteine residue of the SoxY protein as the active site of protein-bound sulfur oxidation of Paracoccus pantotrophus GB17

AbstractFour proteins of Paracoccus pantotrophus are required for hydrogen sulfide-, sulfur-, thiosulfate- and sulfite-dependent horse heart cytochrome c reduction. The lack of free intermediates suggested a protein-bound sulfur oxidation mechanism. The SoxY protein has a novel motif containing a cysteine residue. Electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry of the SoxYZ protein revealed one mass for SoxZ and different masses for SoxY, indicating native SoxY (10 977 Da) and SoxY with additional masses of +32, +80, +112 and +144 Da, suggesting addition of sulfur, sulfite, thiosulfate and thioperoxomonosulfate. Reduction of SoxY removed the additional masses, indicating a thioether or thioester bond. N-Ethylmaleimide inhibited thiosulfate-oxidation and the kinetics suggested a turn-over-dependent mode of action. These data were evidence that the sulfur atom to be oxidized was covalently linked to the thiol moiety of the cysteine residue of SoxY and the active site of sulfur oxidation

Zwei-Farben Zwei-Photonen Mikroskopie

The work presented deals with the development and first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM). The final goal achieved with this technique are UV fluorescence images of cells. Fluorescence is excited by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon corresponds energetically to an one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. In this work deals with fundamental experiments towards the realization of a 2c2pLSM as well as first measurements of living MIN-6 cells are presented. Other examples presented for the significance of this method are the monitoring of the binding of biotin to avidin and the monitoring the enzymatic digestion of BSA by elastine and subtilisine.Die Arbeit behandelt die Entwicklung und erstmalige Realisierung eines Zwei-Farben Zwei-Photonen Mikroskops (2c2pLSM). Ziel dieser Technik ist es die Aufnahme mikroskopischer Bilder von UV-Fluoreszenz lebender Zellen zu ermöglichen. Dabei wird Fluoreszenz mit Hilfe einer Zwei-Farben Zwei-Photonen Absorption angeregt. Für die Fluoreszenzanregung werden die Fundamentale und die ersten Harmonisch Angeregte eines Ti:Sa-Femtosekundenlasers benutzt. Die gleichzeitige Absorption eines 800 nm Photons und eines 400 nm Photons ist dabei energetisch äquivalent zu einer Ein-Photonen-Absorption eines 266 nm Photons. Durch diese Technik wird die effektive Anregungswellenlänge die mit einem Ti:Sa-Laser erreicht werden kann in den UV-Bereich erweitert. Zusätzlich zu den bekannten Vorteilen eines Multiphotonenmikroskops wie intrinsische 3D Auflösung, reduzierter Photozerstörung und hoher Eindringtiefe ermöglicht 2c2pLSM die Verwendung herkömmlicher Mikroskopobjektive mit hoher numerischer Apertur für UV-Imaging. Die oben genannte effektive Anregungswellenlänge von 266 nm deckt sich dabei sehr gut mit dem Anregungsspektrum von Tryptophan. Damit stellt 2c2pLSM ein hervorragendes Handwerkszeug für die bildgebende Untersuchung von intrinscher Proteinfluoreszenz da, deren Ursache hauptsächlich Tryptophanfluoreszenz ist. Dadurch kann eine natürlich vorkommende Fluoreszenzlebensdauersonde benutzt werden um Proteinreaktionen und Proteinkonformationsänderungen zu beobachten. Diese Arbeit behandelt fundamentale Experimente mit Ziel auf die Entwicklung eines 2c2pLSM und erste Anwendungen dieser Mikroskopietechnik auf lebende MIN-6 Zellen gezeigt. Desweiteren werden weitere Beispiele für die Bedeutsamkeit dieser Technik vorgestellt, wie labelfreie Beobachtung der Bindung von Biotin an Avidin sowie die labelfreie Beobachtung der enzymatischen Abbaureaktion von BSA durch Elastase und Subtilisin

Two-Photon Spectroscopy of Wildtype-Plants and Mutants of Arabidopsis thaliana

Die Photosynthese ist einer der bedeutesten enzymatischen Prozesse. Da die Sonneneinstrahlung auf Pflanzen während eines Tages und über das Jahr starken Schwankungen unterliegt, haben sich neben den eigentlichen Prozessen des Lichtsammelns auch Regulationsmechanismen ausgebildet, um die Pflanze vor der Beschädigung durch überschüssige Energie zu schützen. Sowohl für die Lichtabsorption als auch für diese Schutzreaktionen sind die Wechselwirkungen der Pflanzenpigmente, Chlorophylle und Carotinoide von entscheidender Bedeutung. Die Prozesse der Lichtabsorption und der anschließende Energietransfer in Pflanzen laufen auf Piko- oder sogar Femtosekundenzeitskalen ab. Für einen effektiven Schutz muss auch der Energietransfer der Regulationsmechanismen auf ähnlich schnellen Zeitskalen ablaufen. Durch kombinierte Einphotonenanregung der Chlorophylle und Zweiphotonenanregung der Carotinoide mit jeweiliger Detektion der Chlorophyllfluoreszenz können die Wechselwirkungen der in der Pflanze vorliegenden Pigmente sowohl im helladaptierten Zustand als auch im dunkeladaptierten Zustand untersucht werden. So können Rückschlüsse auf die Energietransferwege in Pflanzen gewonnen werden, um so den Ablauf des nicht-photochemischen Quenchens zubestimmen. Die Messungen wurden in vivo an Blättern des Wildtyps der biologischen Modellpflanze Arabidopsis thaliana und an charakterisierten Mutanten von Arabidopsis thaliana durchgeführt. Diese Mutanten besitzen Veränderungen in der Carotinoidbiosynthese oder bei der Ausbildung von Photosystemuntereinheiten. Die Ergebnisse der Messungen deuten daraufhin, dass nicht-photochemisches Quenchen in zwei unterschiedlich schnellen Prozessen abläuft, die von verschiedenen Carotinoiden und Proteinkomplexen abhängt. Dabei konnte gezeigt werden, dass die ermittelte Transfereffizienz gut mit den in der Literatur postulierten Mechanismen korreliert und als ein Maß für nicht-photochemisches Quenchen genutzt werden kann.Photosynthese is one of the most important enzymatic processes. During a day as well as during a year solar irradiation strongly fluctuates. Thus plants have developed beside a light harvesting process also a regulation process to protect plants of damages due to excess energy. The light harvesting processes and also the regulation process are dependend on the interactions of pigments, chlorophylls and carotenoids, formed in plants. These processes and the resulting energy transfer in plants proceed in pico- or even femtosecond time scales. Hence an effective protection can only be assured by a regulation process on a similar time scale. The combination of one photon excitation of chlorophylls and two photon excitation of carotinoids with a respective detection of chlorophyll fluorescence leads to investigation on the interactions of the pigments excisting in plants in the light adapted state as well as in the dark adapted state. Thus conclusions on the energy transfer can be made, to assign non-photochemical quenching. The experiments were done in vivo with leaves of the wildtyp of Arabidopsis thaliana and with leaves of characterized mutants of Arabidopsis thaliana. These mutants have variances in the biosyntheses of carotenoids or in the build up of protein subunits. The results indicate that non-photochemical quenching proceeds in two different processes, which depend on various carotenoids and protein complexes. Furthermore it was shown that the determined transfer efficiencies can be correlated with already postulated mechanisms in literature and, moreover, can also be used as measure for non-photochemical quenching

Förderung von länger verweilenden Kindern in der Schuleingangsphase:eine empirische Analyse auf der Basis von Einzelfallstudien in jahrgangsbezogen und jahrgangsgemischt organisierten Lerngruppen

Seit dem Schuljahr 2005/06 können Schüler in Nordrhein-Westfalen ein bis drei Jahre, entsprechend ihres Lerntempos und -fortschrittes, in der Schuleingangsphase verweilen. In der pädagogisch-didaktischen Diskussion wird gerade ein 3. Schulbesuchsjahr in der Schuleingangsphase als Chance für Kinder mit Lernschwierigkeiten gesehen. Im Fokus der vorliegenden Arbeit steht die Beantwortung der Fragen: Wie werden länger verweilende Kinder in der Schuleingangsphase gefördert? Wie nutzen länger verweilende Kinder die Förderangebote? Wie wirkt sich diese Förderung auf die Lernprozesse der verweilenden Kinder aus? Im Rahmen dieser Arbeit wurden länger verweilende Kinder aus jahrgangsgemischten und jahrgangsbezogenen Lerngruppen beobachtet, um mögliche Unterschiede und Gemeinsamkeiten in der Förderung auf Grund der Organisationsform erheben zu können. Die Arbeit ist als hypothesengenerierende Fallstudie angelegt und ordnet sich in den Bereich der empirisch-qualitativen Grundschulforschung ein.<br

Drift chamber with a c-shaped frame

We present the construction of a planar drift chamber with wires stretched between two arms of a c-shaped aluminium frame. The special shape of the frame allows to extendthe momentum acceptance of the COSY-11 detection system towards lower momenta without suppressing the high momentum particles. The proposed design allows for construction of tracking detectors covering small angles with respect to the beam, which can be installed and removed without dismounting the beam-pipe. For a three-dimensional track reconstruction a computer code was developed using a simple algorithm of hit preselection.Comment: submitted to Nucl. Instr. & Meth

Interaction between Sox proteins of two physiologically distinct bacteria and a new protein involved in thiosulfate oxidation

AbstractOrganisms using the thiosulfate-oxidizing Sox enzyme system fall into two groups: group 1 forms sulfur globules as intermediates (Allochromatium vinosum), group 2 does not (Paracoccus pantotrophus). While several components of their Sox systems are quite similar, i.e. the proteins SoxXA, SoxYZ and SoxB, they differ by Sox(CD)2 which is absent in sulfur globule-forming organisms. Still, the respective enzymes are partly exchangeable in vitro: P. pantotrophus Sox enzymes work productively with A. vinosum SoxYZ whereas A. vinosum SoxB does not cooperate with the P. pantotrophus enzymes. Furthermore, A. vinosum SoxL, a rhodanese-like protein encoded immediately downstream of soxXAK, appears to play an important role in recycling SoxYZ as it increases thiosulfate depletion velocity in vitro without increasing the electron yield

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

BACKGROUND: SoxAX enzymes initiate microbial oxidation of reduced inorganic sulfur compounds. Their catalytic mechanism is unknown. RESULTS: Cyanide displaces the CysS(-) ligand to the active site heme following reduction by S(2)O(4)(2-) but not Eu(II). CONCLUSION: An active site heme ligand becomes labile on exposure to substrate analogs. SIGNIFICANCE: Elucidation of SoxAX mechanism is necessary to understand a widespread pathway for sulfur compound oxidation. SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E(m)) at pH 7.0 of approximately +210, -340, and -400 mV for the His/Met, His/Cys(-), and active site His/CysS(-)-ligated heme, respectively. Exposing SoxAX to S(2)O(4)(2-), a substrate analog with E(m) ~-450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E(m) ~-1140 mV), allows cyanide to displace the cysteine persulfide (CysS(-)) ligand to the active site heme. This provides the first evidence for the dissociation of CysS(-) that has been proposed as a key event in SoxAX catalysis

SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

<p>Abstract</p> <p>Background</p> <p><it>SET-NUP214 </it>fusion resulting from a recurrent cryptic deletion, del(9)(q34.11q34.13) has recently been described in T-cell acute lymphoblastic leukemia (T-ALL) and in one case of acute myeloid leukemia (AML). The fusion protein appears to promote elevated expression of <it>HOXA </it>cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for <it>SET-NUP214 </it>expression to find model systems that might help to elucidate the cellular function of this fusion gene.</p> <p>Results</p> <p>Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the <it>SET(TAF-</it>Iβ)-<it>NUP214 </it>fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two <it>TAF-</it>I isoforms revealed that the cell lines also expressed <it>TAF-</it>Iα-<it>NUP214 </it>mRNA. Results of fluorescence in situ hybridization (FISH) and array-based copy number analysis were both consistent with del(9)(q34.11q34.13) as described. Quantitative genomic PCR also confirmed loss of genomic material between <it>SET </it>and <it>NUP214 </it>in both cell lines. Genomic sequencing localized the breakpoints of the <it>SET </it>gene to regions downstream of the stop codon and to <it>NUP214 </it>intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein.</p> <p>Conclusion</p> <p>Cell lines LOUCY and MEGAL express the recently described <it>SET-NUP214 </it>fusion gene. Of special note is that the formation of the <it>SET </it>exon 7/<it>NUP214 </it>exon 18 gene transcript requires alternative splicing as the <it>SET </it>breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for <it>SET-NUP214 </it>studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.</p

Low-Energy \Lambda-\p Scattering Parameters from the pp→pK+Λpp \to pK^+\Lambda Reaction

Constraints on the spin-averaged $\Lambda p$ scattering length and effective range have been obtained from measurements of the $pp\to pK^+\Lambda$ reaction close to the production threshold by comparing model phase-space Dalitz plot occupations with experimental ones. The data fix well the position of the virtual bound state in the $\Lambda p$ system. Combining this with information from elastic $\Lambda p$ scattering measurements at slightly higher energies, together with the fact that the hyperdeuteron is not bound, leads to a new determination of the low energy $\Lambda p$ scattering parameters.Comment: 18 pages, 7 figure

Experimental results on strangeness production in proton-proton collisions at COSY

The production of K+ and K- mesons in elementary proton-proton collision has been investigated at the Cooler Synchrotron COSY in Juelich. A high quality proton beam with low emittance and small momentum spread permitted to study the creation of these mesons very close to the kinematical threshold. The energy dependence of the total cross section is investigated using internal beam facilities providing a high accuracy particle momentum determination as well as an external non-magnetic detection setup with a large geometrical acceptance. The determination of the four-momentum vectors for all ejectiles of each registered event gives the complete kinematical information allowing to study the interaction of the outgoing particles. Results on the performed studies of the pp --> pp K+ K-, pp --> p Lambda K+ and pp --> p Sigma0 K+ reactions will be presented and their relevance to the interpretation of heavy ion collisions will be discussed.Comment: 8 pages, 6 figures, plenary talk at 6th International Conference On Strange Quarks in Matter: '2001 - A Flavorspace Odyssey' (SQM2001), Frankfurt, Germany, September 25-29, 2001, to be published in J. Phys. G: Nucl. Part. Phy