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Membrane-Phloretin Interaction, Infrared Raman, ESR Spectroscopy The transport inhibitor phloretin was bound to human red cell membrane and the concomitant structural changes were observed by spectroscopic methods. By the spin labeling method a decrease in fluidity of the membrane was found at 1 and 10 |iM concentrations of the reagent. This result was obtained with the 2-(3-Carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, and the 2-(14-Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl lipid spin labels. Infrared spectroscopy of modified membranes revealed an intensity increase of the POO~ band at about 1250 cm-1. Moreover, a shift of the peak at 1050 cm -1 to 1100 cm-1 was observed in the presence of phloretin. Raman spectroscopy of the membranes did not contradict the results found with infrared and ESR spectroscopy: In the phloretin modified membrane we observed a lack of the band at 1085 cm-1, which leads to suggest that the POO" and/or C-C regions are less fluid. Changes of the extracted red cell membrane lipids were less characteristic, and the results differed from those found in red cell membrane.
The technique of site-specific fluorescence labelling with Tetramethylrhodaminemaleimide (TMRM) in combination with two electrode voltage-clamp technique (TEVC), an approach that has been named voltage clamp fluorometry (VCF), has been used in this work to study the Na,K-ATPase. The TMRM dye has the ability to attach covalently to cysteine residues and it responds to changes in the hydrophobicity of its local environment. We exploited this property using a construct of the Na-pump in which the native, extracellularly accessible cysteines were removed and cysteine residues were introduced by site-directed mutagenesis in specific positions of the Na-pump. In this way it was possible to detect site-specific conformational rearrangements of the Na-pump in a time-resolved fashion within a native membrane environment. In particular this technique allows to resolve reactions with low electrogenicity that cannot be satisfactorily analyzed with purely electrophysiological techniques and to identify the conformations of the enzyme under specific ionic composition of the measuring buffers. We used VCF to study the influence that several cations like Na+, K+, NMG+, TEA+ and BTEA+ exert on the distribution of the Na,K-ATPase between several enzymatic intermediates and on some of the reactions related to cation transport. To this end we utilized the mutants N790C in the loop M5-M6 and the mutant E307C, T309C, L311C and E312C in the loop M3-M4. From the correspondence of the fluorescence changes with the activation and inhibition of pumping current, by K+ and ouabain respectively, and from the fact that in Na+/Na+ exchange conditions the voltage distribution of charge movement and fluorescence changes evoked by voltage jumps are in reasonable agreement we conclude that through the fluorescence signals measured from these mutants, we can indeed monitor conformational changes linked to transport activity of the enzyme. For the mutants N790 and L311, it was found that the Na+ dependence of the amplitude and kinetics of the fluorescence signal associated with the E1P-E2P transition is in agreement with the prediction of an access channel model describing the regulation of the access of extracellular Na+ to its binding site. In particular for the mutants E307 and T309 it was found that in Na+/Na+ exchange conditions, the conformational change tracked by the fluorescence was much slower than the charge relaxation at hyperpolarized potentials while the kinetics was very similar at depolarized potentials. This implies that at hyperpolarized potentials the conformational change connected to the E1P-E2P transition does not give a large contribution to the electrogenicity of the process which is also consistent with the access channel model. On the mutant N790C it was found that the external pH does not seem to have any effect on the E1P-E2P equilibrium even if it seems to modulate the fluorescence quantum yield of the dye. Fluorescence quenching experiments with iodide and D2O indicate that at hyperpolarized potentials the local environment of the mutant N790C, experiences a small change in the accessibility to water without major changes in the local electrostatic field ...
Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complex membrane protein complexes. The enzyme contributes substantially to oxidative energy-conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. Here, we report the X-ray structure of mitochondrial complex I at 3.6- 3.9 Å resolution describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the ‘deactive’ form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site rendering support for a two-state stabilization-change mechanism of protonpumping.
A metal–organic framework (MOF) material, [Zn2(adc)2(dabco)] (adc = anthracene-9,10-dicarboxylate, dabco = 1,4-diazabicyclo[2.2.2]octane), the fluorescence of which depends on the loading of its nanopores, was synthesized in two forms: as free-flowing nanocrystals with different shapes and as surface-attached MOFs (SURMOFs). For the latter, we used self-assembled monolayers (SAMs) bearing functional groups, such as carboxylate and pyridyl groups, capable of coordinating to the constituents of the MOF. It could be demonstrated that this directed coordination also orients the nanocrystals deposited at the surface. Using two different patterning methods, i.e., microcontact printing and electron-beam lithography, the lateral distribution of the functional groups could be determined in such a way that the highly localized deposition of the SURMOF films became possible.
Metallorganische Netzwerke (engl. metal-organic frameworks, MOFs) sind eine neuartige Klasse mikro/mesoporöser Materialien, für die eine Vielzahl von möglichen Anwendungen demonstriert werden konnte. Das Ziel dieser Arbeit besteht in der Synthese von MOF Mikro/Nanopartikeln sowie der Herstellung von sogenannten Oberflächen-deponierten MOFs (engl. surface-attached metal-organic frameworks, SURMOFs). MOF Partikel mit kontrollierbarer Morphologie und Größe wurden unter milden Bedingungen synthetisiert. Um MOFs als Sensoren, intelligente Membrane, oder in nanotechnologischen Bauelementen verwenden zu können, ist die Integration auf der jeweiligen Oberfläche wichtig. Daher beschäftigt sich der Großteil dieser Arbeit mit der kontrollierten Abscheidung von SURMOFs auf verschiedenartigen Trägermaterialien. Etliche interessante Eigenschaften (z.B. die Fluoreszenz in Abhängigkeit von der Gegenwart von Gastmolekülen und die dynamische Gasadsorptionskapazität) der SURMOFs wurden untersucht.
The Na+/proline transporter of E. Coli (PutP) is responsible for the uptake of proline which is subsequently used not only as a carbon and nitrogen source and a constituent of proteins but also as a particularly effective osmoprotectant. However, for a long time there was little known about the single steps in the reaction cycle of this transporter and only few details about its structure-function relationship are available. Aim of the present work was to achieve a deeper understanding about the kinetic properties of the Na+/proline transporter and to get insights into the structure-function relationship of the substrate binding. To answer these questions different techniques were used. By using the novel SSM technique combining the preparation of PutP proteoliposomes it was possible to demonstrate for the first time the electrogenic substrate binding to PutP transporter. Due to rapid solution exchange measurements on the SSM it was additionally possible to obtain time resolved information about the kinetic details of the cytoplasmic substrate binding sites which were not available by previous steady state and equilibrium binding measurements. Pre-steady-state charge translocation was observed after rapid addition of one or both of the cosubstrates Na+ and/or proline to the PutP-WT proteoliposomes adsorbed on the SSM. Thereby it was possible to link the observed electrical signals with the binding activity of PutP. The observed Na+ and/or proline induced charge displacement were assigned to an electrogenic Na+ and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k > 50 s-1 proceeding the rate limiting step of the reaction cycle. Furthermore, based on the kinetic analysis of the electrical signals obtained from the measurements of PutP on SSM, the following characteristics of the substrates binding in PutP were deduced: (1) both Na+ and proline can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails; while at sufficiently high concentrations, each substrate can bind in the absence of the other; (2) substrate binding is electrogenic not only for Na+, but also for the uncharged cosubstrate proline. The charge displacement associated with Na+ binding and proline binding is of comparable size and independent of the presence of the respective cosubstrate. In addition, it was concluded that Na+ accesses its binding site through a high-field access channel resulting in a charge translocation, whereas the binding of the electroneutral proline induces a conformation alteration involving the displacement of charged amino acid residue(s) of the protein; (3) Na+ and proline binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate; (4) proline binding proceeds in a two step process: low affinity (~ 0.9 mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition; (5) membrane impermeable PCMBS inhibits both Na+ and proline binding to the inside-out orientated PutP transporter, indicating that rather than selectively blocking a specific binding site, PCMBS probably locks the enzyme in an inactive state. The possible targets for this SH-reagent are cysteines 281 and 344 located close to the cytoplasmic surface of the protein. Beyond it, transient electrical currents of PutP were also observed on the BLM after rapid addition of proline in the presence of Na+. This was possible by combining the conventional BLM technique with high-speed flash-photolysis of caged-proline. Indeed the signals on the BLM indicate the detection of a different underlying reaction process in comparison to the data achieved by the SSM technique. This has paved the way for supplemental information about the reaction cycle since it was possible to assign the flash-photolysis BLM signals to the proline binding step followed by the internalization of Na+ and proline into the liposome. Thereby it was found, that the presence of Na+ is indispensable and the time constant for the process is ~ 63 ms. Moreover, structure-function information about the Na+ and proline binding sites of PutP was obtained by investigating the functionally important amino acid residues Asp55, Gly63 and Asp187 with site-directed mutagenesis and the combined SSM technique. One finding is that the mutated proteins PutP-D55C and PutP-G63C showed no activity on the SSM. Therefore, it can be assumed that either both Asp55 and Gly63 are crucial for the structure of PutP protein, or they are located at or close to the Na+ and proline binding sites. Furthermore, the results obtained from PutP-D187N and PutP-D187C mutants on SSM suggest that Asp187 of PutP is likely to be involved in the Na+ binding at the cytoplasmic side of the backward running carrier. Taken together the results of the present work have substantially broadened the known picture of the Na+/proline transporter PutP thereby several steps of the reaction cycle were elucidated, and moreover, valuable insights into the structure-function relationship of the transporter have become available.
Der Schwerpunkt der vorliegenden Arbeit lag in der Synthese und strukturellen Charakterisierung von sandwichartig aufgebauten kupferhaltigen Organosiloxanen. Diese sollten nach Möglichkeit kristalline Eigenschaften aufweisen und ein interessantes magnetisches Verhalten zeigen. Es galt, die Beziehungen zwischen molekularer Struktur und magnetischen Eigenschaften herauszuarbeiten, um auf der Basis experimenteller Daten dem maßgeschneiderten Design neuer molekularer Magnete näher zu kommen. .... Die in der hier vorgelegten Arbeit erzielten Ergebnisse belegen, dass der Weg zur gezielten Erzeugung molekularer Magnete erfolgreich beschritten wurde. Es wird weiteren Arbeiten vorbehalten bleiben, Cluster der nun vorliegenden Art chemisch so zu verknüpfen, dass daraus polymere Ketten oder Netzwerke entstehen. Deren magnetisches Verhalten lässt erwarten, dass damit möglicherweise neue Materialien zugänglich werden, die dem Anspruch eines molekularen Magneten voll gerecht werden.
Reciprocal t(9;22) ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment
(2009)
Background: t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome – Ph+) determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL) or chronic myeloid leukemia (CML). The "minor" breakpoint in Ph+ ALL encodes p185BCR/ABL from der22 and p96ABL/BCR from der9. The "major" breakpoint in CML encodes p210BCR/ABL and p40ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96ABL/BCR and p40ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. Methodology: All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming - spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. Principal Findings: Both p96ABL/BCR and p40ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96ABL/BCR and to a minor extent p40ABL/BCR forced the B-cell commitment of SL-cells and UCBC. Conclusions/Significance: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.
Unlimited self-renewal is an absolute prerequisite for any malignancy, and is the ultimate arbiter of the continuous growth and metastasis of tumors. It has been suggested that the self-renewal properties of a tumor are exclusively contained within a small population, i.e., the so-called cancer stem cells. Enhanced self-renewal potential plays a pivotal role in the development of leukemia. My data have shown that APL associated translocation products PML/RARalpha and PLZF/RARalpha increased the replating efficiency of mouse lin-/Sca1+ hematopoietic stem cells (HSCs). This effect is partly mediated by induction of gamma–catenin which is an important mediator of the Wnt signaling pathway and has been shown to be up regulated by the AML associated translocation products(AATPs). Suppression of gamma–catenin by siRNA can abrogate the increased replating efficiency induced by AATPs. Transduction of gamma–catenin in lin-/Sca1+ HSCs led to increased replating efficiency and the expression of stem cell markers Sca1 and c-kit. Additionally it induced accelerated cell cycle progression of mouse bone marrow HSCs. Transduction/transplantation mouse models have shown that ectopic expression of gamma–catenin in HSCs led to acute myeloid leukemia without maturation. These data suggest important roles of Wnt signaling pathway in the leukemogenesis induced by PML/RARalpha, PLZF/RARalpha and AML1/ETO. In contrast to AATPs, CML and Ph+-ALL associated translocation products p185(BCR-ABL) and p210(BCR-ABL) did not affect the self-renewal potential of hematopoietic stem/progenitor cells. However my studies indicated that their reciprocal translocation products p40(ABL/BCR) and p96(ABL/BCR) actually increased the replating efficiency of hematopoietic stem/progenitor cells. The effect is stronger when induced by p96(ABL/BCR) than by p40(ABL/BCR). It is very intriguing that p96(ABL/BCR) can activate Wnt signaling and up regulate the expression of HoxB4. Transduction/transplantation mouse model has shown that p40(ABL/BCR) and p96(ABL/BCR) both have their own leukemogenic potential. Given the fact that leukemic stem cells maintain the growth of tumor and are the origin of relapse, the cure of leukemia is dependent on the eradication of the leukemic stem cell and abrogation of aberrantly regulated self-renewal capability. Both t-RA and As2O3 have been shown to induce complete remission in APL patients with PML/RARalpha translocation product. However, t-RA as a single agent achieves completeremission (CR) but not complete molecular remissions (CMR). Therefore, virtually all patients will experience a relapse within a few months. In contrast to t-RA, As2O3 as a single agent is able to induce CR as well as CMR followed by long-term relapse-free survival in about 50% of APL patients even if relapsed after treatment with t-RA-containing chemotherapy regimens. Nothing is known about the mechanisms leading to the complete different clinical outcomes by the two compounds although both have been shown to induce differentiation of blast cells, proliferation arrest, induction of apoptosis and degradation of PML/RARalpha. We investigated the effect of t-RA and arsenic on PML/RARalpha-expressing cell population with stem cell capacity derived from the APL cell line NB4 as well as Sca1+/lin- murine bone marrow cells. We found that t-RA did not reduce the replating efficiency in PML/RARalpha- and PLZF/RARalpha-infected Sca1+/lincells whereas it selected small compact colonies representing very early progenitor cells. T-RA was unable to reduce the capacity to form colony forming units-spleen (CFU-S) of Sca1+/lin-cells expressing PML/RARalpha, additionally t-RA did not impair the capability of engraftment of NB4 cells in NOD/SCID mouse. On the contrary to t-RA, As2O3 abolished the aberrant self-renewal potential of Sca1+/lin- cells expressing PML/RARalpha. As2O3 not only abolished the replating efficiency of PML/RARalpha positive cells but also completely abrogated the ability of PML/RARalpha-positive HSC to produce CFU-S in vivo. On the contrary to As2O3, t-RA increased the absolute cell number and the percentage of cells in the side population with respect to the whole cell population in NB4 cells. Taken together these data suggest that arsenic but not all-trans retinoic acid overcomes the aberrant stem cell capacity of PML/RARalpha positive leukemic stem cells. My data prove for the first time that there is a direct relationship between the capacity of compounds to effectively target the LSC and their capacity to eradicate the leukemia, and, thereby, to induce complete molecular remission and long-term relapse-free survival. Thus, in order to increase the curative potential of leukemia therapies, future studies need to include the effect of given compounds on the stem cell compartment to determine their ability to eradicate the LSC.
The title compound, C16H14N4, features an aromatic ring with two 2,2´-dicyanopropyl residues in positions 1 and 3, which are located above and below the ring plane. The two residues differ in their conformation with respect to the aromatic ring: whereas one of the Cmethyl-C-Cmethylene-Caromatic torsion angles is gauche [68.93 (12)°], the other one is fully staggered [177.63 (9)°]. The crystal structure is stabilized by C-H...N hydrogen-bonding interactions. Key indicators: single-crystal X-ray study; T = 173 K; mean σ(C–C) = 0.002 Å; R factor = 0.037; wR factor = 0.101; data-to-parameter ratio = 15.0.