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ABSTRACT: Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle-regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NDelta253), or the conserved Spindly box (DeltaSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.
Molecular biology of the cell 06/2010; 21(12):1968-81. · 5.98 Impact Factor
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ABSTRACT: The hormone hepcidin is produced mainly in the liver in response to iron loading and inflammation and secreted into the circulation as a 25-amino acid peptide. The 84-amino acid prohormone undergoes limited proteolytic cleavage at a conserved proprotein convertase (PC) recognition site. In addition to the 25-amino acid hepcidin, N-terminally truncated isoforms of lower biological activity are found in plasma and urine. Here we show that a redundant system of proprotein convertases cleaves prohepcidin at the predicted site releasing active hepcidin-25 from the proprotein. In addition to furin mediated cleavage of prohepcidin, we found prohepcidin peptidase activity of proprotein convertases PC5/6, PC7/LPC, PC1/3 and PC2 which was specific for the release of hepcidin-25 from prohepcidin as shown by mass spectrometry. In native tissue extracts, a calcium-dependent prohepcidin peptidase activity is present specifically releasing the 25-mer hepcidin isoform from the recombinant prohormone. In contrast, the 20-mer isoform of hepcidin is generated by a calcium-independent tissue activity which cleaves the 25-mer peptide but has no activity on the entire prohormone. This finding demonstrates the presence of an additional peptidase in this inactivation mechanism for hepcidin. An inhibitor of prohepcidin cleavage was designed and synthesized from d-amino acids (QRRRRR). Biochemical studies indicated that this is a potent and generic inhibitor of prohepcidin cleavage. Biochemical and inhibitor studies of endogenous tissue peptidase activities support the implication of proprotein convertases in the activation of hepcidin. Inactivation of the peptide hormone by N-terminal truncation is mediated by other distinct peptidases, which appear to act sequentially to initial release of hepcidin-25 from the proprotein.
Blood Cells Molecules and Diseases 06/2009; 43(2):169-79. · 2.35 Impact Factor
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ABSTRACT: Graphitic nanofibres (GNFs), 100-200 nm in diameter and 5-20 microm in length have been modified in order to yield different affinities (Cu2+ and Fe3+ loaded immobilized metal affinity chromatography (IMAC) as well as cation and anion exchange materials) for the extraction of a range of biomolecules by their inherited hydrophobicity and the hydrophilic chemical functionalities, obtained by derivatization. Modified GNFs have for the first time been employed as carrier materials for protein profiling in material-enhanced laser desorption/ionization (MELDI) for the enrichment and screening of biofluids. For that purpose, the derivatized GNF materials have comprehensively been characterized regarding surface area, structural changes during derivatization, IMAC, as well as ion exchange and protein-loading capacity and recovery. GNF derivatives revealed high protein-binding capacity (2,000 microg ml(-1) for insulin) and ideal sensitivities, resulting in a detection limit of 50 fmol microl(-1) (for insulin), which is crucial for the detection of low abundant species in biological samples. Compared to other MELDI carrier materials, sensitivity was enhanced on GNF derivatives, which might be ascribed to the fact that GNFs support desorption and ionization mechanisms and by absorbing laser energy in addition to matrix.
Amino Acids 09/2008; 37(2):341-8. · 3.25 Impact Factor
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ABSTRACT: A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.
Analytical Chemistry 09/2008; 80(19):7467-72. · 5.86 Impact Factor
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Frank R Kloss,
Muhammed Najam-Ul-Haq,
Matthias Rainer,
Robert Gassner,
Günter Lepperdinger,
Christian W Huck, Günther Bonn,
Frederik Klauser,
Xianjie Liu,
Norbert Memmel,
Erminald Bertel,
Jose A Garrido,
Doris Steinmüller-Nethl
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ABSTRACT: Nanocrystalline diamond (NCD) has recently been successfully utilized in a variety of life science applications. NCD films are favorable and salubrious substrates for cells during cultivation. Therefore NCD has also been employed in tissue engineering strategies. NCD as reported in this contribution was grown by means of a modified hot-filament chemical vapor deposition technique, which results in less than 3% sp2-hybridization and yields grain sizes of 5-20 nm. After production the NCD surface was rather hydrophobic, however it could be efficiently refined to exhibit more hydrophilic properties. Changing of the surface structure was found to be an efficient means to influence growth and differentiation capacity of a variety of cells. The particular needs for any given cell type has to be proven empirically. Yet flexible features of NCD appear to be superior to plastic surfaces which can be hardly changed in quality. Besides its molecular properties, crystal structural peculiarities of NCD appear to influence cell growth as well. In our attempt to facilitate, highly specialized applications in biomedicine, we recently discovered that growth factors can be tightly bound to NCD by mere physisorption. Hence, combination of surface functionalization together with further options to coat NCD with any kind of three-dimensional structure opens up new avenues for many more applications. In fact, high through-put protein profiling of early disease stages may become possible from serum samples, because proteins bound to NCD can now be efficiently analyzed by MALDI/TOF-MS. Given these results, it is to be presumed that the physical properties and effective electrochemical characteristics of NCD will allow tailoring devices suitable for many more diagnostic as well as therapeutic applications.
Journal of Nanoscience and Nanotechnology 01/2008; 7(12):4581-7. · 1.56 Impact Factor
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Journal of Near Infrared Spectroscopy 01/2008; 16:211-221. · 1.01 Impact Factor
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ABSTRACT: Investigation of the genetic background of complex traits is the focus of recent interest, as several common diseases or the individual response to treatments of various illnesses have not yet been explored. These studies require the development and implementation of reliable and large-scale genotyping methods. In this report, we introduce an efficient technique based on PCR-restriction fragment length sequence variation technique for the analysis of the -360CG and -201CT single-nucleotide sequence variations in the deoxycytidine kinase gene.
A multicapillary gel electrophoresis instrument was used for the size determination of the generated DNA fragments. A healthy Hungarian population of 100 individuals was investigated to determine allele and genotype frequencies for the 2 sequence variations of interest.
We found that the occurrence of the minor allele is rather low, i.e., the frequency of both the -360G and -201T variants is 1%.
Our technique can readily facilitate the analysis of these important sequence variations in other ethnic groups to clarify the role of these sequence variations in conjunction with arabinosylcytosine treatment in acute myeloid leukemia.
Clinical Chemistry 10/2006; 52(9):1756-62. · 7.91 Impact Factor
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Doris Steinmüller-Nethl,
Frank R Kloss,
Muhammed Najam-Ul-Haq,
Matthias Rainer,
Karin Larsson,
Christian Linsmeier,
Gottfried Köhler,
Christine Fehrer,
Günter Lepperdinger,
Xianjie Liu,
Norbert Memmel,
Erminald Bertel,
Christian W Huck,
Robert Gassner, Günther Bonn
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ABSTRACT: Nano-crystalline diamond (NCD)-coated surfaces were efficiently functionalized with bone morphogenetic protein-2 (BMP-2) by means of physisorption. Due to their randomly oriented texture, NCD-coated surfaces appear to bind complex molecules firmly. Applying various highly sensitive analytical methods, the interaction was found extremely stable. The strength of the experimentally measured adherence between BMP-2 and NCD was further corroborated by theoretical calculations. Oxygen treatment rendered NCD hydrophilic by the appearance of surface oxygen containing groups. This particular NCD surface exhibited even higher binding energies towards BMP-2 than the hydrophobic surface, and this surface was also favoured by cultured cells. Most importantly in this context, bound BMP-2 was found fully active. When cultured on BMP-2-treated NCD, osteosarcoma cells strongly up-regulated alkaline phosphatase, a specific marker for osteogenic differentiation. Hence, this simple method will allow generating highly versatile surfaces with complex biomimetic coatings, essentials for novel medical devices and implants as well as for innovative scaffolds in tissue engineering.
Biomaterials 10/2006; 27(26):4547-56. · 7.40 Impact Factor
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ABSTRACT: Crude rice bran oil contains tocopherols (vitamin E), carotenoids (vitamin A), and phytosterols, which possess antioxidant activities and show promising effects as preventive and therapeutic agents. The aim of this work was to establish methods and to compare C18 and C30 silica stationary phases in order to separate and detect tocopherols, carotenoids, and gamma-oryzanol in one single run. Comparing RP-LC on silica C18 and C30, higher resolution between all target compounds was obtained using the C30 stationary phase. Methanol was used as eluent and the elution strength was increased by the addition of tert-butyl methyl ether for highly hydrophobic analytes such as gamma-oryzanol. Detection was accomplished by diode array detection from 200 to 500 nm. Absorbance maxima were found at 295 nm for tocopherols, 324 nm for gammaoryzanol, and 450 nm for carotenoids. Furthermore, compounds were characterized and identified on the basis of their UV-spectra. Both RP systems were coupled to MS (LC-MS) by using an atmospheric pressure chemical ionization interface.
Journal of Separation Science 10/2005; 28(14):1712-8. · 2.73 Impact Factor
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ABSTRACT: This review summarizes the use of capillary electrophoresis (CE) coupled to mass spectrometry (MS) for the analysis of phenolic compounds and its latest developments. Special attention is paid to the different interfaces. The instrumental setups are discussed and demonstrated in a high number of real applications.
Electrophoresis 05/2005; 26(7-8):1319-33. · 3.30 Impact Factor
