Thrombospondin-1 (TSP-1) is a multidomain protein that has been implicated in cell adhesion, motility, and growth. Some of these functions have been localized to the three thrombospondin type 1 ...repeats (TSRs), modules of ∼60 amino acids in length with conserved Cys and Trp residues. The Trp residues occur in WXXW patterns, which are the recognition motifs for protein C-mannosylation. This modification involves the attachment of an α-mannosyl residue to the C-2 atom of the first tryptophan. Analysis of human platelet TSP-1 revealed that Trp-368, -420, -423, and -480 are C-mannosylated. Mannosylation also occurred in recombinant, baculovirally expressed TSR modules from Sf9 and “High Five” cells, contradictory to earlier reports that such cells do not carry out this reaction. In the course of these studies it was appreciated that the TSRs in TSP-1 undergo a second form of unusual glycosylation. By using a novel mass spectrometric approach, it was found that Ser-377, Thr-432, and Thr-489 in the motif CSX(S/T)CG carry the O-linked disaccharide Glc-Fuc-O-Ser/Thr. This is the first protein in which such a disaccharide has been identified, although proteinO-fucosylation is well described in epidermal growth factor-like modules. Both C- andO-glycosylations take place on residues that have been implicated in the interaction of TSP-1 with glycosaminoglycans or other cellular receptors.
The beam diagnostic system of the ATLAS detector comprises two diamond sensor based devices. The innovative Beam Conditions Monitor (BCM) is aimed at resolving background from collision particles by ...sub-ns time-of-flight measurement. The Beam Loss Monitor (BLM) is a clone of the LHC machine BLM system, replacing ionization chambers with diamond sensors. BCM uses 16 1 × 1 cm 2 0.5 mm thick polycrystalline chemical vapor deposition (pCVD) diamond sensors arranged in 8 positions at a radius r ≈ 55 mm, ~ 1.9 m up- and downstream the interaction point. Time measurements at 2.56 GHz sampling rate are performed to distinguish between collision and shower particles from beam incidents. A FPGA-based readout system performs real-time data analysis and interfaces the results to ATLAS and the LHC beam permit system. The diamond sensors, the detector modules and their readout system are described. Results of performance with LHC beams of increasing energy and intensity including timing separation of collisions from beam related background are be presented, and beam abort algorithms discussed. BLM utilizes 12 pCVD diamond sensors close to the beam pipe at z ≈ ±3.5 m. The radiation induced currents in the sensors are read by the LHC machine developed readout, averaging the current over various time constants from 40 μs to 84 s. Exceeding a preset threshold for any of the readings drops the beam permit and aborts the LHC beam. Both systems provide post-mortem data dump of approximately 1000 LHC turns prior to the anomalous condition, allowing to diagnose its development and to refine the BCM algorithms and BLM thresholds. The systems were employed in various modalities from the first physics LHC run in November 2009, and are adapting their performance to balance between the need to protect the sensitive ATLAS Inner Detector, and yet allow efficient operation of the collider.
beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The ...specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.
The ATLAS beam conditions monitor Mikuz, M.; Cindro, V.; Dolenc, I. ...
2007 IEEE Nuclear Science Symposium Conference Record,
2007-Oct., Letnik:
3
Conference Proceeding
Odprti dostop
The ATLAS beam conditions monitor (BCM) was developed as a stand-alone device allowing separation of LHC p-p collisions from background events induced on beam gas or by beam accidents. This ...separation can be achieved by timing coincidences between two detector rings placed symmetrically around the interaction point at z=plusmn184 cm and r=55 mm (eta~4.2). The expected LHC lifetime dose at this position exceeds 0.5 MGy and 10 15 particles/cm 2 . Each ring is composed of four detector modules each having two pCVD diamond sensors read out in parallel by fast current amplifiers yielding 3 ns wide pulses with 1 ns rise time and baseline restoration in 10 ns. The device is aimed at single particle counting, with sub-bunch-crossing timing and pulse height information, providing a flexible and versatile tool to monitor beam conditions as well as to provide a coarse measurement of bunch-by-bunch luminosities in ATLAS. Eleven detector modules have been fully tested and assembled. Tests performed range from full characterization of diamond sensors to full module tests with electron sources and pion test-beams. Recent test-beam results in the CERN SPS show a module median signal to noise of 11:1 for minimum ionizing particles incident at 45deg, using the envisaged digitization electronics. The best eight modules have now been installed on the ATLAS pixel support frame. One of the modules was irradiated in steps to 1 and 3 times 10 14 p/cm 2 at the CERN PS and its performance evaluated in test-beams this fall, followed by a further irradiation to 10 15 p/cm 2 next year.
The low energy dissociation technique electron capture dissociation has been applied to a series of thrombospondin and properdin derived O-fucosylated glycopeptides. Followed by capture of an ...electron by multiply protonated precursor ions M+
nH
n+
reduced odd electron radical cations M+
nH
(
n
−1)
+ were generated. The latter mainly fragment by cleavage of the NCα bonds of the peptide chain without loss of the labile sugar moiety allowing an unambiguous assignment of the glycosylation site. Apart from peptide backbone cleavages, side chain losses of aminocarbonylmethyl and aminocarbonylmethylthiyl radicals from carboxyamidomethylated cysteins are observed. The NCα bond cleavage is greatly reduced on both sides of alkylated Cys. However, fragment ions that are formed by secondary fragmentations of z-type radical cations containing N-terminal cystein give rise to even electron z
SCH
2CONH
2 ions. The potential of the high mass accuracy for the identification of the protein modification topology has been fully explored.
The final chemical structure of a newly synthesized protein is often only attained after further covalent modification. Ideally,
a comprehensive proteome analysis includes this aspect, a task that is ...complicated by our incomplete knowledge of the range
of possible modifications and often by the lack of suitable analysis methods. Here we present two recently discovered, unusual
forms of protein glycosylation, i.e . C- mannosylation and O- fucosylation. Their analysis by a combined mass spectrometric approach is illustrated with peptides from the thrombospondin
type 1 repeats (TSRs) of the recombinant axonal guidance protein F-spondin. Nano-electrospray ionization tandem-mass spectrometry
of isolated peptides showed that eight of ten Trp residues in the TSRs of F-spondin are C- mannosylated. O- Fucosylation sites were determined by a recently established nano-electrospray ionization quadrupole time-of-flight tandem-mass
spectrometry approach. Four of five TSRs carry the disaccharide Hex-dHex- O- Ser/Thr in close proximity to the C- mannosylation sites. In analogy to thrombospondin-1, we assume this to be Glc-Fuc- O -Ser/Thr. Our current knowledge of these glycosylations will be discussed.
Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these ...proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.
We search for CP violation in neutral charm meson decays using a data sample with an integrated luminosity of 966 fb–1 collected with the Belle detector at the KEKB e+e– asymmetric-energy collider. ...The asymmetry obtained in the rate of D0 and D0¯ decays to the π0π0 final state, –0.03±0.64(stat)±0.10(syst)%, is consistent with no CP violation. This constitutes an order of magnitude improvement over the existing result. Here, we also present an updated measurement of the CP asymmetry in the D0→K$^{0}_{S}$π0 decay: ACP(D0 → K$^{0}_{S}$π0) = –0.21±0.16(stat)±0.07(syst)%.
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