Mol. Cell. Proteomics

Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe

Identification of T-cell receptor a-chain genes in the chicken

T-cell receptor (TCR) -chain (TCR) and ß-chain (TCRß) genes are well characterized in mammals, while only TCRß genes have been identified in other vertebrates. To identify avian TCR genes, we used monoclonal anti-CD3 antibodies to isolate chicken TCR for peptide sequence analysis. Degenerate oligonucleotide probes were then used to isolate a candidate TCR cDNA clone that hybridized with a 1.7-kb mRNA species present only in ß T cells and in tissues populated by these cells. Southern blot analysis revealed gene rearrangement in thymocytes and ß T-cell lines. The TCR cDNA candidate encoded an openreading frame of 275 amino acids, the predicted variable (V)-, joining (J)-, and constant (C)-region amino acid sequences of which shared 40%, 60%, and 25% homology with corresponding mammalian sequences. A single C gene and 25 V genes were identified by using region-specific probes. The V cDNA probe isolated from a Vß1+ cell line reacted with transcripts from one of five Vß2+ cell lines, suggesting shared use of V genes by Vß1+ and Vß2+ T cells and the existence of other V gene families. A genomic V sequence was flanked by classical recombination signal sequences but, unlike previously defined V genes, the leader and V region were encoded by a single exon. The data indicate evolutionary conservation of the basic TCR gene structure in birds and mammal

Bupivacaine crystal deposits after long-term epidural infusion

The case of a 45-year-old male patient (body weight 52kg, height 1.61m) with a locally invasive gastric carcinoma infiltrating into the retroperitoneal space is reported. Because of severe cancer pain a tunnelled thoracic epidural catheter (EC) was placed at thoracic spinal level 7/8 and a local anesthetic (LA) mixture of bupivacaine 0.25 % and morphine 0.005 % was infused continuously at 6mlh−1. To optimize pain therapy the concentration was doubled (bupivacaine 0.5 %, morphine 0.01 %) 3 months later but the infusion rate was reduced to 3mlh−1 thus the total daily dose did not change. The patient died 6 months after initiation of the epidural analgesia from the underlying disease. The total amount of bupivacaine infused was 69g and of morphine 1.37g. The patient never reported any neurological complications. The autopsy revealed large white crystalline deposits in the thoracic epidural space which were identified as bupivacaine base by infrared spectrometry. Morphine could not be detected. A histological examination showed unreactive fatty tissue necrosis within the crystalline deposits but nerve tissue could not be identified. It is concluded that the bupivacaine crystalline deposits arose due to precipitation but the clinical significance with regard to sensory level and neuraxial tissue toxicity is unknow

Neutron Irradiation of Mg11B2 : From the Enhancement to the Suppression of Superconducting Properties

In this letter we present the effect of neutron irradiation up to fluences of 3.9 1019 n/cm2 on the superconducting properties of MgB2. In order to obtain a disorder structure homogeneously distributed, the experiment was carried out on bulk samples prepared with the 11B isotope. Up to fluences of 1018 n/cm2 the critical temperature is slightly diminished (36 K) and the superconducting properties are significantly improved; the upper critical field is increased from 13.5 T to 20.3 T at 12 K and the irreversibility field is doubled at 5 K. For larger neutron fluences the critical temperature is suppressed down to 12 K and the superconducting properties come out strongly degraded.Comment: 13 pages, 4 figures. Submitted to Appl.Phys.Let

Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches

Enhanced flux pinning in neutron irradiated MgB2

We study the effect of neutron irradiation on the critical current density Jc of isotopically pure polycrystalline Mg11B2 samples. For fluences in the range 1017-1018 cm-2, Jc is enhanced and its dependence on magnetic field is significantly improved: we demonstrate that, in this regime, point-like pinning centers are effectively introduced in the system proportionally to the neutron fluence. Instead, for larger fluences, a strong suppression of the critical temperature accompanied by a decrease of both the upper critical field Bc2 and Jc is found.Comment: 13 pages, 3 igure

Global alignment of protein-protein interaction networks by graph matching methods

Aligning protein-protein interaction (PPI) networks of different species has drawn a considerable interest recently. This problem is important to investigate evolutionary conserved pathways or protein complexes across species, and to help in the identification of functional orthologs through the detection of conserved interactions. It is however a difficult combinatorial problem, for which only heuristic methods have been proposed so far. We reformulate the PPI alignment as a graph matching problem, and investigate how state-of-the-art graph matching algorithms can be used for that purpose. We differentiate between two alignment problems, depending on whether strict constraints on protein matches are given, based on sequence similarity, or whether the goal is instead to find an optimal compromise between sequence similarity and interaction conservation in the alignment. We propose new methods for both cases, and assess their performance on the alignment of the yeast and fly PPI networks. The new methods consistently outperform state-of-the-art algorithms, retrieving in particular 78% more conserved interactions than IsoRank for a given level of sequence similarity. Availability:http://cbio.ensmp.fr/proj/graphm\_ppi/, additional data and codes are available upon request. Contact: [email protected]: Preprint versio

RNA polymerase III subunit architecture and implications for open promoter complex formation

Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine crosslinking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization

A hybrid column generation and simulated annealing algorithm for direct aperture optimization.

The purpose of this work was to develop a hybrid column generation (CG) and simulated annealing (SA) algorithm for direct aperture optimization (H-DAO) and to show its effectiveness in generating high quality treatment plans for intensity modulated radiation therapy (IMRT) and mixed photon-electron beam radiotherapy (MBRT). The H-DAO overcomes limitations of the CG-DAO with two features improving aperture selection (branch-feature) and enabling aperture shape changes during optimization (SA feature). The H-DAO algorithm iteratively adds apertures to the plan. At each iteration, a branch is created for each field provided. First, each branch determines the most promising aperture of its assigned field and adds it to a copy of the current apertures. Afterwards, the apertures of each branch undergo an MU-weight optimization followed by an SA-based simultaneous shape and MU-weight optimization and a second MU-weight optimization. The next H-DAO iteration continues the branch with the lowest objective function value. IMRT and MBRT treatment plans for an academic, a brain and a head and neck case generated using the CG DAO and H DAO were compared. For every investigated case and both IMRT and MBRT, the H-DAO leads to a faster convergence of the objective function value with number of apertures compared to the CG-DAO. In particular, the H DAO needs on average half the apertures to reach the same objective function value as the CG DAO for a specifically selected number of apertures. The average aperture areas are 27% smaller for H-DAO than for CG-DAO leading to a slightly larger discrepancy between optimized and final dose. However, a dosimetric benefit remains. The H-DAO was successfully developed and applied to IMRT and MBRT. The faster convergence with number of apertures of the H-DAO compared to the CG-DAO allows to select a better compromise between plan quality and number of apertures