Calcineurin activity assay measurement by liquid chromatography-tandem mass spectrometry in the multiple reaction monitoring mode.

Background: Blood concentrations of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus are currently measured to monitor immunosuppression in transplant patients. The measurement of calcineurin (CN) phosphatase activity has been proposed as a complementary pharmacodynamic approach. However, determining CN activity with current methods is not practical.

Mapping cyclosporine-induced changes in protein secretion by renal cells using stable isotope labeling with amino acids in cell culture (SILAC).

Nephrotoxicity is an adverse event that strongly limits the use of the immunosuppressant cyclosporine in solid organ transplantation and the precise molecular mechanisms underlying this toxicity remain unclear. MS-based proteomic analysis of the secretome of HEK-293 renal cells exposed to cyclosporine was performed to identify changes in protein secretion, as a first step to discover potential biomarkers of such nephrotoxicity. To detect and quantify the perturbed proteins in the culture medium we used SILAC and nano-scale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry.

Untargeted screening of urinary peptides with liquid chromatography coupled to hybrid linear-ion trap tandem mass spectrometry.

We formerly developed and applied a liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry technique for the detection and identification of exogenous compounds in clinical and forensic toxicology. In this study, we aimed to adapt this technique to the detection and identification of the constituents of the urinary peptidome. After solid-phase extraction, separation was performed using gradient reversed-phase liquid chromatography.

Tuning of natural killer cell reactivity by NKp46 and Helios calibrates T cell responses.

Natural killer (NK) cells are lymphocytes involved in antimicrobial and antitumoral immune responses. Using N-ethyl-N-nitrosourea mutagenesis in mice, we identified a mutant with increased resistance to viral infections because of the presence of hyperresponsive NK cells. Whole-genome sequencing and functional analysis revealed a loss-of-function mutation in the Ncr1 gene encoding the activating receptor NKp46.

Quantitative proteomic analysis of cyclosporine-induced toxicity in a human kidney cell line and comparison with tacrolimus.

The calcineurin-inhibitors (CNIs) cyclosporine (CsA) and tacrolimus (TAC) remain the pillars of modern immunosuppression regimens used in solid organ transplantation. Nephrotoxicity is an adverse effect that limits their successful use. The precise molecular mechanisms underlying this nephrotoxicity remain unclear.

B7-H6/NKp30 interaction: a mechanism of alerting NK cells against tumors.

Natural killer (NK) cells are lymphocytes of the innate immune system that sense target cells through a panel of activating and inhibitory receptors. Together with NKG2D, the natural cytotoxicity receptors (NCRs) are major activating receptors involved in tumor cell detection. Although numerous NKG2D ligands have been identified, characterization of the molecules interacting with the NCRs is still incomplete.

Amino acid substitutions at the major insertion loop of Candida albicans sterol 14alpha-demethylase are involved in fluconazole resistance.

Background: In the fungal pathogen Candida albicans, amino acid substitutions of 14alpha-demethylase (CaErg11p, CaCYP51) are associated with azole antifungals resistance. This is an area of research which is very dynamic, since the stakes concern the screening of new antifungals which circumvent resistance. The impact of amino acid substitutions on azole interaction has been postulated by homology modeling in comparison to the crystal structure of Mycobacterium tuberculosis (MT-CYP51).

A new strategy for faster urinary biomarkers identification by Nano-LC-MALDI-TOF/TOF mass spectrometry.

Background: LC-MALDI-TOF/TOF analysis is a potent tool in biomarkers discovery characterized by its high sensitivity and high throughput capacity. However, methods based on MALDI-TOF/TOF for biomarkers discovery still need optimization, in particular to reduce analysis time and to evaluate their reproducibility for peak intensities measurement. The aims of this methodological study were: (i) to optimize and critically evaluate each step of urine biomarker discovery method based on Nano-LC coupled off-line to MALDI-TOF/TOF, taking full advantage of the dual decoupling between Nano-LC, MS and MS/MS to reduce the overall analysis time; (ii) to evaluate the quantitative performance and reproducibility of nano-LC-MALDI analysis in biomarker discovery; and (iii) to evaluate the robustness of biomarkers selection.

Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix x marschlinsii).

Two APETALA2 domain transcription factors were characterized first in angiosperms, and, recently, in several gymnosperms. These proteins are involved in several processes, from flowering to embryogenesis in Arabidopsis thaliana. We extrapolated this result to hybrid larch (Larixxmarschlinsii Coaz) resulting from a cross between European (Larix decidua) and Japanese (Larix kaempferi) larches.

Generation of llama single-domain antibodies against methotrexate, a prototypical hapten.

Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L.

A novel human glycosyltransferase: primary structure and characterization of the gene and transcripts.

We report the identification and primary structure of a novel human glycosyltransferase, B3GTL (beta3-glycosyltransferase-like). The 498 residue protein consists of a short cytoplasmic N-terminal "tail" (residues 1-4), a single transmembrane domain with type II topology (residues 5-28), a "stem" region (residues 29-260), and a catalytic domain (residues 261-498). The genomes of Anopheles gambiae, Drosophila melanogaster, and Caenorhabditis elegans encode potential orthologs which share 31-39% sequence identity with B3GTL, as well as the following features: a conserved catalytic domain containing a triple aspartate motif (DDD) at its core, a conserved pattern of cysteine residues, a C-terminal KDEL-like motif, and conserved residues and motifs that affiliate this novel group with a family of beta3-glycosyltransferases (GT31 in the CAZY classification).

Crystal structure of the human natural killer cell activating receptor KIR2DS2 (CD158j).

Killer cell Ig-like receptors (KIRs) regulate the function of human natural killer and T cell subsets. A feature of the KIR locus is the clustering of homologous genes encoding for inhibitory and activating KIR. Inhibitory and activating KIR differ for ligand specificities and/or affinities.

An efficient synthesis of GDP-hexanolamine, a key tool for the purification of fucosyltransferases.

A new efficient synthesis of GDP-hexanolamine from hexanolamine is reported with an overall yield of 71%. The pyrophosphate formation, the key step of this preparation, was achieved through a sequential GMP activation procedure based on polytrifluoroacetylation of GMP followed by activation of the phosphate group by 1-methylimidazole.

A model of photoprobe docking with beta1,4-galactosyltransferase identifies a possible carboxylate involved in glycosylation steps.

A molecular docking study has been performed on the interaction of beta1,4-galactosyltransferase with an acceptor site photoprobe. This is based on an acceptor site peptide fragment which was recently identified by the use of a photoprobe. The present model strongly suggests that the carboxylate group of Asp318 could be involved in the activation of the acceptor sugar 4-OH for the efficient galactosyltransfer.

Bovine alpha1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases.

alpha1,3-galactosyltransferase (alpha3GalT, EC 2.4.1.

Implications of hemolin glycosylation and Ca2+-binding on homophilic and cellular interactions.

Insects are useful models for the study of innate immune mechanisms because of their lack of antibodies and receptors involved in adaptive immune response. Nevertheless, hemolin cloned from moths is a soluble and membrane associated Ig-related molecule that is up-regulated during immune response [Lanz-Mendoza, H. & Faye, I.

Crystal structures of the bovine beta4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose.

beta1,4-galactosyltransferase T1 (beta4Gal-T1, EC 2.4.1.

Molecular cloning and bacterial expression of a general odorant-binding protein from the cabbage armyworm Mamestra brassicae.

A cDNA clone encoding a general odorant-binding protein (GOBP2) was isolated from antennal RNA of Mamestra brassicae by reverse transcription-PCR (RT-PCR) and RACE-PCR. The cDNA encoding the GOBP2 was further used for bacterial expression. Most of the recombinant GOBP2 (>90%) was found to be insoluble.

Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations.

Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.

An inactive pancreatic lipase-related protein is activated into a triglyceride-lipase by mutagenesis based on the 3-D structure.

Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di- and tri-glycerides; phospholipids (PC); etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.

Crystal structure of hemolin: a horseshoe shape with implications for homophilic adhesion.

Hemolin, an insect immunoglobulin superfamily member, is a lipopolysaccharide-binding immune protein induced during bacterial infection. The 3.1 angstrom crystal structure reveals a bound phosphate and patches of positive charge, which may represent the lipopolysaccharide binding site, and a new and unexpected arrangement of four immunoglobulin-like domains forming a horseshoe.

In vivo calcineurin crystals formed using the baculovirus expression system.

Calcineurin is a heterodimeric phosphatase involved in the signal transduction of antigen-activated T cells. Coexpression of its two subunits, the regulatory subunit from human and the catalytic subunit from Neurospora crassa in cultured insect cells using the baculovirus expression system results in the formation of very large crystals in the cytoplasm. The crystals are formed initially in vesicles, but their subsequent growth appears to be uninhibited and continues without the need of an enclosing membrane until the host cell lyses.

Crystal structure at 2.2 A resolution of the MHC-related neonatal Fc receptor.

The three-dimensional structure of the rat neonatal Fc receptor (FcRn) is similar to the structure of molecules of the major histocompatibility complex (MHC). The counterpart of the MHC peptide-binding site is closed in FcRn, making the FcRn groove incapable of binding peptides. A dimer of FcRn heterodimers seen in the crystals may represent a receptor dimer that forms when the Fc portion of a single immunoglobulin binds.

Investigation of the interaction between the class I MHC-related Fc receptor and its immunoglobulin G ligand.

The neonatal Fc receptor (FcRn) is structurally similar to class I major histocompatibility molecules. FcRn transports maternal immunoglobulin G (IgG) from ingested milk into the blood. IgG is bound at the pH of milk (pH 6.

The class I major histocompatibility complex related Fc receptor shows pH-dependent stability differences correlating with immunoglobulin binding and release.

Maternal immunoglobulin G (IgG) in milk is transported to the bloodstream of newborn rodents via an Fc receptor (FcRn) expressed in the gut. The receptor shows a striking structural similarity to class I major histocompatibility complex (MHC) molecules, being composed of a related heavy chain and the identical light chain (beta 2-microglobulin). FcRn binds IgG at the pH of milk in the proximal intestine (pH 6.