Activity-Dependent Photoaffinity Labeling of Metalloproteases.

Metalloproteases, notably members of the matrix metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) families play crucial roles in tissue remodeling, the liberation of growth factors and cytokines from cell membranes (shedding) and cell-cell or cell-matrix interactions. Activity of MMPs or ADAMs must therefore be tightly controlled in time and space by activation of pro-enzymes upon appropriate stimuli and inhibition by endogenous tissue inhibitors of metalloproteases (TIMPs) or α-macroglobulin to prevent irreversible tissue damage due to excessive degradation or uncontrolled release of potent inflammatory mediators, such as tumor necrosis factor-α (TNF-α).Although there is a wide range of methods to measure the amount of metalloproteases based on immunological approaches, relatively little is known about the activation status of a given enzyme at any given time and location.

Outwitting EF-Tu and the ribosome: translation with d-amino acids.

Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of d-amino acids (d-aa) into polypeptides.

Quantification of matrix metalloprotease-9 in bronchoalveolar lavage fluid by selected reaction monitoring with microfluidics nano-liquid-chromatography-mass spectrometry.

Quantitative protein analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the selected reaction monitoring (SRM) mode was used to quantify matrix metalloprotease-9 (MMP-9; ∼90 kDa) in bronchoalveolar lavage fluid (BALF) from patients having undergone lung transplantation. We developed an SRM assay for microfluidics-based nanoLC-MS/MS on a triple quadrupole mass spectrometer based on two signature peptides. Samples were prepared by chloroform-methanol precipitation followed by trypsin digestion in the presence of stable-isotope-labeled internal peptide standards.

Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase σ54 and response regulator LipR.

Initial analysis has shown that the transcription of the Pseudomonas alcaligenes lipA gene, which encodes an extracellular lipase, is governed by the LipQR two-component system consisting of sensor kinase LipQ and DNA-binding regulator LipR. This study further analyzes lipA gene expression and demonstrates that the RNA polymerase σ54 is involved in the transcription. Purified LipR has an ATPase activity that is stimulated by the presence of lipA promoter DNA.

Paclitaxel binding to the fatty acid-induced conformation of human serum albumin--automated docking studies.

Paclitaxel (Taxol((R))) binding to the conformation of human serum albumin assumed in the presence of long-chain fatty acids was studied by automated docking. Reduced binding affinities at both the primary and secondary sites were predicted, compared to those characterizing the interaction with the fatty acid-free protein. The baccatin core of paclitaxel was found to play a more important role than its C13 side chain in determining the ligand binding mode as well as in contributing to the overall binding energy at the primary site.

Proteome analysis of chemically induced mouse liver tumors with different genotype.

Mouse liver tumors frequently harbor mutations in Ha-ras, B-raf, or Ctnnb1 (encoding beta-catenin). We conducted a proteome analysis with protein extracts from normal mouse liver and from liver tumors which were induced by a single injection of N-nitrosodiethylamine (DEN) as initiator followed by multiple injections of two different polychlorinated biphenyls (PCBs) as tumor promoters, or corn oil as a control. Liver tumors were stratified into two classes: they were either mutated in Ctnnb1 and positive for the marker glutamine synthetase (GS(+)), or they lacked Ctnnb1 mutations and were therefore GS-negative (GS(-)).

Paclitaxel binding to human serum albumin--automated docking studies.

A computational approach was used to study the interaction of the potent anticancer drug paclitaxel (Taxol) with human serum albumin. The primary and secondary binding sites were located at the interface of subdomains IIA and IIIA, and in the cleft between domains I and III of the protein, respectively. The C13 side chain and the baccatin core of paclitaxel were found to contribute approximately equally to the binding energy at the primary site, whereas the binding mode appears to be governed by the C13 side chain.

Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues.

GCase (glucosylceramidase) from Paenibacillus sp. TS12, a family 3 glycosidase, hydrolyses the beta-glycosidic linkage of glucosylceramide with retention of anomeric configuration via a two-step, double-displacement mechanism. Two carboxyl residues are essential for catalysis, one functioning as a nucleophile and the other as a general acid/base catalyst.

Chemo- and radiation sensitivity of xenografted acute lymphoblastic leukemias--correlation to the expression of multidrug resistance proteins.

The aim of our study was to characterise, for the first time, the chemo- and radiation sensitivity of seven pediatric acute lymphoblastic leukemias xenotransplanted into immunodeficient NOD/SCID mice and to correlate the findings with the expression of three drug resistance proteins, P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1) and lung resistance protein (LRP). Mice were treated with single drugs used in clinical protocols: daunorubicin, doxorubicin, cyclophosphamide, vincristine, cytarabine, asparaginase and methotrexate. Two ALL samples, established from primarily diagnosed patients, responded to 5 or 6 of the tested cytostatics, respectively, while 3 out of 5 ALLs from relapse patients were only sensitive towards 2-4 drugs tested.