Covalent binding of the 13C-labeled skin sensitizers 5-chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI) to a model peptide and glutathione

The reactivity of 4-[13C]- and 5-[13C]-5-chloro-2-methylisothiazol-3-one (MCI) and 2-methylisothiazol-3-one (MI) towards a model peptide and glutathione was followed by 13C and 1H[13C] NMR spectroscopy. Both molecules were found to react with GSH but in addition MCI was found to react with histidine and lysine to form adducts of a different nature. Reaction with histidine led to stable substitution adducts through an addition-elimination reaction at position 5 while reaction with lysine led to the formation of open adducts of the thioamide or amide type.     

Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair

Chromosome stability depends on accurate chromosome segregation and efficient DNA double-strand break (DSB) repair. Sister chromatid cohesion, established during S phase by the protein complex cohesin, is central to both processes. In the absence of cohesion, chromosomes missegregate and G2-phase DSB repair fails. Here, we demonstrate that G2-phase repair also requires the presence of cohesin at the damage site. Cohesin components are shown to be recruited to extended chromosome regions surrounding DNA breaks induced during G2. We find that in the absence of functional cohesin-loading proteins (Scc2/Scc4), the accumulation of cohesin at DSBs is abolished and repair is defective, even though sister chromatids are connected by S phase generated cohesion. Evidence is also provided that DSB induction elicits establishment of sister chromatid cohesion in G2, implicating that damage-recruited cohesin facilitates DNA repair by tethering chromatids.     

Analysis of kavalactones from Piper methysticum (kava-kava)

The chemical analysis and quality control of both Piper methysticum G. Forster (kava-kava) and extracts obtained by aqueous acetone or aqueous methanol as well as supercritical fluid extraction are reviewed. In the last two decades various procedures concerning the separation and detection of kavalactones have been routinely carried out by gas chromatography (without previous derivatization of kavalactones) and high performance liquid chromatography but most of them are not validated or only partially validated. Recently, analyses by supercritical fluid chromatography and micellar electrokinetic chromatography have also been reported. Both gas chromatography and high performance liquid chromatography can be used for the analysis of kavalactones with some advantages and disadvantages for each method. Using gas chromatography analysis, methysticin and yangonin, which are two of the major components, are generally not separated. In addition, the high temperature of the injection port caused the decomposition of methysticin. Concerning high performance liquid chromatography analyses, the reversed-phase is generally better because highly reproducible with a very low detection limit for all compounds even if the quantitative analysis of the kavalactones by liquid chromatography needs to be carried out in the absence of light to prevent the cis/trans isomerisation of yangonin.     

Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures

N-Methyl-d-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A₂ inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A₂ activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 µM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.     

Hrs and endocytic sorting of ubiquitinated membrane proteins

Endocytosed receptors are either recycled to the plasma membrane or trapped within intralumenal vesicles of multi-vesicular bodies for subsequent degradation in lysosomes. How the cell is able to sort receptors in endosomes has so far been largely unknown. The hepatocyte growth factor regulated tyrosine kinase substrate, Hrs, is an essential protein that has been implicated in cell signalling and intracellular membrane trafficking. Very recently, several reports have demonstrated a role for Hrs in endocytic sorting of ubiquitinated membrane proteins. Here, we review current knowledge about how Hrs recognises ubiquitinated cargo that is destined for lysosomal degradation, and how Hrs may act as a key regulator of the molecular machinery involved in receptor sorting and multivesicular body formation.     

Primary sequence determinants responsible for site-selective dephosphorylation of the PDGF beta-receptor by the receptor-like protein tyrosine phosphatase DEP-1

Site-selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor-like protein tyrosine phosphatase DEP-1 site-selectively dephosphorylates the PDGF beta-receptor. DEP-1 dephosphorylation of original and chimeric phospho-peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at -4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position -1 and a preference against a basic residue at position +3. DEP-1 site-selective dephosphorylation of PDGF beta-receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions -1 and +3.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.