C-propeptide region of human pro alpha 1 type 1 collagen interacts with thioredoxin

Thioredoxin (TRX) is one of major components of thiol reducing systems. To investigate the molecular mechanism of TRX function in the lung tissue, we screened a human lung epithelial cell cDNA library for TRX-binding protein by yeast two-hybrid systems. We isolated a plasmid containing C-propeptide region of human pro alpha 1 type 1 collagen (CP-pro alpha 1(1)). CP-pro alpha 1(1) stably binds to wild type TRX but not to mutant TRX, in which redox-active cysteine residues are substituted. Failure of the interaction of mutant TRX with CP-pro alpha 1(1) was confirmed in yeast two-hybrid systems. The CP-pro alpha 1(1)/TRX interaction was increased by dithiothreitol treatment, but was markedly inhibited by hydrogen peroxide or diamide treatment. These data showed that the reducing status of TRX active site cysteine residues is important for the TRX-CP-pro alpha 1(1) interaction, indicating that collagen biosynthesis is under the regulation of TRX-dependent redox control.     

A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion.     

Chemical cross-linking of peptides derived from RecA with single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine

We report the first example of chemical cross-linking of 5-formyl-2'-deoxyuridine containing oligonucleotides with oligopeptides through a Schiff base formation. Twenty amino acid residue peptides investigated here were derived from the DNA binding site of RecA protein. We have demonstrated that the lysine residue placed at the 6th or 8th position from the N-terminus of the peptide directly contacts with DNA.     

Structural determination of an exocellular mannan from Rhodotorula mucilaginosa YR-2 using ab initio assignment of proton and carbon NMR spectra

The synthesis of 3-azido-3-deoxy, 3-amino-3-deoxy and 3-N-tert-butyloxycarbonyl-3-deoxy derivatives of 2-acetamido-2-deoxy-alpha,beta-D-mannose (N-acetyl-alpha,beta-D-mannosamine, ManNAc), is presented. The 3-azido-3-deoxy- and 3-N-tert-butyloxycarbonyl compounds were further characterised as their peracetates. A preliminary study has found that these C-3 nitrogen-substituted derivatives of ManNAc not to be substrates for Neu5Ac aldolase.     

Synthesis of a set of di- and tri-sulfated galabioses

Among cell-adhesion molecules, L-selectin recognizes sulfated sLe(x) with relatively low affinity. Here, we aimed at artificial mimics by synthesizing a set of di- and tri-sulfated galabioses, which may surpass the affinity of sulfated sLe(x). As a strategy to obtain 3',6',6-tri-O-sulfogalabioses, regioselective reductive cleavage of 4,6- and 4',6'-di-O-benzylidenegalabioses was employed. Two suitably protected galactose precursors were conjugated to yield alpha and beta anomers (48 and 18%, respectively) by using a pentenyl galactoside donor and iodinium di-sym-collidine perchlorate as the catalyst. For synthesizing the 3',6-di-O-sulfogalabiose, however, a trichloroacetimidate donor was superior (52%) to the pentenyl one (30%).     

Cyclic AMP regulates the calcium transients released from IP(3)-sensitive stores by activation of rat kappa-opioid receptors expressed in CHO cells

We analyzed intracellular Ca(2+)and cAMP levels in Chinese hamster ovary cells expressing a cloned rat kappa opioid receptor (CHO-kappa cells). Although expression of kappa(kappa)-opioid receptors was confirmed with a fluorescent dynorphin analog in almost all CHO-kappa cells, the kappa-specific agonists, U50488H or U69593, induced a Ca(2+) transient only in 35% of the cells. The Ca(2+) response occurred in all-or-none fashion and the half-maximal dosage of U50488H (812.1nM) was higher than that (3.2nM) to inhibit forskolin-stimulated cAMP. The kappa-receptors coupled to G(i/o)proteins since pertussis toxin significantly reduced the U50488H actions on intracellular Ca(2+) and cAMP. The Ca(2+) transient originates from IP(3)-sensitive internal stores since the Ca(2+) response was blocked by a PLC inhibitor (U73122) or by thapsigargin depletion of internal stores while removal of extracellular Ca(2+) had no effect. Interestingly, application of dibutyryl cAMP (+ 56.2%) or 8-bromo-cAMP (+ 174.7%) significantly increased the occurrence of U50488H-induced Ca(2+) mobilization while protein kinase A (PKA) inhibitors, Rp-cAMP (-32.3%) or myr-psi PKA (-73.9%) significantly reduced the response. Therefore, it was concluded that cAMP and PKA activity can regulate the Ca(2+) mobilization. These results suggest that the kappa receptor-linked cAMP cascade regulates the occurrence of kappa-opioid-mediated Ca(2+) mobilization.     

Suppressive effect of caffeine on hepatitis and apoptosis induced by tumor necrosis factor-alpha, but not by the anti-Fas antibody, in mice

Tumor necrosis factor (TNF)-alpha-induced hepatitis and apoptosis, as respectively assessed by serum enzyme activities and hepatic DNA fragmentation were effectively suppressed by a single force-feeding of caffeine (100 mg/kg) 1.5 h before injecting the drug. In contrast, caffeine had no significant effect on anti-Fas antibody-induced hepatitis and apoptosis. These results suggest that caffeine differentially affected TNF-alpha receptor- and Fas-mediated hepatitis and apoptosis.     

Interaction of Vesl-1L/Homer 1c with syntaxin 13

Vesl-1S/Homer 1a, reported originally as Vesl/Homer, was isolated as a synaptic plasticity-regulated gene. The expression of Vesl-1S/Homer 1a is regulated during long-term potentiation in the hippocampus. Vesl-1L/Homer 1c, which appears to be formed by a splicing event, shares the N-terminus with Vesl-1S/Homer 1a and also contains additional amino acids at the C-terminus. The short form and the long form of the family members both interact with group 1 metabotropic glutamate receptors (mGluRs). We herein report the identification of syntaxin 13 as a molecule that interacts with Vesl-1L using yeast two-hybrid screening. Syntaxin 13 is a member of the syntaxin family and is regarded as soluble N-ethylmaleimide-sensitive attachment proteins receptors (SNAREs) in the endosomal membranes. The interaction of Vesl-1L and syntaxin 13 was biochemically confirmed by in vitro binding assays. The coexpression of the two proteins in the transfected cells resulted in a colocalization in the intracellular vesicle-like structures. We thus propose that the association of Vesl-1L with syntaxin 13 plays a role in the translocation of Vesl-1L to the intracellular organelles.