SummaryA platelet glycoprotein lb-binding protein (GPIb-BP) was isolated from the snake venom of Bothrops jararaca. Jararaca GPIb-BP showed a single band with Mr of 30,000, and two distinct bands with Mr. of 17,000/13,000 under non-reducing and reducing conditions, respectively, on SDS-polyacrylamide gel electrophoresis. Jararaca GPIb-BP itself induced neither platelet aggregation nor serotonin release from platelets, but specifically bound to GPIb (40,629 ± 2,521 molecules per normal platelet, with Kd 39.1 ± 2.4 nM at saturation). The purified venom protein completely inhibited ristocetin- or botrocetin-induccd von Willebrand factor (vWF) binding, and blocked the bovine vWF binding to GPIb, with IC50 values ranging from 28 to 42 nM, without affecting the platelet aggregation induced by ADP or α-thrombin. 1251-jararaca GPIb-BP binding to GPIb was not altered by the presence of human α-thrombin. Jararaca GPIb-BP at a final concentration of 104 nM totally abolished vWF-dependent shear- induced platelet aggregation (SIPA) at a high shear stress, but had no effect on SIPA at a low shear stress. Reduced and S-carboxyamidomethylated jararaca GPIb-BP lost its inhibitory activity on SIPA. The NH2-terminal amino acid sequences of the subunits revealed a high degree of homology with those of several Ca2+-dependent lectins, especially to those of two functionally opposite venom proteins, botrocetin (a vWF-modulator) and alboaggregin-B (a GPIb- modulator).

Resorcylic acid lactones (RALs) constitute a family of polyketide mycotoxins with a variety of biological activities.1) Representative examples are the heat shock protein 90 inhibitor radicicol and the estrogen agonist zearalenone. Hypothemycin ( Fig. 1), a RAL containing a cis-enone moiety that initially did not reveal any particularly interesting activity, was later shown to inhibit ras-transformation and T cell activation. [2][3][4] Hypothemycin and other closely related cis-enone RALs gained attention as compounds that irreversibly inhibit certain protein kinases such as mitogen-activated protein kinase kinase (MEK), extracellular signalf-regulated kinase (ERK) and TAK1, but not RAF, protein kinase C (PKC) or protein kinase A (PKA). [5][6][7][8] From their structures and irreversible mode of action, cis-enone RALs were predicted to form stable Michael addition products with cysteine residues of protein kinases. Bioinformatics studies revealed that a cysteine residue that immediately precedes the highly conserved Asp-Phe-Gly (DFG) motif is the cis-enone RAL binding site.9) This cysteine is conserved in 9% (46/ 510) of the protein kinases in the human kinome.Although different kinases contain the RAL target cysteine, cis-enone RALs have been isolated as selective inhibitors of MEK. 7,8) Furthermore, cis-enone RALs share various biological properties with MEK inhibitors. Sonoda et al. identified hypothemycin as a ras-signaling inhibitor.3) We reported that the MEK inhibitor U0126 reverses ras-mediated transformation.10) Hypothemycin and MEK inhibitors showed strong activity in cell lines with the activating BRAF V600E mutation.11) Sonoda et al. reported that the suppression of ras-induced transformation was associated with accelerated cyclin D1 degradation, 3) and a MEK inhibitor caused a loss of cyclin D expression in BRAF mutants. 11)Here, we compared the effects of hypothemycin and MEK inhibitors on signaling pathways in cells. Our results show that even though the RAL target cysteine is not confined to the components of the MEK-ERK pathway, in cellular settings hypothemycin preferentially blocks the MEK-ERK axis with sufficient selectivity to impair transformed phenotypes of cancer cells requiring this pathway. The resorcylic acid lactone hypothemycin has been shown to inactivate protein kinases by binding to a cysteine conserved in 46 protein kinases, including mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK) and platelet-derived growth factor receptor (PDGFR). We assessed the selectivity of hypothemycin in cellular contexts. Hypothemycin normalized the morphology and inhibited anchorage-independent growth of Ki-ras transformed normal rat kidney (NRK) cells with selectivity and potency comparable to or greater than that of the MEK inhibitor U0126. In Ki-ras-transformed and phorbol 12-myristate 13-acetate (PMA)-treated NRK cells, hypothemycin blocked ERK activation but showed a minimal effect on autophosphorylation of protein kinase D1 (PKD1), another kinase contain...

Our previous studies demonstrated that the trimethyltin chloride (TMT) causes the granule cell loss in the dentate gyrus (DG) of adult mouse, with being regenerated in the dentate granule cell after the neuronal loss. To elucidate the involvement of peroxynitrite in proliferation of neural stem/progenitor cells (NPCs) after neuronal degeneration, we evaluated the expression of 3-nitrotyrosine (3-NT, a product of tyrosine nitration by peroxynitrite) in the newly generated cells following neurodegeneration in the DG. Mice were given TMT to prepare slices for immunostaining using antibody against nestin (NPCs marker) and 3-NT. Cells positive for nestin and 3-NT markedly increased in the DG on day 3 after TMT treatment. In vitro experiments using the NPCs isolated from the DG on day 3 post-TMT, exposure to apocynin (NADPH oxidase inhibitor) or L-NAME (nitric oxide synthase inhibitor) significantly attenuated the cell proliferation. However, KT5823 (G kinase inhibitor) did not affect it. These results support the possibility that peroxynitrite promotes proliferative activity of the NPCs generated following neuronal degeneration in the DG.