Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Identification of enhanced serine kinase activity in insulin resistance

Insulin receptor substrate (IRS) proteins play a crucial role as signaling molecules in insulin action. Serine phosphorylation of IRS proteins has been hypothesized as a cause of attenuating insulin signaling. The current study investigated serine kinase activity toward IRS-1 in several models of insulin resistance. An in vitro kinase assay was developed that used partially purified cell lysates as a kinase and glutathione S-transferase fusion proteins that contained various of IRS-1 fragments as substrates. Elevated serine kinase activity was detected in Chinese hamster ovary/insulin receptor (IR)/IRS-1 cells and 3T3-L1 adipocytes chronically treated with insulin, and in liver and muscle of obese JCR:LA-cp rats. It phosphorylated the 526-859 amino acid region of IRS-1, whereas phosphorylation of the 2-516 and 900-1235 amino acid regions was not altered. Phosphopeptide mapping of the 526-859 region of IRS-1 showed three major phosphopeptides (P1, P2, and P3) with different patterns of phosphorylation depending on the source of serine kinase activity. P1 and P2 were strongly phosphorylated when the kinase activity was prepared from insulin-resistant Chinese hamster ovary/IR/IRS-1 cells, weakly phosphorylated by the kinase activity from insulin-resistant 3T3-L1 adipocytes, and barely phosphorylated when the extract was derived from insulin-resistant liver. In contrast, P3 was phosphorylated by the serine kinase activity prepared from all insulin-resistant cells and tissues of animals. P1 and P2 phosphorylation can be explained by mitogen-activated protein kinase activity based on the phosphopeptide map generated by recombinant ERK2. In contrast, mitogen-activated protein kinase failed to phosphorylate the P3 peptide, suggesting that another serine kinase regulates this modification of IRS-1 in insulin-resistant state.     

Role of Vanilloid Receptors in the Capsaicin-Mediated Induction of iNOS in PC12 Cells

The vanilloid receptor 1(VR1) is a nonselective cation channel that is activated by pungent vanilloid compound, extracellular protons, or noxious heat. mRNA of VR1 and vanilloid receptor 1-like receptor (VRL1) were expressed in PC12 cells, and only VRI mRNA was detected in glioma and A10 cell lines. VRI protein was demonstrated in PC12 cells by immunocytochemistry and Western blotting. Capsaicin (CPS), the VRI receptor agonist, led to an increase in intracellular calcium ion, and this effect was blocked by pretreatment with VR1 receptor antagonist capsazepin (CPZ). Treatment of PC12 cells with low concentration of CPS (5-50 microM) increased reactive oxygen species (ROS) production, and inducible nitric oxide synthase (iNOS) was expressed after CPS treatment for 24 h. These CPS-induced changes are inhibited by pretreatment of CPZ. These findings suggest that CPS-induced iNOS expression through the VR1 and/or VRL1-mediated pathway, and this may explain the CPS-mediated physiological and pathological effects in neuron system.     

The cDNA and genomic DNA organization of a novel toxin SHT-I from spider Ornithoctonus huwena

Spider venoms are complex mixtures of neurotoxicpeptides, proteins and low molecular mass organicmolecules. Their neurotoxic activity is due to the interac-tion of the venom components with cellular receptors, inparticular ion channels. Spider venoms have…     

The F-box protein AhSLF-S2 physically interacts with S-RNases that may be inhibited by the ubiquitin/26S proteasome pathway of protein degradation during compatible pollination in Antirrhinum

Self-incompatibility S-locus-encoded F-box (SLF) proteins have been identified in Antirrhinum and several Prunus species. Although they appear to play an important role in self-incompatible reaction, functional evidence is lacking. Here, we provide several lines of evidence directly implicating a role of AhSLF-S(2) in self-incompatibility in Antirrhinum. First, a nonallelic physical interaction between AhSLF-S(2) and S-RNases was demonstrated by both coimmunoprecipitation and yeast two-hybrid assays. Second, AhSLF-S(2) interacts with ASK1- and CULLIN1-like proteins in Antirrhinum, and together, they likely form an Skp1/Cullin or CDC53/F-box (SCF) complex. Third, compatible pollination was specifically blocked after the treatment of the proteasomal inhibitors MG115 and MG132, but they had little effect on incompatible pollination both in vitro and in vivo, indicating that the ubiquitin/26S proteasome activity is involved in compatible pollination. Fourth, the ubiquitination level of style proteins was increased substantially after compatible pollination compared with incompatible pollination, and coimmunoprecipitation revealed that S-RNases were ubiquitinated after incubating pollen proteins with compatible but not with incompatible style proteins, suggesting that non-self S-RNases are possibly degraded by the ubiquitin/26S proteasome pathway. Fifth, the S-RNase level appeared to be reduced after 36 h of compatible pollination. Taken together, these results show that AhSLF-S(2) interacts with S-RNases likely through a proposed SCF(AhSLF-S2) complex that targets S-RNase destruction during compatible rather than incompatible pollination, thus providing a biochemical basis for the inhibition of pollen tube growth as observed in self-incompatible response in Antirrhinum.     

Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells. Specific role for glypican-1 in glioma angiogenesis

Fibroblast growth factor-2 (FGF2) is a potent angiogenic factor in gliomas. Heparan sulfate promotes ligand binding to receptor tyrosine kinase and regulates signaling. The goal of this study was to examine the contribution of heparan sulfate proteoglycans (HSPGs) to glioma angiogenesis. Here we show that all brain endothelial cell HSPGs carry heparan sulfate chains similarly capable of forming a ternary complex with FGF2 and fibroblast growth factor receptor-1c and of promoting a mitogenic signal. Immunohistochemical analysis revealed that glypican-1 was overexpressed in glioma vessel endothelial cells, whereas this cell-surface HSPG was consistently undetectable in normal brain vessels. To determine the effect of increased glypican-1 expression on FGF2 signaling, we transfected normal brain endothelial cells, which express low base-line levels of glypican-1, with this proteoglycan. Glypican-1 expression enhanced growth of brain endothelial cells and sensitized them to FGF2-induced mitogenesis despite the fact that glypican-1 remained a minor proteoglycan. In contrast, overexpression of syndecan-1 had no effect on growth or FGF2 sensitivity. We conclude that the glypican-1 core protein has a specific role in FGF2 signaling. Glypican-1 overexpression may contribute to angiogenesis and the radiation resistance characteristic of this malignancy.