Role of phosphoinositide 3-kinase in the nonselective cation channel activation by endothelin-1/endothelinB receptor

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) in Chinese hamster ovarian cells expressing endothelin(B) receptor (CHO-ET(B)R). These channels can be discriminated using the Ca(2+) channel blockers, LOE 908 and SK&F 96365. LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of NSCC-2. In this study, we investigated the possible role of phosphoinositide 3-kinase (PI3K) in the ET-1-induced activation of NSCCs in CHO-ET(B)R using wortmannin and LY-294002, inhibitors of PI3K. ET-1-induced Ca(2+) influx was partially inhibited in CHO-ET(B)R pretreated with wortmannin or LY-294002. In contrast, addition of wortmannin or LY-294002 after stimulation with ET-1 did not suppress Ca(2+) influx. The Ca(2+) channels activated by ET-1 in wortmannin- or LY-294002-treated CHO-ET(B)R were sensitive to LOE 908 and resistant to SK&F 96365. In conclusion, NSCC-2 is stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated independently of the PI3K pathway. Moreover, PI3K seems to be required for the initiation of the Ca(2+) entry through NSCC-2 but not for its maintenance.     

Effects of nonselective cation channels and PI3K on endothelin-1-induced PYK2 tyrosine phosphorylation in C6 glioma cells

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) in C6 glioma cells. In the present study, we investigated the effects of NSCCs on the ET-1-induced proline-rich tyrosine kinase 2 (PYK2) phosphorylation in C6 glioma cells. In addition, we examined the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced NSCCs activation and PYK2 phosphorylation. The PI3K inhibitors wortmannin and LY-294002 inhibited ET-1-induced Ca²⁺ influx through NSCC-2 but not NSCC-1. On the other hand, addition of these inhibitors after stimulation with ET-1 failed to suppress Ca²⁺ influx through NSCC-2. PYK2 phosphorylation was abolished by blocking Ca²⁺ influx through NSCCs. The PI3K inhibitors blocked the NSCC-2-dependent part of ET-1-induced PYK2 phosphorylation. These results indicate that 1) NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade; 2) PI3K seems to be required for the activation of the Ca²⁺ entry, but not for its maintenance; 3) Ca²⁺ influx through NSCC-1 and NSCC-2 plays an essential role in ET-1-induced PYK2 phosphorylation; and 4) PI3K is involved in the ET-1-induced PYK2 phosphorylation that depends on the Ca²⁺ influx through NSCC-2. endothelin; phosphoinositide 3-kinase; nonselective cation channel; proline-rich tyrosine kinase 2; glioma cell     

Soluble branched beta-(1,4)glucans from Acetobacter species show strong activities to induce interleukin-12 in vitro and inhibit T-helper 2 cellular response with immunoglobulin E production in vivo

An extracellular polysaccharide, AC-1, produced by Acetobacter polysaccharogenes is composed of beta-(1,4)glucan with branches of glucosyl residues. We found that AC-1 showed a strong activity to induce production of interleukin-12 P40 and tumor necrosis factor-alpha by macrophage cell lines in vitro. Cellulase treatment completely abolished the activity of AC-1 to induce tumor necrosis factor-alpha production by macrophages, whereas treatment of AC-1 with polymyxin B or proteinase did not affect the activity. Results of experiments using toll-like receptor (TLR) 4-deficient mice and TLR4-transfected human cell line indicated that TLR4 is involved in pattern recognition of AC-1. In vivo administration of AC-1 significantly reduced the serum levels of ovalbumin (OVA)-specific IgE and interleukin-4 production by T cells in response to OVA in mice immunized with OVA. AC-1, a soluble branched beta-(1,4)glucan may be useful in prevention and treatment of allergic disorders With IgE production.     

GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to beta1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029-1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Deltagwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Deltagwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25-32), is due to the properties of Gwt1p itself and not to other membrane components.     

An association of 27- and 40-kDa molecules with glycolipids that bind A-B bacterial enterotoxins to cultured cells

It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than LTp, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins.     

Chemical modification of chitosan. Part 15: synthesis of novel chitosan derivatives by substitution of hydrophilic amine using N-carboxyethylchitosan ethyl ester as an intermediate

The Michael type reaction of chitosan with ethyl acrylate has been investigated. Although this reaction was quite slow in the case of chitosan, the reiteration of the reaction was an effective means for increasing the degree of substitution (DS) of ethyl ester. The N-carboxyethylchitosan ethyl ester as an intermediate was successfully substituted with various hydrophilic amines, although the simultaneous hydrolysis of the ester to carboxylic acid also occurred. Water-soluble chitosan derivatives were obtained by substitution with hydroxyalkylamines and diamines.     

Molecular cloning of a novel transmembrane protein MOLT expressed by mature oligodendrocytes

A novel oligodendrocyte (OL)-specific cDNA was isolated from brain capillary endothelial cells and characterized. The cDNA encodes a protein of 1099 amino acids that contains a signal peptide and a transmembrane domain. The protein was expressed in mature OLs in vivo and in vitro cell cultures and was thus designated as mature OL transmembrane protein (MOLT). RT-PCR analysis showed that MOLT mRNA was expressed in brain, lung, pancreas, and testis. A polyclonal antibody raised against a part of the mouse MOLT reacted specifically with multipolar OLs possessing radially oriented processes that penetrated into the gray matter. More cells were detected in the white matter, and these had longitudinally oriented processes. In a rat OL lineage culture system, oligodendrocyte precursor cells did not initially produce MOLT mRNA and protein, but when they begun to differentiate into mature OLs, they started expressing MOLT. Consequently, MOLT may function as OLs become mature and may serve as a cell-surface marker for OL differentiation.     

Characterization of Ca2+ channels involved in ET-1-induced transactivation of EGF receptors

The purpose of this study was to demonstrate the involvement of Ca(2+) influx through voltage-independent Ca(2+) channels (VICCs) in endothelin-1 (ET-1)-induced transactivation of epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) using the Ca(2+) channel blockers LOE-908 and SK&F-96365 in rabbit internal carotid artery vascular smooth muscle cells. ET-1-induced EGFR PTK transactivation was completely inhibited by AG-1478, which is a specific inhibitor of EGFR PTK. In the absence of extracellular Ca(2+), the magnitude of EGFR PTK transactivation was near the basal level. Based on sensitivity to nifedipine, which is a specific blocker of voltage-operated Ca(2+) channels (VOCCs), VOCCs have minor roles in EGFR PTK transactivation. In contrast, Ca(2+) influx through VICCs plays an important role in EGFR PTK transactivation. Moreover, based on the sensitivity of VICCs to SK&F-96365 and LOE-908, VICCs were shown to consist of two types of Ca(2+)-permeable nonselective cation channels (NSCCs), which are designated NSCC-1 and NSCC-2, and a store-operated Ca(2+) channel. In summary, Ca(2+) influx through VICCs plays an essential role in ET-1-induced EGFR PTK transactivation in rabbit internal carotid artery vascular smooth muscle cells.     

Epithelia-mesenchyme interaction plays an essential role in transdifferentiation of retinal pigment epithelium of silver mutant quail: localization of FGF and related molecules and aberrant migration pattern of neural crest cells during eye rudiment formation

Homozygotes of the quail silver mutation, which have plumage color changes, also display a unique phenotype in the eye: during early embryonic development, the retinal pigment epithelium (RPE) spontaneously transdifferentiates into neural retinal tissue. Mitf is considered to be the responsible gene and to function similarly to the mouse microphthalmia mutation, and tissue interaction between RPE and surrounding mesenchymal tissue in organ culture has been shown to be essential for the initiation of the transdifferentiation process in which fibroblast growth factor (FGF) signaling is involved. The immunohistochemical results of the present study show that laminin and heparan sulfate proteoglycan, both acting as cofactors for FGF binding, are localized in the area of transdifferentiation of silver embryos much more abundantly than in wild-type embryos. More intense immunohistochemical staining with FGF-1 antibody, but not with FGF-2 antibody, is also found in the neural retina, RPE, and choroidal tissue of silver embryos than in wild-type embryos. HNK-1 immunohistochemistry revealed that clusters of HNK-1-positive cells (presumptive migrating neural crest cells) are frequently located around the developing eyes and in the posterior region of the silver embryonic eye. Finally, chick-quail chimerical eyes were made by grafting silver quail optic vesicles to chicken host embryos: in most cases, no transdifferentiation occurs in the silver RPE, but in a few cases, transdifferentiation occurs where silver quail cells predominate in the choroid tissue. These observations together with our previous in vitro study indicate that the silver mutation affects not only RPE cells but also cephalic neural crest cells, which migrate to the eye rudiment, and that these crest cells play an essential role in the transdifferentiation of RPE, possibly by modifying the FGF signaling pathway. The precise molecular mechanism involved in RPE-neural crest cell interaction is still unknown, and the quail silver mutation is considered to be a good experimental model for studying the role of neural crest cells in vertebrate eye development.     

5'-,3'-inverted thymidine-modified antisense oligodeoxynucleotide targeting midkine. Its design and application for cancer therapy

Oligodeoxynucleotides modified at both 5'- and 3'-ends with inverted thymidine (5'-,3'-inverted T) were introduced as new reagents for antisense strategies. These modifications were performed to make the oligodeoxynucleotides resistant to nucleases. The effectiveness of these oligodeoxynucleotides was evaluated in terms of inhibition of synthesis of midkine (MK), a heparin-binding growth factor, and consequent inhibition of growth of CMT-93 mouse rectal carcinoma cells. 5'-,3'-Inverted T antisense MK suppressed synthesis of MK by CMT-93 cells and their growth in culture. Furthermore, 5'-,3'-inverted T oligodeoxynucleotides exhibited less cytotoxicity and better stability than phosphorothioate oligodeoxynucleotides. When 5'-,3'-inverted T antisense MK was mixed with atelocollagen, and injected into CMT-93 tumors pregrown in nude mice, tumor growth was markedly suppressed as compared with tumors injected with sense controls. The suppressive effect of 5'-,3'-inverted T antisense MK on tumor growth was stronger than that of phosphorothioate antisense MK. These findings indicated the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides.