PubMed Health⌕ Search

Biomedical subjects

I Shin

Publications and source records attributed to I Shin.

At least 19 recordsLinked to original sources
PubMed2001

D609-sensitive tyrosine phosphorylation is involved in Fas-mediated phospholipase D activation.

Both Fas and PMA can activate phospholipase D via activation of protein kinase Cbeta in A20 cells. Phospholipase D activity was increased 4 fold in the presence of Fas and 2.5 fold in the presence of PMA. The possible involvement of tyrosine phosphorylation in Fas-induced activation of phospholipase D was investigated. In five minute after Fas cross-linking, there was a prominent increase in tyrosine phosphorylated proteins, and it was completely inhibited by D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). A tyrosine kinase inhibitor, genistein, can partially inhibit Fas-induced phospholipase D activation. There were no effects of genistein on Fas-induced activation of PC-PLC and protein kinase C. These results strongly indicate that tyrosine phosphorylation may in part account for the increase in phospholipase D activity by Fas cross-linking and D609 can block not only PC-PLC activity but also tyrosine phosphorylation involved in Fas-induced phospholipase D activation.

Animals↗
PubMed2001

A structural motif of acetylcholinesterase that promotes amyloid beta-peptide fibril formation.

Acetylcholinesterase (AChE) has been found to be associated with the core of senile plaques. We have shown that AChE interacts with the amyloid beta-peptide (Abeta) and promotes amyloid fibril formation by a hydrophobic environment close to the peripheral anionic binding site (PAS) of the enzyme. Here we present evidence for the structural motif of AChE involved in this interaction. First, we modeled the docking of Abeta onto the structure of Torpedo californica AChE, and identified four potential sites for AChE-Abeta complex formation. One of these, Site I, spans a major hydrophobic sequence exposed on the surface of AChE, which had been previously shown to interact with liposomes [Shin et al. (1996) Protein Sci. 5, 42-51]. Second, we examined several AChE-derived peptides and found that a synthetic 35-residue peptide corresponding to the above hydrophobic sequence was able to promote amyloid formation. We also studied the ability to promote amyloid formation of two synthetic 24-residue peptides derived from the sequence of a Omega-loop, which has been suggested as an AChE-Abeta interacting motif. Kinetic analyses indicate that only the 35-residue hydrophobic peptide mimics the effect of intact AChE on amyloid formation. Moreover, RP-HPLC analysis revealed that the 35-residue peptide was incorporated into the growing Abeta-fibrils. Finally, fluorescence binding studies showed that this peptide binds Abeta with a K(d) = 184 microM, independent of salt concentration, indicating that the interaction is primarily hydrophobic. Our results indicate that the homologous human AChE motif is capable of accelerating Abeta fibrillogenesis.

Acetylcholinesterase↗
PubMed2001

Expression of P27(KIP1) is prognostic and independent of MYCN amplification in human neuroblastoma.

Amplification of the MYCN gene is significantly associated with an unfavorable prognosis and rapid progression in human neuroblastoma tumors. One potential mechanism by which MYCN may cause these effects is by deregulating cell proliferation. Tissue culture experiments support a model in which MYC genes stimulate cell cycle progression by antagonizing the function of the cell cycle inhibitor p27(kip1). In culture, activation of MYC induces both sequestration of p27(kip1) by cyclin D complexes and its subsequent proteolytic degradation. We have tested whether this model applies to human neuroblastoma in a retrospective study of 100 primary tumor biopsy samples from neuroblastoma patients with a documented follow-up. Consistent with this hypothesis, MYCN-amplified tumors express high levels of both cyclin A and proliferating cell nuclear antigen, 2 marker proteins of cell proliferation. Further, expression levels of p27(kip1) are of prognostic significance in human neuroblastoma patients. Similar to tissue culture systems, p27(kip1) is sequestered by cyclin D complexes in a subset of human neuroblastoma samples. Surprisingly, however, expression levels of p27(kip1) are prognostic independent of MYCN amplification, and tumors that have an amplified MYCN gene do not express elevated levels of D-type cyclins or contain significantly lower levels of p27(kip1). Our data do not support a model in which regulation of p27(kip1) function is an important mechanism by which amplified MYCN deregulates cell proliferation in neuroblastoma.

Biomarkers, Tumor↗
PubMed2001

Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1.

The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.

Active Transport, Cell Nucleus↗
PubMed2000

NMR studies on novel antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin.

Artemisinin and its derivatives, which have been known as antimalarial drugs, have also demonstrated their cytotoxicity against tumor cells. It has been proposed that antitumor activity depends on the lipophilicity of functional group on artemisinin derivatives. Solution structures of two artemisinin derivatives as antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin, were determined by NMR spectroscopy to elucidate structure-activity relationship. According to biological assay, antitumor efficiencies are not dependent upon lipophilicity. Instead, these compounds demonstrated their distinctive structural features of boat/chair conformation and capability to interact with receptors, as they have different efficiencies on antitumor activity. Especially, carboxypropyl moiety or carbonyl moiety in artemisinin derivatives influences the conformation and stability of ring structure. Although the detailed mechanism of antitumor activity by artemisinin derivatives has not been addressed, we suggest that antitumor activity is not determined only with lipophilicity and that artemisinin derivatives have specific target proteins in each type of cancer.

Animals↗
PubMed2000

Ras GTPase is essential for fas-mediated activation of phospholipase D in A20 cells.

We have previously reported that Fas cross-linking resulted in an increase in phospholipase D activity in A20 murine cells (J.-S. Han et al., Arch. Biochem. Biophys. 367, 233-239, 1999). In an attempt to explore the Fas downstream factor contributing to the activation of phospholipase D, we have investigated the possible involvement of a small GTP biding protein Ras in signaling events that were triggered by Fas cross-linking. Upon adenoviral expression of dominant negative mutant of Ras (N17Ras), an increase in phospholipase D activity by anti-Fas monoclonal antibody was diminished. Also, the Fas downstream signaling events triggered by Fas cross-linking such as the activation of phosphatidylcholine-specific phospholipase C, the increase in diacylglycerol level, and the translocation of protein kinase C to membrane fraction were all reduced by N17Ras expression. When parallel experiments were performed with manumycin-A, a Ras farnensyltransferase inhibitor, almost identical inhibitory effects on Fas downstream signaling were exhibited. These data suggest that Ras GTPase is essential in transmitting phospholipase D activation signal induced by Fas cross-linking and is located at phosphatidylcholine-specific phospholipase C upstream in Fas signaling cascades.

Adenoviridae↗
PubMed2000

Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos.

A full set of SP6 promoter variants with all possible single substitutions at positions -17 to +5 was constructed. Transcription activities of these variants were individually measured in vivo and in vitro to determine the contribution of each base pair to the promoter activity. The in vivo activity was measured indirectly by transcriptional interference of the replication of promoter-bearing plasmids. This activity depends most highly on residues -11, -9, -8, -7, and +1 (initiation site). All substitutions at -11, -9, -8, and -7 abolished formation of closed complexes, except for A-8C. These residues are involved in base-specific interactions with the polymerase, and the substitutions exhibit the same strong inhibition in vitro. In contrast, the in vitro activities of some other variants, measured on linearized templates, were different from those in vivo. Some variants at -13, -4, and -2, among others, showed exceptionally higher activities in vivo than in vitro, supporting the possibility that these residues are involved in postbinding steps, including template melting and bending. The A-3T variant showed much lower activity in vivo than in vitro, but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymerase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each base pair to the activity.

Bacteriophages↗
PubMed2000

Phosphatidylcholine-specific phospholipase C-mediated induction of phospholipase D activity in Fas-expressing murine cells.

We have previously reported that Fas cross-linking resulted in the activation of phosphatidylcholine-specific phospholipase C (PC-PLC) and the subsequent activation of protein kinase C (PKC) and phospholipase D (PLD) in A20 cells. In an attempt to correlate the existence of PC-PLC activity and activation of PLD by Fas activation among various Fas-expressing murine cell lines, we have investigated the effect of anti-Fas monoclonal antibody on PC-PLC and PLD activities in A20, P388D1 and YAC-1 cell lines. Upon treatment of anti-Fas monoclonal antibody to these three cell lines, the activation of PLD was only observed in A20 cells. When the effect of anti-Fas monoclonal antibody on PKC and PC-PLC activities in Fas-expressing clones were investigated, the activation of PKC and PC-PLC was detected only in A20 clones. Results presented here also show that exogenous addition of Bacillus cereus PC-PLC activates PC hydrolysis, PKC and PLD in all three murine cell lines. These findings suggest that the activation of PC-PLC is a necessary requirement for the activation of PLD by Fas cross-linking and cell lines devoid of functional PC-PLC activity could exhibit enhanced PLD activity by exogenous addition of PC-PLC.

Animals↗
PubMed2000

Ceramide does not inhibit protein kinase C beta-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells.

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) betaI and betaII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC beta-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.

Animals↗
PubMed1999

Fas-mediated activation of phospholipase D is coupled to the stimulation of phosphatidylcholine-specific phospholipase C in A20 cells.

The activation of phospholipase D in murine B cell lymphoma A20 cells treated with anti-Fas monoclonal antibody has been investigated. Fas cross-linking resulted in a both dose- and time-dependent increases in phospholipase D activity. There was a nearly maximum saturated rise in phospholipase D activity at the dose of 200 ng/ml anti-Fas monoclonal antibody showing a fourfold increase within 3 h. Fas activation also caused an approximately twofold increase of phosphatidylcholine-specific phospholipase C activity and 1,2-diacylglycerol release, which could be blocked by 30 min pretreatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 (50 microgram/ml). Pretreatment of D609 also effectively inhibited the translocation of protein kinase C betaI and betaII from the cytosol to the membrane and the activation of phospholipase D induced by Fas cross-linking, suggesting that 1, 2-diacylglycerol released from the cellular phosphatidylcholine pool through phosphatidylcholine-specific phospholipase C plays a major role in protein kinase C/phospholipase D activation. Anti-Fas monoclonal antibody failed to elicit phosphoinositide-specific phospholipase C activation and any changes in the intracellular Ca2+ level in A20 cells, indicating that the phosphoinositide-mediated pathway is not involved in this Fas signaling. Therefore, these results suggest that Fas-mediated phospholipase D activation may be a consequence of primary stimulation of phosphatidylcholine-specific phospholipase C and that phospholipase D may play a role in Fas cross-linking signaling downstream from phosphatidylcholine-specific phospholipase C.

Animals↗
PubMed1999

Lysophosphatidic acid increases intracellular H2O2 by phospholipase D and RhoA in rat-2 fibroblasts.

We have investigated the possible roles of phospholipase D (PLD) and RhoA in the production of intracellular H2O2 and actin polymerization in response to lysophosphatidic acid (LPA) in Rat-2 fibroblasts. LPA increased intracellular H2O2, with a maximal increase at 30 min, which was blocked by the catalase from Aspergillus niger. The LPA-stimulated production of H2O2 was inhibited by 1-butanol or PKC-downregulation, but not by 2-butanol. Purified phosphatidic acid (PA) also increased intracellular H2O2 and the increase was inhibited by the catalase. The role of RhoA was studied by the scrape-loading of C3 transferase into the cells. The C3 toxin, which inhibited stress fiber formation stimulated by LPA, blocked the H2O2 production in response to LPA or PA, but had no inhibitory effect on the activation of PLD by LPA. Exogenous H2O2 increased F-actin content by stress fiber formation. In addition, catalase inhibited actin polymerization activated by LPA, PA, or H2O2, indicated the role of H2O2 in actin polymerization. These results suggest that LPA increased intracellular H2O2 by the activation of PLD and RhoA, and that intracellular H2O2 was required for the LPA-stimulated stress fiber formation.

1-Butanol↗
PubMed1999

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane.

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

Amino Acid Sequence↗
PubMed1999

Roles of RhoA and phospholipase A2 in the elevation of intracellular H2O2 by transforming growth factor-beta in Swiss 3T3 fibroblasts.

We have investigated the mechanisms by which transforming growth factor-beta (TGF-beta) increased intracellular H2O2 in Swiss 3T3 fibroblasts. Increase of intracellular H2O2 by TGF-beta was maximal at 30 min and blocked by catalase from Aspergillus niger. Scrape-loading of C3 transferase, which down-regulated RhoA, inhibited the production of H2O2 in response to TGF-beta. TGF-beta stimulated release of arachidonic acid, which was completely inhibited by mepacrine, a phospholipase A2 inhibitor. Mepacrine also blocked the increase of H2O2 by TGF-beta. In addition, arachidonic acid increased intracellular H2O2. Furthermore, TGF-beta stimulated stress fibre formation, which was blocked by catalase, without membrane ruffling. Catalase also inhibited stimulation of thymidine incorporation by TGF-beta. These results suggested that TGF-beta increased intracellular H2O2 through RhoA and phospholipase A2, and also suggested that intracellular H2O2 was required for the stimulation of stress fibre formation and DNA synthesis in response to TGF-beta.

3T3 Cells↗
PubMed1999

Effects of ceramide, the Fas signal intermediate, on apoptosis and phospholipase D activity in mouse ovarian granulosa cells in vitro.

Although recent studies have demonstrated that ovarian follicular atresia occurs by apoptosis of granulosa cells, the intracellular signaling pathways involved in apoptotic cell death are still poorly characterized. We examined the role of ceramide as a candidate intracellular mediator of Fas-mediated signaling in cultured granulosa cells. Expression of Fas antigen was demonstrated by Western blot of granulosa cell lysates and immunostaining of cultured granulosa cells. Exposure of granulosa cells to anti-Fas monoclonal antibody (anti-Fas mAb) resulted in significant sphingomyelin hydrolysis, which was accompanied by a progressive increase in endogenous levels of ceramide. The addition of exogenous C6-ceramide induced drastic morphological change, including nuclear fragmentation and typical apoptotic DNA degradation. Furthermore, both anti-Fas mAb and C6-ceramide decreased phospholipase D (PLD) activity and diacylglycerol (DAG) concentrations in a time- or a dose-dependent manner. In addition, treatment with phorbol 12-myristate 13-acetate completely attenuated the ceramide-induced inhibition of PLD activity and partially suppressed ceramide-induced apoptosis. These results indicate that the Fas/ceramide signaling pathway might play a role in granulosa cell apoptosis and suggest that the PLD/DAG pathway might be cross-linked to the Fas/ceramide pathway in apoptotic processes of granulosa cells.

Animals↗