Biomedical subjects

Taeg-Kyu Kwon

Publications and source records attributed to Taeg-Kyu Kwon.

8 recordsLinked to original sources

PubMed2005

Inhibitory modulation of ATP-sensitive potassium channels by gallate-ester moiety of (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenolic substance found in green tea, is well recognized to be beneficial for human health. However, it is still controversial as to what dose of this compound is indeed good for human health. Though some recent studies have interestingly reported various beneficial effects of EGCG in cell culture system, however, plasma levels of EGCG attainable by oral regular intake in humans are normally in nanomolar range. However, potential side effects of EGCG when administered parenterally at higher concentration have not been thoroughly tested. Here, we evaluated the effect of EGCG on ATP-sensitive potassium (K(ATP)) channels expressed in Xenopus oocytes. EGCG inhibited the activity of the Kir6.2/SUR1 and Kir6.2DeltaC36 channels with IC(50) of 142+/-37 and 19.9+/-1.7microM, respectively. Inhibition of EGCG was also observed in Kir6.2/SUR2A or Kir6.2/SUR2B channels. Notably, (-)-epicatechin-3-gallate (ECG), another major polyphenolic substance in green tea, was found to reduce the channel activity with greater potency than EGCG. In contrast to EGCG and ECG, which have the gallic acid-ester moiety in their own structures, (-)-epigallocatechin and (-)-epicatechin exhibited very weak inhibition of the K(ATP) channel. Collectively, these results suggest that the gallate-ester moiety of epicatechins may be critical for inhibiting the K(ATP) channel activity via the pore-forming subunit Kir6.2 and this may be a possible mechanism by which green tea extracts or EGCG may cause unexpected side effects at micromolar plasma level.

Adenosine Triphosphate↗

PubMed2004

Pleckstrin homology domains of phospholipase C-gamma1 directly interact with beta-tubulin for activation of phospholipase C-gamma1 and reciprocal modulation of beta-tubulin function in microtubule assembly.

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains, an N-terminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-gamma1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified beta-tubulin as a binding protein of both PLC-gamma1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of alpha- and beta-tubulin heterodimers in all eukaryotic cells. PLC-gamma1 and beta-tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolyzing activity of PLC-gamma1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that beta-tubulin activates PLC-gamma1. Furthermore, indirect immunofluorescent microscopy showed that PLC-gamma1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-gamma1 is involved in spindle fiber formation. The effect of PLC-gamma1 in microtubule formation was assessed by overexpression and silencing PLC-gamma1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-gamma1 showed higher microtubule densities than controls, whereas PLC-gamma1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-gamma1 and beta-tubulin transmodulate each other, i.e. that PLC-gamma1 modulates microtubule assembly by beta-tubulin, and beta-tubulin promotes PLC-gamma1 activity.

Animals↗

PubMed2004

Point mutations in the split PLC-gamma1 PH domain modulate phosphoinositide binding.

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal (PH(1)) and a split PH domain (nPH(2) and cPH(2)). We previously reported that the split PH domain of PLC-gamma1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P(2), we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-gamma1 nPH(2) domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-gamma1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-gamma1 molecules showed reduced PI(4,5)P(2) hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH(1) and nPH(2) domains are responsible for membrane-targeted translocation of PLC-gamma1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro(500) and His(503) are critical for binding of PLC-gamma1 to one of its substrates, PI(4,5)P(2) in the membrane.

Amino Acid Sequence↗

PubMed2004

Taurine increases glucose sensitivity of UCP2-overexpressing beta-cells by ameliorating mitochondrial metabolism.

A low-taurine diet during fetal or early postnatal life causes abnormal pancreatic beta-cell development. Tissue and plasma taurine concentrations can also be low in diabetic patients. We examined the effect of taurine on impaired glucose responses in diabetic rat beta-cells adenovirally overexpressing uncoupling protein (UCP)2, which is upregulated in obesity-related type 2 diabetes. We found that taurine pretreatment restored the ATP-to-ADP (ATP/ADP) ratio and glucose-stimulated insulin secretion in UCP2-infected islets. ATP-sensitive K(+) channel sensitivity to dihydroxyacetone, another insulin secretagogue, was similar in both UCP2-infected and control beta-cells. In freshly isolated mitochondria from UCP2-overexpressing insulin-secreting (INS)-1 beta-cells, methyl pyruvate-mediated mitochondrial Ca(2+) increase was significantly ameliorated by taurine. A mitochondrial Ca(2+) uniporter blocker, ruthenium red, inhibited the action of taurine. This study suggests that taurine enhances the glucose sensitivity of UCP2-overexpressing beta-cells, probably by increasing mitochondrial Ca(2+) influx through the Ca(2+) uniporter, thereby enhancing mitochondrial metabolic function and increasing the ATP/ADP ratio.

Adenosine Diphosphate↗

PubMed2003

Mutations of the BAK gene are infrequent in advanced gastric adenocarcinomas in Koreans.

The bcl-2 homologue antagonist/killer (BAK) is a potently apoptosis-inducing gene and plays an important role in modulating apoptosis in epithelial cells. We have analyzed the mutation of the entire coding region of BAK gene in 107 Korean advanced gastric adenocarcinomas by polymerase chain reaction-single strand conformation polymorphism and sequencing. Homozygous deletions were not found in these samples. Only three cases of 107 gastric adenocarcinomas (2.8%) exhibited the BAK mutations. Two of them exhibited missense mutations and the remaining one had a silent mutation. All of these mutations were exclusively detected in exon 2. Mutations in the BAK gene were observed only in advanced gastric adenocarcinomas with extensive metastases of regional lymph nodes. The data presented here suggest that the mutations of BAK gene rarely occurred in advanced gastric adenocarcinomas.

Adenocarcinoma↗

PubMed2003

Sodium orthovanadate potentiates EGCG-induced apoptosis that is dependent on the ERK pathway.

Epigallocatechin-3-gallate (EGCG) is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis. In the present study, we determined the effect of vanadate, a potent inhibitor of tyrosine phosphatase, on EGCG-induced apoptosis. Investigation of the mechanism of EGCG or vanadate-induced apoptosis revealed induction of caspase 3 activity and cleavage of phospholipase-gamma1 (PLC-gamma1). Furthermore, vanadate potentiated EGCG-induced apoptosis by mitogen-activated protein kinase (MAPK) signaling pathway. Treatment with EGCG plus vanadate for 24h produced morphological features of apoptosis and DNA fragmentation in U937 cells. This was associated with cytochrome c release, caspase 3 activation, and PLC-gamma1 degradation. EGCG plus vanadate activates multiple signal transduction pathways involved in coordinating cellular responses to stress. We demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in EGCG plus vanadate-induced apoptosis in U937 cells. Elevated ERK activity that contributed to apoptosis by EGCG plus vanadate was supported by PD98059 and U0126, chemical inhibitor of MEK/ERK signaling pathway, prevented apoptosis. Taken together, our finding suggests that ERK activation plays an active role in mediating EGCG plus vanadate-induced apoptosis of U937 cells and functions upstream of caspase activation to initiate the apoptotic signal.

Anticarcinogenic Agents↗

PubMed2003

Immunohistochemical analysis for histopathological subtypes in pediatric medulloblastomas.

Medulloblastomas occurring in children represent a histological spectrum of varying anaplasia and nodularity. In order to determine whether immunohistochemical markers might be useful parameters in subclassifying these tumors, 17 pediatric medulloblastomas, including nine diffuse/non-anaplastic, four diffuse/anaplastic, three nodular/non-anaplastic and one nodular/anaplastic subtypes, were studied. In the present report, we investigate the expression of neural cell adhesion molecule (NCAM), nerve growth factor receptor (NGFR), neurofilament (NF), synaptophysin (SYN), glial fibrillary acidic protein (GFAP), S100, Bcl-2, and Ki-67 by using the immunohistochemistry against specific antibodies. This study showed that NGFR, NF, GFAP and S100 were not detected in anaplastic subtypes of medulloblastomas (0/5), while non-anaplastic subtypes were mainly expressed within the nodules. All 17 tumors were reactive for NCAM, SYN and Bcl-2. In addition, Ki-67 labeling indices for anaplastic subtypes (39.0 +/- 7.42%) were significantly higher than that of non-anaplastic medulloblastomas (11.4 +/- 8.04%; P < 0.0001). These results suggest that immunohistochemical markers are a useful adjunct in characterizing subtypes of pediatric medulloblastomas.

Adolescent↗

PubMed2002

Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity.

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.

1-Phosphatidylinositol 4-Kinase↗