• The Journal of Korean Medicine
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• v.21 no.4
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• pp.64-72
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• 2000

Objectives : This study was designed to investigate the neuroprotective effect of Hwansodan(Huanshao-dan) on the apoptosis induced by withdrawal of neurotrophic support. Methods : PCl2 pheochromocytoma cells have been used extensively as a model for studying the cellular and molecular effects of neuronal cells. The viability of cells was measured by MTT assay. We used DNA fragmentation and caspase 3-like protease activation assay. Results : The water extract of Hwansodan(Huanshao-dan) significantly showed protective effects on serum and glucose deprivation-induced apoptotic death. Hwansodan(Huanshao-dan) also prevents DNA fragmentation and caspase 3-like protease activation, representing typical neuronal apoptotic phenomena in PCl2 pheochromocytoma cells and induces tyrosine phosphorylation of proteins around 44 kDa, which was identified as ERK1 with electrophoretic gel mobility shift by Western blot. In addition, MAPK kinase(MEK) inhibitor PD98059 and Ras inactivator, ${\alpha}-hydroxyfarnesylphosphonic$ acid attenuated the neuroprotective effects of Hwansodan(Huanshao-dan) in serum-deprived PCl2 cells. Conclusions : These results indicate that Ras/MEK/ERK signaling pathway plays a key role in neuroprotective effects of Hwansodan(Huanshao-dan) in serum-deprived PCl2 cells. Taken together, we suggest the possibility that Hwansodan(Huanshao-dan) might provide a neurotrophic-like activity in PCl2 cells.

• YAKHAK HOEJI
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• v.49 no.5
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• pp.428-436
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• 2005

DNA topoisomerase I(TOP1) helps the control of DNA replication, transcription and recombination by assist­ing breaking and rejoining of DNA double strand. Camptothecin (CPT) and its derivative, topotecan, are known to inhibit TOP1 by intercalating into TOP1-DNA complex. Recently various non-CPT intercalators are synthesized for a new class of TOP1 inhibitors. In this study, six compounds isolated from Poria cocos were investigated for their interaction with TOP1­DNA complex using the flexible docking program, FlexiDock. The binding modes were analyzed and compared with the TOP1 inhibition activities. The compounds that showed potent activity were intercalated between the + 1/-1 base pairs of DNA, located near the active site phosphotyrosine723 and formed hydrogen bonds with active site residues. On the other hand, compounds with no activity were not docked at all. The binding modes were well correlated with the inhibition activity, suggesting the possibility that potent inhibitors can be designed from the information presented by the docking study.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.135-136
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• 2001

The Tk-ptp gene encoding a protein tyrosine phosphatase (PTPase) from the hyperthermophilic archaeon Thermococcus kodakaraensis KODI was cloned and sequenced. Sequence analysis indicated that Tk-ptp encoded a protein consisting 147 amino acid residues (16,953 Da). The wild type and the mutants were expressed in Escherichia coli cells as His-tagged fusion proteins and examined for enzyme characteristics. Tk-PTP possessed two unique features that were not found in eucaryal and bacterial counterparts. First, the recombinant Tk-PTP showed the phosphatase activity not only for the phosphotyrosine but also phosphoserine. Second, the conserved Asp (Asp-63), which was considered to be a critical residue, was not involved in catalysis. In order to know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate protein. Proteins of 120, 60 and 53 kDa were isolated specifically from KODI cell lysates by affinity chromatography with Tk-PTP-C93S. It is suggested that these proteins are tyrosine-phosphorylated substrates of Tk-PTP.

• Fisheries and Aquatic Sciences
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• v.21 no.2
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• pp.4.1-4.5
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• 2018

Insulin-like growth factors (IGFs), along with IGF-binding protein and IGF receptor, are well-known regulators in the growth and survival of vertebrates. In this study, we investigated the involvement of IGFs and protein variation during embryonic development of the olive flounder (Paralichthys olivaceus). Morphological stages were divided into six main developments as blastula, gastrula, cephalization, cranial regionalization, tail lift, and hatch. During embryonic development, protein variation was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization quadrupole time-of-flight mass spectrometry/mass spectrometry. In addition, the mechanism of signaling of IGF-I receptor was examined using immuno-blot analysis. We found marked changes in protein expression at four stages of embryonic development and identified proteins as belonging to the vitellogenin 2 family. As development progresses, expression of IGF-II, phosphotyrosine, and phospho-Akt increased, while expression of growth factor receptor-bound protein 2 (GRB2) and one of guanine-nucleotide-binding proteins (Ras) decreased. These results provide basic information on the IGF system in the embryonic development of the olive flounder.

• IMMUNE NETWORK
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• v.6 no.2
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• pp.67-75
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• 2006

Background: Cytotoxic function of killer cells is inhibited by specific recognition of class I MHC molecules on target cells by inhibitory killer Ig-like receptors (KIR) expressed on NK cells and some cytotoxic T cells. The inhibitory effect of KIR is accomplished by recruitment of SH2-containing protein tyrosine phosphatase (SHP) to the phosphotyrosine residues in the cytoplasmic tail. Methods: By in vitro coprecipitation experiments and transfection analysis, we investigated the association of KIR with an adaptor protein Shc in Jurkat T cells. Results: The cytoplasmic tail of KIR appeared to associate with an adaptor protein Shc in Jurkat T celilysates. Similar in vitro experiments showed that phosphorylated KIR cytoplasmic tail bound SHP-1 and Shc in Jurkat T cell lysates. The association of KIR with Shc was further confirmed by transfection analysis in 293T cells. Interestingly, however, Shc appeared to be replaced by SHP-2 upon engagement of KIR in 293T cells. Conclusion: Our data indicate that KIR associate with an adaptor protein Shc in Jurkat T cells, and suggest that KIR might have an additional role which is mediated by this adaptor protein.

• Molecules and Cells
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• v.42 no.3
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• pp.245-251
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• 2019

Ependymal cells constitute the multi-ciliated epithelium, which lines the brain ventricular lumen. Although ependymal cells originate from radial glial cells in the perinatal rodent brain, the exact mechanisms underlying the full differentiation of ependymal cells are poorly understood. In this report, we present evidence that the Anks1a phosphotyrosine binding domain (PTB) adaptor is required for the proper development of ependymal cells in the rodent postnatal brain. Anks1a gene trap targeted LacZ reporter analysis revealed that Anks1a is expressed prominently in the ventricular region of the early postnatal brain and that its expression is restricted to mature ependymal cells during postnatal brain development. In addition, Anks1a-deficient ependymal cells were shown to possess type B cell characteristics, suggesting that ependymal cells require Anks1a in order to be fully differentiated. Finally, Anks1a overexpression in the lateral wall of the neonatal brain resulted in an increase in the number of ependymal cells during postnatal brain development. Altogether, our results suggest that ependymal cells require Anks1a PTB adaptor for their proper development.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.2
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• pp.181-189
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• 2008

The aim of this study was to evaluate the effects of $Ca^{2+}$, $HCO_3{^-}$ and BSA on the in vitro capacitation-associated protein tyrosine phosphorylation, hyperactivation and acrosome reaction in guinea pig sperm. Caudal epididymal sperm were incubated in four different groups: modified TALP (Tyrode's albumin lactate pyruvate) or TALP without one of the medium constituents ($Ca^{2+}$, $HCO_3{^-}$ and BSA). After incubation for the required time (0 h, 0.5 h, 1 h, 3 h, 5 h, and 7 h), sperm were removed for further experiment. The capacitation effect was assessed by CTC (Chlortetracycline) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7 h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum. Moreover, an absence of $Ca^{2+}$ or $HCO_3{^-}$ inhibited in vitro hyperactivation and acrosome reaction and decreased the phosphorylation of the proteins throughout the period of in vitro capacitation. However, an absence of BSA could not influence these processes if substituted by polyvinyl alcohol (PVA) in the medium.

• International Journal of Oral Biology
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• v.37 no.4
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• pp.189-195
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• 2012

Resistance to the induction of apoptosis is a possible mechanism by which tumor cells can survive anti-neoplastic treatments. Melanoma is notoriously resistant to anti-neoplastic therapy. Previous studies have demonstrated focal adhesion kinase (FAK) overexpression in melanoma cell lines. Given its probable role in mediating resistance to apoptosis, many researchers have sought to determine whether the downregulation of FAK in melanoma cells would confer a greater sensitivity to anti-neoplastic agents. Genistein is a known inhibitor of protein-tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhibiting the PTK-mediated signaling pathway. This present study was undertaken to investigate the effect of genistein on the expression of FAK and cell cycle related proteins in the G361 melanoma cell line. Genistein was found to have a preferential cytotoxic effect on G361 melanoma cells over HaCaT normal keratinocytes. Genistein decreased the expression of 125 kDa phosphotyrosine kinase and the FAK protein in particular. Genistein treatment did not affect the expression of p53 in G361 cells in which p21 is upregulated. The expression of cyclin B and cdc2 was downregulated by genistein treatment. Taken together, our data indicate that genistein induces the decreased proliferation of G361 melanoma cells via the inhibition of FAK expression and regulation of cell cycle genes. This suggests that the use of genistein may be a viable approach to future melanoma treatments.

• Toxicological Research
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• v.20 no.3
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• pp.179-185
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• 2004

Helicobacter pylori (H. pylori) infects more than half of the people in the world as a major microbe to cause most of gastric diseases. Recently, cytotoxin associated-antigen A (CagA) is believed as one of the most important virulence factors of H. pylori. Molecular toxicological pathway of CagA is necessary to investigate for understanding the pathological and toxicological aspects of H. pylori, since this virulence protein harasses intercellular processes of host cells to get profit for the survival of H. pylori. CagA is coded from cag pathogenicity island (cag PAI) and translocated into host cells by Type 4 secretion system (TFSS). Tyrosine phosphorylation of CagA targets Src homology 2-containing phosphotyrosine phosphatase (SHP-2) to form a CagA-SHP-2 complex. This complex depends on the similarity of sequence between EPIYA motif and Src homology 2 domain (SH2 domain) of CagA. The generation of growth factors is an essential role of CagA in protecting and healing gastric mucosa for the survival of H. pylori. On the other hand, the activation of IL-8 by CagA induces neutrophils generating inflammation and free radicals. Indeed, free radicals are well known carcinogen to induce DNA damage. In addition, the transduction of mitogen-activation signal by CagA is one of the interesting features to understand how to cause cancer. The relationship between cancer and inflammation with CagA was mainly discussed in this review.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5741-5745
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• 2013

Background: Heat-shock protein70 (HSP70) are intracellular protein chaperones, with emerging evidence of their association with various diseases. We have previously reported significantly elevated plasma-HSP70 (pHSP70) in pancreatic cancer. Current methods of pHSP70 isolation are ELISA-based which lack specificity due to cross-reactivity by similarities in the amino-acid sequence in regions of the protein backbone resulting in overestimated HSP70 value. Materials and Methods: This study was undertaken to develop a methodology to capture all isoforms of pHSP70, while further defining their tyrosine and serine phosphorylation status. Results: The methodology included gel electrophoresis on centrifuged supernatant obtained from plasma incubated with HSP70 antibody-coupled beads. After blocking non-specific binding sites, blots were immunostained with monoclonal-antibody specific for human-HSP70, phosphoserine and phosphotyrosine. Conclusions: Our novel immunocapture approach has distinct advantages over the commercially available methods of pHSP70 quantification by allowing isolation of molecular aggregates of HSP70 with additional ability to precisely distinguish phosphorylation state of HSP70 molecules at serine and tyrosine residues.