• Radiation Oncology Journal
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• v.19 no.2
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• pp.171-180
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• 2001

Purpose : Expression of TIMP, intrinsic inhibitor of MMP, is regulated by signal transduction in response to genotoxins and is likely to be an important step in metastasis, angiogenesis and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP expression and its mechanism in head and neck cancer cell lines. Materials and Methods : Human head and neck cancer cell lines established at Asan Medical Center were used and radiosensitivity $(D_0)$, radiation cytotoxicity and metastatic potential were measured by clonogenic assay, n assay and invasion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and expression of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promoter region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC-HN-1 and -HN-9 cells with or without expression vector Ras, then the cells were exposed to radiation or PMA, PKC activator. EMSA was peformed with oligonucleotide (-59/-53 element and SP1) of TIMP1 promoter. Results : $D_0$ of HN-1, -2, -3, -5 and -9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. n assay confirmed cell viability, over $94\%$ at 24hrs, 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuously. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN-1 and HN-9 cell lines but after 10 Gy irradiation, it was increased in all cell lines. At 48hrs after irradiation, it was increased in HN-1 but decreased in HN-9 cells. But the change in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN-1 was induced by PMA but in HN-9 cell lines, it was suppressed. Wild type Ras induced the TIMP-1 transcription by 20 fold and 4 fold in HN-1 and HN-9 respectively. The binding activity to -59/-53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines. Conclusions : We observed the difference of expression and activity of TIMPs between radiosensitive and radioresistant cell line and the different signal transduction pathway between in these cell lines may contribute the different radiosensitivity. Further research to investigate the radiation response and its signal pathway of TIMPs is needed.

• The Korean Journal of Nuclear Medicine
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• v.38 no.4
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• pp.325-329
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• 2004

Purpose: Usefulness of mouse liver S9 fraction was evaluated for the measurement of the metabolites in the in vitro metabolism study of $^{18}F$-labeled radiotracers. Materials and Methods: Mouse liver S9 fraction was isolated at au early step in the course of microsome preparation. The in vitro metabolism studies were tarried out by incubating a mixture containing the radiotracer, S9 fraction and NADPH at $37^{\ciirc}C$, and an aliquot of the mixture was analyzed at the indicated time points by radio-TLC. Metabolic defluorination was further confirmed by the incubation with calcium phosphate, a bone mimic. Results: The radiotracer $[^{18}F]1$ underwent metabolic defluorination within 15 min, which was consistent with the results of the in vivo method and the in vitro method using microsome. Radiotracer $[^{18}F]2$ was metabolized to three metabolites including $4-[^{18}F]fluorobenzoic$ acid within 60 min. It is likely that the one of these metabolites at the origin of radio-TLC was identical with the one that obtained from the in vivo and in vitro (microsome) method. Compared with the in vitro method using microsome, the method using S9 fraction gave a similar pattern of the metabolites but with a different ratio, which can be explained by the presence of cytosol in the S9 fraction. Conclusion: These results suggest that the findings of the in vitro metabolism studies using S9 fraction can reflect the in vivo metabolism of novel radiotracers in the liver. Moreover, this method can be used as a tool to determine metabolic defluorination along with calcium phosphate absorption method.

• Korean Journal of Veterinary Research
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• v.36 no.4
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• pp.783-794
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• 1996

It has been suggested that ion transport systems are intimately involved in mediating the effects of growth regulatory factors on the growth of a number of different types of animal cells in vivo. The functional importance of the apical membrane $Na^+/H^+$ antiporter in the renal proximal tubule is evidenced by estimates that this transporter mediates the reabsorption of approximately one third of the filtered load of sodium and the bulk of the secretion of hydrogen ions. This study was designed to investigate the pathway utilized by IGF-I in regulating sodium transport in primary cultured renal proximal tubule cells. Results were as follows : 1. $Na^+$ was observed to accumulate in the primary cells as a function of time. Raising the concentration of extracellular NaCl induced an decrease in $Na^+$ uptake compared with control cells in a dose dependent manner. The rate of $Na^+$ uptake into the primary cells was about two times higher in the absence of NaCl($40.11{\pm}1.76pmole\;Na^+/mg\;protein/min$) than in the presence of 140mM NaCl($17.82{\pm}0.94pmole\;Na^+/mg\;protein/min$) at the 30 minute uptake. 2. $Na^+$ uptake was inhibited by IAA($1{\times}10^{-4}M$) or valinomycin($5{\times}10^{-6}M$) treatment($50.51{\pm}4.04$ and $57.65{\pm}2.27$ of that of control, respectively). $Na^+$ uptake by the primary proximal tubule cells was significantly increased by ouabain($5{\times}10^{-5}M$) treatment($140.23{\pm}3.37%$ of that of control). When actinomycin D($1{\times}10^{-7}M$) or cycloheximide($4{\times}10^{-5}M$) was applied, $Na^+$ uptake was decreased to $90.21{\pm}2.39%$ or $89.64{\pm}3.69%$ of control in IGF-I($1{\times}10^{-5}M$) treated cells, respectively. 3. Extracellular cAMP decreased $Na^+$ uptake in a dose-dependent manner($10^{-8}-10^{-4}M$). IBMX($5{\times}10^{-5}M$) also inhibited $Na^+$ uptake. Treatment of cells with pertussis toxin(50pg/ml) or cholera toxin($1{\mu}g/ml$) inhibited $Na^+$ uptake. Extracellular PMA decreased $Na^+$ uptake in a dose-dependent manner(1-100ng/ml). 100 ng/ml PMA concentration significantly inhibited $Na^+$ uptake in IGF-I treated cells. However, staurosporine($1{\times}10^{-7}M$) had no effect on $Na^+$ uptake. When PMA and staurosporine were added together, the inhibition of $Na^+$ uptake was not observed. In conclusion, sodium uptake in primary cultured rabbit renal proximal tubule cells was dependent on membrane potentials and intracellular energy levels. IGF-I stimulates sodium uptake through mechanisms that involve some degree of de novo protein and/or RNA synthesis, and cAMP and/or PKC pathway mediating the action mechanisms of IGF-I.

• Journal of Life Science
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• v.19 no.4
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• pp.520-525
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• 2009

Early onset of Batten disease (EBD), one of the most lethal neurodegenerative storage disorders of childhood, is caused by inactivating mutations in the Ceroid Lipofuscinosis, Neuronal (CLN1) gene. Neurogranin, a calmodulin-binding protein, is expressed in the brain and participates in the protein kinase C (PKC) signaling pathway. While oxidative stress is the suggested cause of neurodegeneration in EBD, its molecular mechanism(s) remains obscure. In this research, we examined the levels of neurogranin in the brain mRNA of wild-type (WT) mice and EBD knockout (KO) mice, as well as the proteins. We also performed neuronal cultures to measure the expression levels of neurgranin and phosphorylated-neurogranin with or without oxidative stress inducers and anti-oxidants. Results showed that neurogranin in both EBD KO mice brain mRNA and protein extracts decreased in an age dependent manner. However, high amounts of phosphorylated-neurogranin were detected in the 6-month brain. This pattern was also confirmed by cultured neurospheres samples. Moreover, neurospheres treated with $H_2O_2$, an oxidative stress inducer, showed increased phosphorylated-neurogranin patterns. Interestingly, this pattern returned to normal status when treated with N-acetyl-L-cystein, an anti-oxidant, after $H_2O_2$ treatment was performed. Our results suggest that the phosphorylation of neurogranin is affected by oxidative stress status in EBD, and appropriate anti-oxidant treatment will relieve hyper-phosphorylation of neurogranin.

• The Korean Journal of Physiology
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• v.30 no.2
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• pp.209-218
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• 1996

Acetylcholine (ACh) activates the inwardly rectifying muscarinic $K^{+}$ channel in rat atrial cells via pertussis toxin (PTX)-sensitive G-protein ($G_k$) coupled with the muscarinic receptor (mAChR). Although this $K^{+}\;(K_{ACh})$ channel function has reported to be modulated by the phosphorylation process, a kinase and phosphatase involved in these processes are still unclear. Since either PKA or PKC was not effective on this ATP-modulation, the present study examined the possible involvement of the protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) in the function of the $K_{ACh}$ Channel. In the inside-out (I/O) patch preparation excised from the adult rat atrial cell, when activated by 10 ${\mu}M$ ACh in the pipette and 100 ${\mu}M$ GTP in the bath, the mean open time (${\tau}_{o}$) and the channel activity ($K_{ACh}$) was 1.13 ms (n=5) and 0.19 (n=6), respectively. Following the application of 1 mM ATP into the bath, ${\tau}_{o}$ increased by 34% (1.54 ms, n=5) and $K_{ACh}$ by 66% (0.28, n=6). Channel function elevated by ATP was lasted after washout of ATP. However, this ATP-induced increase in the $K_{ACh}$ channel function did not occur in pretreated cells with genistein ($50{\sim}100 {\mu}M$), a selective PTK inhibitor, but occurred in pretreated cells with equimolar daidzein, a negative control of the genistein. On the contrary, PTP which acts on tyrosine residue conversely reversed both ATP-induced increased ${\tau}_{o}$ by 32% (1.20 ms, n=3) and $K_{ACh}$ by 41% (0.15, n=3), respectively. Taken together, these results suggest that $K_{ACh}$ channel may, at least partly, be regulated by the tyrosyl phosphorylation, although it is unclear where this process exerts on the muscarinic signal transduction pathway comprising the mAChR-$G_{k}$-the $K_{ACh}$ channel.

• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.712-719
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• 1998

Thyroid function is mainly regulated through cAMP and phophatidylinositol, and it is well known that TSH-stimulated thyroxine ($T_4$) release is inhibited by catecholamine from mouse thyroids via the ${\alpha}_1$-adrenoceptor stimulation. Previous study has established that the inhibition of $T_4$ release by ${\alpha}_1$-adrenoceptor stimulation results in activated protein kinase C (PKC). The purpose of this study was to determine if ion transport systems are involved in the inhibition of $T_4$ release elicited by ${\alpha}_1$-adrenergic agonist in mouse thyroids. TSH-, IBMX- and cAMP analogue-stimulated $T_4$ release were significantly inhibited by methoxamine, R59022 (diacylglycerol kinase inhibitor), and MDL (adenylate cyclase inhibitor). TSH-stimulated $T_4$ release could be inhibited by Bay K 8644 and cyclopiazoic acid, but not by verapamil and tetrodotoxin. The addition of nifedipine ($Ca^{2+}$ channel blocker), tetrodotoxin and lidocaine ($Na^+$ channel blockers), but not amiloride (EIPA) and ryanodine, completely blocked the inhibitory effects of methoxamine on $T_4$ release. TSH-stimulated $T_4$ release was also inhibited by benzamil ($Na^+-Ca^{2+}$ exchange inhibitor). TSH-, IBMX- and cAMP-stimulated $T_4$ release were inhibited by methoxamine or R59022, these effects were reversed by nifedipine. but not by verapamil. Furthermore, nifedipine reversed the inhibitory effects of benzamil and R59022 on TSH-stimulated $T_4$ release. These data suggest that the observed ${\alpha}_1$-adrenoceptor-mediated inhibition of $T_4$ release in mouse thyroids is the result of an increase in intracellular $Na^+$ or $Ca^{2+}$ effected via activation of fast $Na^+$ or nifedipine-sensitive $Ca^{2+}$ channels, and that $Na^+-Ca^{2+}$ exchange may play an important role in reducing thyroid hormone by increasing intracellular $Ca^{2+}$.

• Journal of Life Science
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• v.30 no.9
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• pp.826-833
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• 2020

Phospholipase C gamma (PLCγ) has critical roles in receptor tyrosine kinase- and non-receptor tyrosine kinase-mediated cellular signaling relating to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] to produce inositol 1,4,5 trisphosphate (IP₃) and diacylglycerol (DAG), which promote protein kinase C (PKC) and Ca²⁺ signaling to their downstream cellular targets. PLCγ has two isozymes called PLCγ1 and PLCγ2, which control cell growth and differentiation. In addition to catalytically active X- and Y-domains, both isotypes contain two Src homology 2 (SH2) domains and an SH3 domain for protein-protein interaction when the cells are activated by ligand stimulation. PLCγ also contains two pleckstrin homology (PH) domains for membrane-associated phosphoinositide binding and protein-protein interactions. While PLCγ1 is widely expressed and appears to regulate intracellular signaling in many tissues, PLCγ2 expression is restricted to cells of hematopoietic systems and seems to play a role in the regulation of immune response. A distinct mechanism for PLCγ activation is linked to an increase in phosphorylation of specific tyrosine residue, Y783. Recent studies have demonstrated that PLCγ mutations are closely related to cancer, immune disease, and brain disorders. Our review focused on the physiological roles of PLCγ by means of its structure and enzyme activity and the pathological functions of PLCγ via mutational analysis obtained from various human diseases and PLCγ knockout mice.

• Journal of the Korean Chemical Society
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• v.56 no.2
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• pp.274-289
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• 2012

The purposes of this study were to analyze instructional strategies of science teachers, science teachers' orientations toward science teaching by the reason which instructional strategies was used in middle school science classes, and the relations among PCK elements for suggesting a direction of improvement of PKC models. For this purpose, we selected three of middle school teachers as participants who had various teaching experience periods. Semi-structured interviews and classroom observations were gathered for data. From the data collected, we analyzed the type of instructional strategies of science teachers. On the base of these, we identified characteristics of the teachers' orientations toward teaching science. From the reason that instructional strategies was used, we could ascertain that knowledge of science curriculum and knowledge of students' learning which was component of PCK crucially affected instructional strategies of science teachers. Therefore we assured that analysis of practical instructional strategies of science teachers that showed through science instruction was the most effective method that could find out science teacher's orientation of teaching science internalized, and that knowledge of science curriculum and knowledge of students' learning was the basic component of PCK that formed instructional strategies of science teachers. On the basis of the result, a necessity for improvement of PCK models was presented.

• Development and Reproduction
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• v.2 no.2
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• pp.165-171
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• 1998

p62 is a novel cytoplsmic protein that binds to SH2 domain of p56$^{lck}$, lymphocyte-specific protein tyrosine kinase, and the expression of p62 was observed in most tissues. In addition p62 interacts with various proteins including ubiquitin and atypical PKC isoform, indicating its diverse biological role in different tissues. However, little is known about functional connection between p62 and its binding proteins. In the present study, a novel cellular protein, p62 has been shown to bind to 14-3-3 $\tau$ isoform that is specific for T cells. Moreover, overexpression of p62 in T cells caused to delay onset of UV-induced apoptosis characterized by DNA fragmentation and breakdown of poly (ADP-ribose) polymerase (PARP). Lately, 14-3-3 proteins have been shown to mediate survival signal via interacting proapoptotic Bad protein in the Iymphocyte. These results suggested the presence of p62-mediated regulatory mechanism during apoptosis in T cells, in which activation-induced apoptotic signal could be interfered by p62 and 14-3-3 protein.n.

• Journal of Life Science
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• v.12 no.4
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• pp.452-459
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• 2002

Neutrophil apoptosis is a constitutive process that can be enhanced or delayed by various stimuli. In this study, effect of brefeldin A (BFA), which affects biological process of secretion, on constitutive and delayed apoptosis of neutrophils was investigated. Neutrophil apoptosis was determined after culturing for 20 hr in vitro by morphological changes, annexin V staining and DNA electrophoresis. BFA increased the constitutive apoptotic rate of neutrophils in dose-dependent manner. The delay of apoptosis induced by granulocyte macrophage-colony stimulating factor and lipopolysaccharide was also blocked by 10 $\mu$M of BFA. However, this effect of BFA was less marked when neutrophils were treated with dexamethasone, interleukin-8, or dibutyryl-cAMP. Moreover, the delay of neutrophil apoptosis induced by rottlerin, a specific inhibitor of protein kinase C-$\delta$ was significantly abrogated by BFA. Although BFA-induced apoptosis was not blocked by the caspase-3 inhibitor, zDEVD-fmk, expression levels of myeloid cell leukemia-1 (Mcl-1) were down-regulated by BFA. These results suggest that derangement of vesicular protein transport may be involved in the apoptosis of neutrophils, and that the action of BFA on apoptosis is dependent on changes in the expression of Mcl-1.