• The Korean Journal of Zoology
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• v.36 no.4
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• pp.439-451
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• 1993

Protein kinase C isozymes in a human prostate adenocarcinoma PC-3 cell line were characterized. Immunoreactive bands and immunocytochemical stains were obsenred in PC-3 cells with antibodies raised against protein kinase C ${\alpha}$, ${\beta}$, ${\gamma}$, $\delta$, $\varepsilon$, and ζ types, respectively. Protein kinase C ${\alpha}$ corresponded to a immunoreactive band at a molecular weight of 80,000-dalton, whereas molecular weights of other immunoreactive isozvmes of protein kinase C were detected at 68,000-dalton. Protein kinHse C $\delta$ and ζ antibodies detected additional bands at 55,000-dalton and 80,000-dalton, respectively Immunocvtochemical study confirmed the results of the immunoblotting experiments qualitatively: all six protein kinase C isozymes were detected in the cytoplasm of PC-3 cells. Translocation of protein kinase C in PC-3 cells were also examined with phorbol 12-myristate 13-acetate (PMA), bryostatin 2, diolein, and 1-oleoyl-2-acetyl glycerol (OAG). Differential reactions of protein kinase C isozvmes to these activators were obsenred. When PC-3 cells were treated with 10mM bryostatin 2, protein kinase C isozyme u was translocated into the nucleus, whereas s type was translocated into the plasma membrane and the nucleus. Protein kinase C ${\alpha}$ and ζ types were translocated into the nucleus following the treatment with 101M diolein, whereas protein kinase C ${\alpha}$, ${\beta}$, ${\gamma}$, and $\varepsilon$ types were translocated into the nucleus by the treatment with 10mM OAG. Protein kinase C ${\alpha}$ and $\varepsilon$ types were translocated into the nucleus in the presence of 100nM PMA. Protein kinase C $\delta$ type was translocated to the nuclear membrane by these activators, however, only PMA-induced translocation was inhibited by protein kinase C inhibitor, 1-(5-isoquinolinesulfonyll-2-methvlpiperazine dihvdrochloride (H7) . H7 inhibited translocation of protein kinase C ${\alpha}$ type induced by PMA, ${\beta}$ type by OAG and s type by PMA and OAG, whereas it did not affect translocations induced by bryostatin and diolein, respectively. These results suggest that there exist six isoformes of protein kinase C (${\alpha}$, ${\beta}$, ${\gamma}$, $\delta$, $\varepsilon$ and ζ types) in PC-3 cells and that each of these isozvmes distinctivelv reacts to bryostatin, diolein, OAG and PMA, in part due to an altered molecular size and conceivably discrete binding site(s).

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.2
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• pp.134-140
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• 1997

The monoclonal antibody against a novel protein, which was induced in the roots of rice seedlings treated with NaCl, was produced. Oryzea sativa L. cv. Annapruna grains were sown on clean sands and grown for 10days and then the seedlings were soaked in 50mM NaCl aqueous solution. In 48hrs of the NaCl treatment, the roots were collected and homogenated with liquid nitrogen and extraction buffer. The homogenate was centrifuged and to the supernatant 75％ ammonium sulfate was added to pre-cipitate proteins. From these proteins a novel protein was purified through DEAE-ion chromatography and FPLC(Phenyl column). This protein appeared as a single band in the native electrophoresis. Using this protein as antigen, monoclonal antibody was produced. Five cell lines that secreted antibodies specifically bound to this protein were constructed.

• Journal of Microbiology
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• v.37 no.4
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• pp.229-233
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• 1999

The mitochondrial plasmid pMLP1 from a white-rot fungus, Pleurotus ostreatus, is a double-stranded DNA containing 381 bp terminal inverted repeat (TIR) whose 5'-ends are covalently bound by terminal proteins. The plasmid contains two major open reading frames (ORFs), encoding putative DNA and RNA polymerases, and a minor ORF encoding a small, highly basic protein. To identify the DNA binding activity that recognizes the TIR region of pMLP1, gel retardation assays were performed with mitochondrial extracts. A specific protein binding to a region between 123 and 248 nt within TIR was observed. We examined whether the gene product of mORF1 bindes to this region specifically. E. coli cell extract which contains an overproduced mORF1 protein formed a complex specific to the region between 123 and 248 nt. Inclusion of mORF1 protein in the specific complex formed between P. ostreatus mitochondrial extract and TIR was confirmed by a supershift assay using polyclonal antibodies against the mORF1 protein. Our result suggest that the product of mORF1 may function as a terminal region recognition factor (TRF), recognizing an internal region in TIR.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.5
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• pp.545-549
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• 1998

The influence of refeeding with either vitamin, mineral, fibre of water on protein synthesis and mRNA content in the liver and breast muscle of fasted chicks was investigated. At 15 d of age, chicks were fasted for 2 d and then refed either vitamin, mineral, fibre or water. The fractional synthesis rate (FSR) of protein was measured after 30 min of refeeding by using a large dose injection of L - 2, $6[^3H]$ phenylalanine. In the liver, FSR was reduced by fasting and tended to increase but not significantly by refeeding with vitamin or mineral. FSR was not affected by refeeding with fibre or water. There was no influence of fasting and refeeding on ribosomal capacity (the RNA : protein ratio) and ribosomal efficiency (total protein synthesised per total RNA). The absolute synthesis rate (ASR) of liver protein and hepatic mRNA content were reduced by fasting and unchanged by refeeding. In the muscle, FSR, ASR and mRNA content were significantly decreased by fasting and not recovered by refeeding with either vitamin, mineral, fibre or water. It concluded that vitamin, mineral, fibre and water have little capacity to stimulate liver and muscle protein synthesis reduced by fasting.

• Tuberculosis and Respiratory Diseases
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• v.44 no.2
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• pp.338-348
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• 1997

Background : Endotoxin, the component of outermembrane of gram negative organism, plays an important role in the initiation and amplification of inflammatory reaction by its effects on inflammatory cells. Until recently, there have been continuing efforts to delinate the mechanisms of the signal trasduction pathway of endotoxin stimuli on inflammatory cells. By uncovering the mechanisms of signal transduction pathway of endotoxin stimuli, we can expect to have tools to control the excessive inflammatory responses which sometimes may be fatal to the involved host. It was generally accepted that endotoxin exerts its inflammatory effects through inflammatory cytokines that are produced by endotoxin-stimulated inflammatory cells and there were some reports on the importance of protein kinase C and protein tyrosine kinase activation in the production of inflammatory cytokines by endotoxin So we evaluated the effect of pretreatment of protein kinase C inhibitors (H7, Staurosporin) and protein tyrosine kinase inhibitors(Herbimycin, Genistein) on the endotoxin-stimulated cytokines(IL-8 & TNF-$\alpha$) mRNA expression. Method : Peripheral blood monocytes were isolated from healthy volunteers by Ficoll-Hypaque density gradient method and purified by adhesion to 60mm Petri dishes. Endotoxin(LPS 100ng/ml) was added to each dishes except one control dish, and each endotoxin-stimulated dishes was preincubated with H7, Staurosporin(protein kinase C inhibitor), Herbimycin or Genistein(protein tyrosine kinase inhibitor) respectively except one dish. Four hours later the endotoxin stimulation, total RNA was extracted and Northern blot analysis for IL-8 mRNA and TNF-$\alpha$ mRNA was done. Result : Endotoxin stimulation increased the expression of IL-8 mRNA and TNF-$\alpha$ mRNA expression in human peripheral blood monocyte as expected and the stimulatory effect of endotoxin on TNF-$\alpha$ mRNA expression was inhibited by protein kinase C inhibitors(H7, Staurosporin) and protein tyrosine kinase inhibitors (Herbimycin, Genistein). The inhibitory effect of each drugs was increased with increasing concentration. The stimulatory effect of endotoxin on IL-8 mRNA was also inhibited by H7 and protein tyrosine kinase inhibitors (Herbimycin, Genistein) dose-dependently but not by Staurosporin. Conclusion : Protein kinase C and protein tyrosine kinase are involved in the endotoxin induced signal transduction pathway in human peripheral blood monocyte.

• Molecules and Cells
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• v.24 no.2
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• pp.276-282
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• 2007

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin ($Ca^{2+}/CaM$)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about $Ca^{2+}/CaM$-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative $Ca^{2+}$-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a $Ca^{2+}$-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM $Mn^{2+}$. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other $Ca^{2+}/CaM$-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and $Ca^{2+}/CaM$-dependent protein kinase), increasing the concentration of calmodulin to more than $3{\mu}M$ suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis $Ca^{2+}/CaM$-dependent protein kinase which is presumably involved in CaM-mediated signaling.

• Toxicological Research
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• v.31 no.2
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• pp.191-201
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• 2015

We evaluated the inhibitory effect of Pueraria lobata root ethanol extract (PLREE) on lipid accumulation during 3T3-L1 differentiation to adipocytes by measuring the intracellular expression of adipogenic, lipogenic, and lipolytic markers and lipid accumulation. The total polyphenol and flavonoid content of PLREE were 47 and 29 mg/g, respectively. The electron donating capacity of PLREE at $1,000{\mu}g/mL$ was 48.8%. Treatment of 3T3-L1 preadipocytes with 100, 250, or $500{\mu}g/mL$ PLREE for 8 days dose-dependently promoted the differentiation of 3T3-L1 cells. In contrast, the lipid content of PLREE-treated cells was significantly reduced by 7.8% (p < 0.05), 35.6% (p < 0.001), and 42.2% (p < 0.001) following treatment with 100, 250, and $500{\mu}g/mL$ PLREE, respectively, as compared to differentiated control cells. PLREE upregulated peroxisome proliferator-activated receptor ${\gamma}$ mRNA and protein, and sterol regulator element-binding protein-1c mRNA levels, but did not affect CCAAT/enhancer binding-protein ${\beta}$ and ${\alpha}$ mRNA levels. PLREE also downregulated acetyl-CoA carboxylase mRNA and protein, fatty acid synthase (FAS) protein, and leptin mRNA levels, but did not affect FAS mRNA expression. PLREE upregulated adipose triglyceride lipase mRNA and protein expression, and hormone-sensitive lipase (HSL) protein expression, but did not affect HSL mRNA expression. In conclusion, we found that PLREE enhanced adipogenesis, but reduced lipogenesis, resulting in decreased lipid accumulation in 3T3-L1 cells.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.2
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• pp.236-244
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• 2006

This study was designed to determine the effects of daidzein (DE) on hepatic lipid metabolism in chicks fed with low protein (LP) diet based on casein. In experiment 1, the male chicks were fed with one of the three levels of dietary protein containing 10.95%, 21.9% and 43.8% protein content for 2 days. In experiment 2, the chicks were fed one of the three levels of protein with or without DE at 1,000 mg/kg diet for 2 days. Experiment 3 was conducted to compare DE (LP+DE) with estradiol (LP+E2) in chicks fed with LP diet for 7 days. Plasma lipid profiles, hepatic lipid profiles, activities of hepatic malic enzyme and isocitrate dehydrogenase (ICDH) were measured. Transcriptions of hepatic fatty acid synthase, apolipoprotein-B (APO-B), and fructose bisphosphatase mRNA were measured by RT-PCR. Increasing dietary protein levels markedly decreased the concentrations of plasma triglycerides, hepatic total lipids, hepatic TG, and the mRNA transcriptions while the increased dietary protein levels increased hepatic ICDH activities in experiment 1. In experiment 2, the effects of dietary protein levels on blood and hepatic lipid content were more prominent than those of the additional DE. Interestingly, plasma TG levels were affected by DE supplementation (p<0.05). In experiment 3, DE inhibited APO-B mRNA expressions and stimulated the accumulation of lipid in the liver through mechanisms different from E2. In this study, we demonstrate that DE has beneficial effects on blood lipid profiles, but that it inhibits APO-B mRNA transcription and aggravates the fatty liver induced by LP diet in chicks.

• BMB Reports
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• v.37 no.2
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• pp.229-233
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• 2004

A haemagglutinating protein from the saline extracts of Kalanchoe crenata leaves, which agglutinate all human blood types, was purified to homogeneity by ion-exchange chromatography on a DEAE-Cellulose column followed by gel filtration on a Sephadex G-100 column. The purified protein showed one band, both in non-denaturing PAGE and SDS-PAGE. The $M_r$ that was determined by SDS-PAGE was 44,000 Da and that estimated from gel filtration was 47,000. Treatment of the haemagglutinating protein with 5 mM EDTA diminished the haemagglutinating activity to 50% of the original level. The addition of divalent cations, 10 mM $Mg^{2+}$, 10 mM $Mn^{2+}$, or 10 mM $Ba^{2+}$, totally restored and enhanced the activity. The protein showed maximum activity over the 3-7 pH range and was heat-resistant. It was also a glycoprotein containing about 1.5% carbohydrate.

• Progress in Medical Physics
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• v.11 no.1
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• pp.29-38
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• 2000

The signal transduction pathway for most neurotransmitter induced inhibition of $Ca^{2+}$ channels in sympathetic neurons involves a G-protein mediated, membrane-delimited mechanism without the participation of any known protein kinase. However, activation of protein kinase C (PKC) has been proposed as one of the intracellular mechanisms mediating some neurotransmitter induced $Ca^{2+}$ channel inhibition. In the present study, we investigated the effects of phorbol-12, 13-dibutyrate (PDBu) on $Ca^{2+}$ channel currents of acutely dispersed neurons from adult rat superior cervical ganglion (SCG) neurons using whole cell variant of the patch clamp technique. PDBu (500 nM), the activator of PKC, increased $Ca^{2+}$ channel currents and retarded the deactivation of tail currents. The effects of PDBu were voltage dependent and the maximal increase in the current amplitudes was observed between -10 to 10 mV (n=4). PDBu attenuated $Ca^{2+}$ current inhibition induced by norepinephrine (NE), which modulates $Ca^{2+}$ channels via a pertussis toxin (PTX)-sensitive pathway. Inhibition of PDBu by staurosporine (1 $\mu$M) blocked the effects of PDBu on current amplitudes and NE-induced G-protein mediated inhibition of $Ca^{2+}$ currents. Further experiment should be done to know if G-protein or $Ca^{2+}$ channel itself is the target of PKC phosphorvlation.phosphorvlation.