• Journal of Microbiology and Biotechnology
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• v.16 no.4
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• pp.524-530
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• 2006

Two protease inhibitors of 67 and 18 kDa, respectively, were purified from the eggs of glass fish, Liparis tanakai, by affinity chromatography and electro-elution method. The high molecular weight (HMW) protein was purified with a specific inhibitory activity, yield, and purity of 18.46 U/mg, 0.07%, and 131.86 fold, respectively, and was further characterized: Optimal temperature and pH for inhibitory activity of the HMW glassfish egg protease inhibitor were $40^{\circ}C$ and pH 6, respectively, and it was stable between $5^{\circ}C\;and\;50^{\circ}C$ in the pH range of 5-6 with maximal stability at pH 6. It was shown to be a competitive inhibitor against papain with an inhibition constant $(K_i)$ of 97.02 nM. Moreover, the 67 kDa protein inhibited cathepsin, a cysteine protease, more effectively than did an egg-white protease inhibitor. The HMW glassfish egg protease inhibitor was classified as a member of the family III (kininogen).

• Archives of Pharmacal Research
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• v.26 no.11
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• pp.937-942
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• 2003

Nitric oxide(NO) induces apoptosis in human osteoblasts. Treatment with exogenous NO donors, SNAP (S-Nitroso-N-acelylpenicillamine) and SNP (sodium nitroprusside), to MG-63 osteoblasts resulted in apoptotic morphological changes, as shown by a bright blue-fluorescent condensed nuclei and chromatin fragmentation by fluorescence microscope of Hoechst 33258-staining. The activities of caspase-9 and the subsequent caspase-3-like cysteine proteases were increased during NO-induced cell death. Pretreatment with Z-VAD-FMK (a pancaspase inhibitor) or Ac-DEVD-CHO (a specific caspase-3 inhibitor) abrogated the NO-induced cell death. The NO donor markedly activated JNK, a stress-activated protein kinase in the human osteoblasts. This study showed that the inhibition of the JNK pathway markedly reduced NO-induced cell death. But neither PD98059 (MEK inhibitor) nor SB203580 (p38 MAPK inhibitor) had any effect on NO-induced death. Taken together, these results suggest that JNK/SAPK may be related to NO-induced apoptosis in MG-63 human osteoblasts.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.655-660
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• 1999

Sphingomyelinase (SMase EC:3.l.4.l2) has been suggested to play important roles in the cell cycle, differentiation, apoptosis, inflammation, and the regulation of eukaryotic stress responses. SMase inhibitors may be a powerful tool to elucidate and regulate these cellular responses in which SMase involves. We first isolated an SMase inhibitor, named sphinin, from a strain of soil actinomycetes, F50970. Sphinin inhibited Mg/sup 2+/ -dependent neutral SMase from chicken embryo at 1.2 ㎍/㎖ of IC/sub 50/ Sphinin also inhibited acidic SMase, but it had no inhibitory activity on PI-PLC and PC-PLC, suggesting that sphinin is a specific inhibitor of SMase. The strain F50970 was identified as a Streptomyces sp. by its spiral spore chain, LL-diaminopimelic acid, menaquinone patterns of MK-9 (H'6) and MK-9 (H'8), FA-2c type of fatty acid pattern, and other morphological, physiological, and cultural characteristics.

• Archives of Pharmacal Research
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• v.27 no.12
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• pp.1258-1262
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• 2004

Phosphodiesterase (PDE) 4 is an enzyme that degrades intracellular cAMP. In the present study, the effect of rolipram, a specific phosphodiesterase (PDE) 4 inhibitor, on osteoclast formation was investigated. Rolipram induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. This activity was not observed in the absence of calvarial osteoblasts, suggesting that calvarial osteoblasts are likely target cells of rolipram. Osteoclast formation by rolipram was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for the osteoclast differentiation factor, TNF-related activation-induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL). Northern blot analysis revealed the effect of rolipram to be associated with the increased expression of TRANCE mRNA in mouse calvarial osteoblasts. Collectively, these data indicate that PDE4 inhibitor up-regulates the TRANCE mRNA expression in osteoblasts, which in turn controls osteoclast formation.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.3
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• pp.175-182
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• 2022

Translocation of azurophil granules is pivotal for bactericidal activity of neutrophils, the first-line defense cells against pathogens. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances bactericidal activity of human neutrophils via increasing translocation of azurophil granules. However, the precise mechanism of LPC-induced azurophil granule translocation was not fully understood. Treatment of neutrophil with LPC significantly increased CD63 (an azurophil granule marker) surface expression. Interestingly, cytochalasin B, an inhibitor of action polymerization, blocked LPC-induced CD63 surface expression. LPC increased F-actin polymerization. LPC-induced CD63 surface expression was inhibited by both a Rho specific inhibitor, Tat-C3 exoenzyme, and a Rho kinase (ROCK) inhibitor, Y27632 which also inhibited LPC-induced F-actin polymerization. LPC induced Rho-GTP activation. NSC23766, a Rac inhibitor, however, did not affect LPC-induced CD63 surface expression. Theses results suggest a novel regulatory mechanism for azurophil granule translocation where LPC induces translocation of azurophil granules via Rho/ROCK/F-actin polymerization pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.1
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• pp.51-58
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• 2021

Oxidative stress-induced neurodegeneration is one of several etiologies underlying neurodegenerative disease. In the present study, we investigated the functional role of histone methyltransferase G9a in oxidative stress-induced degeneration in human SH-SY5Y neuroblastoma cells. Cell viability significantly decreased on H2O2 treatment; however, treatment with the G9a inhibitor BIX01294 partially attenuated this effect. The expression of neuron-specific genes also decreased in H2O2-treated cells; however, it recovered on G9a inhibition. H2O2-treated cells showed high levels of H3K9me2 (histone H3 demethylated at the lysine 9 residue), which is produced by G9a activation; BIX01294 treatment reduced aberrant activation of G9a. H3K9me2 occupancy of the RE-1 site in neuron-specific genes was significantly increased in H2O2-treated cells, whereas it was decreased in BIX01294-treated cells. The differentiation of H2O2-treated cells also recovered on G9a inhibition by BIX01294. Consistent results were observed when used another G9a inhibitor UCN0321. These results demonstrate that oxidative stress induces aberrant activation of G9a, which disturbs the expression of neuron-specific genes and progressively mediates neuronal cell death. Moreover, a G9a inhibitor can lessen aberrant G9a activity and prevent neuronal damage. G9a inhibition may therefore contribute to the prevention of oxidative stress-induced neurodegeneration.

• International Journal of Oral Biology
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• v.45 no.4
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• pp.225-231
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• 2020

Glial cells, including astrocytes and microglia, interact closely with neurons and modulate pain transmission, particularly under pathological conditions. In this study, we examined the excitability of substantia gelatinosa (SG) neurons of the spinal dorsal horn using a patch clamp recording to investigate the roles of microglial activation in the nociceptive processes of rats. We used xanthine/xanthine oxidase (X/XO), a generator of superoxide anion (O2·-), to induce a pathological pain condition. X/XO treatment induced an inward current and membrane depolarization. The inward current was significantly inhibited by minocycline, a microglial inhibitor, and fluorocitrate, an astrocyte inhibitor. To examine whether toll-like receptor 4 (TLR4) in microglia was involved in the inward current, we used lipopolysaccharide (LPS), a highly specific TLR4 agonist. The LPS induced inward current, which was decreased by pretreatment with Tak-242, a TLR4-specific inhibitor, and phenyl N-t-butylnitrone, a reactive oxygen species scavenger. The X/XO-induced inward current was also inhibited by pretreatment with Tak-242. These results indicate that the X/XO-induced inward current of SG neurons occurs through activation of TLR4 in microglial cells, suggesting that neuroglial cells modulate the nociceptive process through central sensitization.

• YAKHAK HOEJI
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• v.37 no.4
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• pp.420-426
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• 1993

To effectively purify of IL-1 inhibitor from human febrile urine, we have established monoclonal antibody that reacts with human recombinant interleukin l$\beta$(IL-1$\beta$). The antibody, designated ON-1, was highly specific to IL-1$\beta$ and no cross-reaction with other cytokines(IL-l$\alpha$ and IL-4) was observed. As the results of ELISA inhibition assay and Western blotting method, it was further identified that ON-1 had high binding specificity with IL-1$\beta$. IL-1 receptor binding material from febrile patient urine was effectively purified with affinity column chromatography which conjugated with ON-1. This urinary material inhibited the thymocyte proliferation in a dosedependent manner. IL-l$\beta$ induced thymocyte proliferation activity was inhibited to 67.3% at 6 $\mu\textrm{g}$ of the purified urinary material. The result may suggest that this urinary material the purified urinary material. The result may suggest that this urinary material will have antagonic effect on IL-1 action mechanism and act IL-l$\beta$ inhibitor.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.132-134
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• 2005

The CC chemokine, monocyte chemoattractant protein-1 (MCP-1), plays a crucial role in the initiation of atherosclerosis and has direct effects that promote angiogenesis. To develop a specific inhibitor for MCP-1-induced angiogenesis, we performed in vitro selection employing phage display random peptide libraries. Most of the selected peptides were found to be homologous to the second extracellular loops of CCR2 and CCR3. We synthesized the peptide encoding the homologous sequences of the receptors and tested its effect on the MCP-1 induced angiogenesis. Surface Plasmon Resonance measurements demonstrated specific binding of the peptide to MCP-1 but not to the other homologous protein, MCP-3. Flow cytometry revealed that the peptide inhibited the MCP-1 binding to THP-1 monocytes. Moreover, CAM and rat aortic ring assays showed that the peptide inhibited MCP-1 induced angiogenesis. Our observations indicate that the MCP-1-binding peptide exerts its anti-angiogenic effect by interfering with the interaction between MCP-1 and its receptor.

• YAKHAK HOEJI
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• v.49 no.1
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• pp.109-113
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• 2005

In the present study treatment of IBMX, a phosphodiesterase (PDE) inhibitor, alone induced osteoclast formation in co-cultures of mouse bone marrow cells and calvarial osteoblasts. However, treatment of IBMX in combination with prostaglandin $E_2\;(PGE_2)$ inhibited osteoclast formation in a dose-dependent manner. Among various isozyme-specific PDE inhibitors, a PDE4 specific inhibitor, rolipram, showed similar effects as IBMX on osteoclast formation. To address the involvement of cyclic adenosine monophosphate (cAMP) in osteoclast formation, cAMP concentration in calvarial osteoblasts was investigated. When calvarial osteoblasts were co-cultured with IBMX alone or in combination with $PGE_2$, the patterns of cAMP concentration in calvarial osteoblasts were differ each other suggesting that cAMP in calvarial osteoblasts subtly regulates osteoclast formation.