• Journal of Periodontal and Implant Science
• /
• v.35 no.2
• /
• pp.427-436
• /
• 2005

Objective: MMP-8 is a neutrophil enzyme and its level increases in some inflammatory diseases, including periodontal disease. We knew that the lipopolysaccharide of E.coli(E-LPS) induced MMP-8 release from human neutrophils. E-LPS is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor(TLR). In the present study, we investigated whether MMP-8 release by E-LPS is induced via CD14-TLR pathway and the cellular mechanism of MMP-8 release in human neutrophils. Material and methods: Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, anti-TLR2 and anti-TLR4 or several inhibitors of microtubules and microfilaments and then incubated with E-LPS. The cells were treated TPCK and E-LPS simultaneously. The MMP-8amount in the culture medium was determined using ELISA. Results: E-LPS increased MMP-8release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR4 but not by anti-TLR2 antibodies. The inhibitors of microtubule and microfilament polymerization significantly decreased E-LPS-induced MMP-8release. TPCK inhibited E-LPS-induced MMP-8 release. Conclusion: These results suggest that MMP-8 release is induced by E-LPS via the CD14-TLR4 signal pathway in human neutrophils and may be depedent on microtubule and microfilament systems and $NF-{\kappa}B$ pathway.

• The Korean Journal of Zoology
• /
• v.36 no.3
• /
• pp.342-346
• /
• 1993

The protein level of cytoskeletons in cultured myoblasts was found to gradually decrease during the course of myogenesis. This decrease, however, could be prevented by treatiag the ceils with calpeptin (benzyloxycarbonyl-Leu-nLeu-H), a cell penetrating inhibitor of calpain. In contrast, E-64, which also is a potent inhibitor of calpain but can not be transported into the cells, showed little or no effect. In addition, the treatment of calpeptin was found to stabilize a number of specific cytoskeletal proteins from degradation but without any effect on the pattern of total cells proteins. Furthermore, calpeptin, but not E-64, blocked myoblast fusion in a dose-dependent manner. These results suggest that calpain is responsible for the myogenic time-dependent loss of cytoskeletal proteins and that the degradative process is associated with myoblast fusion. These results also suggest that the differential effects of the calpain inhibitors depend on the permeabIlity of the drugs across the cell membrane.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 2006.04a
• /
• pp.123-135
• /
• 2006

Alzheimer's disease (AD) is the most common form of dementia in the aging population and is clinically characterized by a progressive loss of cognitive abilities. Pathologically, it is defined by the appearance of senile plaques - extracellular insoluble, congophilic protein aggregates composed of amyloid $\beta$ (A$\beta$) and neurofibrillary tangles (NFTs) - inyracellular lesions consisting of paired helical filaments from hyperphosphorylated cytoskeletal tau protein as described by Alois Alzheimer a century ago. These hallmarks still serve as the major criteria for a definite diagnosis of the disease. Consequently, one of the key strategy for drug development in this disease area focuses on reducing the concentration of cerebral A$\beta$ plaque by using substances that inhibit A$\beta$ fibril formation. We focused on developing inhibitors by synthesizing several kinds of aromatic molecules. The synthetic compounds were initially screened to evaluate the effective compound by tioflavin T fluorescence assay. The selected effective compounds were tested cytotoxicity and protective effect from A$\beta$-induced neuronal toxicity by cell based MTT assay with HT22 hippocampal neurons. The BBB permeability on effectors was also tested in in vitro co-culture model(HUVEC/C6 cell line). The behavior test wea carried out in mutant APP/PS1 transgenic mouse model of Alzheimer's disease. And inhibition of A$\beta$ fibril formation by the effective compound was monitored with transmitted electron microscopic images.

• Biomedical Science Letters
• /
• v.10 no.3
• /
• pp.179-186
• /
• 2004

We examined a role of proteasome, the non-lysosomal multicatalytic protease complex,on the differentiation of chick embryonic myoblasts in culture. The peptide hydrolyzing activities of proteasome were found to change; the hydrolyzing activity against N-succinyl-Leu-Leu- Val- Tyr-7 -amido-4-methy1coumarin (SLLVY-AMC) was prominent and increased with myogenic differentiation. Proteasome inhibitors, N-carbobenzoxy-Leu-Leu-norvalinal (MG115) and N-carbobenzoxy-Ile-Glu (O-t-butyl)-Ala-Leucinal (PSI), blocked membrane fusion of myoblasts as well as the SLLVY-AMC hydrolyzing activity. Those inhibitory activities of the agents occurred in parallel, but were reversible and both cell fusion and the peptidase activity were restored when the agents were withdrawn from the culture medium. On the other hand, the agents caused accumulation of the ubiquitinylated proteins in the cytoskeletal proteins. These results suggest that each of the peptide hydrolyzing activities of proteasome is independently regulated during the myogenic differentiation and the chymotrypsin-like activity may play an important role in that process.

• Journal of Embryo Transfer
• /
• v.31 no.2
• /
• pp.131-136
• /
• 2016

Since the first success of animal cloning, somatic cell nuclear transfer presented various ideas in many research areas such as regenerative medicine. However, SCNT embryos has poor survival rate. Therefore, numerous researches carried out to enhance the developmental capability of porcine nuclear transfer embryos. Cytochalasin B, demecolcine, latrunculin A, cycloheximide and 6-dimethylaminopurine are efficient chemicals treated in post-activation procedure to increase the efficiency of SCNT. This review study is aim to investigate the effects of these chemicals applied to post-activation in porcine SCNT. Cytochalasin B, demecolcine, latrunculin A are cytoskeletal manuplators inhibit extrusion of pseudo-polar body. Cytochalasin B and demecolcine showed considerably higher blastocyst formation proportion (26-28%) compared to when they are not treated (16%). And when latrunculin A was treated for postactivation, blastocyst formation proportion was increased in SCNT embryos exposed to LA (38%) than those in control (14%). On the other hand, cycloheximide and 6-dimethylaminopurine are protein synthesis and kinase inhibitors. And they help to maintain $Ca^{2+}$ fluctuation in oocytes. Cleavage and blastocyst rates of NT embryos were increased when they were exposed to CHX (16.9% and 5.4% with no CHX).And 6-DMAP also showed higher blastocyst formation (21.5% compared to 15.7%, control). Although all these chemicals have different mechanisms, they showed developmental competence enhancement in NT embryos. However, there are only few studies comparing each chemical's post-activation effect. Therefore, further research and study should be conducted to find optimal chemical for improving the efficiency of SCNT.

• The Korean Journal of Pharmacology
• /
• v.29 no.2
• /
• pp.165-174
• /
• 1993

$Ca^{2+}$ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these $Ca^{2+}-dependent$ effects is unclear. Since cytoskeletal proteins are rapidly degraded by $Ca^{2+}-dependent $ proteinases (calpains) in vitro and in vivo, we investigated whether $Ca^{2+}-induced$ pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors EST (etyl (+)-(2S,3S)-3-[(S)-methyl-1-(3-methlbutylcarbamoyl)-butylcarbamoyl]2-oxiranecarboxylate) and MDL28170 (carbobenzoxyl-Val-Phe-H) to block the $Ca^{2+}$ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a signiciant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST $(5\;or\;20{\mu}M)$ to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 $(20\;{\mu}M)$ showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 nM and 300 nM concentrations, respectively, of A23187. EST $(20\;{\mu}M)$ did not block the A23187-induced rise of $[Ca^{2+}]_{i}$ as measured with fura-2. These results show that $Ca^{2+}-induced$ pruning of neurites in isolated hippocampal pyramidal neurons is mediated by calpains.