• Journal of Life Science
• /
• v.26 no.3
• /
• pp.282-288
• /
• 2016

Protein-protein interactions regulate the subcellular localization and function of receptors, enzymes, and cytoskeletal proteins. Proteins containing the postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain have potential to act as scaffolding proteins and play a pivotal role in various processes, such as synaptic plasticity, neural guidance, and development, as well as in the pathophysiology of many diseases. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, has a scaffolding function in the clustering of surface receptors, organization of signaling complexes, and coordination of cytoskeletal dynamics. However, the cellular function of MUPP1 has not been fully elucidated. In the present study, a yeast two-hybrid system was used to identify proteins that interacted with the N-terminal PDZ domain of MUPP1. The results revealed an interaction between MUPP1 and Wdpcp (formerly known as Fritz). Wdpcp was identified as a planar cell polarity (PCP) effector, which is known to have a role in collective cell migration and cilia formation. Wdpcp bound to the PDZ1 domain but not to other PDZ domains of MUPP1. The C-terminal end of Wdpcp was essential for the interaction with MUPP1 in the yeast two-hybrid assay. This interaction was further confirmed in a glutathione S-transferase (GST) pull-down assay. When coexpressed in HEK-293T cells, Wdpcp was coimmunoprecipitated with MUPP1. In addition, MUPP1 colocalized with Wdpcp at the same subcellular region in cells. Collectively, these results suggest that the MUPP1-Wdpcp interaction could modulate actin cytoskeleton dynamics and polarized cell migration.

• Journal of Life Science
• /
• v.26 no.11
• /
• pp.1313-1319
• /
• 2016

Cancer is a complex disease heterogeneously composed of various types of cells including cancer stem-like cells responsible for relapse and chemoresistance in the tumor microenvironment. The conventional two-dimensional cell culture-based platform has critical limitations for representing the heterogeneity of cancer cells in the three-dimensional tumor niche in vivo. To overcome this insufficiency, three-dimensional cell culture methods in a scaffold-dependent or -free physical environment have been developed. In this study, we improved and simplified the HCT-8 colon cancer cell-based spheroid culture protocol and evaluated the relationship between cancer stemness and responses of chemosensitivity to 5- Fluorouracil (5-FU), a representative anticancer agent against colon cancer. Supplementation with defined growth factors in the medium and the culture dish of the regular surface with low attachment were required for the formation of constant-sized spheroids containing $CD44^+$ and $CD133^+$ colon cancer stem cells. The chemo-sensitivities of $CD44^+$ cancer stem cells in the spheroids were much lower than those of $CD44^-$ non-stem-like cancer cells, indicating that the chemoresistance to 5-FU is due to the stemness of colon cancer cells. Taken together, the inflammation and oncogenic gut environment-sensitive HCT-8 cell-based colon cancer spheroid culture and comparative evaluation using the simplified model would be an efficient and applicable way to estimate colon cancer stemness and pharmaceutical response to anticancer drugs in the realistic tumor niche.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.28 no.6
• /
• pp.511-519
• /
• 2006

Autogenous bone grafts have been considered the gold standard for maxillofacial bony defects. However, this procedure could entail a complicated surgical procedure as well as potential donor site morbidity. Possibly the best solution for bone-defect regeneration is a tissue engineering approach, i.e. the use of a combination of a suitable scaffold with osteogenic cells. A major source of osteogenic cells is the bone marrow. Bone marrow-derived mesenchymal stem cells are multipotent and have the ability to differentiate into osteoblastic, chondrocytic, and adipocytic lineage cells. However, the isolation of cells from bone marrow has someproblems when used in clinical setting. Bone marrow aspiration is sometimes potentially more invasive and painful procedure and carries of a risk of morbidity and infection. A minimally invasive, easily accessible alternative would be cells derived from periosteum. The periosteum also contains multipotent cells that have the potential to differentiate into osteoblasts and chondrocytes. In the present study, we evaluated the osteogenic activity and mineralization of cultured human periosteal-derived cells. Periosteal explants were harvested from mandibule during surgical extraction of lower impacted third molar. The periosteal cells were cultured in the osteogenic inductive medium consisting of DMEM supplemented with 10% fetal calf serum, 50g/ml L-ascorbic acid 2-phosphate, 10 nmol dexamethasone and 10 mM -glycerophosphate for 42 days. Periosteal-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 14 of culture period, then decreased in intensity during the culture period. ALP mRNA expression increased up to day 14 with a decrease thereafter. Osteocalcin mRNA expression appeared at day 7 in culture, after that its expression continuously increased in a time-dependent manner up to the entire duration of culture. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. In conclusion, our study showed that cultured human periosteal-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix. As the periosteal-derived cells, easily harvested from intraoral procedure such as surgical extraction of impacted third molar, has the excellent potential of osteogenic capacity, tissue-engineered bone using periosteal-derived cells could be the best choice in reconstruction of maxillofacial bony defects.

• Journal of Chest Surgery
• /
• v.37 no.6
• /
• pp.482-491
• /
• 2004

Background: The xenogenic or allogenic valves after in Vitro repopulation with autologous cells or in vivo repo-pulation after acellularization treatment to remove the antigenicity could used as an alternative to synthetic polymer scaffold. In the present study, we evaluated the process of repopulation by recipient cell to the acellu-larized xenograft treated with NaCl-SDS solution and grafted in the right ventricular outflow tract. Material and Method: Porcine pulmonary valved conduit were treated with. NaCl-SDS solution to make the grafts acellularized and implanted in the right ventricular outflow tract of the goats under cardiopulmonary bypass. After evaluating the functions of pulmonary valves by echocardiography, goats were sacrificed at 1 week, 1 month, 3 months, 6 months, and 12 months after implantation, respectively. After retrieving the implanted valved conduits, histopathologic examination with Hematoxylin-Eosin, Masson' trichrome staining and immunohistochemical staining was performed. Result: Among the six goats, which had been implanted with acellularized pulmonary valved conduits, five survived the expected time period. Echocardiographic examinations for pulmonary valves revealed good function except mild regurgitation and stenosis. Microscopic analysis of the leaflets showed progressive cellular in-growth, composed of fibroblasts, myofibroblasts, and endothelial cells, into the acellularized leaflets over time. Severe inflammatory respon-se was detected in early phase, though it gradually decreased afterwards. The extracellular matrices were regenerated by repopulated cells on the recellularized portion of the acellularized leaflet. Conclusion: The acellularized xenogenic pulmonary valved conuits were repopulated with fibroblasts, myofibroblasts, and endothelial cells of the recipient and extracellullar matrices were regenerated by repopulted cells 12 months after the implantation. The functional integrity of pulmonary valves was well preserved. This study showed that the acellularized porcine xenogenic valved conduits could be used as an ideal valve prosthesis with long term durability.

• Journal of Periodontal and Implant Science
• /
• v.31 no.1
• /
• pp.1-23
• /
• 2001

Chitosan is biodegradable natural polymer that has been demonstrated its ability to improve wound healing, and calcium metaphosphate(CMP) is a unique class of phosphate minerals having a polymeric structure. In this study, chitosan/CMP and platelet derived growth factor(PDGF-BB) loaded chitosan/CMP sponges were developed, and the effect of the sponges on bone regeneration and their possibility as scaffolds for bone formation by three-dimensional osteoblast culture were examined. PDGF-BB loaded chitosan/CMP sponges were prepared by freeze-drying of a mixture of chitosan solution and CMP powder, and soaking in a PDGF-BB solution. Fabricated sponge retained its 3-dimensional porous structure with $100-200\;{\mu}m$ pores. The release kinetics of PDGF-BB loaded onto the sponge were measured in vitro with $^{125}I-labeled$ PDGF-BB. In order to examine their possibility as scaffolds for bone formation, fetal rat calvarial osteoblastic cells were isolated, cultured, and seeded into the sponges. The cell-sponge constructs were cultured for 28 days. Cell proliferation, alkaline phosphatase activity were measured at 1, 7, 14 and 28 days, and histologic examination was performed. In order to examine the effect on the healing of bone defect, the sponges were implanted into rat calvarial defects. Rats were sacrificed 2 and 4 weeks after implantation and histologic and histomorphometrical examination were performed. An effective therapeutic concentration of PDGF-BB following a high initial burst release was maintained throughout the examination period. PDGF-BB loaded chitosan/CMP sponges supported the proliferation of seeded osteoblastic cells as well as their differentiation as indicated by high alkaline phosphatase activities. Histologic findings indicated that seeded osteoblastic cells well attached to sponge matrices and proliferated in a multi-layer fashion. In the experiments of implantation in rat calvarial defects, histologic and histomorphometric examination revealed that chitosan/CMP sponge promoted osseous healing as compared to controls. PDGF-BB loaded chitosan/CMP sponge further echanced bone regeneration. These results suggested that PDGF-BB loaded chitosan/CMP sponge was a feasable scaffolding material to grow osteoblast in a three-dimentional structure for transplantation into a site for bone regeneration.

• Journal of Life Science
• /
• v.25 no.1
• /
• pp.101-112
• /
• 2015

A type of cell junction that is formed between different parts within the same cell is called autotypic cell junction. Autotypic junction proteins form tight junctions found between membrane lamellae of a cell, especially in myelinating glial cells. Some of them have postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domains, which interact with the carboxyl (C)-terminal PDZ-binding motif of other proteins. PDZ domains are protein-protein interaction modules that play a role in protein complex assembly. The PDZ domain, which is widespread in bacteria, plants, yeast, metazoans, and Drosophila, allows the assembly of large multi-protein complexes. The multi-protein complexes act in intracellular signal transduction, protein targeting, and membrane polarization. The identified PDZ domain-containing proteins located at autotypic junctions include zonula occludens-1 (ZO-1), ZO-2, pals-1-associated tight junction protein (PATJ), multi-PDZ domain proteins (MUPPs), membrane-associated guanylate kinase inverted 2 (MAGI2), and protease-activated receptor (PAR)-3. PAR-3 interacts with atypical protein kinase C and PAR-6, forming a ternary complex, which plays an important role in the regulation of cell polarity. MAGI2 interacts with ${\alpha}$-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) receptor at excitatory synapses. PATJ is detected in paranodal loops associated with claudin-1. On the other hand, MUPP1 is found in mesaxons and Schmidt-Lanterman incisures with claudin-5. ZO-1, ZO-2, and PAR-3 are found at all three sites. Different distributions of PDZ domain-containing proteins affect the development of autotypic junctions. In this review, we will describe PDZ domain-containing proteins at autotypic tight junctions in myelinating Schwann cells and their roles.

• Journal of Life Science
• /
• v.25 no.5
• /
• pp.594-600
• /
• 2015

Protein-protein interactions have a critical role in the regulation of many cellular functions. Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain is one of domains that mediate protein-protein interactions. PDZ domains typically bind to the specific motif at the carboxyl (C)-terminal end of partner proteins. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, serves a scaffolding function for structure proteins and signaling proteins, but the cellular function of MUPP1 has not been fully elucidated. We used the yeast two-hybrid system to identify proteins that interact with PDZ domains of MUPP1. We found an interaction between MUPP1 and muskelin. Muskelin was recently identified as a GABA_A receptor (GABA_AR) α1 subunit binding protein and known to have a role in receptor endocytosis and degradation. Muskelin bound to the 3^rd PDZ domain, but not to other PDZ domains of MUPP1. The C-terminal end of muskelin was essential for the interaction with MUPP1 in the yeast two-hybrid assay. When co-expressed in HEK-293T cells, muskelin but not the C-terminal deleted muskelin was co-immunoprecipitated with MUPP1. In addition, MUPP1 co-localized with muskelin at the same subcellular region in cells. These findings collectively suggest that MUPP1 or its interacting proteins could modulate GABA_AR trafficking and turnover through the interaction with muskelin.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.38 no.11
• /
• pp.1492-1498
• /
• 2009

In the dermis, fibroblast plays an important role in the turnover of the dermal extracellular matrix. Collagen I and III, which are the most important dermal proteins of the extracellular matrix, function as a stabilizing scaffold of dermal connective tissues, as well as a regulator of differentiation and migration of dermal cells. In this study, we investigated the effect of various nutrients, such as ascorbic acid, silicon, Fe, lysine and proline which function as cofactors or building blocks on collagen synthesis. When the physiological concentrations of ascorbic acid (0-100 ${\mu}M$), silicon (0-50 ${\mu}M$), Fe (0-50 ${\mu}M$), lysine (0-150 ${\mu}M$) and proline (0-300 ${\mu}M$) were treated at HS27 for either 3 or 5 days, 5 day treatment of ascorbic acid at the low concentration (5-10 ${\mu}M$) increased the expression of collagen I and III protein by 115-1300% without increasing cell proliferation. 3 or 5 days treatment of Fe increased the expression of collagen I and III proteins up to 323% in parallel with cell proliferation by 164%. However, cell proliferation and expression of collagen I and III protein in silicon treated HS27 did not differ. Proline and lysine only increased cell proliferation up to 247.9%. Taken together, we demonstrate that the physiological concentrations of ascorbic acid and Fe enhance the expression of collagen I and III protein for treatment of 3 or 5 days.

• Journal of Life Science
• /
• v.33 no.7
• /
• pp.531-537
• /
• 2023

Intracellular and axonal transport is mediated by microtubule-dependent motor proteins, such as kinesins and cytoplasmic dynein. Kinesin moves along the microtubule to the positive end of the microtubule, while dynein moves to the negative end of the microtubule. Kinesin-1 was first identified as a kinesin superfamily protein (KIF) that functions in the intracellular transport of various cargoes, including organelles, neurotransmitter receptors, and mRNA-protein complexes, through interactions between the carboxyl (C)-terminal domain and the cargo. It interacts with other cargoes, but the adapter/scaffold proteins that mediate between kinesin-1 and the cargo have yet to be fully identified. In this study, a yeast two-hybrid screen was used to identify adapter proteins that interact with the C-terminal region of KIF5A. We found an association between the C-terminal region of KIF5A and the cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), originally identified in malignant hamster oral keratinocytes. CDK2AP1 bound to the C-terminal region of KIF5A and did not interact with KIF3A (the motor of kinesin-2), KIF5B, KIF5C, and kinesin light chain 1 (KLC1). The C-terminal region of CDK2AP1 is essential for its interaction with KIF5A. When co-expressed in HEK-293T cells, CDK2AP1 and kinesin-1 co-immunoprecipitated and co-localized in the cells. These results suggest that the KIF5A-CDK2AP1 interaction serves as an adapter protein connecting kinesin-1 and the cargo when kinesin-1 transports cargo in cells.

• Journal of Chest Surgery
• /
• v.41 no.6
• /
• pp.679-686
• /
• 2008

Background: Cellular remnants in the bioprosthetic heart valve are known to be related to a host's immunologic response and they can form the nidus for calcification. The extracellular matrix of the decellularized valve tissue can also be used as a biological scaffold for cell attachment, endothelialization and tissue reconstitution. Thus, decellularization is the most important part in making a bioprosthetic valve and biological caffold. Many protocols and agents have been suggested for decellularization, yet there ave been few reports about the effect of a treatment with hypotonic solution prior to chemical or enzymatic treatment. This study investigated the effect of a treatment with hypotonic solution and the appropriate environments such as temperature, the treatment duration and the concentration of sodium dodecylsulfate (SDS) for achieving proper decellularization. Material and Method: Porcine aortic valves were decellularized with odium dodecylsulfate at various concentrations (0.25%, 0.5%), time durations (6, 12, 24 hours) and temperatures ($4^{\circ}C$, $20^{\circ}C$)(Group B). Same the number of porcine aortic valves (group A) was treated with hypotonic solution prior to SDS treatment at the same conditions. The duration of exposure to the hypotonic solution was 4, 7 and 14 hours and he temperature was $4^{\circ}C$ and $20^{\circ}C$, respectively. The degree of decellularization was analyzed by performing hematoxylin and eosin staining. Result: There were no differences in the degree of decellularization between the two concentrations (0.25% 0.5%) of SDS. Twenty four hours treatment with SDS revealed the best decellularization effect for both roups A and B at the temperature of $4^{\circ}C$, but there was no differences between the roups at $20^{\circ}C$. Treatment with hypotonic solution (group A) showed a better ecellularization effect at all the matched conditions. Fourteen hours treatment at $4^{\circ}C$ ith ypotonic solution prior to 80S treatment revealed the best decellularization effect. The treatment with hypotonic solution at $20^{\circ}C$ revealed a good decellularization effect, but his showed significant extracellular matrix destruction. Conclusion: The exposure of porcine heart valves to hypotonic solution prior to SDS treatment is highly effective for achieving decellularization. Osmotic treatment with hypotonic solution should be considered or achieving decellularization of porcine aortic valves. Further study should be carried out to see whether the treatment with hypotonic solution could reduce the exposure duration and concentration of chemical detergents, and also to evaluate how the structure of the extracellular matrix of the porcine valve is affected by the exposure to hypotonic solution.