• Journal of Life Science
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• v.27 no.8
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• pp.864-872
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• 2017

Equine chorionic gonadotropin (eCG) consists of highly glycosylated ${\alpha}-$ and ${\beta}-subunits$ and is a unique member of the gonadotropin family, because it elicits the response characteristics of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in species other than the horse. To directly assess the biological function of $rec-eCG{\beta}/{\alpha}$, we constructed mammalian expressing vectors of equine luteinizing hormone/chorionic gonadotropin receptors (eLH/CGR). The activity of $rec-eCG{\beta}/{\alpha}$ in vitro assayed in transient transfected CHO-K1 cells and in stably transfected PathHunter Parental cells with eLH/CGR was investigated. $rec-eCG{\beta}/{\alpha}$ was efficiently secreted in the CHO-K1 suspension cell media, and the quantity detected was about 200 mIU/ml from 1 to 7 days after transfection. In the western blot analysis, the $rec-eCG{\beta}/{\alpha}$ protein was broadly identified to be about 40~45 kDa molecular weight. The cAMP stimulation in CHO-K1 cells expressing eLH/CGR was determined to evaluate the activity of $rec-eCG{\beta}/{\alpha}$. The cAMP concentration increased in direct proportion to the concentration of the $rec-eCG{\beta}/{\alpha}$. The $EC_{50}$ value in the transient transfected CHO-K1 cells was $8.1{\pm}6.5ng$. The stable cell lines of eLH/CGR were established in the PathHunter Parental cells expressing ${\beta}-arrestin$. We found that $rec-eCG{\beta}/{\alpha}$ had full LH activity in the PathHunter Parental cells expressing eLH/CGR. The $EC_{50}$ value in transient and stable cells was $5.0{\pm}4.7ng/ml$ and $4.5{\pm}5.2ng/ml$, respectively. These results suggest that $rec-eCG{\beta}/{\alpha}$ has a biological activity in a cell expressing eLH/CGR. These stable cells expressed in PathHunter Parental cells could be useful for elucidating the functional mechanisms of deglycosylated $rec-eCG{\beta}/{\alpha}$ mutants.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.5
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• pp.446-456
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• 2005

Nerve growth factor(NGF) has a critical role in peripheral nerve regeneration. The aim of this study is to construct a well-functioning hNGF-$\beta$ recombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-$\beta$ gene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-$\beta$ which was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-$\beta$ recombinant adenovirus. After production of recombinat adenovirus in a large scale, its transfection efficiency, expression, and function were evaluated using cell lines or primarily cultured cells of HEK293 cells, Schwann cells, fibroblast(NIH3T3) and myocyte(CRH cells). GFP expression was observed in 90% of infected cells compared to uninfected cells. Total mRNA isolated from hNGF-$\beta$ recombinat adenoviru infected cells showed strong RT-PCR band, however, LacZ recombinant adenovirus infected or uninfected cells did not. NGF quantification by ELISA showed a maximal release of 18.865 +/- 0.31ng/mL at 4th day. PC-12 cells exposed to media with hNGF-$\beta$ recombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-$\beta$ recombinant adenovirus and induced over expression of NGF successfully in nonneuronal and neuronal cells. Following these result, it is expected to develop an improved treatment strategy peripheral nerve regeneration using the hNGF-$\beta$ gene transfected cells.

• Animal cells and systems
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• v.8 no.2
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• pp.125-133
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• 2004

We have extended our previous work that cross-linking CD4 molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$We have extended our previous work that cross-linking CD$4^+$ molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$ T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD$4^+$T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4 molecules. The CD$4^+$cross-linking failed to induce effector cell proliferation or the transcription of TNF${\beta}$ Upregulation of TNF${\beta}$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF${\beta}$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased p$56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD4T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4molecules. The CD4 cross-linking failed to induce effector cell proliferation or the transcription of TNF$\beta$. Upregulation of TNF$\beta$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF$\beta$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased $56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.

• Development and Reproduction
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• v.3 no.1
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• pp.59-67
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• 1999

To investigate the role of interleukin-l$\beta$ (IL-1$\beta$) in the embryonic development, in vivo and in vitro expression patterns of IL-1$\beta$ gene in the preimplantation mouse embryos were examined by RT-PCR, and the effects of explanted mouse ovi-duct and uterine epithelial cells on the expression of IL-1$\beta$ gene in the pleimplantation mouse embryos were examined by co-culture. IL-1$\beta$ mRNA was detected in the embryos from 4-cell stage to blastocyst stage in vivo and from morula stage to hatching blastocyst stage in vitro. This transcript was not detected from the GV stage to late 2-cell stage in vivo, and not at the 4-cell and 8-cell stages in vitro. For the co-culture of late 2-cell embryos with the explanted mouse oviduct and uterine epithelial cells, oviducts and uterine epithelial cells were isolated at 48 hour alter the hCG injection. The explanted oviduct and uterine epithelial cells in co-culture groups facilitated the IL-1$\beta$ gene expression of the mouse embryos in comparison with the control. Taken together these results suggest that the presence of IL-1$\beta$ plays an important role in preimplantation embryonic development. In addition, the up-regulation of IL-1$\beta$ gene expression by the explanted oviduct and uterine epithelial cells demonstrates that embryonic expression of IL-l$\beta$ gene may be regulated by the interaction with oviductal and uterine factor (s).

• Applied Microscopy
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• v.19 no.2
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• pp.119-137
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• 1989

It has been shown in this and earlier investigation that the turtle bladder mucosa has three main cell types on their mucosal surface. They are the granular cells, ${\alpha}$ CA cells, and ${\beta}$ CA cells. The three major transport mechanisms that occurs in the turtle bladder are sodium reabsorption, proton secretion, and bicarbonate secretion. In the present work the trans-port mechanisms by bladder epithelial cells of freshwater turtle, Pseudemys scripta, are summarized as follows. 1. The granular cells play an important role in sodium transport, while the ${\alpha}$ and ${\beta}$ CA cells do not appear to play a determining role in sodium transport. 2. It appears that the active sodium transport in the granular cells occurs in two-step process, implying that first, sodium diffuses into the cells, followed by an energy-dependent efflux step, which is catalyzed by the ouabain-sensitive Na-K ATPase. 3. The ${\alpha}$ type of CA cells are responsible for the proton secretion using the proton pump on the apical plasma membrane, while the ${\beta}$ type of CA cells are believed to be responsible for bicarbonate secretion. 4. When looked at under freeze-fracture electron microscopy, the apical plasma membrane of ${\alpha}$ cells have a characteristic population of rod-shaped intramembranous particles which are believed to be components of the proton pumps. Conversely, ${\beta}$ type of CA cells show rod-shaped particles in their basolateral plasma membranes, which is consistent with the proton absorptive, bicarbonate secretory mechanism. 5. In the turtle bladder, the ${\alpha}$ and ${\beta}$ type of cells are believed to be both responsible for proton transport, but in opposite directions.

• Journal of Sasang Constitutional Medicine
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• v.21 no.3
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• pp.138-153
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• 2009

1. Objectives: Yeolda-Hanso tang (YH) has long been used as traditional herbal formula in Korea as various diseases. Now we modified Yeolda-Hanso tang (YH) for neurodegenerative diseases treatment and named New-Yeolda-Hanso tang (NYH). We investigated neuroprotective effects of NYH on NGF-differentiated PC12 cells cytotoxicity induced by $\beta$-Amyloid peptide (A$\beta$25-35) and evaluated the ability of NYH to prevent and treat for neurodegenerative diseases via autophagy enhancement. 2. Methods and Results: 1) Protective effect of NYH on PC12 cells cytotoxity induced by A$\beta$25-35. PC12 cells survival was measured by MTT and lactate dehydrogenase (LDH) assay. $20{\mu}M$ $\beta$-Amyloid peptide (A$\beta$25-35) induced cytotoxicity on NGF-differentiated PC12 cells. NYH attenuated the cytotoxic effects of A$\beta$25-35 in a dose-dependent manner. 2) Pharmacological induction of Autophagy by NYH in PC12 cells Autophagy induction and activation was measured by immunoblot assay. Marker of autophagy, LC3 II expression and the ratio of LC3-II/I was slightly increased in the protein treated with YH, and significantly augmented in the protein treated with NYH. NYH-induced increase of LC3-II protein level was inhibited by 3MA. 3) Induction of Autophagy by NYH on A$\beta$25-35-induced injury in PC12 cells In MTT assay, $100{\mu}g/ml$ re-treated NYH attenuated $20{\mu}M$ A$\beta$25-35-induced cytotoxicity in PC12 cells. Protection effect of NYH was blocked by autophagy inhibitor 3MA. In immunoblot assay, $1200{\mu}g/ml$ pre-treated NYH activated autophagy in $20{\mu}M$ A$\beta$25-35-induced cytotoxicity in PC12 cells. The observed effect was partially blocked by 3MA. 3. Conclusions: All the results indicated that NYH possesses neuroprotective potential partially mediated by autophagy enhancement and NYH may be considered to be a promising new herbal formula to prevent and treat for neurodegenerative diseases including Alzheimer's disease (AD).

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.65-75
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• 2003

Objectives: To investigate the role of TGF (Transforming growth factor-$\beta$) involved in the paracrinic communication during decidualization between UEC (uterine epithelial cells) and USC (uterine stromal cells), we have employed a co-culture system composed of human endometrial epithelial and stromal cells in defined hormonal conditions. Design: In the co-culture, endometrial epithelial cells cultured in the matrigel-coated cell culture insert are seeded on top of the endometrial stromal cells cultured within a collagen gel. The co-culture was maintained for 48 hours under the following hormonal conditions: progesterone dominant condition (100 nM P4 and 1 nM E2) or estrogen-dominant condition (100 nM E2 and 1 nM P4). 10 ng/ ml HGF and/or 10 ng/ml TGF-$\beta$1 are added. Methods: RT-PCR is utilized to detect mRNAs quantitatively. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining are utilized to detect proteins in the tissue. Results: Prolactin mRNA is expressed in the co-cultured stromal cells under the progesterone dominant condition. TGF-$\beta$1 and its receptors are expressed in both the co-cultured epithelial and stromal cells irrespective of the steroid present, which is in contrast with no or negligible expression of TGF-$\beta$1 or its receptor in cells separately cultured. Both estrogen and progesterone significantly elevate the concentration of hepatocyte growth factor (HGF) in the conditioned medium of the co-culture with the value of 4, 325 pg/ml in E2-dominant and 2, 000 pg/ml in P4-dominant condition compare to 150 pg/ml in no hormone. In separately cultured stromal cells, administration of HGF induces the expression of TGF receptor 1 in both hormonal conditions, but induction of TGF receptor 2 is only manifest in the P4-dominant condition. Administration of TGF-$\beta$ and HGF directly induce the decidualization marker prolactin mRNA in separately cultured stromal cells. Conclusion: It is likely that steroid hormones induces prolactin mRNA indirectly by promoting the cell to cell communication between the stromal and the epithelial cells. TGF-$\beta$ and HGF are two possible paracrine mediators in the human endometrial decidualization.

• YAKHAK HOEJI
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• v.41 no.2
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• pp.260-267
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• 1997

To establish prediabetes in vitro model concerning the etiology of IDDM(Insulin Dependent Diabetes Mellitus) in cellular level we have designed prediabetes in vitro models in pa ncreatic beta cells. HIT-T15, RINm5F and isolated rat islets were chosen as pancreatic beta cells, and streptozotocin (STZ) used as diabetogenic agent. Degree of beta cell destruction to establish prediabetic in vitro model was determined by cell proliferation and insulin release using thymidine uptake and radio immuno assay. When HIT-T15 and RINm5F cells were treated with STZ, the degree of cell deterioration was dependent upon the origin and passage number of beta cells, and in the case of isolated islets STZ showed the more sensitivity than above two beta cell lines. The concentration and exposure time of STZ treatment to establish prediabetes in vitro model in beta cell lines and isolated rat islets were 2 ~ 10mM, 30 min. and 1 ~ 5mM, 30 min., respectively.

• Environmental Mutagens and Carcinogens
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• v.25 no.3
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• pp.104-109
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• 2005

[ $7{\beta}$ ]-Hydroxycholesterol (cholest-5-ene-3, 7-diol, $7{\beta}$-OHC) showed the cytotoxicity on human cervical carcinoma cells (HeLa), $10{\mu}M$ of 50% inhibitory concentration. We evaluated $7{\beta}$-OHC as the possibility of radiation sensitizer. The combination effect of $7{\beta}-OHC\;and\;{\gamma}$-irradiation was measured using colony forming assay and flow cytometer with propidium iodide and $DiOC_6$ stained cells, respectively. The combined treatment of $7{\beta}-OHC\;and\;{\gamma}$-irradiation did not show significant enhancing effects on HeLa cells.

• Journal of Integrative Natural Science
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• v.6 no.3
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• pp.125-137
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• 2013

Promising evidence suggests that amyloid beta peptide ($A{\beta}$), a key mediator in age-dependent neuronal and cerebrovascular degeneration, activates death signalling processes leading to neuronal as well as non-neuronal cell death in the central nervous system. A major cellular event in $A{\beta}$-induced apoptosis of non-neuronal cells, including cerebral endothelial cells, astrocytes and oligodendrocytes, is mitochondrial dysfunction. The apoptosis signalling cascade upstream of mitochondria entails $A{\beta}$ activation of neutral sphingomyelinase, resulting in the release of ceramide from membrane sphingomyelin. Ceramide then activates protein phosphatase 2A (PP2A), a member in the ceramide-activated protein phosphatase (CAPP) family. PP2A dephosphorylation of Akt and FKHRL1 plays a pivotal role in $A{\beta}$-induced Bad translocation to mitochondria and transactivation of Bim. Bad and Bim are pro-apoptotic proteins that cause mitochondrial dysfunction characterized by excessive ROS formation, mitochondrial DNA (mtDNA) damage, and release of mitochondrial apoptotic proteins including cytochrome c, apoptosis inducing factor (AIF), endonuclease G and Smac. The cellular events activated by $A{\beta}$ to induce death of non-neuronal cells are complex. Understanding these apoptosis signalling processes will aid in the development of more effective strategies to slow down age-dependent cerebrovascular degeneration caused by progressive cerebrovascular $A{\beta}$ deposition.