• The Korean Journal of Pharmacology
• /
• v.27 no.2
• /
• pp.155-166
• /
• 1991

In order to evaluate a potential role of mitochondria in the mediation of toxicity of paraquat (PQ), submitochondrial particle and microsome of mouse liver were compared by oxygen radical generation and lipid peroxidation. With NADH in submitochondrial particle and NADPH in microsome as electron donors, PQ stimulated production of superoxide anion and $H_2O_2$ in both fractions. Under the same conditions, PQ enhanced the generation of ethylene from methional suggestiong stimulation of OH production by PQ. But these effects by PQ were somewhat lower in submitochondrial particle than in microsome. In addition, lipid peroxidation(measured as MDA production) was stimulated by PQ in both fractions. The stimulation of lipid peroxidation in both fractions seemed to occur by the same mechanism probably through perferryl ion. This was supported by the following findings: i) The lipid peroxidation in both fractions was partially inhibited by SOD and completely inhibited by DETAPAC(an iron chelator) but not by catalase or OH scavenger. ii) Addition of $ADP-Fe^{3+}$ further increased PQ-induced lipid peroxidation but decreased ethylene production from methional suggesting no correlation between OH production and lipid peroxidation. The redox-cycling of PQ in mitochondria appeared to be linked to NADH dehydrogenase, not to CoQ since all of the observed stimulations by PQ in submitochondrial particle were inhibited by p-hydroxymercuribenzoate(a NADH dehydrogenase inhibitor) but not affected by other respiratory chain blockers. The above results demonstrate that redox-cycling properties of PQ leading to oxygen radical generation and lipid peroxidation can also occur in mitochondria in the same manner as in microsome. Therefore, the observed actions of PQ in mitochondria suggest that mitochondria may also contribute to toxicity of this drug in vivo.

• Clinical and Experimental Reproductive Medicine
• /
• v.31 no.2
• /
• pp.133-139
• /
• 2004

Objective: To investigate the association of FSH receptor (FSHR) polymorphism at position 680 with outcomes of controlled ovarian hyper-stimulation for IVF-ET in Korean women. Design: Genetic polymorphism analysis. Materials and Methods: The FSHR polymorphism was analyzed by PCR-RFLP in 172 ovulatory women below the age of 40 year. Patients with polycystic ovary syndrome, endometriosis, or previous history of ovarian surgery were excluded. Results: Genotype distribution was 41.9% for the Asn/Asn, 47.7% for the Asn/Ser, and 10.5% for the Ser/Ser FSHR genotype group. There was no difference in age of subjects and infertility diagnosis between genotype groups. When the patients were grouped according to their FSHR genotype, the basal levels of FSH (day 3) were significantly different among the three groups ($6.0{\pm}0.3\;IU/L$ (mean $\pm$ SEM), $5.8{\pm}0.3\;IU/L$, and $8.6{\pm}1.2\;IU/L$ for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, p=0.002). The Ser/Ser group showed a higher total doses of gonadotropins required to achieve ovulation induction, and a lower serum estradiol levels at the time of hCG administration compared with other two groups, but the differences were of no statistical significance. The numbers of oocytes retrieved were significantly different among the three groups ($8.6{\pm}0.8$, $9.9{\pm}0.6$, and $6.3{\pm}0.9$, for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, p=0.049). Clinical pregnancy rates were 42.4%, 25.9%, and 29.4% for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively. Conclusion: Homozygous Ser/Ser genotype of FSHR polymorphism at position 680 was associated with decreased ovarian response to gonadotropin stimulation for IVF-ET.

• Korean Journal of Animal Reproduction
• /
• v.25 no.2
• /
• pp.191-198
• /
• 2001

The purpose of this study was to investigate the effects of embryo sources such as in vivo vs. in vitro produced blastocyst, and culture systems on the membrane permeability and viability of bovine blastocyst following GMP vitrification. To produce in vivo embryos, six cows were superovulated by administration of follicle stimulation hormone (FSH) and prostaglandin $F_{2{\alpha}}$(PG $F_{2{\alpha}}$). in vitro embryos were produced by two different culture systems, oviduct co-culture (OCS) and defined culture system (HECM-6; DCS). Ovaries were picked up at a local slaughterhouse and transported to laboratory in 3$0^{\circ}C$ saline within 2 h. Ovaries were washed with same saline three times and then placed in saline on warm plate adjusted at 3$0^{\circ}C$ during aspiration. The blastocysts produced were assigned for membrane permeability and viability following GMP vitrification. The membrane permeability of blastocysts was checked in 0.5 M sucrose solution on warm plate at 35$^{\circ}C$ for 0, 2, 5 and 7 min, respectively. Then the diameters (width and length) of embryo cytoplasms were measured by a eyepiece meter, and they were converted to their volume by 4/3 $\pi$ $r^3$. The blastocysts were cryopreserved by GMP vitrification method, where they were sequentially placed into vitrification solution before being loaded into GMP vessels and immersed into L$N_2$ within 20 to 25 sec. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in HM and TCM-199 for 5 min each, and then cultured in TCM 199 supplemented with 10% FCS for 24 or 48 h. The volume change of in vivo blastocyst at 0, 2, 5 and 7 min (100, 37.1, 34.3 and 31.6%) was significantly more shrunk than those of in vitro blastocysts derived from OCS (100, 59.8, 48.9, 47.9%) and DCS (100, 57.2, 47.3 and 46.9%) (P＜0.05). The viability of post-thaw blastocyst derived from in vivo (93.6%) was also significantly different from those in OCS and DCS (81.9 and 83.6%; P＜0.05). In the present culture system, the morphology of embryos produced in vitro was similar to that of in vivo embryos, but the quality in membrane permeability and post-thaw viability showed a big difference from their sources as in vivo or in vitro derived from OCS and DCS. The results indicated that the quality of in vivo embryos in membrane permeability and post-thaw viability was better than those of in vitro embryos derived from OCS or DCS.

• Journal of Periodontal and Implant Science
• /
• v.25 no.3
• /
• pp.720-732
• /
• 1995

The use of transforming growth $factor-{\beta}1$ which functions as a potent biologic mediator regulating numerous activities of wound healing has been suggested for the promotion of periodontal regeneration. The mitogenic effects of transforming growth $factor-{\beta}1$ on human periodontal ligament cells and human gingival fibroblasts were evaluated by determining the incorporation of $[^3H]-thymidine$ into DNA of the cells dose-dependently. Cells were prepared with primary cultured fibroblasts and periodontal ligament cells from humans, and used in experiments were the fourth or sixth subpassage. Cells were seeded with serum free Dulbecco's modified Eagle medium containing 0.1% bovine serum albumine. The added concentrations of transforming growth $factor-{\beta}1$ were 0.25, 0.5, 1, 2.5, 5ng/ml and transforming growth $factor-{\beta}1$ were added to the quiescent cells for 24hours, 48hours, 72hours. They were labeled with lnCi/ml $[^3H]$ thymidine for the last 24hour of the each culture. The results were presented as the mean counts per minute (CPM) per well and S.D. of four determinations. The results were as follows. : The DNA synthetic activity of human gingival fibroblasts was increased dose-dependently by transforming growth $factor-{\beta}1$ at 24 hours, 48 hours and 72 hours. The maximum mitogenic effects were at the 48 hour application of transforming growth $factor-{\beta}1$. The DNA synthetic activity was generally more decreased at the 72 hour application than at the 48 hour the application of transforming growth $factor-{\beta}1$. The DNA synthetic activity of human periodontal ligament cells was increased dose-dependently by transforming growth $factor-{\beta}1$ at 24 hours and 48 hours. But the DNA synthetic activity was decreased at 5ng/ml of the 72 hour application. The maximum mitogenic effects were also at the 48 hour application of transforming growth $factor-{\beta}1$. The DNA synthetic activity of human periodontal ligament cells was generally more decreased at the 72 hour application than at the 48 hour application of transforming growth $factor-{\beta}1$. In the comparision of DNA synthetic activity between the human gingival fibroblasts and human periodontal ligament cells, the human gingival fibroblasts had more activity than the human periodontal ligament cells at all time application with the concentration of transforming growth $factor-{\beta}1$. In conclusion, transforming growth $factor-{\beta}1$ has an important roles in the stimulation of DNA synthesis in human periodontal ligament cells and human gingival fibroblasts, which means an increase in collagen synthesizing cells and thus, may be useful for clinical application in periodontal regenerative procedures.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.27 no.4
• /
• pp.327-334
• /
• 1994

This study was undertaken to clarify the effect of killing methods on the morphological and histological changes of plaice, Paralichthys olivaceus muscle at early stage after killing. Killed samples by the three different methods were stored at $5^{\circ}$, and the changes in breaking strength of muscle, morphological observation of myofibrils and histological observation of extracellular spaces through storage were monitored. Samples killed by electrifying in sea water showed the maximum value of breakin strength immediately after killing and then it dropped significantly(p<0.05) until 2.5hrs passed. Breaking strength of samples killed by spiking at the head instantly and dipping in sea water including anesthetic rose steadily over 10hrs and 15hrs after killing, respectively. In myofibrills prepared from dorsal muscles immediately after spiking at the head instantly, A-band, H-band, I-band, and Z-line in sarcomere were clearly distinguishable each other. Due to muscle contraction by electrical stimulation, it was impossible to distinguish H-band from I-band observed in sarcomere immediately after killing for samples killed by electrifying. But, in the cases of samples killed by spiking and dipping, H-band could be observed dimly until 10hrs and 15hrs storage. No extracellular space was observed among muscle cells immediately after spiking at the head instantly. Samples killed by spiking at the head instantly and dipping in sea water including anesthetic showed extracellular spaces among all muscle cells after 15hrs and 25hrs storage, respectively. The other hand, samples killed by electrifying in sea water (110V, 30sec.) showed a few extracellular spaces immediately after killing and then it showed extracellular spaces among all muscle cells after 2.5hrs storage.

• Archives of Pharmacal Research
• /
• v.27 no.4
• /
• pp.407-414
• /
• 2004

The expression of CYP3A4 gene is induced by a variety of structurally unrelated xenobiotics including the antibiotic rifampicin, pregnenolone 16-carbonitrile (PCN), and endogenous hormones, that might mediate through steroid and xenobiotic receptor (SXR) system. The molecular mechanisms underlying regulation of CYP3A4 gene expression have not been understood. In order to gain the insight of the molecular mechanism of CYP3A4 gene expression, study has been undertaken to investigate if the histone deacetylation is involved in the regulation of CYP3A4 gene expression by proximal promoter in human hepatoma HepG2 cells. Also we have investigated to see if SXR is involved in the regulation of CYP3A4 proximal promoter activity in human hepatoma HepG2 cells. HepG2 cells were transfected with a plasmid PCYP3A4-Luc containing ${\~}1kb$ of the CYP3A4 proximal promoter region (-863 to +64 bp) in front of a reporter gene, luciferase, in the presence or absence of pSAP-SXR. In HepG2 cells, CYP3A4 inducers, such as rifampicin, PCN and RU486 showed minimal stimulation of CYP3A4 proximal promoter activity in the absence of SXR and histone deacetylase (HDAC) inhibitors. 4-Dimethylamino-H-[4-(2-hydroxycarbamoylvinyl)benzyl]benzamide (IN2001), a new class HDAC inhibitor significantly increased CYP3A4 proximal promoter activity over untreated control cells and rifampicin concomitant treatment with IN2001 increased further CYP3A4 proximal promoter activity that was stimulated by IN2001 The results of this study demon-strated that both HDAC inhibitors and SXR are essential to increase of CYP3A4 proximal promoter activity by CYP3A4 inducers such as PCN, rifampicin, and RU486. Especially SXR seems to be important for the dose dependent response of CYP3A4 inducing chemicals to stimulate CYP3A4 proximal promoter activity. Also this data suggested that HDAC inhibitors seemed to facilitate the CYP3A4 proximal promoter to be activated by chemicals.

• IMMUNE NETWORK
• /
• v.1 no.3
• /
• pp.250-259
• /
• 2001

To determine if initial infection with Mycobacterium tuberculosis changes the balance of cytokines between T cells and macrophages, we evaluated interferon (IFN)-${\gamma}$), interleukin-12 (IL)-12, and tumor necrosis factor (TNF)-${\alpha}$ productions by peripheral blood mononuclear cells (PBMC) from 15 untreated active pulmonary tuberculosis (TB) patients and 12 healthy tuberculin reactors (HTR). Freshly isolated PBMC were stimulated with Triton X-100 solubilized protein (TSP), 30-kDa or purified protein derivatives (PPD) antigen for 6, 18 and 96 hours. IL-12 p40 production by antigen-stimulated PBMC from TB patients was significantly decreased compared with that in HTR. In addition, IFN-${\gamma}$ production was significantly depressed in TB patients than that in HTR at a 96-hr stimulation. However, TNF-${\alpha}$ production was significantly higher in antigen-stimulated PBMC from TB than that of HTR. A pronounced increase in IFN-${\gamma}$ protein followed neutralization of IL-10 in early TB patients. However, neutralization of TNF-${\alpha}$ did not significantly alter IFN-${\gamma}$ induction in PBMC from TB patients. There were no significantly differences in the cytokine productions among three proteins, TSP, 30-kDa or PPD antigen. These results indicate that development of TB may be strongly associated with dysregulated productions of IL-12, IFN-${\gamma}$ and TNF-${\alpha}$, during the initial immune responses to M. tuberculosis. Further understanding of operative cytokine networks during human immune cell responses to protein antigens of M. tuberculosis may improve strategies for vaccine development.

• Journal of Embryo Transfer
• /
• v.10 no.1
• /
• pp.65-72
• /
• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• The Korean Journal of Pharmacology
• /
• v.18 no.2
• /
• pp.79-87
• /
• 1982

Higenamine(dl-demethylcoclaurine, dl-1-(4-hydroxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrah-ydroisoquinoline hydrochloride), which has recently been isolated from Aconite root by Drs. Kosuge and Yokota, has known to be the main cardiotonic component of the Aconite root. The present study was undertaken to investigate the effects of Higenamine on the calcium binding and release and ATPase activity of fragmented cardiac sarcoplasmic reticulum under in vitro condition. The calcium binding and release of sarcoplasmic reticulum were measured by using the double-beam spectrophotometer and the calcium sensitive dye, murexide. In the presence of $10^{-4}{\sim}5{\times}10^{-3}M$ of Higenamine, the maximal calcium binding and the initial binding rate of porcine cardiac sarcoplasmic reticulum were inhibited dose dependently by up to 43%. However, the calcium release from cardiac sarcoplasmic reticulum, which was loaded with $Ca^{++}(50{\mu}M)$, was stimulated in dose dependent manner. When incubated in the medium of 20 mM Tris-maleate(pH 7.0), 100 mM KCl, 10 mM $MgCl_2,\;0.05mM\;CaCl_2\;and\;0.014{\sim}1\;mM\;Tris-ATP\;at\;30^{\circ}C$ in the presence of Higenamine $(10^{-4}{\sim}5{\times}10^{-3}M)$, both $Ca^{++}-and\;Mg^{++}-ATPase$ of sarcoplasmic reticulum were inhibited non-competitively by Higenamine and values of $K_i$ were 4.896 mM and 6.875 mM respectively. It is suggested from the above findings that the cardiotonic effects of Higenamine might be partially explained by the inhibition of calcium binding and the stimulation of calcium release from the sarcoplasimic reticulum which may increase the free intracellular calcium that is available in the contraction of the cardiac muscle fiber.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.14
• /
• pp.5635-5641
• /
• 2015

Background: Cirrhosis is regarded as a possible end stage of many liver diseases, including viral infection. It occurs when healthy liver tissue becomes damaged and is replaced by scar tissue and finally may lead to hepatocellular carcinoma. Interferons (IFNs)are two general categories, type I and II. Type I includes one beta interferon and over 20 different alpha interferons. Alpha interferons are very similar in how they work, interacting with other proteins on cells like receptors. The main objective of this study was to compare Mx gene productivity post different cell line treatment with imported and Egyptian biosimilar locally produced IFNs, as well as the efficacy of those tested IFNs. Also, an assessment was made of sensitivity of different cell lines as alternatives to that recommended for evaluation of antiviral activity. Materials and Methods: Different cell lines (Vero, MDBK and Wish) were employed to evaluate cytotoxicity using the MTT assay. Antiviral activity was evaluated compared with standard IFN against VSV, Indiana strain -156, on tested rh-IFNs (imported; innovated and Egyptian biosimilar locally produced IFNs) in the pre-treated cell lines previously mentioned. The virus was propagated in the Wish cell line as recommended. Finally we estimated up-regulation of the Mx gene as a biomarker. Results: Data recorded revealed that test IFNs were safe in test cell lines. Viability was around 100%. Locally tested interferon did not realize the international potency limits, while the imported one was accepted compared with the standard IFN. These results were the same either using infectivity titer reduction assay or crystal violet staining of residual non- infected cells. Mx protein production was cell type related and confirmed by the detected Mx gene expressed in imported and locally produced IFN pre-treated cell lines. The expression of the gene was arranged in the order of Vero> wish > MDBK for the imported IFN, while for the Egyptian biosimillar locally produced one it was MDBK> Vero> wish. With regard to the antiviral activity there was a significant difference of imported IFN potency compared with the locally produced IFN (P<0.05), the IFN potential (antiviral activity) was not cell line related and showed non-significant difference for each separate product. Conclusions: Vero cells can be used as an alternative cell line for evaluation of IFN potency in case of unavailable USP recommended cell lines. Alternative potency evaluation assay could be used and proved significant difference in IFN potency in case of local and imported agents. Evaluation of antiviral activity could be used in parallel to viral infectivity reduction assay for better accuracy. Mx gene can be used as a marker for IFN potential.