• The Korean Journal of Physiology and Pharmacology
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• 제21권2호
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• pp.233-239
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• 2017

Intracellular calcium ($Ca^{2+}$) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide ($H_2O_2$) on intracellular $Ca^{2+}$ accumulation in mouse pancreatic acinar cells. Perfusion of $H_2O_2$ at $300{\mu}M$ resulted in additional elevation of intracellular $Ca^{2+}$ levels and termination of oscillatory $Ca^{2+}$ signals induced by carbamylcholine (CCh) in the presence of normal extracellular $Ca^{2+}$. Antioxidants, catalase or DTT, completely prevented $H_2O_2$-induced additional $Ca^{2+}$ increase and termination of $Ca^{2+}$ oscillation. In $Ca^{2+}$-free medium, $H_2O_2$ still enhanced CCh-induced intracellular $Ca^{2+}$ levels and thapsigargin (TG) mimicked $H_2O_2$-induced cytosolic $Ca^{2+}$ increase. Furthermore, $H_2O_2$-induced elevation of intracellular $Ca^{2+}$ levels was abolished under sarco/endoplasmic reticulum $Ca^{2+}$ ATPase-inactivated condition by TG pretreatment with CCh. $H_2O_2$ at $300{\mu}M$ failed to affect store-operated $Ca^{2+}$ entry or $Ca^{2+}$ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial $Ca^{2+}$ uniporter blocker, failed to attenuate $H_2O_2$-induced intracellular $Ca^{2+}$ elevation. These results provide evidence that excessive generation of $H_2O_2$ in pathological conditions could accumulate intracellular $Ca^{2+}$ by attenuating refilling of internal $Ca^{2+}$ stores rather than by inhibiting $Ca^{2+}$ extrusion to extracellular fluid or enhancing $Ca^{2+}$ mobilization from extracellular medium in mouse pancreatic acinar cells.

• Journal of Korean Neurosurgical Society
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• 제46권5호
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• pp.472-478
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• 2009

Objective : Hyaluronidase (HAse), a degrading enzyme of hyaluronic acid (HA), is highly expressed in patients with malignant glioma. The purpose of this study was to verify whether HAse is related to the invasion of glioma cells. We also investigated if glioma cells with higher mobility in 2-dimensioal (2-D) method have also higher mobility at 3-dimensional (3-D) environment. Methods : Malignant glioma cell lines (U87MG, U251MG, U343MG-A, and U373MG) were used, and their HAse expressions were evaluated by HA zymography. The migration ability was evaluated by simple scratch technique. The invasiveness of each cell lines was evaluated by Matrigel invasion assay and HA hydrogel invasion assay. In HA hydrogel invasion assay, colonies larger than $150\;{\mu}m$ were regarded as positive ones and counted. Statistical analysis of migration ability and invasion properties of each cell lines was performed using t-test. Results : In scratch test to examine migration ability of each cell lines, U87MG cells were most motile than others, and U343MG-A least motile. The HAse was expressed in U251MG and U343MG-A cell lines. However, U87MG and U373MG cell lines did not express HAse activity. In Matrigel invasion assay, the cell lines expressing HAse (U251MG and U343MG-A) were more invasive in the presence of HA than HAse deficient cell lines (U87MG and U373MG). In HA hydrogel invasion assay, the HAse-expressing cell lines formed colonies more invasively than HAse-deficient ones. Conclusion : Malignant Glioma cells expressing HAse were more invasive than HAse-deficient ones in 3-dimensional environment. Therefore, it might be suggested that invasion of malignant gliomas is suppressed by inhibition of HAse expression or HA secretion. Additionally, the ability of 2-D migration and 3-D invasion might not be always coincident to each other in malignant glioma cells.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.915-921
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• 2001

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.

• 한국미생물·생명공학회지
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• 제26권5호
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• pp.406-412
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• 1998

Trichoderma sp. C-4 유래의 endoglucanase 유전자(egl6)를 ADH1 promoter 하류에 연결하여 구성적 발현 plasmid인 pVT-C4를 제작하였다. 이를 3종의 S. cerevisiae숙주세포(YNN27, 2805, SEY2102)에 형질전환시켜 각 숙주세포당 80개의 형질전환체를 시험배양하여, 균체증식과 endoglucanase 발현량이 뛰어난 형질전환 효모 균주를 선별하였다. 3종의 재조합 효모를 YPD 배지에서 회분 배양했을 때, YNN27 균주가 가장 낮은 균체농도(OD$_{600}$=19)를, 2805 균주가 가장 높은 균체농도(OD$_{600}$=33)를 보였으며, 총 발현된 endoglucanase 활성은 균체농도에 의존적인 양상으로 나타나 YNN27의 경우 586 unit/l, SEY2102의 경우 1023 unit/l, 2805의 경우 1142 unit/l 순서로 증가하였다 비활성(균체농도당 endoglucanase활성 )은 세 균주 모두 31~34 unit/l/OD$_{600}$ 값을 보여 단위 균체당 발현능 차이는 거의 없었다. Plasmid 안정성은 배양 72시간에서 세 균주 모두 80% 이상의 높은 값을 보였다. 발현된 endoglucanase의 분비 국재성은 세포밖 배지에 약 80%, periplasmic space 잔존 활성이 11~13%, 세포질 분획에서의 활성이 8~10% 정도로 세 균주에서 모두 비슷한 분포를 보였다. 즉, egl6의 분비신호는 S. cerevisiae에서도 매우 효율적으로 분비 기능을 발휘하였다. 재조합 endoglucanase는 nondenaturing-PAGE 상에서 전형적인 당단백질의 disperse band를 두개 보였다. 세가지 숙주세포에 따른 재조합 endoglucanase활성 band들의 이동상의 차이는 거의 나타나지 않고, 당쇄부가 양상은 야생형보다 훨씬 더 복잡하고 불균일하게 발생했음을 알 수 있었다.수 있었다.

• 한국가축번식학회지
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• 제17권2호
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• pp.165-171
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• 1993

Ornithine decarboxylase(ODC)가 polyamine 생합성에 주요 효소로 역할을 하지만 difluoromethylornithine(DFMO)은 polyamine 합성에 억제자로 작용하고 있다. Cycling crossbed gilt들을 무작위로 두 그룹으로 분배하였다(6/group). Indwelling silicone catheter를 모든 동물의 경정맥에 수술을 통해 이식하였다. DFMO는 생리식염수(20mg/ml)에 용해하여 매일 80mg/kg을 i.m.으로 주사하였으며 대조구에게는 같은 양의 생지식염수만을 주사하였다. DFMO는 estrous cycle day 16일부터 21일 혹은 발정때까지 하루 3번(08:00, 16:00, 24:00h) 주사하였다. Day 14일 부터 마지막 DFMO주사 이틀 후까지 하루 한번씩 10ml의 혈액을 채취하였다. Day 16일부터 21일까지 매일 다른 gilt로부터 8시간 동안(08:00∼16:00h) 15분 간격의 주기로 window 혈액을 채취하였다. Serum으로부터 progesteron(P4), Estradiol(E2), LH 및 FSH를 측정하였다. P4와 E2는 DFMO처리에 관계없이 follicular phase동안에 전형적인 profile을 보였다. DFMO처리는 발정직전의 LH농도를 저하시켰지만 (p<0.01), FSH에는 아무런 영향을 미치지 않았다. 결론적으로 gilt에 있어서 DFMO는 LH분비에 억제적인 영향을 미치는 한편, P4, E2 및 FSH에는 별로 영향을 미치지 않음이 나타났다.

• Journal of Nutrition and Health
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• 제34권8호
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• pp.896-904
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• 2001

Conjugated linoleic acid(CLA) is a group of positional and geometric isomers of linoleic acid(LA) and exhibits anticarcinogenic activity in multiple experimental animal models. Cis-9,trns-11(c9t11) and trans-10,cis-12(t10c12) CLA are the principal isomers found in foods. The present study was performed to determine whether CLA and the two isomers inhibits HT-29 cell proliferation and to assess whether such an effect was related to changes in secretion of eicosanoids. Cells were incubated in serum-free medium with various concentrations(0 to 20$\mu$M) of CLA or LA. CLA inhibited cell proliferation in a dose-dependent manner, with maximal inhibition(70 $\pm$ 1%) observed at 20$\mu$M concentration after 96 hours. However, LA had no effect at the same concentration range. To compare the ability of c9f11 and t10c12 to inhibit cell proliferation, cells were incubated with increasing concentrations(0 to 4$\mu$M) of these isomers. T10c12 inhibited cell proliferation in a dose-dependent manner. A 66 $\pm$ 2% decrease in cell number was observed within 96 hours after addition of 4$\mu$M t10c12. By contrast, c9t11 had no effect. The concentrations of CLA and the two isomers in the plasma membrane were increased when they were added to the incubation medium. However, they did not alter the levels of arachidonic acid in plasma membrane. To assess whether the proliferation inhibiting effect of CLA was related to changes in eicosanoid production, prostaglandin E$_2$(PGE$_2$) and leukotriene B$_4$(LTB$_4$) concentrations in conditioned media were estimated by a competitive enzyme immunoassay. Both CLA and t10c12 increased the production of materials reactive to PGE$_2$ and LTB$_4$ antibodies in a dose-dependent manner. By contrast, c9t11 had no effect. These results indicate that inhibition of HT-29 cell proliferation by CLA is attributed to the effect of the t10v12 isomer. The materials reactive to PGE$_2$ and LTB$_4$ antibodies may inhibit growth stimulatory effect of arachidonic acid-derived eicosanoids on HT-29 cell proliferation.

• 대한한방소아과학회지
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• 제29권3호
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• pp.65-79
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• 2015

Objectives : In this study, effects of haepyoijintang (HIJ) on the increase in airway epithelial mucosubstances of rats and ATP-, PMA-, EGF- or TNF-${\alpha}$-induced MUC5AC mucin production and gene expression from human airway epithelial cells were investigated. Methods : Hypersecretion of airway mucus was induced by exposure of rats to $SO_2$ during 3 weeks. Effect of orally-administered HIJ during 2 weeks on increase in airway epithelial mucosubstances from tracheal goblet cells of rats was evaluated using histopathological analysis after staining the epithelial tissue with PAS-alcian blue. Possible cytotoxicity of HIJ was evaluated by examining the potential damage of kidney and liver functions by measuring serum GOT/GPT activities and serum BUN and creatinine concentrations of rats and the body weight gain during experiment, after administering HIJ orally. At the same time, the effect of HIJ on ATP-, PMA-, EGF- or TNF-${\alpha}$-induced MUC5AC mucin production and gene expression from human airway epithelial cells (NCI-H292) were investigated. Confluent NCI-H292 cells were pretreated for 30 min in the presence of HIJ and treated with ATP ($200{\mu}M$), PMA (10 ng/ml), EGF (25 ng/ml) or TNF-${\alpha}$ (0.2 nM) for 24 hrs, to evaluate the effect of HIJ both on ATP-, PMA-, EGF- or TNF-${\alpha}$-induced MUC5AC mucin production using enzyme-linked immunosorbent assay (ELISA) and on gene expression by the same inducers using reverse transcription-polymerase chain reaction (RT-PCR). Results : (1) HIJ decreased the amount of intraepithelial mucosubstances of trachea of rats. (2) HIJ did not show renal and hepatic toxicities and did not affect body weight gain of rats during experiment. (3) HIJ significantly inhibited ATP-, PMA-, EGF-, and TNF-${\alpha}$-induced MUC5AC mucin productions from NCI-H292 cells. (4) HIJ significantly inhibited ATP-, PMA-, EGF-, and TNF-${\alpha}$-induced MUC5AC mucin gene expression from NCI-H292 cells. Conclusions : The result from the present study suggests that HIJ might control the production and gene expression of airway mucin observed in various respiratory diseases accompanied by mucus hypersecretion and do not show in vivo toxicity to liver and kidney functions after oral administration. Effect of HIJ with their diverse components should be further investigated using animal experimental models that can reflect the pathophysiology of airway diseases through future studies.

• 한방안이비인후피부과학회지
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• 제22권2호
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• pp.1-18
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• 2009

Objective : Moutan Cortex (the root bark of Paeonia suffruticosa Andr.) is widely used in oriental medicine as a remedy for inflammation. However, as yet there is no clear explanation of how MC(Moutan Cortex) affects the production of inflammatory cytokine. This study was to determine the effects of Essence extracted MC on the mast cell-mediated inflammatory responses. Method : We observed the effect of MC on compound 48/80-induced histamine release of rat peritoneal mast cells and the effect of administering MC on PCA in rat. We measured the amount of inflammatory cytokine production induced by the phorbol myristate acetate (PMA) plus calcium ionophore(A23187) in the human mast cell line (HMC-1) incubated with various concentrations of MC. The TNF-$\alpha$ protein levels were analysised by Western blot. The TNF-$\alpha$, IL-6 and IL-8 secreted protein levels were measured by the ELISA assay. The TNF-$\alpha$, IL-6 and IL-8 mRNA levels were measured by the RT-PCR analysis. NF-$\kappa$B, phospho-I$\kappa$B and MAPKs were exmined by Western blot analysis. The NF-$\kappa$B promoter activity was examined by luciferase assay. Result : 1. Enzyme immunoassay indicated that MC suppressed histamine secretion of rat peritoneal mast cells. 2. In PCA dependent on IgE, MC had anti-allergic effect of the internal surface of rat skin. 3. Western blot indicated that MC decreased TNF-$\alpha$ protein levels. 4. ELISA indicated that MC decreased TNF-$\alpha$, IL-6 but MC had no significant effect on IL-8 in HMC-1 cells. 5. RT-PCR indicated that MC decreased TNF-$\alpha$, IL-8 but MC had no significant effect on IL-6 in HMC-l cells. 6. Western blot indicated that MC suppressed the induction of MAPKs, NF-$\kappa$B & phospho-I$\kappa$B activity in HMC-1 cells. 7. Luciferase assay indicated that MC suppressed the PMA plus A23187-induced NF-$\kappa$B promoting activityin HMC-1 cells. Conclusion : In this study, we have found that MC is an inhibitor of NF-$\kappa$B, MAPKs & cytokines on the mast cell-mediated inflammatory responses.

• 치위생과학회지
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• 제14권2호
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• pp.223-229
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• 2014

본 연구에서는 체내 염증지표 변화를 모니터링 함으로써 새로운 천연추출물인 polycan 및 calcium gluconate 복합제제의 치주질환 개선 효과를 평가하고자 하였으며, 다음과 같은 결론을 얻었다. 1. 치은열구액 내 $IL-1{\beta}$는 대조군과 시험군 모두 시간에 따른 유의한 차이는 없었으나(p>0.05), 4주째 시험군이 대조군보다 낮았다(p<0.05). 2. 치은열구액 내 MMP-8과 $TNF-{\alpha}$는 4주째 시험군에서 초기에 비해 감소하였으며, 시험군이 대조군보다 낮았다. 3. 혈액 내 MMP-8은 4주째 시험군에서 유의하게 감소하였고(p<0.05), 대조군에 비해 통계적으로 유의하게 낮았다(p<0.05). 그러나 혈액 내 CRP는 유의한 변화가 관찰되지 않았다(p>0.05). 이상의 결과들을 종합해 보았을 때, polycan을 함유한 calcium gluconate 복합제 복용이 전신적인 염증성 생체지표에 영향을 미쳐 치은열구액 내의 염증매개물질들을 감소시킴으로써 치은건강에 긍정적인 효과를 나타내는 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.135-141
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• 1997

The early studies demonstrated that the relative amount of FSH was important for stimulating normal ovarian activity and demonstrated the existence of a threshold level for FSH, above which follicular growth was activated. It was found that only a modest increase in circulating FSH level above the threshold (between 10 and 30%) was required to stimulate folliculogenesis. In addition, FSH is primary responsible for initiating estradiol production through the activation of the aromatase enzyme system in granulosa cells, follicular secretion and growth. LH on the other hand, plays a supportive role in ovarian steroidogenesis, stimulating the ovarian thecal cells to produce androgen, the precursor for estradiol synthesis. But there is now an increasing number of reports in the literature demonstrating an adverse effect of LH on fertility and miscarriage in infertile and fertile women. So HP-FSH is the drug of a highly purified FSH preparation which has a higher specific activity and far fewer impurities than FSH. This study was performed to evaluate the efficacy and safety of HP-FSH administered (SC; subcutaneous) versus FSH(IM; intramuscular) for ovulation induction. 20 candidates patients for ovulation induction were participated. All patients underwent pituitary desensitizing with a long gonadotropin-releasing hormone (GnRH) agonist protocol and ovulation induction was started with HP-FSH SC (10 patients; group I) or FSH IM (10 patients; group II). After ovulation, outcome of ovulation induction and local reaction of injection site were compared. There were no difference of outcome of ovulation in two groups except pregnancy rate/embryo transfer. Group I had a higher pregnancy rate/ embryo transfer than Group II (44.4% Vs 28.6%). Pain, redness, tenderness, bruising and itching when the injection received on the first 5 days of treated (50 SC and 50 IM injections) were assessed. There were no significant difference (P>0.05) in the incidence of tenderness, bruising and itching between the IM and SC injection. But IM injection (FSH) had a tendency of higher above incidence. The number of reports of pain, redness were significantly increased in IM injection group (P<0.05). These results indicate that SC administration of HP-FSH has been shown to be as effect for superovulation as traditional gonadotropins, with an improved safety profile due to the removal of extaneous proteins.