• The Korean Journal of Physiology and Pharmacology
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• 제23권3호
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• pp.219-227
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• 2019

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when co-expressed with CaM and $CaM{\triangle}N$. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ${\triangle}EF$-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

• Molecules and Cells
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• 제46권7호
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• pp.441-450
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• 2023

β-Catenin (Ctnnb1) has been shown to play critical roles in the development and maintenance of epithelial cells, including the retinal pigment epithelium (RPE). Ctnnb1 is not only a component of intercellular junctions in the epithelium, it also functions as a transcriptional regulator in the Wnt signaling pathway. To identify which of its functional modalities is critically involved in mouse RPE development and maintenance, we varied Ctnnb1 gene content and activity in mouse RPE lineage cells and tested their impacts on mouse eye development. We found that a Ctnnb1 double mutant (Ctnnb1^dm), which exhibits impaired transcriptional activity, could not replace Ctnnb1 in the RPE, whereas Ctnnb1^Y654E, which has reduced affinity for the junctions, could do so. Expression of the constitutively active Ctnnb1^∆ex3 mutant also suppressed the development of RPE, instead facilitating a ciliary cell fate. However, the post-mitotic or mature RPE was insensitive to the loss, inactivation, or constitutive activation of Ctnnb1. Collectively, our results suggest that Ctnnb1 should be maintained within an optimal range to specify RPE through transcriptional regulation of Wnt target genes in the optic neuroepithelium.

• Molecules and Cells
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• 제20권2호
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• pp.196-200
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• 2005

The neurofibromatosis type2 (NF2) tumor suppressor gene product, merlin, is structurally related to the ezrin-radixin-moesin (ERM) family of proteins that anchor the actin cytoskeleton to specific membrane proteins and participate in cell signaling. However, the basis of the tumor suppressing activity of merlin is not well understood. Previously, we identified a role of merlin as an inhibitor of the Ras-ERK signaling pathway. Recent studies have suggested that phosphorylation of merlin, as of other ERM proteins, may regulate its function. To determine whether phosphorylation of merlin affects its suppression of Ras-ERK signaling, we generated plasmids expressing full-length merlin with substitutions of serine 518, a potential phosphorylation site. A substitution that mimics constitutive phosphorylation (S518D) abrogated the ability of merlin to suppress effects of the Ras-ERK signaling pathway such as Ras-induced SRE transactivation, Elk-mediated SRE transactivation, Ras-induced ERK phosphorylation and Ras-induced focus formation. On the other hand, an S518A mutant, which mimics nonphosphorylated merlin, acted like wild type merlin. These observations show that mimicking merlin phosphorylation impairs not only growth suppression by merlin but also its inhibitory action on the Ras-ERK signaling pathway.

• 한국미생물·생명공학회지
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• 제26권2호
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• pp.179-186
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• 1998

본 연구에서는 dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM과 dextransucrase constitutive mutants인 B-742CB 및 B-1355C에서 얻은 dextransucrase 및 alternansucrase를 이용하여 maltose, lactose, gentiobios 및 raffinose의 수용체반응을 수행하였다. B-512FMCM의 경우 수용체로서 maltose와 gentiobiose는 설탕에 대한 농도비가 1/1이하일 때 수용체에 ${alpha}$l-6으로 D-glucose가 결합한 일련의 올리고당을 생성하였고, 9/1이상에서는 단 두 가지의 수용체산물로서 maltose의 경우에는 panose와 isomaltosyl maltose를, gentiobiose의 경우에는 6$^2$-${alpha}$-D-glucopyranosyl gentiobiose와 isomaltosyl gentiobiose만을 생성하였다. 그러나 lactose는 유일한 산물인 2$^1$-${alpha}$-D-glycopyranosyl lactose만을 생성하였다. 효소양의 변화에 대해서 maltose와 gentiobiose의 수용체반응에서 생성된 수용체산물(올리고당)의 수와 양은 효소양이 증가함에 따라 증가하였으나 lactose의 경우에는 효소양의 증가에 대해 수용체산물 양의 변화가 크지 않았다. B-742CB dextransucrase와 B-1355C alternansucrase를 사용하면 B-512FMCM dextransucrase에서는 단 한가지만의 수용체산물을 생산하였던 lactose와 raffinose를 수용체로 하여 여러 가지의 수용체산물(올리고당)을 생산하였다.

• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2002년도 Current Trends in Toxicological Sciences
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• pp.36-42
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• 2002

Diesel exhaust (DE) has been recognized as a noxious mutagen and/or carcinogen, because its components can form DNA adducts. Mechanisms governing the susceptibility to DE and the efficiency of such DNA adduct formation require clarification. The transcription factor Nrf2 is essential for inducible and/or constitutive expression of a group of detoxification and antioxidant enzymes, and we hypothesized that the nrf2 gene knockout mouse might serve as an excellent model system for analyzing DE toxicity. To address this hypothesis, lungs from nrf2(-/-) and nrf2(+/-) mice were examined for the production of xenobiotic-DNA adducts after exposure to DE (3 $mg/m^{3}$ suspended particulate matter) for 4 weeks. Whereas the relative adduct levels (RAL) were significantly increased in the lungs of both nrf2(+/-) and nrf2(-/-) mice upon exposure to DE, the increase of RAL in the lungs from nrf2(-/-) mice exposed to DE were approximately 2.3-fold higher than that of nrf2(+/-) mite exposed to DE. In contrail, cytochrome P4501Al mRNA levels in the nrf2(-/-)mouse lungs were similar to those in the nrf2(+/-) mouse lungs even after exposure to DE, suggesting that suppressed activity of phase II drug-metabolizing enzymes is important in giving ise to the increased level of DNA adducts in the Nrf2-null mutant mouse subjected to DE. Importantly, severe hyperplasia and accumulation of the oxidative DNA adduct 8-hydroxydeoxyguanosine were observed in the bronchial epidermis of nrf(-/-) mite following DE exposure. These results demonstrate the increased susceptibility of the nrf2 germ line mutant mouse to DE exposure and indicate the nrf2 gene knockout mouse nay represent a valuable model for the assessment of respiratory DE toxicity.

• Animal Bioscience
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• 제35권3호
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• pp.399-409
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• 2022

Objective: Follicle-stimulating hormone (FSH) is the central hormone involved in mammalian reproduction, maturation at puberty, and gamete production that mediates its function by control of follicle growth and function. The present study investigated the mutations involved in the regulation of FSH receptor (FSHR) activation. Methods: We analyzed seven naturally-occurring mutations that were previously reported in human FSHR (hFSHR), in the context of equine FSHR (eFSHR); these include one constitutively activation variant, one allelic variant, and five inactivating variants. These mutations were introduced into wild-type eFSHR (eFSHR-wt) sequence to generate mutants that were designated as eFSHR-D566G, -A306T, -A189V, -N191I, -R572C, -A574V, and -R633H. Mutants were transfected into PathHunter EA-parental CHO-K1 cells expressing β-arrestin. The biological function of mutants was analyzed by quantitating cAMP accumulation in cells incubated with increasing concentrations of FSH. Results: Cells expressing eFSHR-D566G exhibited an 8.6-fold increase in basal cAMP response, as compared to that in eFSHR-wt. The allelic variation mutant eFSHR-A306T was not found to affect the basal cAMP response or half maximal effective concentration (EC₅₀) levels. On the other hand, eFSHR-D566G and eFSHR-A306T displayed a 1.5- and 1.4-fold increase in the maximal response, respectively. Signal transduction was found to be completely impaired in case of the inactivating mutants eFSHR-A189V, -R572C, and -A574V. When compared with eFSHR-wt, eFSHR-N191I displayed a 5.4-fold decrease in the EC₅₀ levels (3,910 ng/mL) and a 2.3-fold decrease in the maximal response. In contrast, cells expressing eFSHR-R633H displayed in a similar manner to that of the cells expressing the eFSHR-wt on signal transduction and maximal response. Conclusion: The activating mutant eFSHR-D566G greatly enhanced the signal transduction in response to FSH, in the absence of agonist treatment. We suggest that the state of activation of the eFSHR can modulate its basal cAMP accumulation.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.579-588
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• 2015

Fungi of the genus Pestalotiopsis have drawn attention for their capability to produce an array of bioactive secondary metabolites that have potential for drug development. Here, we report the determination of a polyketide derivative compound, pestalotiollide B, in the culture of the saprophytic fungus Pestalotiopsis microspora NK17. Structural information acquired by analyses with a set of spectroscopic and chromatographic techniques suggests that pestalotiollide B has the same skeleton as the penicillide derivatives, dibenzodioxocinones, which are inhibitors of cholesterol ester transfer protein (CETP), and as purpactins A and C', inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Strain NK17 can make a fairly high yield of pestalotiollide B (i.e., up to 7.22 mg/l) in a constitutive manner in liquid culture. Moreover, we found that a putative histone deacetylase gene, designated as hid1, played a role in the biosynthesis of pestalotiollide B. In the hid1 null mutant, the yield of pestalotiollide B increased approximately 2-fold to 15.90 mg/l. In contrast, deletion of gene hid1 led to a dramatic decrease of conidia production of the fungus. These results suggest that hid1 is a modulator, concerting secondary metabolism and development such as conidiation in P. microspora. Our work may help with the investigation into the biosynthesis of pestalotiollide B and the development for new CETP and ACAT inhibitors.

• 한국미생물·생명공학회지
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• 제48권3호
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• pp.296-302
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• 2020

Rapamycin, derived from Streptomyces rapamycinicus, is an important bioactive compound having a therapeutic value in managing Parkinson's disease, rheumatoid arthritis, cancer, and AIDS. Because of its pharmaceutical activity, studies over the past decade have focused on the biosynthesis of rapamycin to enhance its yield. In this study, the effect of rapG on rapamycin production was investigated. The rapG expression vector was constructed by utilizing the integration vector pSET152 under the control of the erythromycin resistance gene (ermE^⁎), a strong constitutive promoter. The rapamycin yield of wild type (WT) and WT/rapG overexpression mutant strains, under fermentation conditions, was analyzed by high-performance liquid chromatography (HPLC). Our results revealed that overexpression of rapG increased rapamycin production by approximately 4.9-fold (211.4 mg/l) in MD1 containing 15 g/l of glycerol, compared to that of the WT strain. It was also found that Illicium verum powder (10 g/l), containing shikimic acid, enhanced rapamycin production in both WT and WT/rapG strains. Moreover, the amount of rapamycin produced by the WT/rapG strain was statistically higher than that produced by the WT strain. In conclusion, the addition 15 g/l glycerol and 15 g/l I. verum powder produced the optimal conditions for rapamycin production by WT and WT/rapG strains.

• KSBB Journal
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• 제14권6호
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• pp.713-717
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• 1999

Lipomyces starkeyi ATCC 74054를 UV로 돌연변이하여 선발한 JLC26의 효소적인 특성을 알아보았다. 효소는 70% ammonium sulfate로 침전시키고, CM-Sepharose column chromatography로 부분 정제하였다. 이때 얻어진 각 효소 specific activity는 amylase 활성의 경우는 5367 unit/mg이었고 dextranase 활성의 경우는 3045 unit/mg이었다. 효소의 최적 pH와 온도는 5.5와 37$^{\circ}C$였으며, pH 2.5-5.5와 4-55$^{\circ}C$에서 안정성을 보였다. 부분 정제된 효소는 amylase와 dextranase 활성을 동시에 보이는 100 KDa의 크기를 가지는 하나의 단백질이었다. 이 효소는 maltotriose 기질과 반응할 때 disproportionation reaction 현상을 보여 가지구조를 지닌 panose와 ${\alpha}(1{\rightarrow}6)$glucosylmaltotriose을 생산하였다. 이 균은 starch 배지에서 키워 amylase와 dextranase 활성을 동시에 지니는 효소를 생산함으로써 dextran과 mutan은 구성된 치태를 분해하는 효소재료로의 이용성이 기대된다.

• The Korean Journal of Physiology and Pharmacology
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• 제22권4호
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• pp.457-465
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• 2018

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.