• 한국수정란이식학회지
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• 제24권4호
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• pp.259-263
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• 2009

Salivary lipocalin (SAL1) is a member of the lipocalin protein family that has a property to associate with many lipophilic molecules and was identified as pheromone-binding protein in pigs. Our previous study has shown that SAL1 is expressed in the uterine endometrium in a cell type- and implantation stage-specific manner and secreted into the uterine lumen in pigs. However, function of SAL1 in the uterus during pregnancy in pigs is still not known. To understand physiological function of SAL1 in the uterine endometrium during pregnancy in pigs, it needs to elucidate the ligand(s) for SAL1. Thus, to identify the ligand for SAL1 in the porcine uterus, we collected uterine luminal fluid from pigs on day 12 of pregnancy by flushing with PBS. Proteins from the uterine luminal fluid were separated by ion exchange chromatography and gel filtration. Fractions containing SAL1 protein were pooled and concentrated. Immunoblot analysis confirmed successful purification of SAL1. Then, we extracted lipids from the purified SAL1 protein and analyzed the lipids by liquid chromatography-mass spectrometry, and predicted to be steroid hormones and prostaglandins as SAL1 ligands. Results in this study showed that SAL1 protein in the uterine secretions has a small lipophilic molecule as a natural ligand. Further characterization of ligand extracted from purified SAL1 will be useful for understanding physiological function of SAL1 during pregnancy and its application to increase the pregnancy rate in pigs.

• Reproductive and Developmental Biology
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• 제28권1호
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• pp.45-52
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• 2004

To search a new porcine pheromonal odorants, this research for biostimulation and role of pheromone was augmented by means of "control system technologies" to offer a potentially useful and practical way to improve reproductive efficiency in livestock species. Therefore the 13 physicochemical parameters such as similarity indice (S), hydrophobicity (logP) and van der Waals molecule volume (MV) etc. of 54 steroid analogues, which are analogous of substrate molecules, 5$\alpha$-androst-16-en-3-one (P1) and 5$\alpha$-androst-16-en-3-ol (P2) of lipocalin as receptor of pig pheromones were calculated and discussed. The physicochemical properties of these steroid analogues were mainly followed by steric dissimilar of A and D ring in steroid nucleus. And we found that from correlation with S values and MV constants of molecules, the more MV constants are small, the more S values tend to approach 1. Based on this results, the S-values of 4-androsten-3,17-dione (P1-1) and 5 $\alpha$ -androstan-3-one (P2-1) were 1.0, respectively. The two compounds of them were chosen because they showed the same value each other at a side of hydrophobicity, molar refractivity and molecular volume. It is expected that the new two compounds will be able to substitute for P1 and P2, porcine pheromonal odorants.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
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• pp.148.2-148.2
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• 2013

We prepared hydrophobic PDMS-coated porous silica as pre-concentration adsorbent for chemical warfare agents (CWAs). Since CWAs can be harmful to human even with a small amount, detecting low-concentration CWAs has been attracting attention in defense development. Porous silica is one of the promising candidates for CWAs pre-concentration adsorbent since it is thermally stable and its surface area is sufficiently high. A drawback of silica is that adsorption of CWAs can be significantly reduced due to competitive adsorption with water molecule in air since silica is quite hydrophilic. In order to solve this problem, hydrophobic polydimethylsiloxane (PDMS) thin film was deposited on silica. Adsorption and desorption of chemical warfare agent (CWA) simulants (Dimethylmethylphosphonate, DMMP and Dipropylene Glycol Methyl Ether, DPGEM) on bare and PDMS-coated silica were studied using temperature programed desorption (TPD) with and without co-exposing of water vapor. Without exposure of water vapor, desorbed amount of DMMP from PDMS-coated silica was twice larger than that from bare silica. When the samples were exposed to DMMP and water vapor at the same time, no DMMP was desorbed from bare silica due to competitive adsorption with water. On the other hand, desorbed DMMP was detected from PDMS-coated silica with reduced amount compared to that from the sample without water vapor exposure. Adsorption and desorption of DPGME with and without water vapor exposing was also investigated. In case of bare silica, all the adsorbed DPGME was decomposed during the heating process whereas molecular DPGME was observed on PDMS-coated silica. In summary, we showed that hydrophobic PDMS-coating can enhance the adsorption selectivity toward DMMP under humid condition and PDMS-coating also can have positive effect on molecular desorption of DPGME. Therefore we propose PDMS-coated silica could be an adequate adsorbent for CWAs pre-concentration under practical condition.

• Biomolecules & Therapeutics
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• 제28권1호
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• pp.98-103
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• 2020

Marfan syndrome (MFS), a connective tissue disorder caused by mutations in the fibrillin-1 (Fbn1) gene, has vascular manifestations including aortic aneurysm, dissection, and rupture. Its vascular pathogenesis is assumed to be attributed to increased transforming growth factor β (TGFβ) signaling and blockade of excessive TGFβ signaling has been thought to prevent dissection and aneurysm formation. Here, we investigated whether galunisertib, a potent small-molecule inhibitor of TGFβ receptor I (TβRI), attenuates aneurysmal disease in a murine model of MFS (Fbn1^C1039G/+) and compared the impact of galuninsertib on the MFS-related vascular pathogenesis with that of losartan, a prophylactic agent routinely used for patients with MFS. Fbn1^C1039G/+ mice were administered galunisertib or losartan for 8 weeks, and their ascending aortas were assessed for histopathological changes and phosphorylation of Smad2 and extracellular signal-regulated kinase 1/2 (Erk1/2). Mice treated with galunisertib or losartan barely exhibited phosphorylated Smad2, suggesting that both drugs effectively blocked overactivated canonical TGFβ signaling in Fbn1^C1039G/+ mice. However, galunisertib treatment did not attenuate disrupted medial wall architecture and only partially decreased Erk1/2 phosphorylation, whereas losartan significantly inhibited MFS-associated aortopathy and markedly decreased Erk1/2 phosphorylation in Fbn1^C1039G/+ mice. These data unexpectedly revealed that galunisertib, a TβRI inhibitor, showed no benefits in aneurysmal disease in MFS mice although it completely blocked Smad2 phosphorylation. The significant losartan-induced inhibition of both aortic vascular pathogenesis and Smad2 phosphorylation implied that canonical TGFβ signaling might not prominently drive aneurysmal diseases in MFS mice.

• Biomolecules & Therapeutics
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• 제28권5호
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• pp.482-489
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• 2020

G protein-coupled receptor kinase 5 (GRK5) has been considered as a potential target for the treatment of heart failure as it has been reported to be an important regulator of pathological cardiac hypertrophy. To discover novel scaffolds that selectively inhibit GRK5, we have identified a novel small molecule inhibitor of GRK5, KR-39038 [7-((3-((4-((3-aminopropyl)amino)butyl)amino)propyl)amino)-2-(2-chlorophenyl)-6-fluoroquinazolin-4(3H)-one]. KR-39038 exhibited potent inhibitory activity (IC₅₀ value=0.02 µM) against GRK5 and significantly inhibited angiotensin II-induced cellular hypertrophy and HDAC5 phosphorylation in neonatal cardiomyocytes. In the pressure overload-induced cardiac hypertrophy mouse model, the daily oral administration of KR-39038 (30 mg/kg) for 14 days showed a 43% reduction in the left ventricular weight. Besides, KR-39038 treatment (10 and 30 mg/kg/day, p.o.) showed significant preservation of cardiac function and attenuation of myocardial remodeling in a rat model of chronic heart failure following coronary artery ligation. These results suggest that potent GRK5 inhibitor could effectively attenuate both cardiac hypertrophy and dysfunction in experimental heart failure, and KR-39038 may be useful as an effective GRK5 inhibitor for pharmaceutical applications.

• 약학회지
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• 제44권5호
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• pp.391-398
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• 2000

Leptin, the product of the ob gene, is a small peptide molecule synthesized by white adipocytes with an important role in the regulation of body fat and food intake. Based on the evidence that synthesis of leptin is regulated by female sex hormone, estrogen, this present study was investigated whether sex hormone precursor DHEA, can regulate obese gene expression in lean and genetically obese (ob/ob) mice. Antiobesity activity of DHEA was evaluated by determining body weight, food consumption, epididymal fat weight and serum levels of cholesterol and triglyceride in ICR, C57BL/6J, and ob/ob mice. The treatment of C57BL/6J lean and obese mice with a diet containing 0.3% and 0.6% DHEA resulted in lowered rates of weight gain in comparison to non-treated mice, although much greater response was found in the obese mice. All other concentrations of DHEA (0.015%, 0.06%, 0.15%, 0.3%) except the highest one(0.6%) showed no significant effects on weight gain in ICR mice. Food consumption was significantly decreased in all mice treated with 0.6% DHEA, whereas it was not decreased in ICR mice at lower concentrations than 0.6% DHEA. DHEA decreased significantly epididymal adipose tissue weight and serum triglyceride levels dose dependently in lean and obese mice. However serum cholesterol levels were decreased at lower concentrations than 0.15% DHEA and increased at concentrations of 0.3% and 0.6% DHEA in lean and obese mice. These increases in serum cholestrol levels at high concentrations of DHEA might result from the fact that DHEA has a cholesterol moiety thereby interfered the assay system. As an approach to elucidate the mechanism for antiobesity activity of DHEA, we examined mRNA levels of obese gene in the adipocyte and obese gene product (leptin) in the serum. The results showed that DHEA did not affect obese gene expression in ICR and C57BL/6J mice. Therefore, we concluded that antiobesity activity of DHEA was not modulated by obese gene expression.

• 한국미생물·생명공학회지
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• 제49권2호
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• pp.148-156
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• 2021

Single-chain antibodies against epidermal growth factor receptor variant III (EGFRvIII) are potentially promising agents for developing antibody-based cancer treatment strategies. We described in our previous study the successful expression of an anti-EGFRvIII scFv antibody in Escherichia coli. However, we could also observe the formation of insoluble aggregates in the periplasmic space, limiting the production yield of the active product. In the present study, we investigated the mechanisms by which growth conditions could affect the expression of the soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures, attempting to maximize production. The secreted scFv molecules were purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and western blot analyses. We used the ImageJ software for protein quantification and determined the antigen-binding activity of the scFv antibody against the EGFRvIII protein. Our results showed that the highest percentage of soluble scFv expression could be achieved under culture conditions that combined low IPTG concentration (0.1 mM), low growth temperature (18℃), and large culture dish surface area. We found moderate-yield soluble scFv production in the culture medium after lactose-mediated induction, which was also beneficial for downstream protein processing. These findings were confirmed by conducting western blot analysis, indicating that the soluble, approximately 30-kDa scFv molecule was localized in the periplasm and the extracellular space. Moreover, the antigen-binding assay confirmed the scFv affinity against the EGFRvIII antigen. In conclusion, our study reveals that low-speed protein expression is preferable to obtain more soluble anti-EGFRvIII scFv protein in an E. coli expression system.

• Genomics & Informatics
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• 제19권1호
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• pp.6.1-6.10
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• 2021

Vascular endothelial growth factor (VEGF) is expressed at elevated levels by most cancer cells, which can stimulate vascular endothelial cell growth, survival, proliferation as well as trigger angiogenesis modulated by VEGF and VEGFR (a tyrosine kinase receptor) signaling. The angiogenic effects of the VEGF family are thought to be primarily mediated through the interaction of VEGF with VEGFR-2. Targeting this signaling molecule and its receptor is a novel approach for blocking angiogenesis. In recent years virtual high throughput screening has emerged as a widely accepted powerful technique in the identification of novel and diverse leads. The high resolution X-ray structure of VEGF has paved the way to introduce new small molecular inhibitors by structure-based virtual screening. In this study using different alkaloid molecules as potential novel inhibitors of VEGF, we proposed three alkaloid candidates for inhibiting VEGF and VEGFR mediated angiogenesis. As these three alkaloid compounds exhibited high scoring functions, which also highlights their high binding ability, it is evident that these alkaloids can be taken to further drug development pipelines for use as novel lead compounds to design new and effective drugs against cancer.

• Biomolecules & Therapeutics
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• 제27권2호
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• pp.216-221
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• 2019

The c-Met protein is a receptor tyrosine kinase involved in cell growth, proliferation, survival, and angiogenesis of several human tumors. Overexpression of c-Met has been found in gastric cancers and correlated with a poor prognosis. Indirubin is the active component of Danggui Longhui Wan, which is a traditional Chinese antileukemic recipe. In the present study, we tested the anti-cancer effects of an indirubin derivative, LDD-1937, on human gastric cancer cells SNU-638. When we performed the in vitro kinase assay against the c-Met activity, LDD-1937 inhibited the activity of c-Met. This result was confirmed by immunoblot and immunofluorescence of phosphorylated c-Met. Immunoblot analysis showed that LDD-1937 decreased the expression of the Erk1/2, STAT3, STAT5, and Akt, downstream proteins of c-Met. In addition, LDD-1937 reduced the cell viability and suppressed colony formation and migration of SNU-638 cells. Furthermore, LDD-1937 induced $G_2/M$ phase arrest in the SNU-638 cells by decreasing the expression levels of cyclin B1 and CDC2. Cleaved-PARP, an apoptosis-related protein, was up-regulated in cells treated with LDD-1937. Overall, this study suggests that LDD-1937 may be a novel small-molecule with therapeutic potential for selectively inhibiting c-Met and c-Met downstream pathways in human gastric cancers overexpressing c-Met.

• Journal of Ginseng Research
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• 제45권1호
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• pp.156-162
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• 2021

Background: It is estimated that 20-30% of ginseng crops in Canada are lost to root rot each harvest. This disease is commonly caused by fungal infection with Ilyonectria, previously known as Cylindrocarpon. Previous reports have linked the virulence of fungal disease to the production of siderophores, a class of small-molecule iron chelators. However, these siderophores have not been identified in Ilyonectria. Methods: High-resolution LC-MS/MS was used to screen Ilyonectria and Cylindrocarpon strain extracts for secondary metabolite production. These strains were also tested for their ability to cause root rot in American ginseng and categorized as virulent or avirulent. The differences in detected metabolites between the virulent and avirulent strains were compared with a focus on siderophores. Results: For the first time, a siderophore N,N',N"-triacetylfusarinine C (TAFC) has been identified in Ilyonectria, and it appears to be linked to disease virulence. Siderophore production was suppressed as the concentration of iron increased, which is in agreement with previous reports. Conclusion: The identification of the siderophore produced by Ilyonectria gives us further insight into the root rot disease that heavily affects ginseng crop yields. This research identifies a molecular pathway previously unknown for ginseng root rot and could lead to new disease treatment options.