• 한국가축번식학회지
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• 제20권2호
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• pp.215-221
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• 1996

This study was carried out to develop an immunoassay for the diagnosis of the pregnancy and ovarian function of domestic animals. Using 2E92C10 monoclonal antibody(McAb) generated against estrone-3-glucuronide(E1-3-G) and appeared a high cross-reactivity with estrone-3-sulfate(E1-3-S), chemiluminesence immunoassay (CIA) to detect E1-3-S was developed. 2E92C10 McAb cross-reacted with E1-3-S (30%) was purified from ascites fluid using protein G sepharose gel column. The purity of purified antibody fraction was monitored by SDS-PAGE and was better compared to that of crude ascite fluid. The soild and liquid phase CIA for E1-3-S were established utilizing 2E92C10 antibody and E1-3-G-ABEI conjugate used as a tracer. As the results, the titer of 2E92C10 antibody was 5g/ml in soild phase and 1:2000 in liquid phase. The sensitivity on soild and solid phase CIA were about 200 pg/ml. These results indicate that CIA for measurement of E1-3-S was successfully developed by using ant-E1-3-G McAb cross-reacted with E1-3-S and could be usefully used to research this area.

• Molecules and Cells
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• 제25권2호
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• pp.265-271
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• 2008

Single nucleotide polymorphisms (SNPs) are the most common form of human genetic variation. Non-synonymous SNPs (nsSNPs) change an amino acid. Organic anion transporters (OATs) play an important role in eliminating or reabsorbing endogenous and exogenous organic anionic compounds. Among OATs, hOAT4 mediates high affinity transport of estrone sulfate and dehydroepiandrosterone sulfate. The rapid bone loss that occurs in post-menopausal women is mainly due to a net decrease of estrogen. In the present study we searched for SNPs within the exon regions of hOAT4 in Korean women osteoporosis patients. Fifty healthy subjects and 50 subjects with osteoporosis were screened for genetic polymorphism in the coding region of SLC22A11 (hOAT4) using GC-clamp PCR and denaturing gradient gel electrophoresis (DGGE). We found three SNPs in the hOAT4 gene. Two were in the osteoporosis group (C483A and G832A) and one in the normal group (C847T). One of the SNPs, G832A, is an nsSNP that changes the $278^{th}$ amino acid from glutamic acid to lysine (E278K). Uptake of [$3^H$] estrone sulfate by oocytes injected with the hOAT4 E278K mutant was reduced compared with wild-type hOAT4. Km values for wild type and E278K were $0.7{\mu}M$ and $1.2{\mu}M$, and Vmax values were 1.8 and 0.47 pmol/oocyte/h, respectively. The present study demonstrates that hOAT4 variants can causing inter-individual variation in anionic drug uptake and, therefore, could be used as markers for certain diseases including osteoporosis.

• The Korean Journal of Physiology and Pharmacology
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• 제19권4호
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• pp.319-325
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• 2015

Among solute carrier proteins, the organic anion transporters (OATs) play an important role for the elimination or reabsorption of endogenous and exogenous negatively charged anionic compounds. Among OATs, SLC22A9 (hOAT7) transports estrone sulfate with high affinity. The net decrease of estrogen, especially in post-menopausal women induces rapid bone loss. The present study was performed to search the SNP within exon regions of SLC22A9 in Korean females with osteoporosis. Fifty healthy controls and 50 osteoporosis patients were screened for the genetic polymorphism in the coding region of SLC22A9 using GC-clamped PCR and denaturing gradient gel electrophoresis (DGGE). Six SNPs were found on the SLC22A9 gene from Korean women with/without osteoporosis. The SNPs were located as follows: two SNPs in the osteoporosis group (A645G and T1277C), three SNPs in the control group (G1449T, C1467T and C1487T) and one SNP in both the osteoporosis and control groups (G767A). The G767A, T1277C and C1487T SNPs result in an amino acid substitution, from synonymous vs nonsynonymous substitution arginine to glutamine (R256Q), phenylalanine to serine (F426S) and proline to leucine (P496L), respectively. The Km values and Vmax of the wild type, R256Q, P496L and F426S were 8.84, 8.87, 9.83 and $12.74{\mu}M$, and 1.97, 1.96, 2.06 and 1.55 pmol/oocyte/h, respectively. The present study demonstrates that the SLC22A9 variant F426S is causing inter-individual variation that is leading to the differences in transport of the steroid sulfate conjugate (estrone sulfate) and, therefore this could be used as a marker for certain disease including osteoporosis.

• Asian-Australasian Journal of Animal Sciences
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• 제16권9호
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• pp.1254-1260
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• 2003

The purpose of this study firstly was conducted to establish a radioimmunoassay (RIA) of estrone sulfate ($E_1S$), secondly to monitor the reproductive status of dairy cows using their urine samples. Urine and blood samples were collected in series within a day from four pregnant Holstein-friesian cows to evaluate the relationship between $E_1S$ levels in blood and urine with or without urinary creatinine basis. The urine was then collected biweekly from three cows in estrous and those artificially inseminated; collection from pregnant cows was made on a monthly basis. Results indicated that sensitivity for the $E_1S$ RIA was 5 pg/tube and the recovery rate was 100%. The daily urinary creatinine concentrations fluctuated within a day, but changes were slighter in midday, whereas the changes of concentrations of $E_1S$ in urine were relatively smaller. The concentrations of serum $E_1S$ during the estrous cycle were undetectable due to the limitation of assay, but the urinary $E_1S$ level could be measured with no obvious changes during the cycle. The urinary $E_1S$ levels increased remarkably around 7.7 to 8.3 ng/ml, 80 to 100 days after pregnancy but the serum $E_1S$ levels did not elevate until 120 to 150 days. The level of $E_1S$ increased gradually during pregnancy and eventually reached its peak before parturition at around 40 ng/ml and finally decreased to its basal level 2 days postparturition. During pregnancy, $E_1S$ concentrations of urine increased earlier than those in blood. The correlation coefficients between urinary and serum $E_1S$ concentration during pregnancy and postparturm were higher than those adjusted with creatinine (creatinine ratio). The concentrations of $E_1S$ in urine could be maintained unchanged for 8 days storing the samples in room temperature, which was extended to 8 days when the samples were pretreated by boiling for 30 minutes or treated with autoclave. In conclusion urinary $E_1S$ concentrations can be used directly for monitoring the pregnant status and fetal viability of dairy cows and can assist accurate confirmation of pregnancy in cows at least 80 to 100 days after insemination much earlier than by serum $E_1S$.

• Biomolecules & Therapeutics
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• 제20권6호
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• pp.556-561
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• 2012

Steroid sulfatase (STS) is responsible for the conversion of estrone sulfate to estrone that can stimulate growth in endocrine-dependent tumors such as prostate cancer. Although STS is considered as a therapeutic target for the estrogen-dependent diseases, cellular function of STS are still not clear. Previously, we found that tumor necrosis factor (TNF)-${\alpha}$ significantly enhances steroid sulfatase expression in PC-3 human prostate cancer cells through PI3K/Akt-dependent pathways. Here, we studied whether bacterial lipopolysaccharides (LPS) which are known to induce TNF-${\alpha}$ may increase STS expression. Treatment with LPS in PC-3 cells induced STS mRNA and protein in concentration- and time-dependent manners. Using luciferase reporter assay, we found that LPS enhanced STS promoter activity. Moreover, STS expression induced by LPS increased PC-3 tumor cell migration determined by wound healing assay. We investigated that LPS induced IL-6 expression and IL-6 increased STS expression. Taken together, these data strongly suggest that LPS induces STS expression through IL-6 pathway in human prostate cancer cells.

• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2161-2166
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• 2015

Estrogen receptors (ERs) are steroid receptors located in the cytoplasm and on the nuclear membrane. The sequence similarities of human $ER{\alpha}$, mouse $ER{\alpha}$, rat $ER{\alpha}$, dog $ER{\alpha}$, and cat $ER{\alpha}$ are above 90%, but structures of $ER{\alpha}$ may different among species. Estrogen can be agonist and antagonist depending on its target organs. This hormone play roles in several diseases including breast cancer. There are variety of the relative binding affinity (RBA) of ER and estrogen species in comparison to $17{\beta}-estradiol$ (E2), which is a natural ligand of both $ER{\alpha}$ and $ER{\beta}$. The RBA of the estrogen species are as following: diethyl stilbestrol (DES) > hexestrol > dienestrol > $17{\beta}-estradiol$ (E2) > 17- estradiol > moxestrol > estriol (E3) >4-OH estradiol > estrone-3-sulfate. Estrogen mimetic drugs, selective estrogen receptor modulators (SERMs), have been used as hormonal therapy for ER positive breast cancer and postmenopausal osteoporosis. In the postgenomic era, in silico models have become effective tools for modern drug discovery. These provide three dimensional structures of many transmembrane receptors and enzymes, which are important targets of de novo drug development. The estimated inhibition constants (Ki) from computational model have been used as a screening procedure before in vitro and in vivo studies.

• Journal of Pharmaceutical Investigation
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• 제36권4호
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• pp.283-286
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• 2006

The present study aimed to investigate the interaction of nucleoside analogs with human organic anion transporter 1 and 3(hOAT1 and hOAT3) that play a primary role in the tubular uptake of endogenous and exogenous organic anions in the kidney. The interactions of ddC, ara-C, ara-A and ara-U with hOAT1 and hOAT3 were examined using MDCK cells stably overexpressing hOAT1 or hOAT3. Among the tested drugs, ddC showed the highest affinity to hOAT1 with $IC_{50}$ values of 5.2 mM, while ara-A, ara-C and ara-U weakly inhibited the cellular uptake of $[^3H]-PAH$ in MDCK-hOAT1 cells at 1 mM. In contrast, all the tested drugs did not have any inhibition effect on the cellular uptake of $[^3H]-estrone$ sulfate in MDCK-hOAT3 cells over the drug concentration of 0.01-2 mM, implying that they might not interact with hOAT3. Taken all together, the present study suggests that hOAT1 could weakly interact with nucleoside analogues such as ddC, ara-C, ara-A and ara-U but the interaction with hOAT3 during the urinary excretion of these nucleoside analogues may be negligible in the kidney.

• Parasites, Hosts and Diseases
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• 제61권4호
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• pp.428-438
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• 2023

Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis-Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 µM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.