• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.109-115
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• 2005

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughoutthe lifetime. In this study, we analyzed the expression of receptors of $P2Y_{10}$, purinergic receptor families in murine hematopoietic stem cells, hematopoietic progenitor cells. In addition, the biological activity of $P2Y_{10}$ was investigated with B lymphocyte cell line, Ba/F3 in effect to cell growth and cell cycle. From the analysis of expression in hematopoieticstem cell. and progenitor with RT-PCR, $P2Y_{10}$ was strongly expressed in murine hematopoieticstem cells (c-kit+ Sca-l+ Lin-) and progenitor cell population, such as c-kit- Sca-l+ Lin-, c-kit+ Sca-l- Lin- and c-kit- Sca-l- Lin-. To investigate the biological effects by $P2Y_{10}$, retroviral vector from subcloned murine $P2Y_{10}$ cDNA was used fur gene introduction into Ba/F3 cells, and stable transfectant cells were obtained by flow cytometry sorting. In cell proliferation assay, the proliferation ability of $P2Y_{10}$ receptor gene­transfected cells was strongly inhibited, and the cell cycle was arrested at G1 phase. These result suggest that the $P2Y_{10}$ may be involved the biological activity in hematopoietic stem cells and immature B lymphocytes.

• BMB Reports
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• v.51 no.9
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• pp.468-473
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• 2018

Purinergic receptor signaling is increasingly recognized as an important regulator of inflammation. The P2X family purinergic receptors P2X5 and P2X7 have both been implicated in bone biology, and it has been suggested recently that P2X5 may be a significant regulator of inflammatory bone loss. However, a role for P2X5 in periodontitis is unknown. The present study aimed to evaluate the functional role of P2X5 in ligature-induced periodontitis in mice. Five days after placement of ligature, analysis of alveolar bone revealed decreased bone loss in $P2rx5^{-/-}$ mice compared to $P2rx7^{-/-}$ and WT control mice. Gene expression analysis of the gingival tissue of ligated mice showed that IL1b, IL6, IL17a and Tnfsf11 expression levels were significantly reduced in $P2rx5^{-/-}$ compared to WT mice. These results suggest the P2X5 receptor may regulate bone loss related to periodontitis and it may thus be a novel therapeutic target in this oral disease.

• YAKHAK HOEJI
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• v.58 no.1
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• pp.16-20
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• 2014

KR-31064 was developed for the strong angiotensin II receptor antagonist among the one of pyridyl imidazol series compounds. To investigate the receptor-ligand binding characteristics of this nonpeptide antagonist, binding experiments were deployed in various conditions and ex vivo contractile responses were tested toward the standard compound, losartan. Receptor binding experiments with radiolabeled angiotensin II, the $IC_{50}$ value for KR-31064 resulted 0.67 nM without any activities toward type 2 angiotensin II receptor. The comparative potency against losartan was more than 18 fold and the specific activity in type 1 angiotensin II receptor was more than 10,000 fold comparing to the type 2 receptor. Scatchard analysis of saturation binding data showed KR-31064 acted on the receptor in a competitive mode. KR-31064 inhibited the contractile response derived by angiotensin II ($pK_B$: 9.86) similar to that of losartan with decreased maximum signals. As a potent and specific type 1 angiotensin II receptor antagonist, KR-31064 may have possibilities for the development of diagnostic ligands that can be used as tools for various biochemical research experiments and non-invasive diagnostics.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.61-61
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• 1993

사람의 혈액의 백혈구로부터 Polymerase Chain Reaction 방법으로 $\beta$$_2$-adrenergic receptor 유전자를 증폭하였다. 이 증폭된 유전자를 pbluescript KS(＋)에 cloning하였으며, 제한효소지도 작성과 부분적인 염기배열 확인으로 증폭된 유전자가 사람의 $\beta$$_2$-adrenergic receptor 유전자임을 확인하였다. $\beta$$_2$-adrenergic receptor 유전자를 N말단의 아미노산이 21개 제거되도록 결손시킨후 yeast의 pSec5의 Killer toxin signal sequence의 뒤에 in-frame으로 연결하였다. 현재 효모에서의 발현양상 및 발현된 단백질의 활성을 조사중이다.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.340-350
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• 2007

The objective of this study was to determine whether peroxisome proliferator-activated receptor ${\gamma}$(PPAR${\gamma}$ is involved in the regulation of weaning to estrus of primiparous sows. Twelve sows composed of 6 groups of 2 full-sibs in a similar age (325.2 d), body weight (BW; 152.4 kg) and backfat thickness (BFT; 27.0 mm) at start of lactation, were allocated to accept 31 MJ (restricted group, R-group) or 53 MJ (control group, C-group) DE/d treatment, respectively. The experimental results indicated that the low energy intake resulted in excessive losses of BW and BFT during lactation in R-group sows, which may be related to decrease of serum 15-deoxy-${\Delta}^{12,14}$-prostaglandin $J_2$ (15d-$PGJ_2$), a ligand of PPAR${\gamma}$ The obvious peak and the frequency of LH, FSH and estradiol ($E_2$) were only observed in C-group sows. Except for $E_2$ at d 1 and 2, serum FSH, LH and $E_2$ concentrations in R-group were lower than those in C-group sows after weaning. However, the serum progesterone ($P_4$) level in R-group sows was always more than that in C-group. The expression abundances of PPAR${\gamma}$and GnRH receptor (GnRH-R) in pituitary, FSH receptor (FSH-R), LH receptor (LH-R), estrogen receptor (ES-R) and aromatase in ovary of anestrous sows were lower than those of estrous sows. Neither the BFT nor the BW was associated with the mRNA abundance of PPAR${\gamma}$in hypothalamus during lactation. Expressions of PPAR${\gamma}$in pituitary and ovary were affected evidently by the BFT changes and only by the loss of BW of sows during and after lactation. Furthermore, PPAR${\gamma}$mRNA level in ovary was significantly related to the expression abundances of GnRH-R, FSH-R, ES-R and aromatase, and GnRH-R was obviously associated with PPAR${\gamma}$expression in pituitary. However, PPAR${\gamma}$expression in hypothalamus likely has no effects on these genes expression and no obvious difference for all sows. Not serum $E_2$ or $P_4$ alone but the ratios of $E_2$ to $P_4$ and 15d-$PGJ_2$ to $P_4$, and serum FSH and LH were evidently related to PPAR${\gamma}$expression in pituitary and ovary. It is concluded that PPAR${\gamma}$is associated with body conditions, reproduction hormones and their receptor expression, which affected the functions of pituitary and ovary and ultimately the estrus after weaning of primiparous sows.

• IMMUNE NETWORK
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• v.15 no.1
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• pp.44-49
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• 2015

Interactions between microbes and epithelial cells in the gastrointestinal tract are closely associated with regulation of intestinal mucosal immune responses. Recent studies have highlighted the modulation of mucosal immunity by microbe-derived molecules such as ATP and short-chain fatty acids. In this study, we undertook to characterize the expression of the ATP-gated $P2X_7$ receptor ($P2X_7R$) on M cells and its role in gastrointestinal mucosal immune regulation because it was poorly characterized in Peyer's patches, although purinergic signaling via $P2X_7R$ and luminal ATP have been considered to play an important role in the gastrointestinal tract. Here, we present the first report on the expression of $P2X_7R$ on M cells and characterize the role of $P2X_7R$ in immune enhancement by ATP or LL-37.

• Archives of Pharmacal Research
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• v.29 no.11
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• pp.1032-1041
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• 2006

Extracellular adenosine 5'-triphosphate (ATP) affects the function of many tissues and cells. To confirm the biological activity of ATP on human myeloid leukemic cells, F-36P and HL-60, cells were treated with a variety of concentrations of ATP. The stimulation with extracellular ATP induced the arrest of cell proliferation and cell death. from the analysis of Annexin-V staining and caspase activity by flow cytometry. The Annexin-V positive cells in both cell lines were dramatically increased following ATP stimulation. The expression of P2 purinergic receptor genes was confirmed, such as P2X1, P2X4, P2X5, P2X7 and P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y11 in both leukemic cell lines. Interestingly, ATP induced intracellular calcium flux in HL-60 cells but not in F-36P cells, as determined by Fluo-3 AM staining. Cell cycle analysis revealed that ATP treatment arrested both F-36P and HL-60 cells at G1/G0. Taken together, these data showed that extracellular ATP via P2 receptor genes was involved in the cell proliferation and survival in human myeloid leukemic cells, HL-60 and F-36P cells by the induction of apoptosis and control of cell cycle. Our data suggest that treatment with extracellular nucleotides may be a novel and powerful therapeutic avenue for myeloid leukemic disease.

• International Journal of Oral Biology
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• v.31 no.4
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• pp.141-148
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• 2006

The effects of adenosine triphosphate(ATP) on salivary glands have been recognized since 1982. The presence of purinergic recepetors(P2Rs) that mediate the effects of ATP in various tissues, including parotid and submandibular salivary gland, has been supported by the cloning of receptor cDNAs and the expression of the receptor proteins. P2Rs have many subtypes, and the activation of these receptor subtypes increase intracellular $Ca^{2+}$, a key ion in the regulation of the secretion in the salivary gland. The apical pores of taste buds in circumvallate and foliate papillae are surrounded by the saliva from von Ebner salivary gland(vEG). Thus, it is important how the secretion of vEG is controlled. This study was designed to elucidate the roles of P2Rs on salivary secretion of vEG. Male Sprague-Dawley rats (about 200 g) were used for this experiment. vEG-rich tissues were obtained from dissecting $500-1,000\;{\mu}m$ thick posterior tongue slices under stereomicroscope view. P2Rs mRNA in vEG acinar cells were identified with RT-PCR. To observe the change in intracellular $Ca^{2+}$ activity, we employed $Ca^{2+}-ion$ specific fluorescence analysis with fura-2. Single acinar cells and cell clusters were isolated by a sequential trypsin/collagenase treatment and were loaded with $10\;{\mu}M$ fura -2 AM for 60 minutes at room temperature. Several agonists and antagonists were used to test a receptor specificity. RT-PCR revealed that the mRNAs of $P2X_4$, $P2Y_1$, $P2Y_2$ and $P2Y_3$ are expressed in vEG acinar cells. The intracellular calcium activity was increased in response to $10\;{\mu}M$ ATP, a P2Rs agonist, and 2-MeSATP, a $P2Y_1$ and $P2Y_2R$ agonist. However, $300\;{\mu}M\;{\alpha}{\beta}-MeATP$, a $P2X_1$ and $P2X_3R$ agonist, did not elicit the response. The responses elicited by $10\;{\mu}M$ ATP and UTP, a $P2Y_2R$ agonists, were maintained when extracellular calcium was removed. $10\;{\mu}M$ suramin, a P2XR antagonist, and reactive blue 2, a P2YR antagonist, partially blocked ATP-induced response. However, when extracellular calciums were removed, suramin did not abolish the responses elicited by ATP. These results suggest that P2Rs play an important role in salivary secretion of vEG acinar cells and the effects of ATP on vEG salivary secretion may be mediated by $P2X_4$, $P2Y_1$, $P2Y_2$, and/or $P2Y_3$.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.61-66
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• 2011

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used $Ca^{2+}$ imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP ($10\;{\mu}M$) elicited strong but transient $[Ca^{2+}]_i$ increase in a concentration dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking $[Ca^{2+}]_i$ transients were 2MeS-ATP>ATP>>UTP=${\alpha}{\beta}$-MeATP, which was compatible with the subclass of $P2Y_1$ receptor. The $[Ca^{2+}]_i$ transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of $P2Y_1$ selective blocker (MRS 2179; $30\;{\mu}M$). $P2Y_1$ receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, $P2Y_1$ receptor is mainly expressed in a retinoblastoma cell, which elicits $Ca^{2+}$ release from internal $Ca^{2+}$ storage sites via the phospholipase C-mediated pathway. $P2Y_1$ receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic $[Ca^{2+}]_i$ signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

• The Journal of Internal Korean Medicine
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• v.28 no.2
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• pp.262-274
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• 2007

Objective : It has been reported that two-repeats ($IL1RN^{\ast}2$) of interleukin-1 receptor antagonist (IL-1Ra) gene is associated with ischemic stroke, and that Ala allele of the common Pro12Ala polymorphism in $PPAR-{\gamma}2$ isoform is associated with reduced risk for type 2 DM and its complications. The aim of the present study is to assess the association of IL-1Ra and $PPAR-{\gamma}2$ Pro12Ala polymorphism with the presence of ischemic stroke in the case of diabetic and non-diabetic patients. Methods : Genomic DNA was obtained from 373 healthy subjects, 157 DM subjects without ischemic stroke (known DM duration ${\ge}10$ years) and 302 ischemic stroke patients (including with DM). IL-1Ra polymorphism was analysed by polymerase chain reaction (PCR), and $PPAR-{\gamma}2$ polymorphism by restriction fragment length polymorphism after PCR. Results : $IL1RN^{\ast}1/IL1RN^{\ast}2$ genotype was associated with significantly increased risk for DM (OR=2.86, P = 0.0008) and ischemic stroke (OR=2.74, P = 0.0016). Pro/Ala genotype was associated with the reduced risk for DM (OR=0.53, P = 0.0491) and ischemic stroke (OR=0.38, P = 0.0039). They were also associated with the reduced risk for ischemic stroke in the DM patients compared with DM without ischemic stroke (OR=0.25, P = 0.0321). Conclusions : $IL1RN^{\ast}2$ allele could be an accelerating factor, not a predictive marker for ischemic stroke in type 2 DM. The Pro/Ala genotype of $PPAR-{\gamma}2$ Pro12Ala polymorphism may be associated with reduced risk for ischemic stroke with type 2 DM. Therefore it could be a useful predictive marker for ischemic stroke in Korean type 2 DM.