• Parasites, Hosts and Diseases
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• 제46권4호
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• pp.209-216
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• 2008

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.237-242
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• 2009

Pregnancy is a unique event in which a fetus develops in the uterus despite being genetically and immunologically different from the mother, and the underlying mechanisms remain poorly understood. To analyze the differential gene expression profiles in nonpregnant and 7 days post coitus (dpc) pregnant uterus of mice, we performed a global proteomic study by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The uterine proteins were separated using 2-DE, Approximately 1,000 spots were detected on staining with Coomassie brilliant blue. An image analysis using Melanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between pregnant and nonpregnant uterus. Twenty-one spots were identified as differentially expressed proteins, of which 10 were up-regulated proteins such as alpha-fetoprotein, chloride intracellular channel 1, transgelin, heat-shock protein beta-1, and carbonic anhydrase II, while 11 were down-regulated proteins such as X-box binding protein, glutathione S-transferase omega 1, olfactory receptor Olfr204, and metalloproteinase-disintegrin domain containing protein TECADAM. Most of the identified proteins appeared to be related with catabolism, cell growth, metabolism, regulation, cell protection, protein repair, or protection. Our results uncovered key proteins of mouse uterus involved in pregnancy.

• 한방재활의학과학회지
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• 제30권1호
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• pp.13-29
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• 2020

Objectives This study was performed to decide the bone union effect of Hwaweo-jeon on tibia fractured mice. Methods In this study, laboratory experiments were implemented by the stage of in vitro and in vivo. In in vitro, MC3T3-E1 cells were treated with various concentration of Hwaweo-jeon extract (HWJ). To investigate effect of HWJ for osteoblast, relative mRNA expression of 5 substances (alkaline phosphatase [ALP], runt-related transcription factor 2 [Runx2], osteocalcin [OCN], osterix [OSX] and collagen type II alpha 1 chain [Col2a1]) was used as a marker of osteogenesis. In order to determine HWJ's effect for fracture healing, relative gene expression level of ALP, Runx2, OCN, OSX and Col2a1 were used to find out the influence to osteoblast. Furthermore, receptor activator of nuclear factor kappa-B ligand and osteoprotegerin relative mRNA expression were used to estimate the impact to osteoclast. Also, X-ray was used for the purpose of identifying bone union in tibia-fracture mouse model. Results In in vitro experiment, most part of relative mRNA expression were increased compared to control group. In in vivo and in vitro experiment, HWJ induced osteoblast activitation by verifying relative mRNA expression of 5 substances. And in vivo experiment, we can also identify that HWJ triggered osteoclast activation during early stage of tibia fracture. Furthermore, X-ray pictures show noticeable recovery of tibia fracture. Conclusions HWJ extract promotes bone union by facilitating the osteoblast. But, HWJ may occur liver & kidney toxicity over specific concentration. Therefore, when HWJ is applied to human body, doctors have to follow up the liver function test & renal function test of patient.

• Asian-Australasian Journal of Animal Sciences
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• 제33권8호
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• pp.1352-1359
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• 2020

Objective: Lactobacilli in rabbit intestine is rare and its function in rabbit gut health is not fully understood. The present study aimed to evaluate in vivo the probiotic potential of Lactobacillus casei for suckling rabbits. Methods: Two healthy 5-day-old suckling rabbits with similar weights from each of 12 New Zealand White litters were selected and disturbed to control group and treatment group. All rabbits were artificially fed. The treatment group had been supplemented with live Lactobacillus casei in the milk from the beginning of the trial to 13 days of age. At 15 days of age, healthy paired rabbits were slaughtered to collect intestinal samples. Results: i) Oral administration of Lactobacillus casei significantly increased the proportion of Lactobacilli in the total intestinal bacteria (p<0.01) and obviously reduced that of Escherichia-Shigella (p<0.01); ii) treatment increased the length of vermiform appendix (p<0.05); iii) a higher percentage of degranulated paneth cells was observed in the duodenum and jejunum when rabbits administered with Lactobacillus casei (p<0.01); and iv) the expression of toll-like receptor 9, lysozyme (LYZ), and defensin-7-like (DEFEN) in the duodenum and jejunum was stimulated by supplemented Lactobacillus casei (p<0.05). Conclusion: Orally administered Lactobacillus casei could increase the abundance of intestinal Lactobacilli, decrease the relative abundance of intestinal Escherichia-Shigella, promote the growth of appendix vermiform, stimulate the degranulation of paneth cells and induce the expression of DEFEN and LYS. The results of the present study implied that Lactobacillus casei exhibited probiotic potential for suckling rabbits.

• 한방재활의학과학회지
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• 제30권4호
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• pp.1-16
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• 2020

Objectives The main purpose of this study was to evaluate the bone healing effect of Pahyeolsandong-tang (PHT)(Poxiesanteng-tang) extract in tibia fracture-induced mice. Methods PHT was extracted using a solution of 35% ethanol in 60℃ for 8 hours. Mice were randomly divided into 4 groups (normal, control, PHT 50 and PHT 100). Mice of experimental groups were medicated with PHT 50 or 100 mg/kg for 7 to 21 days. To clarify the effect of bone fracture healing, relative messenger RNA (mRNA) expressions of osteocalcin (OCN), runt-related transcription factor 2 (Runx2), osterix (OSX), Sox9, collagen type II alpha 1 chain (Col2a1), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) were examined. Results In in vitro experiment, relative mRNA expression of OCN, Runx2, Col2a1 was significantly increased in PHT treated group to compare with control differentiation group. In in vivo experiment, relative mRNA expression of OCN, Runx2, OSX, Sox9, Col2a1, RANKL, OPG was significantly increased in PHT treated group. Conclusions This study showed that PHT accelerates bone fracture healing through the activation of osteoclasts and osteoblasts. It was showed that PHT significantly promotes osteoblasts differentiation by osteoblast differentiation markers such as OCN, Runx2, Col1a2. Also it was investigated that PHT had stimulatory effect on osteoblasts function through enhancing OCN, Runx2, OSX, Sox9, Col2a1 and, osteoclasts function through enhancing RANKL and OPG markers. PHT effectively promotes bone fracture healing process through activation of osteoblasts and osteoclasts.

• International Journal of Oral Biology
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• 제31권2호
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• pp.45-51
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• 2006

R-type($Ca_v2.3$) calcium channel contributes to pain sensation in peripheral sensory neurons. Six isoforms of $Ca_v2.3$ that result from combinations of presence or deletion of three inserts(insert I and insert in the II-III loop, and insert III in N-terminal regions) have been demonstrated to be present in different mammalian tissues. However, the molecular basis of $Ca_v2.3$ in trigeminal ganglion(TG) neurons is not known. In the present study, we determined which isoforms of $Ca_v2.3$ are expressed in rat TG neurons using the RT-PCR analysis. Whole tissue RT-PCR analyses revealed that only two isoforms, $Ca_v2.3a$ and $Ca_v2.3e$, were present in TG neurons. From single-cell RT-PCR, we found that $Ca_v2.3e$ rather than $Ca_v2.3a$ was the major isoform expressed in TG neurons, and $Ca_v2.3e$ was preferentially detected in small-sized neurons that express nociceptive marker, transient receptor potential vanilloid 1(TRPV1). Our results suggest that $Ca_v2.3e$ in trigeminal neurons may be a potential target for the pain treatment.

• Journal of Ginseng Research
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• 제35권2호
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• pp.200-208
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• 2011

Ginsenoside (G) $Rp_1$ is a ginseng saponin derivative with anti-cancer and anti-inflammatory activities. In this study, we examined the mechanism by which G-$Rp_1$ inhibits inflammatory responses of cells. We did this using a strategy in which DNA constructs containing cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) promoters were transfected into HEK293 cells. G-$Rp_1$ strongly inhibited the promoter activities of COX-2 and iNOS; it also inhibited lipopolysaccharide induced upregulation of COX-2 and iNOS mRNA levels in RAW264.7 cells. In HEK293 cells G-$Rp_1$ did not suppress TANK binding kinase 1-, Toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-${\beta}$ (TRIF)-, TRIF-related adaptor molecule (TRAM)-, or activation of interferon regulatory factor (IRF)-3 and nuclear factor (NF)-${\kappa}$B by the myeloid differentiation primary response gene (MyD88)-induced. However, G-$Rp_1$ strongly suppressed NF-${\kappa}$B activation induced by I${\kappa}$B kinase (IKK)${\beta}$ in HEK293 cells. Consistent with these results, G-$Rp_1$ substantially inhibited IKK${\beta}$-induced phosphorylation of $I{\kappa}B{\alpha}$ and p65. These results suggest that G-$Rp_1$ is a novel anti-inflammatory ginsenoside analog that can be used to treat IKK${\beta}$/NF-${\kappa}$B-mediated inflammatory diseases.

• Archives of Pharmacal Research
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• 제29권11호
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• pp.969-976
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• 2006

Novel chalcones were found as potent inhibitors of interleukin-5 (II-5). 1-(6-Benzyloxy-2-hydroxyphenyl)-3-(4-hydroxyphenyl)propenone (2a, 78.8% inhibition at $50\;{\mu}M,\;IC_{50}=25.3\;{\mu}M$) was initially identified as a potent inhibitor of IL-5. This activity is comparable to that of budesonide or sophoricoside (1a). The benzyloxy group appears to be critical for the enhancement of the IL-5 inhibitory activity. To identify the role of this hydrophobic moiety, cyclohexyloxy (2d), cyclohexylmethoxy (2c), cyclohexylethoxy (2e), cyclohexylpropoxy (2f), 2-methylpropoxy (2g), 3-methylbutoxy (2h), 4-methylpentoxy (2i), and 2-ethylbutoxy (2j) analogs were prepared and tested for their effects on IL-5 bioactivity. Compounds 2c ($IC_{50}=12.6\;{\mu}M$), 2d ($IC_{50}=12.2\;{\mu}M$), and 2i ($IC_{50}=12.3\;{\mu}M$) exhibited the most potent activity. Considering the cLog P values of 2, the alkoxy group contributes to the cell permeability of 2 for the enhancement of activity, rather than playing a role in ligand motif binding to the receptor. The optimum alkoxy group in ring A of 2 should be one that provides the cLog P of 2 in the range of 4.22 to 4.67.

• 약학회지
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• 제47권6호
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• The Korean Journal of Physiology and Pharmacology
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• 제19권4호
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• pp.299-307
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• 2015

Severe acute pancreatitis (SAP) is normally related to multiorgan dysfunction and local complications. Studies have found that local pancreatic renin-angiotensin system (RAS) was significantly upregulated in drug-induced SAP. The present study aimed to investigate the effects of angiotensin II receptors inhibitor valsartan on dual role of RAS in SAP in a rat model and to elucidate the underlying mechanisms. 3.8% sodium taurocholate (1 ml/kg) was injected to the pancreatic capsule in order for pancreatitis induction. Rats in the sham group were injected with normal saline in identical locations. We also investigated the regulation of experimentally induced SAP on local RAS expression in the pancreas through determination of the activities of serum amylase, lipase and myeloperoxidase, histological and biochemical analysis, radioimmunoassay, fluorescence quantitative PCR and Western blot analysis. The results indicated that valsartan could effectively suppress the local RAS to protect against experimental acute pancreatitis through inhibition of microcirculation disturbances and inflammation. The results suggest that pancreatic RAS plays a critical role in the regulation of pancreatic functions and demonstrates application potential as AT1 receptor antagonists. Moreover, other RAS inhibitors could be a new therapeutic target in acute pancreatitis.