• Title/Summary/Keyword: EPO

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Production of Recombinant Human Hyperglycosylated Erythropoietin Using Cell Culture Technology by Improving Sialylation. (Sialic Acid 함량 증가 배양기술에 의한 재조합 인간 다당쇄 에리스로포이에틴의 생산)

  • 박세철;이승오;박만식;김승훈;김준환;송무영;이병규;고인영;강희일
    • Microbiology and Biotechnology Letters
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    • v.32 no.2
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    • pp.142-148
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    • 2004
  • Erythropoietin is a main regulator of human erythropoiesis. Recombinant human erythropoietin (rhEPO) is one of the glycoproteins produced in animal cells, and it has oligo saccharides chains which comprise about 40% of its molecular mass. Because the content of sialic acid can extend circulatory lifetime, the high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the sialylation of rhEPO produced by chinese hamster ovary cell culture was maximized by supplementing the culture medium with N-acetylm-annosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), a sialidase inhibitor. Feeding of 20 mM ManNAc/0.5 mM NeuAc2en into culture medium increased the sialic acid content by nearly tenfold compared with unsupplemented medium. This effect was achieved without affecting the cell growth or product yield. Six erythropoietin fractions differing in sialic acid content, ranging from 11∼15% of EPO, were identified from chinese hamster ovary cell-derived rhEPO by mono Q column chromatography. It was found that, at 20 mM ManNAc/0.5 mM NeuAc2en feeding, productivity of hyper-glycosylated EPO increased up to 50%, compared with the unsupplemented medium.

Production of Transgenic Bovine Embryos Following Nuclear Transfer of Bovine Fetal Fibroblasts Transfected by Foreign Genes (외래유전자를 도입한 소 태아세포의 핵치환에 의한 형질전환 소 수정란 생산)

  • Kil, K.S.;Uhm, S.J.;Kim, E.H.;Chung, H.J.;Kim, T.;Park, H.;Lee, H.T.;Chung, K.S.
    • Korean Journal of Animal Reproduction
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    • v.24 no.4
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    • pp.429-437
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    • 2000
  • This study investigated the successful introduction of genes of erythropoietin (EPO) and enhanced green fluorescent protein (EGFP) in bovine embryos following nuclear transfer of bovine fetal fibroblasts (bFF), which were transfected by retrovirus vector system. Non-starved bFF were, transferred into perivitelline space of enucleated oocytes. The bFF-oocyte units were accomplished by cell to cell fusion and activated with calcium inophore and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CRlaa medium for 8 days. Out of 187 (EPO) and 210 (EGFP) bovine eggs reconstructed by nuclear transfer, 149 (EPO : 80.0%) and 158 (EGFP : 75.2%) embryos were cleaved, and among them 36 (EPO : 24.2%) and 35 (EGFP : 22.2%) embryos developed to the blastocyst stage. Of these blastocysts, 100% integration of EPO gene in 36 embryos was determined by PCR, and 100% expression of EGFP gene in 35 embryos was observed under the fluorescent microscope. This result indicates that bovine oocytes reconstructed by nuclear transfer of transfected bFF can successfully develop to the blastocyst stage. Furthermore, this novel procedure may be presumably an attractive method efficiently to produce the transgenic cattles.

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Ginsenoside Rg1 Induces Apoptosis through Inhibition of the EpoR-Mediated JAK2/STAT5 Signalling Pathway in the TF-1/Epo Human Leukemia Cell Line

  • Li, Jing;Wei, Qiang;Zuo, Guo-Wei;Xia, Jing;You, Zhi-Mei;Li, Chun-Li;Chen, Di-Long
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.6
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    • pp.2453-2459
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    • 2014
  • Ginsenoside Rg1 is one effective anticancer and antioxidant constituent of total saponins of Panax ginseng (TSPG), which has been shown to have various pharmacological effects. Our previous study demonstrated that Rg1 had anti-tumor activity in K562 leukemia cells. The aim of this study was designed to investigate whether Rg1 could induce apoptosis in TF-1/Epo cells and further to explore the underlying molecular mechanisms. Here we found that Rg1 could inhibit TF-1/Epo cell proliferation and induce cell apoptosis in vitro in a concentration and time dependent manner. It also suppressed the expression of EpoR on the surface membrane and inhibited JAK2/STAT5 pathway activity. Rg1 induced up-regulation of Bax, cleaved caspase-3 and C-PAPR protein and down-regulation of Bcl-2 and AG490, a JAK2 specific inhibitor, could enhance the effects of Rg1. Our studies showed that EpoR-mediated JAK2/STAT5 signaling played a key role in Rg1-induced apoptosis in TF-1/Epo cells. These results may provide new insights of Rg1 protective roles in the prevention a nd treatment of leukemia.

Evening primrose oil and hemp seed oil as an ${\gamma}-linolenic$ acid source for broiler;Influence of fatty acid composition of chicken skin, thigh and breast muscle (브로일러에 대한 감마리놀렌산의 급원으로써 달맞이꽃종자유와 삼씨유;닭 껍질, 다리살 및 가슴살 지질의 지방산 조성에 미치는 영향)

  • Park, Byung-Sung;Kang, Hwan-Ku
    • Journal of the Korean Applied Science and Technology
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    • v.24 no.2
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    • pp.196-204
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    • 2007
  • The objective of this study was to determine the effect of dietary oils on the levels of the ${\gamma}-linolenic$ acid in chicken meat lipids. Three hundred ten five, 1-d old, male, Ross strain, broiler chicks were fed for 35 d to compare diets containing evening primrose oil(EPO) and hemp seed oil(HO) to a control diet. Fatty acid composition of lipid from chicken skin, thigh and breast muscle were determined at the end of the trial. The level of ${\gamma}-linolenic$ acid of lipids from chicken meat fed diets containing EPO or HO was significantly higher than that of the control group(p<0.05). The level of ${\gamma}-linolenic$ acid of lipids from chicken skin was highest in the group, which had been fed the EPO 0.85%, followed in order by EPO 0.7%, 0.5%, EPO mixed oil, HO and HO mixed oil. There was a significant difference in the level of ${\gamma}-linolenic$ acid of chicken skin between the control and treatment groups(p<0.05). The level of ${\gamma}-linolenic$ acid of lipids from chicken thigh muscle was also similar to skin, and significantly higher than that of the control group(p<0.05). The level of ${\gamma}-linolenic$ acid of lipids from chicken breast muscle was highest in the group, which had been fed the EPO 0.5%, followed in order by EPO 0.7%, 0.85%, HO 0.5% and HO mixed oil. There was a significant difference in the level of ${\gamma}-linolenic$ acid of chicken breast muscle between the control and treatment groups(p<0.05).

Effect of Subcutaneous Methoxy Polyethylene Glycol-epoetin Beta on Anemia and Nutritional Indices in Peritoneal Dialysis Patients (복막투석 환자에서 Methoxy Polyethylene Glycol-epoetin Beta 피하 투여가 빈혈과 영양지표에 미치는 효과)

  • Kim, Kyung Sook;Lee, Jun Seop;Kim, Hye-Young;Lee, Myung Koo
    • Korean Journal of Clinical Pharmacy
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    • v.22 no.3
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    • pp.202-210
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    • 2012
  • Methoxy polyethylene glycol-epoetin beta (MPG-EPO), a continuous erythropoietin receptor activator, is a new erythropoiesis-stimulating agent with a long half-life. The purpose of this prospective study is to assess the effects of once-monthly subcutaneous MPG-EPO on hematological responses and nutritional status in peritoneal dialysis patients. Forty four patients undergoing stable peritoneal dialysis were enrolled into the study. Darbepoetin alfa therapy, in peritoneal dialysis patients, was converted to the monthly administration of subcutaneous MPG-EPO for 6 months. The starting dose of MPG-EPO was based on the previous weekly dose of darbepoetin alfa. The dose adjustments were performed to maintain the hemoglobin (Hb) levels in a target range of 10.5-11.0 g/dL. If the Hb levels exceeded 11.0 g/dL, MPG-EPO was temporarily interrupted for 1 month. The mean Hb levels were stable with the values of $9.5{\pm}1.1$ g/dL at baseline, and $10.4{\pm}0.9$ g/dL at the 6th month after conversion. The mean differences in the changes of Hb levels between the baseline and the 6th month were $0.9{\pm}1.4$ g/dL, which was statistically significant. However, the mean differences of iron, transferrin saturation and ferritin concentrations were not significant. It did not show significant differences in the changes of the nutritional parameters. These results suggest that the once-monthly subcutaneous administration of MPG-EPO for 6 months effectively maintains the Hb levels and nutritional status in peritoneal dialysis patients. Taken together, the once-monthly subcutaneous administration of MPG-EPO was practical and might improve the clinical compliance for the management of renal anemia in peritoneal dialysis patients.

Effect of dietary gamma-linolenic acid on milk production in cow

  • Park, Chang-Seok;Kim, Sang-Bouym;Kang, Sung-Sik;Kwon, Eung-Gi;Park, Sung-Kwon
    • Korean Journal of Agricultural Science
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    • v.43 no.2
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    • pp.232-239
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    • 2016
  • Recently, research has been focusing on high quality and safer animal production by utilizing natural functional materials. The aim of this study was to evaluate the effect of administration of natural Evening Primrose Oil (EPO) on gamma linolenic acid (GLA) levels in milk from Holstein dairy cows. Quality and quantity of milk as well as blood and fatty acids from Holstein cow fed diets supplemented with 2.7-4% calcium-salted EPO coated with palm stearin oil were analyzed. There was no significant difference in yield and composition of milk between control and EPO treatment. However, EPO treatment lowered blood aspartate aminotransferase (AST), somatic cell count (SCC), and cholesterol levels (p < 0.05) compared to untreated control. Blood urea nitrogen (BUN) level was decreased (p < 0.05) in GLA 1 and GLA 2 group when compared with control group. Non-esterified fatty acids (NEFA) concentration was lower (p < 0.05) in GLA 1 and GLA 2 groups than in control group. The level of GLA in milk was increased in EPO group when compared to control. Therefore, results from the present study demonstrate that supplementary EPO has beneficial effects on cow health, showing a decrease in somatic cell count and levels of blood cholesterol, alanine aminotransferase (ALT), and AST. Furthermore, supplementation of EPO improves milk quality with increased amounts of GLA.

Biological Activity of Human Dimeric Hyperglycosylated Erythropoietin (dHGEPO) Fusion Proteins

  • Naidansuren, Purevjargal;Min, Kwan-Sik
    • Reproductive and Developmental Biology
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    • v.34 no.4
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    • pp.289-297
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    • 2010
  • Erythropoietin (EPO) is a glycoprotein hormone secreted from primarily cells of the peritubular capillary endothelium of the kidney, and is responsible for the regulation of red blood cell production. We constructed and expressed dimeric cDNAs in Chinease hamster ovary (CHO) cells encoding a fusion protein consisting of 2 complete human EPO domains linked by a 2-amino acid linker (Ile-Asp). We described the activity of dimeric hyperglycosylated EPO (dHGEPO) mutants containing additional oligosaccharide chains and characterized the function of glycosylation. No dimeric proteins with mutation at the $105^{th}$ amino acid were found in the cell medium. Growth and differentiation of the human EPO-dependent leukemiae cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of dHGEPO proteins. MIT assay at 24 h increased due to the survival of F36E cells. The dHGEPO protein migrated as a broad band with an average molecular mass of 75 kDa. The mutant, dHGEPO, was slightly higher than the wild-type (WT) dimeri-EPO band. Enzymatic N-deglycosylation resulted in the formation of a narrow band with a molecular mass twice of that of of monomeric EPO digested with an N-glycosylation enzyme. Hematocrit values were remarkably increased in all treatment groups. Pharmacokinetic analysis was also affected when 2.5 IU of dHGEPO were intravenously injected into the tails of the mice. The biological activity and half-life of dHGEPO mutants were enhanced as compared to the corresponding items associated the WT dimeric EPO. These results suggest that recombinant dHGEPO may be attractive biological and therapeutic targets.

Comparison of Dangguibohyel-tang and Erythropoietin on Cyclophosphamide-induced Anemia in Rats (당귀보혈탕(當歸補血湯)과 eryhropoietin이 cyclophosphamide로 유도된 흰쥐의 빈혈에 미치는 영향 비교 연구)

  • Kang Soon-Ah;Chang Mun-Seog;Oh Myung-Sook;Kim Do-Rim;Kim Ji-Sook;Park Seong-Kyu
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.20 no.1
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    • pp.31-36
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    • 2006
  • The aim of this study was to elucidate the mechanism of anemia associated with Dangguibohyel-tang (DBT) in rats. Using cyclophosphamide-induced (30mg/kg BW) anemic rats, changes in weight gain, the levels of red blood cell (RBC), hematocrit (Hct), platelet and hemoglobin (Hgb), serum vitamin B12, ALT(GPT) levels and erythropoietin (EPO) gene expression were monitored, and compared with DBT (1,000mg/kg BW, 14d)-treated and EPO (1,000IU/kg BW, 14d, s.c.)-treated rats. Food efficiency ratio (FER) were 31.6%, in normal group, 28.1% in cyclophosphamide-induced control group, 31.7% in DBT-treated group and 25.1%, in EPO-treated group after 14 days. The levels of red blood cell (RBC), hematocrit (Hct), platelet and hemoglobin (Hgb) of DBT-treated group were significantly higher than those of control. And DBT extract administered group showed dominant effects on the recovery of Hgb level. Serum vitamin B12 and ALT(GPT) levels were significantly increased at DBT-treated groups. EPO gene expression was decreased 91,9% in control group, 79.6% in DBT-treated group and 53.9% in EPO-treated group, respectively. These results suggest that administration of DBT could prevent human patient from chemotherapy derived anemia by improving hematological value and EPO status.

Expression of HSP70 Immunoreactivity in EPO Treated Rat Kidney (콩팥에서 Erythropoietin 투여로 인한 HSP70의 발현 변화)

  • Jung, Ju-Young;Kim, Jin
    • Applied Microscopy
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    • v.37 no.3
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    • pp.167-174
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    • 2007
  • Heat shock protein (HSP) 70 functions as a molecular chaperon and reduces stress-induced denaturation and aggregation of intracellular proteins. Erythropoietin (EPO) plays an important role during acute renal failure repair process by rapidly correcting anemia and enhancing renal tubular regeneration. The purpose of this study was to examine the effect of EPO treatment on renal HSP70 expression. Male Sprague-Dawley rats were injected rHUEPO. Kidney were preserved by in vivo perfusion with paraformaldehyde-lysine-periodate (PLP) and processed for immunohistochemistry and electron microscopy. In control kidney, HSP70 was expressed in the cortex, outer medulla and inner medulla. Especially, HSP immunoreactiviy was mainly founded in descending thin limb of outer medulla and inner medullary collecting duct. In EPO treated kidney, HSP70 expression markedly increased in the descending thin limb of outer medulla and newly detected in cortical collecting duct. Electron microscopy showed the presence of HSP immunoreactivity on the intracelluar vesicles and Golgi complex of descending thin limb and cortical collecting duct. These findings suggest that EPO treatment leads to new production of HSP70 in renal tubular cells, and induction of HSP70 by rHuEPO is causally related to protective function.

3'-Half of the Thrombopoietin cDNA Confers Higher Expression of Erythropoietin at the RNA Level but Not at the Protein Level

  • Kim, Tae-Won;Ji, Jin Woo;Chang, Ho Gun;Kim, Myoung Ok;Ryoo, Zae Young;Park, In Kook;Kim, Sun Jung
    • Molecules and Cells
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    • v.19 no.2
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    • pp.198-204
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    • 2005
  • Both erythropoietin (EPO) and the short-form thrombopoietin (TPO) were expressed at low levels whereas the long-form TPO was expressed at high levels in transgenic animals. To elucidate the role of carboxy-terminal half of the long-form TPO which is absent in the short-form, we generated recombinant TPO or EPO expression vectors which contain or lack the carboxy-terminal half of TPO and examined their expression in the HC11 and 293 cells. The long-form TPO was expressed higher than the short-form regardless of the cell types, transfection modes, and promoters. When 3'-half of the long-form TPO cDNA was placed downstream of the EPO cDNA to act as a 3'-untranslated region, expression of EPO was moderately increased at the RNA level, however, no remarkable increase was observed at the protein level. These results suggest that the low expression of EPO, as like as the short-form TPO, is due to absence of the 3'-half in the full-length TPO that confers stability both at the RNA and protein levels.