• Title/Summary/Keyword: recombinant alkaline phosphatase

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Cloning and Expression of D-amino Acid Oxidise from Trigonopsis variabilis for Cephalosporin C Biotransformation (Cephalosporin C의 생변환을 위한 Trigonopsis variabilis의 D-amino Acid Oxidase 유전자의 클로닝 및 발현)

  • 이진형;정태완
    • KSBB Journal
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    • v.10 no.3
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    • pp.264-270
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    • 1995
  • Trigonopsis variabilis is a strong producer of D-amino acid oxidase that can transform cephalosporin C(ceph C) to ${\alpha}$-keto-adipyl-7-aminocephalosporanic acid(AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated blunt-end PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficiency One of recombinant E. coli transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.

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A Case with Perinatal Hypophosphatasia Caused by the ALPL Mutations (ALPL 유전자의 돌연변이를 가진 양성 주산기 저인산증 1례)

  • Kim, Joonil;Kang, Eungu;Kim, Yoon-Myung;Lee, Beom Hee;Kim, Gu-Hwan;Yoo, Han-Wook
    • Journal of The Korean Society of Inherited Metabolic disease
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    • v.16 no.3
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    • pp.141-147
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    • 2016
  • Hypophosphatasia is caused by the mutations in ALPL, which encodes tissue-nonspecific alkaline phosphatase (TNSALP). It can be inherited either in an autosomal dominant or recessive manner. Clinically, hypophophosphatasia is characterized by skeletal findings similar to those in rickets or osteomalacia, but serum alkaline phosphatase levels are decreased in the affected patients. Hypophosphatasia can be classified into six clinical forms according to age at diagnosis and severity of symptoms: perinatal lethal, infantile, childhood, adult, odontohypophosphatasia, and perinatal benign. As being a very rare disease, only one case has been reported in Korean population. Here we describe a case with perinatal benign hypophosphatasia with recessive ALPL mutations. Bowing of lower legs was detected in prenatal period and low serum alkaline phosphatase level was noted after birth. During the follow-up evaluation for 4.5 years, bone mineralization and legs bowing were improved but the growth retardation was persistent. As the recombinant bone-targeted human TNSALP became available, the clinical improvement of the affected patients is expected including the case described here with this treatment. More efforts are needed to identify the cases affected by hypophosphatasia.

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Studies on the Safety of Recombinant Bovine Somatotropin in Dairy Cow : Effects of ${\gamma}$BST on Hematologic and Blood Chemical Values in Dairy Cow (${\gamma}$BST의 젖소에 대한 안전성 연구 II. 성장호르몬이 젖소의 혈액상 및 혈액화학치에 미치는 영향)

  • Lee Mun-Han;Jin Young-Wha;Lee Chang-Woo
    • Journal of Veterinary Clinics
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    • v.8 no.2
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    • pp.157-170
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    • 1991
  • Effects of recombinant bovine somatotropln(${\gamma}$BST) on hematologie and blood chemical values were investigated in twenty-five multiparous Holstein dairy cows. Recombinant BST was administered by two different routes ; intramusculary(12.5mg and 25mg/day) and subcutaneously(500mg and 750mg) in sustained-release vehicle every 2 weeks beginning 4 weeks postpartum and continuing for 7 months. Whole blood and serum samples were collected 0, 1, 2, 3, 5, and 7 months after beginning of treatments from control and ${\gamma}$BST-administered groups. Hematologic values including RBC, PCV, HB, MCH, MCHC, WBC and differential counts of treatment groups receiving ${\gamma}$BST were similiar to those of control group. Blood chemical values observed were total protein, albumin, A/G ratio, glucose, cholesterol, Ca, Pi, Ca/pi ratio, total bilirubin, creatinine, BUN, alkaline phosphatase, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase. There were no significant differences in blood chemical values of cows administered with ${\gamma}$BST from those of control. Although some blood chemical values were fluctuated at a certain observation period, they were remained within the normal physiological ranges. It is concluded from the observations of these experiments that the dose and dosage froms of ${\gamma}$BST employed in this work might not affect hematologic and blood chemical values in dairy cows under the normal sanitary condition and adequate nutritional balance.

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High-Level Expression of Recombinant Human Bone Morphogenetic Protein-4 in Chinese Hamster Ovary Cells

  • PARK JUNHO;YU SUNGRYUL;YOON JAESEUNG;BAEK KWANGHEE
    • Journal of Microbiology and Biotechnology
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    • v.15 no.6
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    • pp.1397-1401
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    • 2005
  • Bone morphogenetic protein-4 (BMP-4) is a signaling homodimeric molecule that acts as a morphogen to influence cell fate in a concentration-dependent manner. The limited supply of a pure preparation of BMP-4, due to very low level of their expression in vivo, makes it difficult not only to study the biological activities of BMPs, but also to use them as a clinical tool. For a large-scale production of BMP-4, human BMP-4 cDNA was expressed in Chinese hamster ovary (CHO) cells by a recently development vector system, which confers position-independent stable expression of the foreign genes. The CHO cell line expressing recombinant human BMP-4 (rhBMP-4) at the level of $7\;{\mu}g/ml$ could be obtained after stepwise selection with methotrexate. This level of expression is about 70 times higher than those previously reported. The partially processed form of BMP-4 as well as mature form could be detected, when the aliquots of culture media were analyzed by Western blot. The glycosylation pattern and biological activity of the rhBMP-4 were determined by glycosidase treatment and the induction rate of alkaline phosphatase in mouse osteoblastic cells.

Cloning of nif Gene Cluster from Klebsiella pneumoniae and Expression in Escherichia coli (Klebsiella pneumoniae의 nif Gene Cluster의 Cloning 및 Escherichia coli 에서의 발현)

  • 이재선;이성희;심웅섭
    • Korean Journal of Microbiology
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    • v.27 no.1
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    • pp.21-26
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    • 1989
  • The chromosomal DNA of Klebsiella penumoniae KNF1 was partially digested with HindIII. pBR322 was completely digested with same enzyme with additional alkaline phosphatase treatment. Both DNA samples were ligated and transformed into E. coli KO60. Single coloneis of the transformed cells were isolated on N0free agar media. These colonies were ampicillin-resistant and tetracycline-sensitive. When the plasmids in transformants were cured, nitrogen-fixing activities were lost. Therefore, these transformants harbored recombinant plasmid including nif genes of K. pneumoniae. Nitrogenase activity of transformant was higher than or the same as that of K. pneumoniae KNF1.

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Examination of Cytopathic Effect and Apoptosis in Listeria monocytogenes-Infected Hybridoma B-Lymphocyte (Ped-2E9) Line In Vitro

  • Bhunia, Arun-Kumar;Feng, Xiang
    • Journal of Microbiology and Biotechnology
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    • v.9 no.4
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    • pp.398-403
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    • 1999
  • In our previous studies, we reported that hybridoma B-lymphocytes can be used to determine the virulence of Listeria species in an in vitro cytotoxicity assay. Here, we examined the cytopathic effect, i.e., membrane damage and the nature of cell death induced by Listeria monocytogenes on murine hybridoma B-lymphocytes (Ped-2E9). Membrane damage was assessed by microscopic analyses and by measuring the release of intracellular alkaline phosphatase(AP) and lactate dehydrogenase (LDH). Cell death was determined by DNA fragmentation analyses using agarose gel electrophoresis. Infection by listeriolysin O (LLO)-producing L. monocytogenes strains induced substantial amounts of AP and LDH release from Ped-2E9 hybridoma B-cells, suggesting severe membrane damage in these cells, while an LLO-negative L. monocytogenes mutant strain had no effect. An LLO-producing recombinant L. innocua ($prifA^+hly^+$) strain also induced high AP and LDH release and cytopathic changes in Ped-2E9 cells. Light or scanning electron microscopic examination revealed L. monocytogenes mediated membrane destabilization, pore formation, intense cytoplasmic granulation, bleb formation, and lysis of Ped-2E9 cells. LLO-producing L. monocytogenes and L. innocua ($prifA^{+}hly{^}+$) also induced ladder-like DNA fragmentation in Ped-2E9 cells. Collectively, these results suggest that L. monocytogenes, specifically LLO-producing strains, can induce a severe cytopathic effect leading to apoptosis in hybridoma B-lymphocytes (Ped-2E9).

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Effect of BMP-7 on osteoblastic differentiation of rat periodontal ligament cells (백서 치주인대세포의 분화에 대한 Bone morphogenetic protein-7의 영향)

  • Lee, Ho-Jae;Kim, Young-Jun;Chung, Hyun-Ju
    • Journal of Periodontal and Implant Science
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    • v.35 no.3
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    • pp.747-760
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    • 2005
  • Periodontal therapy has dealt primarily with attempts at arresting progression of disease. however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. Recombinant human bone morphogenetic protein-7(rhBMP-7) can differentiate the osteoprogenitor cells and induce bone formation. The purpose of this study was to evaluate the effect of BMP-7 on rat periodontal ligament cells differentiation, in vitro. In the control group, cells was cultured with DMEM media. In the experimental groups, cells were cultured with rhBMP-7 in concentration of 10, 25, 50 and 100 ng/ml. Each group was characterized by examining alkaline phosphatase activity at 3 and 5 days of culture and the ability to produce mineralized nodules of rat calvarial cells at 14 days of culture. Synthesis of type I collagen(COL-I), osteocalcin(OCN), and bone sialoprotein(BSP) was evaluated by RT-PCR at 7 days of culture. Activation of Smad proteins and p38 MAP kinase was determined by western blot analysis of the cell lysates. Alkaline phosphatase activity was significantly increased in the concentration of BMP-7 50 ng/ml and 100 ng/ml compared to the control(p<0.05). The mineralized bone nodule formation was greater with addition of 50 ng/ml and 100 ng/ml BMP-7 than the control(p<0.01). In 7 days' culture, the expressions of COL-I, BSP, and OCN was increased by BMP-7 in concentration of 10 $ng/ml{\sim}100$ ng/ml. In western blot analysis, BMP-7 treated culture cells expressed Smad 1,5,8 in dose-dependent manner, whereas BMP-7 did not activate phosphorylated form of p38 MAP kinase. These result suggested that BMP-7 stimulate rat periodontal ligament cells to differentiate toward osteoblast phenotype and increase bone matrix production by activation of BMP-Smad pathway.

Combined Treatment with Low-Level Laser and rhBMP-2 Promotes Differentiation and Mineralization of Osteoblastic Cells under Hypoxic Stress

  • Heo, Jin-Ho;Choi, Jeong-Hun;Kim, In-Ryoung;Park, Bong-Soo;Kim, Yong-Deok
    • Tissue Engineering and Regenerative Medicine
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    • v.15 no.6
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    • pp.793-801
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    • 2018
  • BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha ($HIF-1{\alpha}$), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

Effects of Diets Supplemented with Recombinant Epidermal Growth Factor and Glutamine on Gastrointestinal Tract Development of Early-weaned Piglets

  • Lee, D.N.;Chang, W.F.;Yu, I.T.;Chiou, Peter W.S.;Weng, C.F.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.4
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    • pp.582-589
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    • 2008
  • This study attempted to determine effects of recombinant porcine epidermal growth factor (pEGF) and glutamine (Gln) supplement on the growth performance and intestinal development of piglets weaned at 14 days of age. A total of ninety-six piglets were allotted to one of four dietary treatments which comprised inclusion of 1.0 mg pEGF supernatant/kg diet or 0.5% Gln both alone and in combination. Each treatment consisted of four replicates with six pigs per pen for a 28 days experimental period. Two pigs per replicate were sacrificed and gastrointestinal tract samples were collected on day 14. Data showed that dietary treatment failed to promote growth performance. On day 14, diets supplemented with pEGF elevated pancreatic chymotrypsin, jejunal alkaline phosphatase, sucrase, lactase and maltase activities (p<0.05), but failed to alter the small intestinal villus morphology, DNA, or protein content of gastrointestinal mucosa. Diets supplemented with Gln increased pancreatic chymotrypsin activity, tended to enhance the protein contents of gastric (p = 0.08) and jejunal mucosa (p = 0.09) but did not influence the serum IgA level or the enzyme activity in the gastrointestinal tract. On day 28, the diets supplemented with Gln increasedt (p<0.05) serum IgA and the proliferation of peripheral blood mononuclear cells by PHA stimulation. However, a combination of pEGF and Gln did not have a synergistic effect on these biomarkers in early-weaned piglets. The results demonstrate that diets supplemented with recombinant pEGF supernatant indeed improve intestinal digestive enzyme activity and diets supplemented with Gln increases the immune response in early-weaned piglets.

A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

  • Lee, Yura;Bae, Kyoung Jun;Chon, Hae Jung;Kim, Seong Hwan;Kim, Soon Ae;Kim, Jiyeon
    • Molecules and Cells
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    • v.39 no.5
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    • pp.389-394
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    • 2016
  • Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders.