• Title/Summary/Keyword: Cell-free protein synthesis

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Transcriptome analysis revealed regulatory mechanisms of light and culture density on free-living sporangial filaments of Neopyropia yezoensis (Rhodophyta)

  • Bangxiang He;Zhenbin Zheng;Jianfeng Niu;Xiujun Xie;Guangce Wang
    • ALGAE
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    • v.38 no.4
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    • pp.283-294
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    • 2023
  • Previous research indicated that free-living sporangial filament keep hollow morph under high-culture density and form bipartite cells under low-culture density, while the following conchospore release was inhibited by high light. Here, we further explored the molecular bases of these affects caused by light and culture density using a transcriptome analysis. Many differentially expressed genes (DEGs) related to carbon dioxide concentration and fixation, photosynthesis, chlorophyll synthesis and nitrogen absorption were upregulated under high-light conditions compared with low-light conditions, indicating the molecular basis of rapid vegetative growth under the former. The stress response- and ion transport-related DEGs, as well as the gene encoding the vacuole formation-brefeldin A-inhibited guanine nucleotide exchange protein (BIG, py05721), were highly expressed under high-density conditions, indicating the molecular basis of the hollow morph of free-living sporangial filaments under high-culture density conditions. Additionally, the brefeldin A treatment indicated that the hollow morph was directly influenced by vacuole formation-related vesicle traffic. Others DEGs related to cell wall components, zinc-finger proteins, ASPO1527, cell cycle and cytoskeleton were highly expressed in the low density with low-light group, which might be related to the formation and release of conchospores. These results provide a deeper understanding of sporangial filaments in Neopyropia yezoensis and related species.

Expression of Bacillus macerans Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae.

  • Kim, Gyu-Yong;Kim, Myeong-Dong;Han, Nam-Su;Seo, Jin-Ho
    • 한국생물공학회:학술대회논문집
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    • 2000.11a
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    • pp.191-193
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    • 2000
  • A whole-cell biocatalyst was constructed by immobilizing an enzyme on the surface of the yeast Saccharomyces cerevisiae. The gene encoding Bacillus macerans cyclodextrin glucanotransferase(CGTase) was fused with the AGA2 gene encoding a small peptide disulfide-linked to the aga1, a cell wall protein of a-agglutinin. The plasmid was introduced S. cerevisiae and expressed in the medium consisting of 10g/L yeast extract, 20g/L peptone, and 20g/L galactose. The activity was detected with the formation of cyclodextrin(CD) from 10g/L soluble starch. Surface display of CGTase was also verified with the halo-test, flow cytometry, and immunofluorescence microscopy. The recombinant S. cerevisiae produced ${\alpha}-cyclodextrin$ more efficiently than the free CGTase by simultaneous fermentation and cyclization as yeast consumes glucose and maltose which are inhibitors for CD synthesis.

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Synthesis and Biological Evaluation of Novel IM3829 (4-(2-Cyclohexylethoxy)aniline) Derivatives as Potent Radiosensitizers

  • Ahn, Jiyeon;Nam, Ky-Youb;Lee, Sae-Lo-Oom;Ryu, Hwani;Choi, Hyun Kyung;Song, Jie-Young
    • Bulletin of the Korean Chemical Society
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    • v.35 no.12
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    • pp.3623-3626
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    • 2014
  • Nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates the expression of over 200 genes of antioxidant and phase II drug-metabolizing enzymes, and is highly expressed in non-small cell lung cancer (NSCLC). Nine derivatives of 4-(2-cyclohexylethoxy)aniline were designed. Our previous study demonstrated that IM3829 increases radiosensitivity of several lung cancer cells in vitro and in vivo. Here, biological effects of IM3829 derivatives (2a-2i) were evaluated. Compound 2g derivative effectively inhibits mRNA and protein expression of Nrf2 and HO-1. In addition, we observed over two fold enhancement in IR-induced cell death, from $2.90{\pm}0.22$ to $6.02{\pm}0.87$, in H1299 cancer cell-line. Among the nine derivatives, compound 2g derivative exhibited the highest enhancement of radiosensitizing effect via inhibition of Nrf2 activity.

Purification and Characterization of Antiviral Protein (AAP29) from the Leaves of Amaranthus mangostanus (참비름 (Amaranthus mangostanus)에서 항바이러스성 단백질 (AAP29)의 분리 및 특성)

  • Yi, Seung-In;Kim, Yeong-Tae;Hwang, Young-Soo;Cho, Kang-Jin
    • Applied Biological Chemistry
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    • v.38 no.6
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    • pp.528-533
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    • 1995
  • An antiviral protein (AAP29) with ribosome-inactivating activity was purified and characterized from the leaves of the Amaranthus mangostanus. Purification was accomplished through crude extraction, ammonium sulfate precipitation, S-Sepharose chromatography, gel filtration, CM-Sepharose chromatography and Blue sepharose chromatography. This protein was about 29.2 kDa and strongly basic with the PI value between 9.0 and 9.6, indicating that AAP29 is similar to Type 1 RIP. The AAP29 showed high thermostability without activity toss even after 20 min at $50^{\circ}C$. In cell free system using rabbit reticulocyte lysate, AAP29 inhibited protein synthesis with an $IC_{50}$, of 0.18 nM. This protein also reduced mosaic symptoms of cucumber mosaic virus (CMV) on tobacco leaves. The N-terminal amino acid sequences of the AAP29 are ADLTFTVTKDGTSQSYXTLXNXWRXW and shows no sequence similarity with any known RIPs.

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Dipeptides Inhibit Melanin Synthesis in Mel-Ab Cells through Down-Regulation of Tyrosinase

  • Lee, Hyun-E;Kim, Eun-Hyun;Choi, Hye-Ryung;Sohn, Uy-Dong;Yun, Hye-Young;Baek, Kwang-Jin;Kwon, Nyoun-Soo;Park, Kyoung-Chan;Kim, Dong-Seok
    • The Korean Journal of Physiology and Pharmacology
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    • v.16 no.4
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    • pp.287-291
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    • 2012
  • This study investigated the effects of proline-serine (PS) and valine-serine (VS) dipeptides on melanogenesis in Mel-Ab cells. Proline-serine and VS significantly inhibited melanin synthesis in a concentration-dependent manner, though neither dipeptide directly inhibited tyrosinase activity in a cell-free system. Both PS and VS down-regulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. In a follow-up study also described here, the effects of these dipeptides on melanogenesis-related signal transduction were quantified. Specifically, PS and VS induced ERK phosphorylation, though they had no effect on phosphorylation of the cAMP response element binding protein (CREB). These data suggest that PS and VS inhibit melanogenesis through ERK phosphorylation and subsequent down-regulation of MITF and tyrosinase. Properties of these dipeptides are compatible with application as skin-whitening agents.

Design, Synthesis and Preliminary Biological Evaluation of a Biotin-S-S-Phosphine Reagent

  • Kang, Dong W.;Kim, Eun J.
    • Bulletin of the Korean Chemical Society
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    • v.35 no.2
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    • pp.383-391
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    • 2014
  • Biotin-S-S-Phosphine was designed and synthesized as a potential tool for a proteomic study of O-GlcNAcmodified proteins. This reagent features a disulfide linker between a triarylphosphine moiety, which allows selective conjugation to azide-containing proteins, and a biotin moiety that can allow easy isolation through its strong affinity toward avidin-coated solid beads. The disulfide linkage within this reagent can allow the easy release of the bound molecules of interest, which is difficult to achieve when a biotin:avidin pair is used alone, by reducing the disulfide bond of the reagent with DTT. Preliminary in vitro biological assays with azidelabeled and unlabeled cell lysates and a pure protein Nup62 showed that the Biotin-S-S-Phosphine reagent is highly reactive toward the free thiol groups of proteins. When a molecular tool with a disulfide linker is applied to the enrichment of the molecules of interest from other species, it is important to block the free-thiols of the sample using exhaustive alkylation prior to the Staudinger ligation reactions to restore the bioorthogonal nature of this reaction.

Effect of Insulin on Differention of Chick Embryonic Neuroblasts Cultured in vitro (배양 계배 신경아세포의 분화에 미치는 insulin의 영향)

  • 이창호;최덕영;박혜경;곽규봉;김혜선;정진하;하두봉
    • The Korean Journal of Zoology
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    • v.34 no.2
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    • pp.209-216
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    • 1991
  • To examine the effed of Insulin on neuronal differentiation, telencephalic neuroblasts from chick embryonic brains were cultured in a serum-free medium. Indirect immunofluorescence microscopic studies revealed that the spedfic protein, MAP-2, was localized in both cell bodies and neurites of developing neuroblasts. Furthermore, treatinent of increasing concentration of Insulin promoted the MAP-2 synthesis as well as the neurite outgrowth activity. Thus, the enhancement of the morphological and biochemical parameters for neuronal differentiation appears to he closely correlated, and the neurotrophic effect of insulin may play a crucial role in neuronal process formation.

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Study of Inhibitory Effect of Melanogenesis and Antioxidant Activity of Agrimonia pilosa Ledeb (선학초 추출물의 멜라닌합성 억제 및 항산화효과)

  • Kim, Dae-Sung;Kim, Yeong-Mok;Woo, Won-Hong;Mun, Yeun-Ja
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.24 no.2
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    • pp.236-241
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    • 2010
  • The purpose of this study was to investigate the mechanism of ethanol extract of Agrimonia pilosa Ledeb. (EAP)-reduced melanogenesis and diphenyl-picryl-hydrazyl (DPPH) radical scavenging activity. Agrimonia pilosa Ledeb., a perennial herbaceous plant, has been used as an antihemorrhagic, anthelminntic, and antiinflammatory agents in Chinese herbal medicine. In the present study, we observed that melanin synthesis and tyrosinase activity of B16F10 cells were significantly decreased by EAP. However, EAP could not suppress tyrosinase activity in the cell-free system, whereas kojic acid directly inhibited tyrosinase activity. Furthermore, EAP decreased the protein expression of tyrosinase, tyrosinase-related prootein 1 (TRP-1), and tyrosinase-related prootein 2 (TRP-2). EAP scavenged DPPH radical up to 41% with 100 ${\mu}g/m{\ell}$ concentration. These results suggest that the hypopigmentary effect of EPA was due to regulation of tyrosinase protein.

THE STUDY ON THE EFFECTS OF THE INSULIN-LIKE GROWTH FACTOR-I ON THE BIOLOGICAL ACTIVITY OF THE HUMAN PERIODONTAL LIGAMENT CELLS (Insulin-like growth factor-I 이 치주인대세포의 생물학적 활성도에 미치는 영향에 대한 연구)

  • Kim, Seong-Jin;Lee, Jae-Mok;Suh, Jo-Young
    • Journal of Periodontal and Implant Science
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    • v.24 no.2
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    • pp.219-237
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    • 1994
  • The ultimate goal of clinical periodontal therapy is to achieve regeneration of a healthy connective tissue reattachment. Conventional therapy including scaling, root planing, gingival curettage, gingivectomy and flap procedures of various types results primarily in repair rather than regeneration of the periodontium. In order for periodontal regeneration to occur, progenitor periodontal ligament cells must migrate to the denuded root surface, attach to it, proliferate and mature into an organized and functional fibrous attachment apparatus. Polypeptide growth factors belong to a class of potent biologic mediators which regulate cell differentiation, proliferation, migration and metabolism. Insulin-like growth factor-I (IGF- I ) of these factors appear to have an important role in periodontal wound healing and bone formation. The purpose of this study is to evaluate the effects of IGF- I on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were obtained from periodontal tissue explants culture of the first premolar tooth extracted for the orthodontic treatment. Cells were cultured in Dulbecco's modified Eagle medium(DMEM) with 10% fetal bovine serum. Fourth to seventh passage cells were plated in 24 well tissue culture plates and medium changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of $[^3H]-thymidine$ into DNA, Protein synthesis was determined by measurement of $[^3H]-proline$ incorporation into collagenase-digestible protein(CDP) and noncollagenous protein(NCP) according to the method of Peterkofsky and Diegelmann (1971), And alkaline phosphatase activity was measured as one parameter of osteoblastic differentiation. The results were as follows : The DNA synthetic activity was increased in a dose-dependent manner with IGF- I except for 0.1ng/ml concentration of IGF- I At the concentration of 10, 100ng/ml, IGF- I significantly increased the DNA synthetic activity(P<0.05) The total protein, collagen and noncollagen synthesis was increased in a dose-dependent manner with IGF- I except for 0.1ng/ml concentration of IGF- I. At the concentration of 1, 10, 100ng/ml, IGF- I significantly increased the total protein, collagen and noncollagen synthesis activity(P<0.95, P<0.001). The % of collagen was not effected according to the concentration of IGF- I. The alkaline phosphatase activity was increased in a dose-, time-dependent manner with IGF- I (10, 100ng/ml). In conclusions, the present study shows that IGF- I has a potentiality to enhance the DNA synthesis of periodontal ligament cells with including the increase of the total protein and collagen synthetic activity. The use of IGF- I to mediate biological stimulation of periodontal ligament cells shows promise for future therapeutic applications.

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