• Journal of Microbiology and Biotechnology
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• 제30권11호
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• pp.1739-1749
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• 2020

Exopolysaccharide produced by the yeast Papiliotrema flavescens, isolated from wine grape berries of Champagne vineyard, was investigated for both chemical and functional characterization. SECMALLS and colorimetric assay analyses showed that the EPS is a high M_W heteropolymer (2.37 × 10⁶ g/mol) majorily consisting of mannose, glucose, xylose and glucuronic acid as monosaccharide constituents, with two substituents (sulphate and phosphate groups), and a minor protein moiety. Structural enchainment of these carbohydrates based on methylation, GC-MS and NMR analyses revealed a linear main backbone built up of α-(1 → 3)-D-mannopyranosyl residues on which are branched side chains consisting of a single β-D-glucopyranosyluronic acid residue and β-(1 → 2)-xylopyranoses (2-5 residues). Suggestion of some xylopyranose side chains containing a mannose residue at the nonreducing terminal end was also proposed. This is first report on EPSs from the grape P. flavescens yeast with such structural characteristics. Furthermore, investigations for valuating the application performance of these EPS in relation with their structural features were carried out in 8% alcohol experiment solutions. Very exceptional viscosifying and foaming properties were reported by comparison with commercial biopolymers such as Arabic, gellan and xanthan gums. The intrinsic properties of the natural biopolymer from this wild grape-associated P. flavescens yeast make it a potential candidate for use in various biotechnology applications.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.475-479
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• 2003

미더덕으로부터 GAGs는 1/60 M sodium phosphate buffer로 $105^{\circ}C$로 열수 추출하는 것이 가장 경제적인 것으로 나타났다. 이렇게 추출한 GAGs의 $SO_4$ 함량은 31.2%, 회분 함량은 22.2%이다 회분 함량의 증가는 sodium phosphate의 사용으로 추정되며, 이것은 무기질 분석에서 Na의 함량이 총 무기질의 47.6%를 차지하고 있다는 것으로 뒷받침한다. 일반 성분, HPLC 분석, 당 분석, 아미노산 분석의 결과로 GAGs의 주된 구조는 glucosamine과 galacturonic acid로 결합되어 있으며, 당과 단백질은 threonine으로 연결되어 있다는 것을 알 수 있다. 화장품 원료 규격에 적합한 제단백은 5.0%, 10.0%, 20.0% TCA(w/v) 처리, 10.0%, 20.0%, 40.0% HCl(v/v) 처리, UF(ultra filteration)를 포함한 10.0% TCA(w/v), 20.0% S-SAS(w/v), 25.0% HCl(v/v) 처리가 가능하며, 이중 5.0% TCA(w/v) 및 10.0% HCl(v/v)의 처리가 가장 경제적이며 효율적이라는 것을 알 수 있었다.

• KSBB Journal
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• 제18권3호
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• pp.180-185
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• 2003

GAGs의 추출 방법 중 효소의 사용으로 추출 효율의 상승과 원료수급의 문제점을 해결할 수 있었다. 그러나 효소 사용에 따른 생산단가의 상승과 단백질 함량의 증가에 따른 화장품 규격을 만족시킬 수 없음으로, 1/60 M sodium phosphate buffer로 105$^{\circ}C$로 열수 추출하는 것이 화장품 규격을 만족시키는 방법으로 나타났다. 이렇게 추출한 GAGs의 SO$_4$ 함량은 31.2%이고 회분의 함량이 22.2%로 GAGs 추출 향상을 위해 사용되어지는 sodium phosphate의 사용으로 회분 함량이 높아졌음을 알 수 있고 무기질 분석에서 Na의 함량이 3.0 mg%로 총 무기질의 47.6%를 차지하고 있었다. 시료를 HPLC로 분석하였을 때 chondroitin sulfate A, C와 동일한 분석시간을 나타내어 chondroitin sulfate의 함유 가능성을 나타내고, glucosamine과 galactose가 78.0% 존재하는 것을 당조성 분석을 통해 확인하였다. 일반 성분, HPLC분석, 당 조성분석과 아미노산 분석결과로 glucosamine과 galacturonic acid가 주가 되어 threonine으로 연결된 구조를 하는 GAGs라는 것을 알 수 있다. 화장품 원료 기준에 맞추기 위해서는 sulfate 함량이 35.0~45.0%, 단백질 함량은 14.0~22.0%로 유지해야하므로 제단백결과 5.0%, 10.0%, 20.0% TCA (w/v) 처리, 10.0%, 20.0%, 40.0% HCI (v/v) 처리, UF(ultra filtration)를 포함한 10.0% TCA (w/v), 20.0% SSA (w/v), 25.0% HCI (v/v) 처리 결과 5.0% TCA (w/v) 및 10.0% HCI (v/v)이 화장품 기준에 적합하며 경제적이며 효율적이라는 것을 알 수 있었다.

• The Plant Pathology Journal
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• 제37권6호
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• pp.555-565
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• 2021

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.372-379
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• 2017

The transport of lysosomal enzymes into the lysosomes depends on the phosphorylation of their chains and the binding of the phosphorylated residues to mannose-6-phosphate receptors. The efficiency of separation depends more on the phosphodiesterases (PDEs) than on the activity of the phosphorylation of mannose residues and can be determined in vitro. PDEs play important roles in regulation of the activation of lysosomes. The expression of proteins was confirmed by western blotting. All PDE4 series protein expression was reduced in high concentrations of rolipram. As a result of observing the fluorescence intensity after rolipram treatment, the lysosomal enzyme was activated at low concentrations and suppressed at high concentrations. High concentrations of rolipram recovered the original function. Antimicrobial activity was not shown in either 10 or $100{\mu}M$ concentrations of rolipram in treated HeLa cells in vitro. However, the higher anticancer activity at lower rolipram concentration was shown in lysosomal enzyme treated with $10{\mu}M$ of rolipram. The anticancer activity was confirmed through cathepsin B and D assay. Tranfection allowed examination of the relationship between PDE4 and lysosomal activity in more detail. Protein expression was confirmed to be reduced. Fluorescence intensity showed decreased activity of lysosomes and ROS in cells transfected with the antisense sequences of PDE4 A, B, C, and D. PDE4A showed anticancer activity, whereas lysosome from cells transfected with the antisense sequences of PDE4 B, C, and D had decreased anticancer activity. These results showed the PDE4 A, B, C, and D are conjunctly related with lysosomal activity.

• Journal of mucopolysaccharidosis and rare diseases
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• 제2권1호
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• pp.13-16
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• 2016

Mucolipidosis (ML) II/III are autosomal recessive diseases caused by deficiency of post-translational modification of lysosomal enzymes. The mannose-6-phosphate (M6P) residue in lysosomal enzymes synthesized by N-acetylglucosamine 1-phosphotransferase (GlcNAc-phosphotransferase) serves as recognition marker for trafficking in lysosomes. GlcNAc-phosphotransferase is encoded by GNPTAB and GNPTG. Mutations in GNPTAB cause severe ML II alpha/beta and the attenuated ML III alpha/beta. Whereas mutations in GNPTG cause the ML III gamma, the attenuated type of ML III variant. For the diagnostic approaches, increased urinary oligosaccharides excretion could be a screening test in clinically suspicious patients. To confirm the diagnosis, instead of measuring the activity of GlcNAc phosphotransferase, measuring the enzymatic activities of different lysosomal hydrolases are useful for diagnosis. The activities of several lysosomal hydrolases are decreased in fibroblasts but increased in serum of the patients. In addition, the sequence analysis of causative gene is warranted. Therefore, the confirmatory diagnosis requires a combination of clinical evaluation, biochemical and molecular genetic testing. ML II/III show complex disease manifestations with lysosomal storage as the prime cellular defect that initiates consequential organic dysfunctions. As there are no specific therapy for ML to date, understanding the molecular pathogenesis can contribute to develop new therapeutic approaches ultimately.

• 한국산학기술학회논문지
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• 제10권7호
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• pp.1766-1772
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• 2009

본 연구에서는 한국인 아동의 우식치아로부터 S. mutans를 분리하고, acid stress하에서 분리한 S. mutans의 내산성 능력과 관련된 단백질을 규명하고자 하였다. 2D gel electrophoresis를 수행한 결과, acid stress동안 elongation factor Ts, hypothetical protein, putative amino acid ABC transporter, adenylate kinase, fructokinase, Putative 40K cell well protein precursor, peptide deformylase, shikimate 5-dehydrogenase, mannose-6-phosphate isomerase, threonine synthase, putative dTDP-glucose-4,6-dehydratase의 발현량이 뚜렷이 증가하였으며 이들 단백질은 acid stress에 관여하는 단백질들로 추정된다.

• Journal of Animal Science and Technology
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• 제50권5호
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• pp.649-656
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• 2008

본 연구는 insulin-like growth factors(IGFs)가 돼지 지방전구세포의 증식과 분화에 미치는 작용기전을 구명하기 위해서 수행하였다. 지방전구세포는 갓난 암퇘지의 등지방에서 분리하였고, serum-deprived 조건하에서 IGFs와 mutant IGFs를 함유시켜 배양했는데 이 mutant IGFs는 IGF-Ⅰ에 비해 type-1 IGF receptor와 insulin receptor에 대한 친화력이 낮다. 50ng/ml의 IGF-Ⅰ, [Leu60]IGF-I, IGF-Ⅱ 및 [Leu27]IGF-Ⅱ를 배양중인 세포에 4일동안 처리했다. IGF-Ⅰ, [Leu60]IGF-I, IGF-Ⅱ 및 [Leu27]IGF-Ⅱ는 돼지 지방전구세포의 증식을 각각 39%, 8%, 25% 및 2% 촉진했다(증가된 세포수에 의해 측정). 이 사실은 IGF-Ⅰ과 IGF-Ⅱ는 type-1 IGF receptor 또는 insulin receptor에 결합을 통해서 지방세포의 증식 촉진을 가져왔음을 나타낸다. 그리고 IGF-Ⅰ, [Leu60]IGF-I, IGF-Ⅱ 및 [Leu27] IGF-Ⅱ는 지방전구세포의 분화를 50%, 17%, 37% 및 30% 각각 촉진시켰다(세포 분화는 glycerol- phosphate dehydrogenase 활성도에 의해 측정했다). IGF-Ⅰ의 type-1 IGF receptor 또는 insulin receptor에의 친화력이 낮아져서 세포 분화 촉진작용을 감소시킨 것이다. 그러나 [Leu27] IGF-Ⅱ의 분화촉진 작용은 IGF-Ⅱ의 그것에 비해 크게 차이가 나지 않았는데, 이 사실은 IGF-Ⅰ과 IGF-Ⅱ는 서로 다른 수용체－매개 작용기전에 의해 세포분화를 촉진시킴을 나타낸다. 즉 IGF-Ⅱ는 type-1 IGF receptor 또는 insulin receptor가 관여하지 않는 작용을 통해 돼지 지방전구세포의 분화를 촉진시켰다. 이 작용은 IGF-Ⅱ가 type-2 IGF receptor(또는 cation- independent mannose-6 phosphate receptor [CIM6P /IGF2 receptor])에 결합을 통해서 이뤄지는 것으로 여겨진다. 위의 결과는 IGF-Ⅱ가 CIM6P/ IGF2 receptor에의 결합을 통해 동물 지방전구세포의 분화를 촉진시킨다는 것을 밝혀낸 최초의 연구이다. 요약하면 이 본 연구는 IGF-Ⅰ과 IGF-Ⅱ는 서로 다른 세포내 receptor가 관여하는 작용기전을 통해 돼지 지방전구세포의 분화를 촉진함을 보여준다.

• Journal of mucopolysaccharidosis and rare diseases
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• 제2권1호
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• pp.5-7
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• 2016

Mucolipidoses II and III alpha/beta (ML II and ML III) are lysosomal disorders in which the essential mannose-6-phosphate recognition marker is not synthesized onto lysosomal hydrolases and other glycoproteins. The disorders are caused by mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase ML II, recognizable at birth, often causes intrauterine growth impairment and sometimes the prenatal "Pacman" dysplasia. The main postnatal manifestations of ML II include gradual coarsening of neonatally evident craniofacial features, early cessation of statural growth and neuromotor development, dysostosis multiplex and major morbidity by hardening of soft connective tissue about the joints and in the cardiac valves. Fatal outcome occurs often before or in early childhood. ML III with clinical onset rarely detectable before three years of age, progresses slowly with gradual coarsening of the facial features, growth deficiency, dysostosis multiplex, restriction of movement in all joints before or from adolescence, painful gait impairment by prominent hip disease. Cognitive handicap remains minor or absent even in the adult, often wheelchair-bound patient with variable though significantly reduced life expectancy. As yet, there is no cure for individuals affected by these diseases. So, clinical manifestations and conservative treatment is important. This review aimed to highlight the extra-skeletal clinical problems in ML II and III.

• 약학회지
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• 제53권3호
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• pp.145-150
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• 2009

We evaluated cytotoxic effect based on the MTT assay and identified altered proteins in 5-FU(fluorouracil) treated HT29 cells using two-dimensional gel electrophoresis and MALDI-TOF/TOF-MS. As proteins inducing apoptosis, siah binding protein 1 and p47 protein isoform a were up-regulated and tumor protein translationally-controlled 1 was down-regulated by 5-FU treatment. And mannose 6 phosphate receptor binding protein 1 controls DNA mismatch repair system was increased. We suggest 5-FU promotes a cytotoxicity under the action of these proteins in colon cancer cells.