• Title/Summary/Keyword: SH3

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Molecular Cloning and NMR Characterization of the Nonreceptor Tyrosine Kinase PTK6 SH3-SH2-Linker Domain

  • Lee, Young-Min;Ahn, Kyo-Eun;Ko, Sung-Geon;Lee, Weon-Tae
    • Bulletin of the Korean Chemical Society
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    • v.30 no.5
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    • pp.1043-1046
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    • 2009
  • Human protein tyrosine kinase-6 (PTK6) is a member of the non-receptor protein tyrosine kinase family and it is found in two-thirds of all breast tumors. Very recently, we proposed that the SH3 domain of PTK6 interacts with the linker region (Linker) between the SH2 and kinase domains, proving that the interaction between SH3 domain and Linker plays an important role in auto-inhibition mechanism. Residues from 1 to 191 corresponding region of SH3-SH2-Linker (SH32L) of PTK6 was cloned into the pET32a expression vector with Tobbaco etch virus (TEV) protease enzyme site by sequence homology and 3D structural model. The purified PTK6-SH32L was determined as a monomer conformation in solution. The amide proton resonances in the $^{15}N-^{1}H$ 2D-HSQC spectrum suggest that PTK6-SH32L possesses disordered structural region of the flexible/unstructured linker region. In addition, the backbone amide proton chemical shifts of the SH3 domain in the PTK6-SH32L differ from that of the independent domain, indicating that intra-molecular interaction between SH3 and Linker in the PTK6-SH32L is present.

Expression, Purification and NMR studies of SH3YL1 SH3 domain

  • Shrestha, Pravesh;Yun, Ji-Hye;Lee, Weon-Tae
    • Journal of the Korean Magnetic Resonance Society
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    • v.14 no.2
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    • pp.105-116
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    • 2010
  • SH3YL1, a novel protein containing one Src homology 3 domain at the carboxyl terminus was first detected in mouse anagen skin cDNA. This protein had a significant homology with YHRO 16c/Ysc 84, the yeast Src homology 3 domain-containing protein. The sequence identity was remarkable at the carboxyl and amino-terminal Src homology 3 domain, suggesting that the novel protein is a mouse homolog of the yeast protein and thus was termed as SH3YL1. SH3YL1 is composed of two domains, a DUF500 at N-termini and a SH3 domain at C-termini. In our study we cloned the SH3 domain in bacterial expression system in Escherichia coli using pET32a vector with TEV protease cleavage site and purified as a monomer using affinity chromatography. The N-terminal poly-Histidine tag was cleaved with TEV protease and target protein was used for backbone studies. Our study showed that SH3 domain primarily consists of $\beta$-sheet which is in consistence with previous result performed on the truncated SH3 domain of SH3YL1.

Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells

  • Kim, Si Won;Lee, Jeong Hyo;Park, Tae Sub
    • Asian-Australasian Journal of Animal Sciences
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    • v.30 no.8
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    • pp.1183-1189
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    • 2017
  • Objective: Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods: Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2) gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7) cells during muscle differentiation. Results: Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO) QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion: We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

Purification and Spectroscopic Characterization of the Human Protein Tyrosine Kinase-6 SH3 Domain

  • Koo, Bon-Kyung;Kim, Min-Hyung;Lee, Seung-Taek;Lee, Weon-Tae
    • BMB Reports
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    • v.35 no.3
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    • pp.343-347
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    • 2002
  • The human protein tyrosine kinase-6 (PTK6) polypeptide that is deduced from the cDNA sequence contains a Src homology (SH) 3 domain, SH2 domain, and catalytic domain of tyrosine kinase. We initiated biochemical and NMR characterization of PTK6 SH3 domain in order to correlate the structural role of the PTK6 using circular dichroism and heteronuclear NMR techniques. The circular dichroism data suggested that the secondary structural elements of the SH3 domain are mainly composed of $\beta$-sheet conformations. It is most stable when the pH is neutral based on the pH titration data. In addition, a number of cross peaks at the low-field area of the proton chemical shift of the NMR spectra indicated that the PTK6 SH3 domain retains a unique and folded conformation at the neutral pH condition. For other pH conditions, the SH3 domain became unstable and aggregated during NMR measurements, indicating that the structural stability is very sensitive to pH environments. Both the NMR and circular dichroism data indicate that the PTK6 SH3 domain experiences a conformational instability, even in an aqueous solution.

Examination of Root Induction Ratio for Regeneration of Alfalfa by Medium Component (알팔파 재분화에서 배지조성에 따른 뿌리 유도율 조사)

  • 김기용;강경민;박근제;배은경;이인애;이병현;곽상수;조진기
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.23 no.2
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    • pp.95-100
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    • 2003
  • The alfalfa (Medicago sativa L.) callus was induced from seeds on SH medium contained $3\;mg/{\ell}$ of 2,4-D. Several regenerated alfalfa plants and many shoots were obtained by procedure of Kim et al. (1999); 1) incubation for 28~30 days on SH medium added $5\;mg/{\ell}$ of NAA and $2\;mg/{\ell}$ of Kinetin, 2) incubation for 3~5 days on SH medium added $11\;mg/{\ell}$ of 2,4-D and $1\;mg/{\ell}$ of kinetin. 3) incubation for 21~25 days on SH medium added $1.6\;g/{\ell}$ of ammonium sulfate and $5.75\;g/{\ell}$ of proline. To increase of root induction ratio on plant regeneration process of alfalfa, root induction ratio was examined on 8 kinds of medium, containing different amount of hormone and SH salt. Root induction ratio was higher on SH medium contained IBA than SH basal medium. In case 1.5 mg of IBA was added in SH medium, root induction ratio was the highest to 56.0% in this study. On the other hand, root induction ratio was higher on SH medium diminished SH salt amount to half volume and addition of IBA makes high root induction ratio, too. Thus, we conclude that the medium for root induction of alfalfa may be added $1.5\;mg/{\ell}$ of IBA and diminished SH salt amount to half volume.

Propolis Inhibits Neurite Outgrowth in Differentiating SH-SY5Y Human Neuroblastoma Cells

  • Kim, Han Bit;Yoo, Byung Sun
    • Toxicological Research
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    • v.32 no.3
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    • pp.239-243
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    • 2016
  • Propolis is a multicomponent, active, complex resinous substance collected by honeybees from a variety of plant sources. We have studied the effect of propolis on neurite outgrowth of SH-SY5Y human neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). Propolis, at a concentration of $3{\mu}g/mL$, had no significant effect on the viability of differentiating SH-SY5Y cells. However, the neurite outgrowth of the differentiating SH-SY5Y cells treated with propolis ($0.3{\sim}3{\mu}g/mL$) for 48 hr was significantly inhibited in a dose-dependent manner. Treatment of RA-stimulated differentiating SH-SY5Y cells with 0.3 to $3{\mu}g/mL$ propolis resulted in decreased level of transglutaminase and 43-kDa growth-associated protein (GAP-43) in a dose-dependent manner. The results indicate that propolis is able to inhibit neurite outgrowth of differentiating SH-SY5Y cells.

Application of Chitosan Preparations for Eco-friendly Control of Potato Late Blight (감자 역병의 친환경 방제를 위한 키토산 제형의 살포)

  • Chang, Taehyun;Kim, Byung Sup
    • Research in Plant Disease
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    • v.18 no.4
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    • pp.338-348
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    • 2012
  • Potato late blight caused by Phytophthora infestans Cooke is one of the major diseases in the cultivation of potatoes in Korea. Effect of chitosan preparations (SH-1 and SH-2) was evaluated on the inhibition of mycelial growth of P. infestans, and protective activity using detached potato leaf assay both in vivo and in the field test. SH-1 and SH-2 were showed protective activity of young plant with control values more than 95% potato late blight by inoculation with pathogens under growth chamber conditions. Mycelial growth was inhibited the radial growth over 74% at a concentration of $300{\mu}g/ml$ of both SH-1 and SH-2. Spraying with SH-1 and SH-2 on the leaves for detached leaf assay reduced disease development. The content of total polyphenol in stem was significantly increased by SH-1 and SH-2 application in the field. In field experiments, foliar application with both SH-1 and SH-2 were significantly reduced the development of late blight on potato plants. Control of late blight disease was obtained with control values of 72% and 53% by application of 1% SH-1 and SH-2, respectively, with 4 times at 7 days interval, and reduced with similar disease control values by application with 3 times at 14 days interval compared with untreated control. SH-1 and SH-2 applications increased the fresh weight of potato, and higher grade potatoes were also increased. The results showed that SH-1 and SH-2 applications can be used as eco-friendly natural fungicide for organic farming for the increase of yields and control of late blight.

Isolation and Identification of Linear Alkylbenzene Sulfonate Degrading Bacteria (Linear Alkylbenzene Sulfonate 분해세균의 분리 및 동정)

  • Lee, Ki-Moo;Choi, Woo-Young
    • Korean Journal of Agricultural Science
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    • v.21 no.1
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    • pp.60-66
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    • 1994
  • Among the various bacterial isolates from municipal sewages which utilized linear alkylbenzene sulfonate (LAS) as a sole source of carbon. 3 potent strains - KL-3, SH-2 and EN-1 - were selected. The strains were classified: KL-3 as a strain belong to the genus Klebsiella; SH-2 Shigella; and EN-1 Enterobacter, respectively. They were grown in a broth containing 200 ppm of LAS, using a laboratory scale fermentor: the bacterial growth reached stationary phase after 2 days with a maximum viability of $10^8cfu/mL$ of the culture; initial rates of LAS degradation were high during the first 24 hours of cultivation (KL-3 and SH-2, approx. 50%; EN-1, 20%); after 1 day a lag period of about 24 hours was observed for all the strains, and thereafter break-down proceeded rapidly; final rates after 7 days were approximately 85% by KL-3, 82% by SH-2 and 75% by EN-1. Adsorption of LAS by the bacterial cell mass was high for the strain SH-2, as Freundlich equation: Y= 0.030X + 0.95 was calculated.

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Coincidence analysis of keywords and MeSH terms in the Korean Journal of Emergency Medical Services (한국응급구조학회지 게재 논문의 중심 단어 분석(2005년-2011년))

  • Lee, Kyoung-Hee;Ham, Young-Lim
    • The Korean Journal of Emergency Medical Services
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    • v.16 no.2
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    • pp.43-51
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    • 2012
  • Purpose : We try to disclose how much the keywords of the papers from the Korean Journal of Emergency Medical Services with Medical Subject Headings(MeSH) terminologies and to understand the major subjects of the recent emergency medical technology research in Korea from keywords. Methods : We analyzed keywords from 524 articles of the Korean Journal of Emergency Medical Services that were published between 2005 and 2011. We investigated frequently used keywords and what percentages of keywords agree with MeSH terms using the MeSH browser. Results : There were on average 3.2 keywords per article. The most frequent key words were AED, Attitude, Cardiopulmonary Resuscitation, CPR, EMT, EMT students, External Defibrillator, Job satisfaction, Knowledge, 119 EMT in order. The number of terms in precise agreement with MeSH headings was 101(19.3%); 327 terms(62.4%) were not found in the MeSH browser and 96 terms(18.3%) partially matched MeSH terms. Conclusion : Many keywords used in the Korean Journal of Emergency Medical Services did not agree with MeSH terms. We conclude that contribution rules should be using MeSH terms and authors should be educated in the proper use of MeSH terms in their research and subsequent publication.

Root Initiation in Cut Birdsfoot trefoil Stems by Treatment of IBA (IBA 처리에 의한 버즈풋 트레포일 줄기 절단면에서 뿌리 분화)

  • 김기용;최기준;성병렬;임용우;임영철;장요순;김원호
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.21 no.1
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    • pp.35-38
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    • 2001
  • When root initiation ratio of cut Birdsfoot trefoil (Lotus corniculatus L.) stems was examined in several medium conditions containing different IBA concentration, the higher IBA contration of medium was elucidated superior then lower IBA concentration. Highest root initiation ratio was confirmed at 2.5 mg/$\ell$ of IBA and the ratio was 90~95%. When the stems from regenerated shoots from callus were treated at 8 kinds of medium for 12 days, the root iniation result was 9 (45%) at 1/2 SH-0 medium, 10 (50%) at SH-0 medium, 10 (50%) at SH-0.5IBA medium, 10 (50%) at SH-1.0IBA medium, 13 (65%) at SH-1.5IBA medium, 14 (70%) at SH-2.0IBA medium, 19 (95%) at SH-2.5IBA medium and 14 (70%) at SH-3.0IBA medium.

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