• Title/Summary/Keyword: $\alpha$-dCTP

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Purification and Characteristic Properties of DNA Polymerase $\alpha$ from Sea-Urchin, Hemicentrotus pulcherrismus (말똥 성게의 DNA Polymerase $\alpha$의 정제와 특성)

  • HA Mi-Suck;RYU Beung-Ho
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.20 no.2
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    • pp.136-145
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    • 1987
  • From the sea-urchin, Hemicentrotus pulcherrismus, we have purified by four column chromatographic steps for DNA polymerase $\alpha$ activity. The molecular weight of DNA polymerase u was determined to be around 137,000-138,000 by Sephadex G-200 gel filtration and SDS-polyacrylamide gel electrophoresis. The purified enzyme had the optimal activity at pH 7.4. This enzyme showed to be a function of the metal ion $K^+,\;Na^+$\;and\;Mg^{2+}$ employed as activators, the optimum $K^+$\;or\;Na^+ concentration were 20 mM or 25mM and the optimum $Mg^{2+}$ concentration was 10 mM. The enzyme activity was inhibited by N-ethyl-maleimide, aphidicolin, cytosine $\beta-D-arabinofuranoside$ 5'-triphoshate (ara CTP) and phosphonoacetic acid.

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WEYL@S THEOREMS FOR POSINORMAL OPERATORS

  • DUGGAL BHAGWATI PRASHAD;KUBRUSLY CARLOS
    • Journal of the Korean Mathematical Society
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    • v.42 no.3
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    • pp.529-541
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    • 2005
  • An operator T belonging to the algebra B(H) of bounded linear transformations on a Hilbert H into itself is said to be posinormal if there exists a positive operator $P{\in}B(H)$ such that $TT^*\;=\;T^*PT$. A posinormal operator T is said to be conditionally totally posinormal (resp., totally posinormal), shortened to $T{\in}CTP(resp.,\;T{\in}TP)$, if to each complex number, $\lambda$ there corresponds a positive operator $P_\lambda$ such that $|(T-{\lambda}I)^{\ast}|^{2}\;=\;|P_{\lambda}^{\frac{1}{2}}(T-{\lambda}I)|^{2}$ (resp., if there exists a positive operator P such that $|(T-{\lambda}I)^{\ast}|^{2}\;=\;|P^{\frac{1}{2}}(T-{\lambda}I)|^{2}\;for\;all\;\lambda)$. This paper proves Weyl's theorem type results for TP and CTP operators. If $A\;{\in}\;TP$, if $B^*\;{\in}\;CTP$ is isoloid and if $d_{AB}\;{\in}\;B(B(H))$ denotes either of the elementary operators $\delta_{AB}(X)\;=\;AX\;-\;XB\;and\;\Delta_{AB}(X)\;=\;AXB\;-\;X$, then it is proved that $d_{AB}$ satisfies Weyl's theorem and $d^{\ast}_{AB}\;satisfies\;\alpha-Weyl's$ theorem.

Functional Characterization of the ${\alpha}$- and ${\beta}$-Subunits of a Group II Chaperonin from Aeropyrum pernix K1

  • Lee, Jin-Woo;Kim, Se Won;Kim, Jeong-Hwan;Jeon, Sung-Jong;Kwon, Hyun-Ju;Kim, Byung-Woo;Nam, Soo-Wan
    • Journal of Microbiology and Biotechnology
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    • v.23 no.6
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    • pp.818-825
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    • 2013
  • We isolated and functionally characterized the ${\alpha}$- and ${\beta}$-subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d-ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at $80^{\circ}C$. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}C$ and $50^{\circ}C$, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

Cutaneous hydration effect of collagen hydrolysate containing collagen tripeptides (콜라겐 트리펩타이드를 고함량으로 함유하는 콜라겐 가수분해물의 피부 보습 효과)

  • Kim, Ae-Hyang;Kim, Yi-Soo;Piao, Zhe;Shin, Yong Chul;Ha, Min Woo
    • Korean Journal of Food Science and Technology
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    • v.50 no.4
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    • pp.420-429
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    • 2018
  • Skin ageing is associated with compromised performance of its fundamental barrier functions, with undesirable changes in appearance. Since this may introduce a detrimental impact on the quality of life, significant effort to discover effective ingredients against ageing is being invested. Recently, collagen hydrolysates containing tripeptides such as GlyPro-Hyp (GPH) have been developed with anticipation of improved effects compared to that of existing collagen hydrolysate-products. To evaluate the cutaneous hydration effect of collagen tripeptides (CTP), meaningful biomarkers in human dermal fibroblasts (HDF) and NC/Nga Tnd mice were analyzed in this study. Increased levels of ceramide kinase, hyaluronic acid, collagen 1A, and hyaluronan synthase-2 (HAS2), and decreased levels of hyaluronidase-1 (HYAL1) and CD44 in HDF cells were demonstrated. Furthermore, significant reduction of transepidermal water loss (TEWL), scratching behavior, HYAL1, $TNF-{\alpha}$ and IL-6 and increased water content and HAS2 were verified by in vivo tests. These results strongly suggest the potential of CTP as a skin hydration agent.

Molecular characterization of diapause-related genes in the bumblebee, Bombus ignitus (호박벌의 휴면 제어 관련 유전자 탐색 및 특성 분석)

  • Hwang, Jae-Sam;Park, In-Gyun;Yun, Eun-Young;Jung, Duck-Oung;Yoon, Hyung Joo;Lee, Sang Beom;Kim, Seong-Ryul;Lee, Joon-Ha;Kim, In-Woo;Kwon, Young Nam
    • Journal of Sericultural and Entomological Science
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    • v.50 no.2
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    • pp.189-193
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    • 2012
  • To find diapause-related genes, we were performed by differential hybridization with three types of [${\alpha}-^{32}P$]dCTP-labeled total cDNA probes synthesized from diapause-prepared, diapause-maintained and diapause-activated stage of Bombus ignitus queen. Nine individual cDNA clones were found to be differentially expressed in diapause-maintained and diapause-activated stage. Among these clones, BIDC9(BIDC ; Bombus ignitus differentially expressed clone) was analyzed through full-length sequencing and expression pattern analysis. This clone was specifically expressed in the thorax organ. The effect of Juvenile hormone analog(JHA) and $CO_2$ treatment was examined. JHA treatment induced the expression of BIDC9 cDNA clone abruptly after 4 day of treatment. $CO_2$ treatment induced also the clone after 2 day of treatment. BIDC9 cDNA was identified as Bombus ignitus diapause gene contained an open reading frame of 1376 bp encoding 255 amino acids.

The Prediction Method of the Small Strain Shear Modulus for Busan Clay Using CPT and DMT (CPT와 DMT를 이용한 부산점토의 최대전단탄성계수 추정방법에 관한 연구)

  • Hong, Sung-Jin;Yoon, Hyung-Ko;Lee, Jong-Sub;Lee, Woo-Jin
    • Journal of the Korean Geotechnical Society
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    • v.25 no.6
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    • pp.5-16
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    • 2009
  • The is study is to evaluate the small strain shear modulus ($G_{max}$) of Busan clay using in-situ penetration tests. A series of dilatometer tests (DMT) and piezocone penetration tests (CPTu) are performed at Busan newport and Noksan sites, and hybrid oedometer tests are also carried out on the specimens obtained from both sites. The $G_{max}$ is evaluated from the shear wave velocity ($V_s$) measured by the bender elements installed at the boundary of oedometer cell. By analyzing these data, the relationship of $G_{max}$ and state variables, such as confined stress and void ratio, is developed. The analysis of lab and in-situ test results reveals that the ratio of $G_{max}$ to $q_t$ is inversely proportional to the plasticity index while the ratio of $G_{max}$ to $E_D$ has a linear relationship with ($I/I_D$)$(p_a/{\sigma}'_v)^{0.5}$. Two correlations suggested in this study, based on CPT and DMT results, appear to provide reasonable predictions of the small strain shear modulus.

Biological Functions of N- and O-linked Oligosaccharides of Equine Chorionic Gonadotropin and Lutropin/Chorionicgonadotropin Receptor

  • Min, K. S.
    • Proceedings of the KSAR Conference
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    • 2000.10a
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    • pp.10-12
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    • 2000
  • Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$-subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$ -subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was. efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to consist of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t63I or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17-fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

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Biological Functions of N- and O-linked Oligosaccharides of Equine Chorionic Gonadotropin and Lutropin/Chorionic Gonadotropin Receptor

  • Min, K.S.
    • Korean Journal of Animal Reproduction
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    • v.24 no.4
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    • pp.357-364
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    • 2000
  • Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$ -subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$-subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t631 or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632~653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agoinst-occupied receptors ~2- and ~17- fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

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Rapid detection of Rifampicin- resistant M, tuberculosis by PCR-SSCP of rpoB gene (결핵균의 rpoB유전자 PCR-SSCP법에 의한 Rifampicin 내성의 신속 진단)

  • Shim, Tae Sun;Yoo, Chul-Gyu;Han, Sung Koo;Shim, Young-Soo;Kim, Young Whan
    • Tuberculosis and Respiratory Diseases
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    • v.43 no.6
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    • pp.842-851
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    • 1996
  • Background : Rifampicin(RFP) is a key component of the antituberculous shon-course chemotherapy and the RFP-resistance is a marker of multi-drug resistant(MDR) M. tuberculosis. rpoB gene encodes the ${\beta}$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. Recent reports show that rpoB gene mutations are the cause of RFP resistance of M. tuberculosis and the main mechanism of rpoB gene mutation is point mutation. And PCR-SSCP is a rapid and easy method for detecting point mutations. So we performed PCR-SSCP of rpoB gene of M. tuberculosis and compared the result with traditional RFP sensitivity test. Method : The 27 RFP sensitive M. tuberculosis culture isolates and 25 RFP resistant isolates were evaluated. The RFP sensitivity test was done at the Korean Tuberculosis istitute. The DNA was extracted by bead beater method and was amplified with primers TR-8 and TR-9 in a 20ul PCR reaction containing 0.1ul(luCi) [${\alpha}-^{32}P$] - dCTP. After amplification, SSCP was done using non-denaturaring polyacrylamide gel electrophoresis. Then direct sequencing was done in cases of different eletrophoretic mobility compared with that of H37Rv. In 19 cases, we compared PCR-SSCP results with patient's clinical course and the results of traditional RFP sensitivity test. Results : 1) All 27 RFP sensitive M. tuberculosis isolates showed the same electrophoretic mobility compared with that of H37Rv. And all 25 RFP resistant M. tuberculosis isolates showed different electrophoretic mobility. 2) The mechanism of rpoB gene mutation of M. tuberculosis is mainly point mutation. 3) The PCR-SSCP results correlate well with traditional RFP sensitivity and patient's clinical response to antituberculous treatment. Conclusion: The PCR-SSCP of rpoB gene is a very sensitive and rapid mehod in detecting RFP- resistant M. tuberculosis.

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