• 한국미생물·생명공학회지
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• 제20권4호
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• pp.422-429
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• 1992

Maltosy-$\beta$-cyclodextrin는 $\beta$-cyclodextrin에 maltose가 $\Alpha$-1,6 glycosidic bond로 결합된 분지환상결합체로서 pullulanase의 역반응(축합반응)을 이용하여 $\beta$-cyclodextrin과 maltose로부터 합성된다. Maltosyl-$\beta$-cycloextrin의 합성수율을 증가시키기 위하여 각종 water activity depressor인 각종 polyol, sugar 그리고 polyethylene glycol(PEG)등의 첨가의 영향을 검토하였다. 가장 적절한 water activity depressor는 PEG 6000로서, 첨가량 10%(w/w)의 경우 maltosyl-$\beta$-cyclodextrin 생성량과 합성수율은 크게 증가하여 3.02g/100ml 와 55.9%(w/w)로서, 첨가하지 않았을 경우보다 약 1.3배 증가하였다. Water activity는 PEG 6000을 20%(w/w) 첨가할 때 원래의 0.966에서 0.914로 감소하였으며, maltosyl-$\beta$-cyclodextrin 합성수율은 water activity에 반비례하여 증가하였다. Pullulanase의 역반응을 이용한 maltosyl-$\beta$-cyclodextrin 합성반응의 각종 열역학적 상수를 평가하였으며, $\Delta$H는 36.788kJ/mol, $\Delta$S는 0.067kJ/moleK, 그리고 $\Delta$G는 14.433kJ/mole이였다. PEG 6000의 분리회수에 적절한 ultrafiltration membrane의 pore size는 3K dalton이었으며, 여과액과 농축액의 적정 분획비는 1.0 : 9.0였다. Maltosyl-$\beta$-cyclodextrin 합성수율의 증가는 첨가한 PEG가 water activity를 감소시켜 합성된 maltosyl-$\beta$-cyclodextrin이 재분해되는 pullulanase이 정반응인 hydrolysis reaction을 억제하여 equilbrium state에 변화를 주기 때문인 것으로 유추된다.

• 한국미생물·생명공학회지
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• 제38권2호
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• pp.158-162
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• 2010

고온균 Thermus thermophilus HJ6 유래의 N-말단 결실 Tod polymerase($\Delta$Tod polymerase)는 온도 감수성 프로모터 (lambda pR and pL)를 포함하는 pJLA503 벡터를 이용하여 대장균에서 발현하였다. N-말단 250개 아미노산이 제거된 $\Delta$Tod polymerase는 5'$\rightarrow$3' exonuclease 활성은 없어지고 DNA 중합반응의 활성은 그대로 유지되었다. $\Delta$Tod polymerase는 $MgCl_2$의 존재 하에서 매우 효율적으로 역전사 반응과 PCR 반응을 수행하였다. 또한 $\Delta$Tod polymerase는 one-step RT-PCR 반응에서 Taq polymerase 보다 높은 cDNA 증폭 효율을 나타내었다.

• Journal of Periodontal and Implant Science
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• 제35권2호
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• pp.345-357
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• 2005

Prostaglandin plays a significant role in the local control of bone metabolism associated with periodontal disease. ${\Delta}^{12}-PGJ_2$ is a natural $PGD_2$ metabolite that is formed in vivo in the presence of plasma. It is known for ${\Delta}^{12}-PGJ_2$ to stimulate calcification in osteoblastic cells. Bone morphogenetic protein(BMP) stimulated osteoblastic differentiation in various types of cells and greatly enhanced healing of bony defects. The purpose of this study was to evaluate the effect of rhEMP-2 on ${\Delta}^{12}-PGJ_2$ induced osteoblastic differentiation and mineralization in vitro. A human osteosarcoma cells line Saos-2 were cultured. In the test groups, 10-7M of ${\Delta}^{12}-PGJ_2$ or mixture of 10-8M of ${\Delta}^{12}-PGJ_2$ and 100ng/ml of rhBMP-2 or 100ng/ml of rhEMP-2 were added to culture media. After 1 day, 2 days and 4 days of culture period, the cell number was measured. Alkaline phosphatase activity was measure at 3 days. Reverse transcription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein at 8 hours, 1 day and 7 days. The ability to produce mineralized nodules in rat osteoblasts(MC3T3-E1) was evaluated at 21 days. The results were as follows : 1. rhEMP-2 or mixture of rhBMP-2 and ${\Delta}^{12}-PGJ_2$ inhibited cell proliferation of human osteosarcoma cells. 2. rhEMP-2 or mixture of rhBMP-2 and ${\Delta}^{12}-PGJ_2$ stimulated alkaline phosphatase activity significantly higher than ${\Delta}^{12}-PGJ_2$ alone. 3. rhBMP-2 or mixture of rhEMP-2 and ${\Delta}^{12}-PGJ_2$ stimulated mineralization compared to ${\Delta}^{12}-PGJ_2$ alone. 4. mRNA of alkaline phosphatase, BMP-2, cbfa 1, Type I collagen were detected in the group treated with ${\Delta}^{12}-PGJ_2$/rhBMP-2, rhBMP-2 alone, ${\Delta}^{12}-PGJ_2$ alone. These results show that mixture of ${\Delta}^{12}-PGJ_2$ and rhBMP-2 causes more bone formation than ${\Delta}^{12}-PGJ_2$ alone while the bone formation effects of mixture of ${\Delta}^{12}-PGJ_2$ and rhBMP-2 are less than those of rhBMP-2 alone. Further researches would be necessary to clarify the interactions of these agents.

• BMB Reports
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• 제40권2호
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• pp.218-225
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• 2007

In Arabidopsis thaliana, the microtubule-associated protein AtMAP65-1 shows various functions on microtubule dynamics and organizations. However, it is still an open question about whether AtMAP65-1 binds to tubulin dimers and how it regulates microtubule dynamics. In present study, the tubulin-binding activity of AtMAP65-1 was investigated. Pull-down and co-sedimentation exp eriments demonstrated that AtMAP65-1 bound to tubulin dimers,at a molar ratio of 1 : 1. Cross-linking experiments showed that AtMAP65-1 bound to tubulin dimers by interacting with $\alpha$-tubulin of the tubulin heterodimer. Interfering the bundling effect of AtMAP65-1 by addition of salt and monitoring the tubulin assembly, the experiment results indicated that AtMAP65-1 promoted tubulin assembly by interacting with tubulin dimers. In addition, five truncated versions of AtMAP65-1, namely AtMAP65-1 $\Delta$N339 (amino acids 340-587); AtMAP65-1 $\Delta$N494 (amino acids 495-587); AtMAP65-1 340-494 (amino acids 340-494); AtMAP65-1 $\Delta$C495 (amino acids 1-494) and AtMAP65-1 $\Delta$C340 (amino acids 1-339), were tested for their binding activities and roles in tubulin polymerization in vitro. Four (AtMAP65-1 $\Delta$N339, $\Delta$N494, AtMAP65-1 340-494 and $\Delta$C495) from the five truncated proteins were able to co-sediment with microtubules, and three (AtMAP65-1 $\Delta$N339, $\Delta$N494 and AtMAP65-1 340-494) of them could bind to tubulin dimers in vitro. Among the three truncated proteins, AtMAP65-1 $\Delta$N339 showed the greatest activity to promote tubulin polymerization, AtMAP65-1 $\Delta$N494 exhibited almost the same activity as the full length protein in promoting tubulin assembly, and AtMAP65-1 340-494 had minor activity to promote tubulin assembly. On the contrast, AtMAP65-1 $\Delta$C495, which bound to microtubules but not to tubulin dimers, did not affect tubulin assembly. Our study suggested that AtMAP65-1 might promote tubulin assembly by binding to tubulin dimers in vivo.

• 한국환경과학회지
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• 제2권4호
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• pp.255-270
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• 1993

녹두 유식물의 자엽에서 엽록소 및 단백질 함량과 $\delta$-aminolevulinate dehydratase(ALAD)활성의 변화에 미치는 polyamine의 영향을 조사하였다. Polyamine은 녹화과정에서 자엽내의 엽록소 생성을 촉진하였으며, 이 효과는 KCl에 의해서 상승되었다. 자엽내의 단백질 함량의 변화 또한 엽록소 함량의 변화와 유사하였다. ALAD활성은 암하에서보다 광선하에서 억제되었으나, 18시간 암처리후의 광조사는 ALAD활성을 증가시켰다. Putre-scine처리에 의한 ALAD활성은 암하에서 촉진효과가 낮았으나 광선하에는 그 활성이 증가되었다. KCl은 암하에서 ALAD활성을 촉진시켰으나 광선하에서는 그 효과가 감소되었다. 또한 polyamine과 KCl의 복합처리에서는 촉진효과가 없었다. 이와같은 결과에서 녹두자엽에서의 색소체발달은 polyamine과 광선에 의해 영향을 받으며, polya- mine은 색소체발달에 중요한 작용을 하는 것으로 사료된다.

• BMB Reports
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• 제30권5호
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• pp.370-377
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• 1997

A novel assay method is described for rapid quantitation of reaction rate of sterol ${\Delta}^7$-reductase (${\Delta}^7$-SR) which catalyzes reduction of the ${\Delta}^7$-double bond of sterols. Of six different organ tissues-liver, small intestine, brain, lung, kidney, and testis-. ${\Delta}^7$-SR activity was detected only in liver (2.30 nmol/min/mg protein) and testis (0.11 nmol/min/mg protein). Using a newly developed method which employs diet-induced enzyme proteins and ergosterol as substrate, we assessed both kinetics ($K_m=210\;{\mu}M$, $V_{max}=1.93\;nmol/min/mg$) and inhibition of the rat hepatic ${\Delta}^7$-SR against well-studied cholesterol lowering agents such as triparanol ($IC_{50}=16\;{\mu}M$). 3-$\beta$-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) ($IC_{50}=5.2\;{\mu}M$), and trans-1.4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) ($IC_{50}=0.25\;{\mu}M$). Of the three well-known AY-9944-sensitive cholesterogenic enzymes (i.e., ${\Delta}^7$-SR, sterol ${\Delta}^8$-isomerase, and sterol ${\Delta}^14$-reductase). ${\Delta}^7$-SR was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound ($K_i=0.109\;{\mu}M$). Substrate specificity studies of the microsomal ${\Delta}^7$-SR indicate that the relative reaction rate for 7-dehydrocholesterol and ergosterol are 5.6-fold and 1.6-fold higher than that for lathosterol. ${\Delta}^7$-SR activity was also modulated by feeding rats a diet supplemented with 0.5% ergosterol (>2.6-fold) in addition to 5.0% cholestyramine plus 0.1% lovastatin ($\simeq$5.0-fold). Finally, microsomal ${\Delta}^7$-SR was solubilized by 1.5% 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and enriched on PEG (0~10%) precipitation, which should be suitable for further purification of the enzyme.

• 대한본초학회지
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• 제28권2호
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• pp.39-44
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• 2013

Objectives : Gambigyeongsinhwan 16 (GGEx16), gambigyeongsinhwan 18 (GGEx18) and gambitongseong capsule are shown to be involved in the regulation of obesity. Therefore, the aim of this study was to compare the reporter activity of anti-obesity genes such as peroxisome proliferator-activated receptor ${\alpha}$ ($PPAR{\alpha}$) and $PPAR{\delta}$ by GGEx16, GGEx18 and gambitongseong capsule. Methods : After NMu2Li liver cells, C2C12 skeletal muscle cells and 3T3-L1 preadipocytes were treated with GGEx16 (1 ${\mu}g/ml$), GGEx18 (1 ${\mu}g/ml$) and different concentrations of gambitongseong capsule, the transactivation of $PPAR{\alpha}$ and $PPAR{\delta}$ was measured by a luciferase reporter gene assay. Results : $PPAR{\alpha}$ reporter gene activity in NMu2Li liver cells and 3T3-L1 preadipocytes was significantly increased by GGEx16, GGEx18 and gambitongseong capsule compared with control, whereas $PPAR{\alpha}$ reporter gene activity in C2C12 skeletal muscle cells was significantly increased by GGEx18 only compared with control. Similarly, $PPAR{\delta}$ reporter gene activity in 3T3-L1 preadipocytes was also significantly increased by GGEx18 compared with control. $PPAR{\delta}$ reporter gene activity in C2C12 skeletal muscle cells was significantly increased by GGEx16 and GGEx18 compared with control although $PPAR{\delta}$ reporter gene activity in NMu2Li liver cells was not changed by these three formulas. Conclusions : These results suggest that all three formulas have the ability to stimulate $PPAR{\alpha}$ and $PPAR{\delta}$ transactivation in animal cell lines with high metabolic rates. In particular, this effects were most prominent in GGEx18-treated cells. In addition, it is likely that GGEx18 may be used as an effective anti-obesity composition.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.33-40
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• 2010

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked $\alpha$- and $\beta$-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (${\Delta}1$, ${\Delta}2$, ${\Delta}3$, ${\Delta}4$, and ${\Delta}5$) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2~3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (${\Delta}l$, ${\Delta}4$, and ${\Delta}5$) were transcripted, but not translated into proteins. Rec-eCG A2 was secreted in much lower amounts than the wild type. Only the rec-eCG ${\Delta}3$ ($\beta$-subunit: $Gln^{94}-Ile^{95}-Lys^{96}{\rightarrow}Ala^{94}-Ala^{95}-Ala^{96}$) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered $eCG{\beta\alpha}$. However, the FSH-like activity of rec-$eCG{\beta\alpha\Delta}3$ was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94~96 amino acid sequences in eCG $\beta$-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

• 한국발생생물학회지:발생과생식
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• 제15권3호
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• pp.215-221
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• 2011

Endocytosis of the Notch ligand, DeltaD, by mind bomb1 is indispensable for activation of Notch in cell fate determination, proliferation, and differentiation during zebrafish neurogenesis. Loss of mind bomb1 activity as an E3 Ubiquitin ligase causes the accumulation of deltaD at the plasma membrane and results in the ectopic neurogenic phenotype by activation of Notch in early zebrafish embryogenesis. However, the regulatory mechanism of deltaD during neurogenesis is not identified yet. This study aims to analyze the pathway of mib1 and deltaD after endocytosis in vivo during zebrafish embryogenesis. Mind bomb1 and deltaD are co-localized into autophagosome and mutant form of mind bomb1 fails to cargo deltaD into autophagosomes. These findings suggest that mind bomb I mediates deltaD regulation by autophagy in an ubiquitin-dependent manner during zebrafish embryogenesis.

• Journal of Microbiology and Biotechnology
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• 제4권2호
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• pp.119-125
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• 1994

Steroid $\Delta^1$-dehydrogenase purified from hydrocortisone-induced cells of Arthrobacter simplex converted various 3-ketosteroids into their corresponding $\Delta^1$-dehydrogenated products. The transformation efficiencies depend upon the chemical structure of the steroids, especially length of the side chain at 17 position and hydroxyl groups at 11 and 17 positions. The Km values for androstenedione, the most favorable substrate examined, and hydrocortisone were 74 ${\mu}M$ and 294 ${\mu}M$, respectively. The optimum temperature and pH of the enzyme reaction were 35$^{\circ}C$ and pH 9, respectively, and the enzyme was relatively stable at the range from 20 to 35$^{\circ}C$ and from pH 5 to 10 after one hour of incubation. The enzyme activity was markedly inhibited in the presence of $Cu^{2+},\;Fe^{3+},\;Hg^{2+},\;Mo^{6+}$ ions, and somewhat inhibited by $Zn^{2+}$ and $Fe^{2+}$. $\alpha,\alpha'$-Dipyridyl that inhibits 9$\alpha$-hydroxylase and accumulates 1,4-androstadiene-3,17-dione from sterols revealed no inhibitory effect on this enzyme. EGTA showed inhibitory effect. $\beta$-Estradiol competitively inhibited the enzyme activity. Chemical modifications of the enzyme were attempted with several reagents. p-Hydroxymer-curibenzoate showed inhibition of the enzyme activity and protection of the substrate. This suggests that cysteine residue may be involved in the active site of the enzyme.