• Title/Summary/Keyword: in vivo and in vitro conversion

Search Result 49, Processing Time 0.026 seconds

Isolation and Characterization of Engineered Nucleoside Deoxyribosyltransferase with Enhanced Activity Toward 2'-Fluoro-2'-Deoxynucleoside

  • Yoo, Yeon-Jin;Choi, Kang-Hyun;Kim, Byoung-Kyun;Choi, Si-Sun;Kim, Eung-Soo
    • Journal of Microbiology and Biotechnology
    • /
    • v.32 no.8
    • /
    • pp.1041-1046
    • /
    • 2022
  • Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40℃) and 4.4-fold (at 50℃) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

The Effects of Ginseng Saponin Fraction on Growth and Siderophore Formation in Eseherichia coli K-12 (인삼사포닌 분획이 Escherichia coli K-12의 성장과 Siderophore 생성에 미치는 영향)

  • 조영동;이용범
    • Journal of Ginseng Research
    • /
    • v.7 no.2
    • /
    • pp.102-107
    • /
    • 1983
  • The effects of saponin, one of major components (Panax ginseng C.A. Meyer), on the growth of E. coli K-12 and the formation of siderphore was observed The following results were obtained. 1. When E. coli was grown on medium containing 1${\times}$10-5%-11${\times}$10-1% of the saponin, the rate of growth was stimulated at 10-1% of the saponin significantly compared to that of control. 2. When E. coli K-12 was grown on medium containing 1${\times}$10-1% of the saponin, the amount of siderphore was two times as much as the control. 3. The growth of E. coli was observed to be dependent on the concentration of siderophore when siderophore was added to medium. 4. The effect of saponin on the formation of siderophore in vitro was observed to reach maximum at 1${\times}$10-3% of the saponin. Such results suggest that the growth rate of E. coli K-12 could be enhanced by ginseng saponin fraction through stimulation of siderphore formation. We have described the fast growth of E. coli, K-12 and B. subtilis, rapid uptake of 14C-glucose, and high level of other metabolites such as lipids and proteins of E. coli, and B. subtilis in medium containing saponing fraction compared to that of microorganisms without saponin fraction.1∼3Such differences were claimed to be due to rapid uptake of 14C-glucose by widened periplasmic region throught unknown mechanism in the prescence of saponin fraction in medium3 and have raised a question whether there is another possible factor, siderophore4(Greek for iron bears), since microorganisms must secure a sufficient amount of iron for normal growth. These are known to be synthesized by the cells under iron-deficient condition and in most case, excreted into the medium5, where they can complex and solubilize any iron present there. It is generally believed that these complexes are then taken into the cells presumably by specific transport systems, thus providing iron for cell metabolism. Within the group of enteric bacteria, only three species (E. coli, S. typhimurium, and A. aerogense) have, so far, been studied in a ny detail. The main iron-binding compound produced by these species is enterochelin, and its role in iron transport is now well established. And biosynthesis of enterochelin from 2, 3- dihydroxybenzoate and serine in the prescence of magnesium ions and ATP was reported6. 2, 3-dihydroxybenzoate was also shown to involve isochorismate and 2, 3-dihydro-2, 3-dihydroxybenzoate as intermediate.7∼11 The present paper deals with the effect of ginseng saponin fraction on growth, the level of enterochelin formation in vivo and the conversion of 2, 3-dihydroxybenzoate and serine into entrochelin in vitro, and entrochelin obtained on the growth in relation to possible explanation of ginseng saponin fraction on the rapid growth of E. coli, K-12.

  • PDF

Effect of Heat-epimerized-catechin-mixture Rich in Gallocatechin-3-gallate on Skin Barrier Recovery (갈로카테킨-3-갈레이트가 풍부한 열전환 카테킨의 피부 장벽 회복에 대한 개선 효과)

  • Kim, Jeong-Kee;Shin, Hyun-Jung;Lee, Sang-Min;Jeon, Hee-Young;Lee, Sang-Jun;Lee, Byeong-Gon
    • Journal of the Society of Cosmetic Scientists of Korea
    • /
    • v.34 no.2
    • /
    • pp.93-99
    • /
    • 2008
  • Until now, (-)-epigallocatechin-3-gallate(EGCG) is known as the most powerful antioxidant among green tea catechins having many beneficial effects on human skin. Considering that the content of catechins is variable according to many conditions such as solvent, temperature and pressure, we prepared the heat-epimerized-EGCG-mixture (HE-EGCG-mix) containing high content of gallocatechin-3-gallate(GCG) by epimerization during autoclaving process and found out its optimal condition for maximizing conversion from EGCG to GCG. To investigate the effects of EGCG and HE-EGCG-mix on skin barrier function, we performed in vivo experiments with hairless mice. We found that HE-EGCG-mix has more potent stimulating activity than EGCG for the production of involucrin 7(INV7) and for recovery of barrier function in SKH-1 mice. Also, we found that GCG stimulates $PPAR-{\alpha}$ transactivation more effectively than EGCG in vitro by transient transfection assay for $PPAR-{\alpha}$ activation activity. These imply that HE-EGCG-mix consisting of high content of GCG should stimulate more efficiently recovery of skin barrier through PPAR-mediated-kerationocyte differentiation than EGCG. In conclusion, our study may provide a possibility that GCG, the C-2 epimer of EGCG, could be a potentially effective agent for development of new cosmetics or health foods for recovery of skin barrier.

Effects of Keratinase on Performance, Nutrient Utilization, Intestinal Morphology, Intestinal Ecology and Inflammatory Response of Weaned Piglets Fed Diets with Different Levels of Crude Protein

  • Wang, D.;Piao, X.S.;Zeng, Z.K.;Lu, T.;Zhang, Q.;Li, P.F.;Xue, L.F.;Kim, S.W.
    • Asian-Australasian Journal of Animal Sciences
    • /
    • v.24 no.12
    • /
    • pp.1718-1728
    • /
    • 2011
  • Two experiments were conducted to investigate the in vitro ability of keratinase to hydrolyze soybean glycinin and ${\beta}$-conglycinin and to evaluate the in vivo effects of keratinase when included in corn-soybean diets with different levels of crude protein and fed to nursery pigs. In experiment 1, a saturated keratinase solution (1 ml) was added to two blank controls of either glycinin or ${\beta}$-conglycinin resulting in the hydrolysis of 94.74% glycinin and 88.89% ${\beta}$-conglycinin. In experiment 2, 190 pigs (8.3${\pm}$0.63 kg BW) were allotted to one of four treatments in a 2${\times}$2 factorial arrangement on the basis of body weight, and sex was balanced among the pens. The effects of crude protein (19 vs. 22%) and keratinase (0 vs. 0.05%) were studied. Each treatment was applied to six pens with seven (two pens) or eight pigs per pen. Pigs were fed the experimental diets for 21 d. Weight gain and feed conversion ratio were improved (p<0.05) with keratinase supplementation while feed intake was reduced (p<0.05). Keratinase supplementation increased (p<0.05) the apparent total tract digestibility of dry matter, energy, crude protein and phosphorus. Keratinase supplementation also increased n-butyric acid in the cecum and colon, lactobacilli and total anaerobe counts in the colon as well as the ratio of villus height to crypt depth in the ileum. Additionally, fecal score, ammonia nitrogen and branch chain volatile fatty acids in the colon, E. coli and total aerobe counts in the colon, crypt depth in the jejunum and ileum as well as serum interleukin-1 and interleukin-6 concentrations were also decreased (p<0.05) by keratinase supplementation. A reduction in dietary crude protein decreased (p<0.05) colon ammonia nitrogen concentration and cecal propionic acid and branch chain volatile fatty acid concentrations. In addition, cecal E. coli counts, colon total anaerobe counts, ileal crypt depth, and serum interleukin-1 and interleukin-6 concentrations were also decreased (p<0.05) with the reduction of dietary crude protein. With the exception of fecal scores, there were no significant interactions between crude protein and keratinase. This study provides evidence that dietary keratinase supplementation improved nursery pig performance by improving intestinal morphology and ecology, thus improving nutrient digestibility and alleviating the inflammatory response.

Alterations and Co-Occurrence of C-MYC, N-MYC, and L-MYC Expression are Related to Clinical Outcomes in Various Cancers

  • Moonjung Lee;Jaekwon Seok;Subbroto Kumar Saha;Sungha Cho;Yeojin Jeong;Minchan Gil;Aram Kim;Ha Youn Shin;Hojae Bae;Jeong Tae Do;Young Bong Kim;Ssang-Goo Cho
    • International Journal of Stem Cells
    • /
    • v.16 no.2
    • /
    • pp.215-233
    • /
    • 2023
  • Background and Objectives: MYC, also known as an oncogenic reprogramming factor, is a multifunctional transcription factor that maintains induced pluripotent stem cells (iPSCs). Although MYC is frequently upregulated in various cancers and is correlated with a poor prognosis, MYC is downregulated and correlated with a good prognosis in lung adenocarcinoma. MYC and two other MYC family genes, MYCN and MYCL, have similar structures and could contribute to tumorigenic conversion both in vitro and in vivo. Methods and Results: We systematically investigated whether MYC family genes act as prognostic factors in various human cancers. We first evaluated alterations in the expression of MYC family genes in various cancers using the Oncomine and The Cancer Genome Atlas (TCGA) database and their mutation and copy number alterations using the TCGA database with cBioPortal. Then, we investigated the association between the expression of MYC family genes and the prognosis of cancer patients using various prognosis databases. Multivariate analysis also confirmed that co-expression of MYC/MYCL/MYCN was significantly associated with the prognosis of lung, gastric, liver, and breast cancers. Conclusions: Taken together, our results demonstrate that the MYC family can function not only as an oncogene but also as a tumor suppressor gene in various cancers, which could be used to develop a novel approach to cancer treatment.

Effect of Dietary Supplementation of Plum or Omija on Growth Performance, Blood Biochemical Profiles and Antioxidant Defense System in Broiler Chickens (육계 사료 내 매실 및 오미자 첨가가 성장, 혈액 생화학 성분 및 항산화 작용에 미치는 영향)

  • Ko, Young-Hyun;Moon, Yang-Soo;Sohn, Sea-Hwan;Jung, Chang-Yong;Jang, In-Surk
    • Korean Journal of Poultry Science
    • /
    • v.39 no.2
    • /
    • pp.121-131
    • /
    • 2012
  • To investigate the effect of dietary supplementation of freeze-dried plum (Prunus mume Siebold and Zucc., PMS) or omija meal (Schizandra chinensis Baill.; SCB) on growth performance, organ weights, blood biochemical profiles and antioxidant defense system, a total of 96, 3-day-old male broiler chickens were assigned to three dietary groups: (1) control diet, (2) control diet supplemented with PMS at 0.2%, (3) control diet supplemented with SCB at 0.2%. In vitro antioxidant activity, plum and omija extracts showed a significantly higher radical scavenging activity (RSA). In particular, omija extract showed much higher RSA than plum extract. Dietary addition of plum or omija did not affect body weight, feed intake, feed conversion and the relative weight of digestive organ in birds. Plasma triglyceride significantly (P<0.05) increased in birds fed the diet supplemented with omija compared with those fed control diet without affecting the other blood biochemical components. Furthermore, reduced form of glutathione (GSH) in the liver or muscle significantly (P<0.05) increased in birds fed the diet fortified with plum and omija. However, the specific activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) and MDA (malondealdehyde) in the intestine, liver and muscle were not altered by dietary antioxidant sources. In conclusion, dietary plum and omija resulted in a positive effect on some antioxidant indicators such as increased in vitro RAS in extracts and in vivo GSH level in the liver and muscle without affecting growth performance. Therefore, dietary addition of 0.2% of plum or omija could be applicable as potential antioxidant sources in broiler chick production.

Primer RNA Synthesis by E. coli RNA Polymerase on the SSB-coated 229-nt ssi Signal of Lactococcal Plasmid pGKV21 (Lactococcal plasmid pGKV21의 SSB-coated 229-nt ssi signal 상에서 E. coli RNA polymerase에 의한 시발체 RNA 합성)

  • Jeong, Jin-Yong;Kim, Eun-Sil;Kim, Sam-Woong;Kang, Ho-Young;Bahk, Jeong-Dong
    • Journal of Life Science
    • /
    • v.19 no.3
    • /
    • pp.305-310
    • /
    • 2009
  • Plasmid pGKV21 contains a 229-nucleotide (nt) single-strand DNA initiation (ssi) signal. Using asymmetric PCR, we prepared a small single-stranded (ss) DNA fragment of the ssi signal and, using the 229-nt ssDNA fragment, determined the requirements of RNA polymerase for priming and DNA-protein interaction. The ssi fragment prepared was able to generate primer RNAs with almost the same efficiency as the $M13{\Delta}lac182/229$ phage DNA. However, the cssi (complementary strand of the ssi signal) fragment could not synthesize primer RNAs. This result suggests that the 229-nt ssi signal functions in a strand specific manner. Gel retardation and DNase I footprinting demonstrated that the synthesized ssi fragment could interact with both E. coli RNA polymerase and SSB protein to synthesize primer RNA. In Escherichia coli [pWVAp], an addition of rifampicin resulted in an accumulation of ssDNA, indicating that the host-encoded RNA polymerase is involved in the conversion of ssDNA to double-stranded plasmid DNA.

Anti-Obesity Effects of Jeju Hallabong Tangor (Citrus kiyomi${\times}$ponkan) Peel Extracts in 3T3-L1 Adipocytes (제주산 한라봉 과피 추출물의 지방세포에서의 항비만 효과)

  • Lim, Heejin;Seo, Jieun;Chang, Yun-Hee;Han, Bok-Kyung;Jeong, Jung-Ky;Park, Su-Beom;Choi, Hyuk-Joon;Hwang, Jinah
    • Journal of the Korean Society of Food Science and Nutrition
    • /
    • v.43 no.11
    • /
    • pp.1688-1694
    • /
    • 2014
  • Jeju Hallabong Tangor (Citrus kiyomi${\times}$ponkan) is a Citrus species with a variety of physiological properties such as anti-oxidant, anti-inflammation, anti-cancer, and anti-obesity. We investigated the anti-obesity effects of Hallabong Tangor peel extracts before (HLB) and after (HLB-C) bioconversion with cytolase based on modulation of adipocyte differentiation and lipid metabolism in 3T3-L1 adipocytes. Treatment with cytolase decreased flavanone rutinoside forms (narirutin and hesperidin) and increased flavanone aglycone forms (naringenin and hesperetin). During adipocyte differentiation, 3T3-L1 cells were treated with 0.5 mg/mL of Sinetrol (a positive control), HLB or HLB-C. Adipocyte differentiation was inhibited in both citrus groups, but not in control and Sinetriol groups. HLB and HLB-C tended to reduce insulin-induced mRNA levels of CCAAT/enhancer-binding protein ${\alpha}$ ($C/EBP{\alpha}$) and sterol regulatory element-binding protein 1c (SREBP1c). Compared to the control and Sinetrol groups, HLB and HLB-C markedly suppressed insulin-induced protein expression of $C/EBP{\alpha}$ and peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$). The HLB and Sinetrol groups, but not HLB-C group, significantly increased adipolytic activity with higher release of free glycerol compared to the control group in differentiated 3T3-L1 adipocytes. These results suggest that bio-conversion of Hallabong Tangor peel extracts with cytolase increases aglycone flavonoids. Irrespective of bioconversion, both Hallabong Tangor peel extracts exert anti-obesity effects that may contribute to prevention of obesity through inhibition of adipocyte differentiation or induction of adipolytic activity.

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
    • /
    • 2016.05a
    • /
    • pp.19-20
    • /
    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

  • PDF