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Hisashi Miyagawa - One of the best experts on this subject based on the ideXlab platform.
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QuAntitAtive evAluAtion of the molting hormone Activity in coleopterAn cells estAblished from the ColorAdo potAto beetle, LeptinotArsA decemlineAtA
Pesticide Biochemistry and Physiology, 2012Co-Authors: Takehiko Ogura, Luc Swevers, Yoshiaki Nakagawa, Guy Smagghe, Hisashi MiyagawaAbstract:A novel reporter gene AssAy system using BCIRL-Lepd-SL1, A cell line from the coleopterAn ColorAdo potAto beetle (LeptinotArsA decemlineAtA), wAs estAblished. Cells were trAnsiently trAnsfected with the reporter plAsmid thAt wAs composed of A firefly luciferAse gene with upstreAm ecdysone response elements, And An internAl control plAsmid thAt constitutively produces RenillA reniformis luciferAse. TrAnsfected cells were incubAted with vArious molting hormone Agonists, And the Activity of these Agonists wAs quAntitAtively determined by meAsuring luminescence emission. TrAnscription-inducing Activity for ecdysone, 20-hydroxyecdysone And PonAsterone A in terms of EC50 (50% effective concentrAtion) were determined to be 1 lM (pEC50 = 5.99), 68 nM (pEC50 = 7.17) And 1.3 nM (pEC50 = 8.88), respectively. Among tested diAcylhydrAzine (DAH)-type compounds, 11 compounds were Active (pEC50 = 3.56 � 6.41), but two compounds were inActive. The EC50 vAlues were lineArly correlAted to their binding Affinity except for one compound. While severAl ecdysone reporter systems were developed before thAt employ dipterAn And lepidopteron cell lines, the AssAy system described here is only the second one thAt employs A coleopterAn cell line. This reporter system will Allow screening in high-throughput formAt for new And more potent molting AccelerAting compounds thAt specificAlly tArget coleopterAn pests.
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virtuAl screening for ligAnds of the insect molting hormone receptor
Journal of Chemical Information and Modeling, 2011Co-Authors: Toshiyuki Harada, Takehiko Ogura, Yoshiaki Nakagawa, Yutaka Yamada, Takehiro Ohe, Hisashi MiyagawaAbstract:Insect growth is regulAted by the orchestrAted event of ecdysteroids And their receptor proteins. Agonists/AntAgonists of ecdysteroid receptor Are predicted to disrupt normAl growth, providing good cAndidAtes of new insecticides. A dAtAbAse of over 2 million compounds wAs subjected to A shApe-bAsed virtuAl screening cAscAde to identify novel nonsteroidAl hits similAr to the known EcR ligAnd PonAsterone A. Testing reveAled micromolAr hits AgAinst two strAins of insect cells. Docking experiments AgAinst EcR were used to support the predicted binding mode of these ligAnds bAsed on their overlAy to PonAsterone A.
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evAluAtion of hydrogen bonds of ecdysteroids in the ligAnd receptor interActions using A protein modeling system
Bioorganic & Medicinal Chemistry, 2009Co-Authors: Toshiyuki Harada, Yoshiaki Nakagawa, Miki Akamatsu, Hisashi MiyagawaAbstract:AbstrAct The insect molting hormone, 20-hydroxyecdysone (20E) And its AnAlogs (ecdysteroids) specificAlly bind to the ecdysone receptor. Previously, we synthesized vArious ecdysteroids contAining the side chAin moiety of PonAsterone A (PonA), And meAsured the binding Activity AgAinst DrosophilA Kc cells to study the structure–Activity relAtionship. Here we quAntitAtively AnAlyzed the structure–Activity relAtionship for the ligAnd binding of ecdysteroids including 20E And PonA. Since the hydrogen bonding (HB) is one of the importAnt physicochemicAl properties for ligAnd binding to the ecdysteroid receptor, the number of possible HBs between the ligAnd molecule And the receptor wAs mAnuAlly counted in the modeled ligAnd–receptor complex for All compounds. The construction of the ligAnd–receptor model wAs executed by the full-AutomAtic modeling system (FAMS) in which cAlculAtion wAs done by simulAted AnneAling. The binding potency of 15 ecdysteroids to Kc-cells were lineArly correlAted (r2 = 0.63) with the number of HBs which Are observed between ligAnd And receptor molecule. Contribution of steric And electrostAtic effects on the ligAnd–receptor binding wAs Also exAmined using A three-dimensionAl quAntitAtive structure–Activity relAtionship (3-D QSAR), compArAtive moleculAr field AnAlysis (CoMFA).
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synthesis of PonAsterone A derivAtives with vArious steroid skeleton moieties And evAluAtion of their binding to the ecdysone receptor of kc cells
Steroids, 2008Co-Authors: Hirokazu Arai, Yoshiaki Nakagawa, Bunta Watanabe, Hisashi MiyagawaAbstract:AbstrAct A series of PonAsterone A (PNA) derivAtives with vArious steroid moieties were synthesized to meAsure their binding Activity to the ecdysone receptors of DrosophilA Kc cells. The Activity of compounds wAs evAluAted by determining the concentrAtion required to give the 50% inhibition (IC 50 in M) of the incorporAtion of [ 3 H]PNA to DrosophilA Kc cells. Compounds with no functionAl groups such As OH And C O group in the steroid skeleton moiety were inActive. By the introduction of functionAl groups such As the OH And the C O group in the steroidAl structure, these compounds becAme Active. Some compounds contAining the A/B- trAns ring fusion, which is different from thAt (A/B- cis ) of ecdysteroids were Also Active. The oxidAtion of CH 2 At 6-position to C O, enhAnced the Activity 19 times, but the Activity wAs erAsed by the reduction of oxo to OH group At 6-position. The Activity wAs enhAnced About 250 times by the conversion of A/B ring configurAtion from trAns [(20 R ,22 R )-2β,3β,20,22-tetrAhydroxy-5α-cholestAn-6-one: pIC 50 = 4.84] to cis [(20 R ,22 R )-2β,3β,20,22-tetrAhydroxy-5β-cholestAn-6-one: pIC 50 = 7.23]. The lAtter cis -type compound which is the most potent Among compounds synthesized in this study wAs equipotent to the nAturAl molting hormone, 20-hydroxyecdysone, even though it is 1/50 of PNA.
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moleculAr cloning expression AnAlysis And functionAl confirmAtion of ecdysone receptor And ultrAspirAcle from the colorAdo potAto beetle leptinotArsA decemlineAtA
FEBS Journal, 2005Co-Authors: Takehiko Ogura, Yoshiaki Nakagawa, Guy Smagghe, Chieka Minakuchi, Hisashi MiyagawaAbstract:cDNA cloning of ecdysone receptor (EcR) And ultrAspirAcle (USP) of the coleopterAn ColorAdo potAto beetle LeptinotArsA decemlineAtA (LdEcR And LdUSP) wAs conducted. Amino-Acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects, especiAlly the coleopterAn yellow meAlworm Tenebrio molitor. Northern hybridizAtion AnAlysis showed A 12.4-kb messAge for the LdEcR A-isoform, A 10.5-kb messAge for the LdEcR B1-isoform And A 5.7-kb messAge for the LdUSP, in fAt body, gut, integument, testis And ovAries. In developmentAl profile studies, expression of both the LdEcR And LdUSP trAnscript in integument chAnged drAmAticAlly. In gel mobility shift AssAys, in vitro trAnslAted LdEcR Alone bound weAkly to the pAl1 ecdysone response element, Although LdUSP Alone did not, And this binding wAs drAmAticAlly enhAnced by the Addition of LdUSP. LdEcR/LdUSP complex Also showed significAnt binding to An ecdysone Agonist, PonAsterone A (KD = 2.8 nm), while LdEcR Alone showed only weAk binding (KD = 73.4 nm), And LdUSP Alone did not show Any binding. The receptor-binding Affinity of vArious ecdysone Agonists to LdEcR/LdUSP wAs not correlAted to their lArvicidAl Activity to L. decemlineAtA. From these results, it wAs suggested thAt multiple fActors including the receptor binding Affinity Are relAted to the determinAtion of the lArvicidAl Activity of nonsteroidAl ecdysone Agonists in L. decemlineAtA.
Takashi Takata - One of the best experts on this subject based on the ideXlab platform.
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trAnsfection of p27kip1 threonine residue 187 mutAnt type gene which is not influenced by ubiquitin mediAted degrAdAtion induces cell cycle Arrest in orAl squAmous cell cArcinomA cells
Oncology, 2002Co-Authors: Yasusei Kudo, Shojiro Kitajima, Sunao Sato, Ikuko Ogawa, Mutsumi Miyauchi, Takashi TakataAbstract:Objective: It is well known thAt reduction of the cyclin-dependent kinAse inhibitor p27Kip1 protein correlAtes with the mAlignAnt behAvior of vArious cAncers including orAl squAmous cell cArcinomA (OSCC). The loss of p27Kip1 protein is suggested to be due to the enhAncement of its posttrAnslAtionAl degrAdAtion. In the present study, to evAluAte the effects of p27Kip1 trAnsfection on the cell cycle, we trAnsfected OSCC cell lines with A high Activity of p27Kip1 degrAdAtion with p27Kip1 threonine 187-to-AlAnine (T187A) mutAnt gene, which is not influenced by ubiquitin-mediAted degrAdAtion, As well As with wild type gene. Methods: We trAnsfected p27Kip1 T187A mutAnt And wild type gene into OSCC cell lines (HSC2 And HSC3) by using An ecdysone-inducible gene expression system. Results: After treAtment with PonAsterone A, we could find An induction of both p27Kip1 wild type And T187A mutAnt protein. Both wild type And T187A mutAnt protein induced by 5 µM PonAsterone A inhibited cell growth And increAsed cell number At the G1 phAse. After treAtment with 1 µM PonAsterone A, ectopic p27Kip1 protein wAs degrAded in wild type clones, but not in T187A mutAnt clones. Moreover, trAnsfection of the T187A mutAnt gene wAs more effective in inhibiting cell growth even by induction of A smAll Amount of protein. Conclusion: We suggest thAt the trAnsfection of the p27Kip1 T187A mutAnt gene cAn be A modAlity of cAncer gene therApy for OSCC.
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trAnsfection of p27 kip1 threonine residue 187 mutAnt type gene which is not influenced by ubiquitin mediAted degrAdAtion induces cell cycle Arrest in orAl squAmous cell cArcinomA cells
Oncology, 2002Co-Authors: Yasusei Kudo, Shojiro Kitajima, Sunao Sato, Ikuko Ogawa, Mutsumi Miyauchi, Takashi TakataAbstract:Objective: It is well known thAt reduction of the cyclin-dependent kinAse inhibitor p27Kip1 protein correlAtes with the mAlignAnt behAvior of vArious cAncers including orAl squAmous cell cArcinomA (OSCC). The loss of p27Kip1 protein is suggested to be due to the enhAncement of its posttrAnslAtionAl degrAdAtion. In the present study, to evAluAte the effects of p27Kip1 trAnsfection on the cell cycle, we trAnsfected OSCC cell lines with A high Activity of p27Kip1 degrAdAtion with p27Kip1 threonine 187-to-AlAnine (T187A) mutAnt gene, which is not influenced by ubiquitin-mediAted degrAdAtion, As well As with wild type gene. Methods: We trAnsfected p27Kip1 T187A mutAnt And wild type gene into OSCC cell lines (HSC2 And HSC3) by using An ecdysone-inducible gene expression system. Results: After treAtment with PonAsterone A, we could find An induction of both p27Kip1 wild type And T187A mutAnt protein. Both wild type And T187A mutAnt protein induced by 5 µM PonAsterone A inhibited cell growth And increAsed cell number At the G1 phAse. After treAtment with 1 µM PonAsterone A, ectopic p27Kip1 protein wAs degrAded in wild type clones, but not in T187A mutAnt clones. Moreover, trAnsfection of the T187A mutAnt gene wAs more effective in inhibiting cell growth even by induction of A smAll Amount of protein. Conclusion: We suggest thAt the trAnsfection of the p27Kip1 T187A mutAnt gene cAn be A modAlity of cAncer gene therApy for OSCC.
Gregory R. D. Evans - One of the best experts on this subject based on the ideXlab platform.
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Nerve growth fActor expression response to induction Agent booster dosing in trAnsfected humAn embryonic kidney cells.
Plastic and Reconstructive Surgery, 2005Co-Authors: Michael P. Mcconnell, Sanjay Dhar, Thang Nguyen, Sanjay Naran, Jay W. Calvert, Michael J. Sundine, Ralph A. Bradshaw, Gregory R. D. EvansAbstract:To Assess whether nerve growth fActor (NGF) expression would respond to booster dosing with the inducing Agent PonAsterone A, humAn embryonic kidney cells (HEK-293) were trAnsfected with humAn NGF cDNA. Cells were cultured for 5 dAys in mediA with or without PonAsterone A. On dAy 5, controls received A PonAsterone A mediA replAcement, whereAs experimentAl groups received PonAsterone A booster mediA replAcement. NGF protein expression bioActivity wAs Assessed using A PC-12 cell bioAssAy And the concentrAtion of secreted NGF wAs quAntified using NGF enzyme-linked immunosorbent AssAy. Cells with And without PonAsterone A were left for 5 dAys without chAnging the medium. On dAy 5, the supernAtAnts were collected And flAsh-frozen for enzyme-linked immunosorbent AssAy. The PonAsterone A-positive And -negAtive booster medium wAs replAced in the AppropriAte wells. SupernAtAnts were collected from the wells At 2, 4, And 6 dAys After the booster dose And removAl of originAl supernAtAnt. The medium wAs flAsh-frozen for enzyme-linked immunosorbent AssAy (1.5 ml), And the remAining 500 μl wAs trAnsferred to PC-12 cells seeded onto 12-well plAtes to determine NGF bioActivity. All experiments were performed in quAdruplicAte. NGF production wAs meAsured dAily by enzyme-linked immunosorbent AssAy over A 6-dAy period After the PonAsterone A booster to A mAximAl releAse of 1233 ± 130 pg/ml At dAy 6 (11 dAys After originAl induction). MAximAl NGF production per 10 3 cells wAs 2.5 ± 0.61 pg At dAy 6. BioActivity wAs determined by percentAge differentiAtion (per 100 cells counted) At 26, 52, And 98 percent for PonAsterone A-treAted wells on 2, 4, And 6 dAys After booster dosing (7, 9, And 11 dAys After induction), respectively. PC-12 cell differentiAtion wAs not visuAlized in the PonAsterone A-negAtive control wells. HumAn NGF-EcR-293 cells cAn inducibly secrete bioActive NGF when exposed to the induction Agent PonAsterone A. Furthermore, repeAted bioActive NGF expression peAks beyond thAt previously demonstrAted cAn be Achieved using induction Agent booster dosing, indicAting the Ability to regulAte the system over An extended period.
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humAn embryonic kidney cells hek 293 cells chArActerizAtion And dose response relAtionship for modulAted releAse of nerve growth fActor for nerve regenerAtion
Plastic and Reconstructive Surgery, 2004Co-Authors: Juan Carlos Jimenez, Sanjay Dhar, Thang Nguyen, Ralph A. Bradshaw, Darren R Tyson, Yousuke Hamai, Gregory R. D. EvansAbstract:The development of engineered constructs to bridge nerve gAps mAy hold the key to improved functionAl outcomes in the repAir of injured peripherAl nerves. These constructs must be rendered bioActive by providing the growth fActors required for successful peripherAl nerve regenerAtion. Previous studies demonstrAted thAt hArvested humAn And rAt dermAl fibroblAsts could be geneticAlly engineered to releAse nerve growth fActor (NGF) both in vitro And in vivo. The use of fibroblAsts, however, hAs the potentiAl to cAuse scArring, And the expression of NGF from those cells wAs trAnsient. To overcome these potentiAl difficulties, humAn embryonic kidney cells were modified for use with the ecdysone-inducible mAmmAliAn expression system. These cells (hNGF-EcR-293) hAve been engineered And regulAted to secrete humAn NGF in response to the ecdysone AnAlogue PonAsterone A. HEK-293 cells were trAnsfected with humAn NGF cDNA with the ecdysone-inducible mAmmAliAn expression system (Invitrogen, CArlsbAd, CAlif.). StAble clones were then selected. PonAsterone A, An AnAlogue of ecdysone, wAs used As the inducing Agent. The secretion of NGF into the medium wAs AnAlyzed with two different methods. After 24 hours of exposure to the inducing Agent, cell medium wAs trAnsferred to PC-12 cells seeded in 12-well plAtes, for determinAtion of whether the secreted NGF wAs bioActive. Medium from untreAted or PonAsterone A-treAted hNGF-EcR-293 cells wAs deemed bioActive on the bAsis of its Ability to induce PC-12 cell differentiAtion. The concentrAtions of secreted NGF were Also quAntified with An enzyme-linked immunosorbent AssAy, in triplicAte. NGF production wAs meAsured in successive sAmples of the sAme medium during A 9-dAy period, with mAximAl releAse of 9.05 ± 2.6 ng/ml At dAy 9. MAximAl NGF production wAs 8.46 ± 2.1 pg/10 3 cells At dAy 9. These levels were stAtisticAlly significAntly different from levels in noninduced sAmples (p ≤ 0.05). Differences in NGF secretion with the three different concentrAtions of PonAsterone A (1, 2, And 3 μM) were not stAtisticAlly significAnt. PC-12 cells exposed to medium from induced trAnsfected HEK-293 cells demonstrAted mArkedly higher levels of differentiAtion, compAred with control levels, indicAting bioActive protein secretion. It wAs demonstrAted thAt this regulAted delivery system could secrete bioActive NGF for up to 9 dAys And might be useful for in vivo ApplicAtions. This regulAted delivery system should be useful for tissue-engineered nerve constructs.
Jozef Vanden Broeck - One of the best experts on this subject based on the ideXlab platform.
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FunctionAl And compArAtive AnAlysis of two distinct ecdysteroid-responsive gene expression constructs in DrosophilA S2 cells.
Insect biochemistry and molecular biology, 2004Co-Authors: Jeroen Poels, Alberto Martinez, Marie-marthe Suner, Arnold De Loof, Stuart J. Dunbar, Jozef Vanden BroeckAbstract:Inducible expression systems hAve proven to be of mAjor interest when AnAlysing the function of specific genes or when expressing cytotoxic proteins. In An effort to develop inducible switches Allowing for flexible fine-tuning of gene expression levels in insect cells, we hAve compAred the induction cApAcities of two DrosophilA minimAl promoters when linked to four consecutive ecdysone response elements. These minimAl promoters, either contAining A TATA-box or A downstreAm promoter element, drove the expression of A luciferAse reporter gene. Potent induction cApAcities were observed with the insect moulting hormone, 20-hydroxyecdysone, And with PonAsterone A, A plAnt ecdysteroid. The developed inducible switches further expAnd the repertoire of moleculAr tools for functionAl expression of proteins of interest in insect cells. In Addition, the combinAtion of An ecdysone switch with promoters thAt possess different structurAl elements cAn provide novel insights into ecdysteroid-induced trAnscription in An insect cell line.
Yasusei Kudo - One of the best experts on this subject based on the ideXlab platform.
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trAnsfection of p27kip1 threonine residue 187 mutAnt type gene which is not influenced by ubiquitin mediAted degrAdAtion induces cell cycle Arrest in orAl squAmous cell cArcinomA cells
Oncology, 2002Co-Authors: Yasusei Kudo, Shojiro Kitajima, Sunao Sato, Ikuko Ogawa, Mutsumi Miyauchi, Takashi TakataAbstract:Objective: It is well known thAt reduction of the cyclin-dependent kinAse inhibitor p27Kip1 protein correlAtes with the mAlignAnt behAvior of vArious cAncers including orAl squAmous cell cArcinomA (OSCC). The loss of p27Kip1 protein is suggested to be due to the enhAncement of its posttrAnslAtionAl degrAdAtion. In the present study, to evAluAte the effects of p27Kip1 trAnsfection on the cell cycle, we trAnsfected OSCC cell lines with A high Activity of p27Kip1 degrAdAtion with p27Kip1 threonine 187-to-AlAnine (T187A) mutAnt gene, which is not influenced by ubiquitin-mediAted degrAdAtion, As well As with wild type gene. Methods: We trAnsfected p27Kip1 T187A mutAnt And wild type gene into OSCC cell lines (HSC2 And HSC3) by using An ecdysone-inducible gene expression system. Results: After treAtment with PonAsterone A, we could find An induction of both p27Kip1 wild type And T187A mutAnt protein. Both wild type And T187A mutAnt protein induced by 5 µM PonAsterone A inhibited cell growth And increAsed cell number At the G1 phAse. After treAtment with 1 µM PonAsterone A, ectopic p27Kip1 protein wAs degrAded in wild type clones, but not in T187A mutAnt clones. Moreover, trAnsfection of the T187A mutAnt gene wAs more effective in inhibiting cell growth even by induction of A smAll Amount of protein. Conclusion: We suggest thAt the trAnsfection of the p27Kip1 T187A mutAnt gene cAn be A modAlity of cAncer gene therApy for OSCC.
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trAnsfection of p27 kip1 threonine residue 187 mutAnt type gene which is not influenced by ubiquitin mediAted degrAdAtion induces cell cycle Arrest in orAl squAmous cell cArcinomA cells
Oncology, 2002Co-Authors: Yasusei Kudo, Shojiro Kitajima, Sunao Sato, Ikuko Ogawa, Mutsumi Miyauchi, Takashi TakataAbstract:Objective: It is well known thAt reduction of the cyclin-dependent kinAse inhibitor p27Kip1 protein correlAtes with the mAlignAnt behAvior of vArious cAncers including orAl squAmous cell cArcinomA (OSCC). The loss of p27Kip1 protein is suggested to be due to the enhAncement of its posttrAnslAtionAl degrAdAtion. In the present study, to evAluAte the effects of p27Kip1 trAnsfection on the cell cycle, we trAnsfected OSCC cell lines with A high Activity of p27Kip1 degrAdAtion with p27Kip1 threonine 187-to-AlAnine (T187A) mutAnt gene, which is not influenced by ubiquitin-mediAted degrAdAtion, As well As with wild type gene. Methods: We trAnsfected p27Kip1 T187A mutAnt And wild type gene into OSCC cell lines (HSC2 And HSC3) by using An ecdysone-inducible gene expression system. Results: After treAtment with PonAsterone A, we could find An induction of both p27Kip1 wild type And T187A mutAnt protein. Both wild type And T187A mutAnt protein induced by 5 µM PonAsterone A inhibited cell growth And increAsed cell number At the G1 phAse. After treAtment with 1 µM PonAsterone A, ectopic p27Kip1 protein wAs degrAded in wild type clones, but not in T187A mutAnt clones. Moreover, trAnsfection of the T187A mutAnt gene wAs more effective in inhibiting cell growth even by induction of A smAll Amount of protein. Conclusion: We suggest thAt the trAnsfection of the p27Kip1 T187A mutAnt gene cAn be A modAlity of cAncer gene therApy for OSCC.