Publications by authors named "René Lafont"

47 Publications

20-Hydroxyecdysone activates the protective arm of the RAAS via the MAS receptor.

J Mol Endocrinol 2021 12 23;68(2):77-87. Epub 2021 Dec 23.

Biophytis, Sorbonne Université - BC9, Paris, France.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) were used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin(1-7), the endogenous ligand of MAS. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using siRNA or pharmacological inhibitors. 17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin(1-7) antagonists. A mechanism involving cooperation between the MAS receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the MAS receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between the effects of angiotensin(1-7) and 20E. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.
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http://dx.doi.org/10.1530/JME-21-0033DOI Listing
December 2021

A comparison between virus- versus patients-centred therapeutic attempts to reduce COVID-19 mortality.

Emerg Microbes Infect 2021 Dec;10(1):2256-2263

Biophytis, Sorbonne Université, Paris, France.

Since December 2019, coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has changed our lives. Elderly and those with comorbidities represent the vast majority of patients hospitalized with severe COVID-19 symptoms, including acute respiratory disease syndrome and cardiac dysfunction. Despite a huge effort of the scientific community, improved treatment modalities limiting the severity and mortality of hospitalized COVID-19 patients are still required. Here, we compare the effectiveness of virus- and patients-centred strategies to reduce COVID-19 mortality. We also discuss the therapeutic options that might further reduce death rates associated with the disease in the future. Unexpectedly, extensive review of the literature suggests that SARS-CoV-2 viral load seems to be associated neither with the severity of symptoms nor with mortality of hospitalized patients with COVID-19. This may explain why, so far, virus-centred strategies using antivirals aiming to inhibit the viral replicative machinery have failed to reduce COVID-19 mortality in patients with respiratory failure. By contrast, anti-inflammatory treatments without antiviral capacities but centred on patients, such as dexamethasone or Tocilizumab, reduce COVID-19 mortality. Finally, since the spike protein of SARS-CoV-2 binds to angiotensin converting enzyme 2 and inhibits its function, we explore the different treatment options focussing on rebalancing the renin-angiotensin system. This new therapeutic strategy could hopefully further reduce the severity of respiratory failure and limit COVID-19 mortality in elderly patients.
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http://dx.doi.org/10.1080/22221751.2021.2006579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8648031PMC
December 2021

A2E-induced inflammation and angiogenesis in RPE cells are modulated by PPAR-α, -β/δ, -γ, and RXR antagonists and by norbixin.

Aging (Albany NY) 2021 09 20;13(18):22040-22058. Epub 2021 Sep 20.

Biophytis, Sorbonne Université, Paris 75005, France.

-retinylidene--retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing angiogenesis and inflammation. A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates also peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR). 9'--norbixin, a di-apocarotenoid is also a ligand of these nuclear receptors (NR). Norbixin inhibits PPAR and RXR transactivation induced by A2E. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and AP-1 transactivation and mRNA expression of the inflammatory interleukins (IL) -6 and -8 and of vascular endothelial growth factor (VEGF) enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression in response to A2E. Selective PPAR-α, -β/δ and -γ antagonists inhibit the expression of IL-6 and IL-8 while only the antagonist of PPAR-γ inhibits the transactivation of NF-κB following A2E exposure. In addition, a cocktail of all three PPARs antagonists and also HX531, an antagonist of RXR reproduce norbixin effects on inflammation. Altogether, A2E's deleterious biological effects could be inhibited through PPAR and RXR regulation. Moreover, the modulation of these NR by norbixin may open new avenues for the treatment of AMD.
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http://dx.doi.org/10.18632/aging.203558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507260PMC
September 2021

20-Hydroxyecdysone, from Plant Extracts to Clinical Use: Therapeutic Potential for the Treatment of Neuromuscular, Cardio-Metabolic and Respiratory Diseases.

Biomedicines 2021 Apr 29;9(5). Epub 2021 Apr 29.

Biophytis, Sorbonne Université, BC9, 4 place Jussieu, 75005 Paris, France.

There is growing interest in the pharmaceutical and medical applications of 20-hydroxyecdysone (20E), a polyhydroxylated steroid which naturally occurs in low but very significant amounts in invertebrates, where it has hormonal roles, and in certain plant species, where it is believed to contribute to the deterrence of invertebrate predators. Studies in vivo and in vitro have revealed beneficial effects in mammals: anabolic, hypolipidemic, anti-diabetic, anti-inflammatory, hepatoprotective, etc. The possible mode of action in mammals has been determined recently, with the main mechanism involving the activation of the Mas1 receptor, a key component of the renin-angiotensin system, which would explain many of the pleiotropic effects observed in the different animal models. Processes have been developed to produce large amounts of pharmaceutical grade 20E, and regulatory preclinical studies have assessed its lack of toxicity. The effects of 20E have been evaluated in early stage clinical trials in healthy volunteers and in patients for the treatment of neuromuscular, cardio-metabolic or respiratory diseases. The prospects and limitations of developing 20E as a drug are discussed, including the requirement for a better evaluation of its safety and pharmacological profile and for developing a production process compliant with pharmaceutical standards.
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http://dx.doi.org/10.3390/biomedicines9050492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146789PMC
April 2021

The complex metabolism of poststerone in male rats.

J Steroid Biochem Mol Biol 2021 09 20;212:105897. Epub 2021 Apr 20.

Biophytis SA, Sorbonne Université, 4 Place Jussieu, F-75005 Paris, France; Sorbonne Université, UPMC (Université Paris 06), IBPS - BIOSIPE, F-75005 Paris, France. Electronic address:

Ecdysteroids are not endogenous to mammals, but are normal components of the food intake of many mammalian species consuming phytoecdysteroid-containing plants. The most frequently encountered phytoecdysteroid is 20-hydroxyecdysone (20E). Several pharmaceutical effects have been observed after ecdysteroid injection or ingestion, but it is not clear to what extent metabolites generated in the mammalian body contribute to these effects. The C21-ecdysteroid poststerone (Post) is a metabolite of 20E in rodents. Post analogues are key intermediates in the metabolism of exogenous ecdysteroids possessing a C20/22-diol. The pharmacokinetics, bioavailability and metabolism of Post have been assessed in male rats after ingestion and injection. The bioavailability of Post is significantly greater than that of 20E and the presence of an efficient entero-hepatic cycle allows Post to be effectively metabolised to a wide range of metabolites which are excreted mainly in the faeces, but also to some extent in the urine. Several of the major metabolites in the bile have been identified unambiguously as 3-epi-poststerone, 16α-hydroxypoststerone, 21-hydroxypoststerone and 3-epi-21-hydroxypoststerone. Conjugates are also present. Parallels are drawn to the metabolism of endogenous vertebrate steroid hormones, to which Post bears more similarity than 20E.
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http://dx.doi.org/10.1016/j.jsbmb.2021.105897DOI Listing
September 2021

Developing new drugs that activate the protective arm of the renin-angiotensin system as a potential treatment for respiratory failure in COVID-19 patients.

Drug Discov Today 2021 05 17;26(5):1311-1318. Epub 2021 Feb 17.

Biophytis, Sorbonne Université - BC9, 4 Place Jussieu, 75005 Paris, France. Electronic address:

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has reached pandemic proportions with negative impacts on global health, the world economy and human society. The clinical picture of COVID-19, and the fact that Angiotensin converting enzyme 2 (ACE2) is a receptor of SARS-CoV-2, suggests that SARS-CoV-2 infection induces an imbalance in the renin-angiotensin system (RAS). We review clinical strategies that are attempting to rebalance the RAS in COVID-19 patients by using ACE inhibitors, angiotensin receptor blockers, or agonists of angiotensin-II receptor type 2 or Mas receptor (MasR). We also propose that the new MasR activator BIO101, a pharmaceutical grade formulation of 20-hydroxyecdysone that has anti-inflammatory, anti-fibrotic and cardioprotective properties, could restore RAS balance and improve the health of COVID-19 patients who have severe pneumonia.
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http://dx.doi.org/10.1016/j.drudis.2021.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888990PMC
May 2021

Beyond AREDS Formulations, What Is Next for Intermediate Age-Related Macular Degeneration (iAMD) Treatment? Potential Benefits of Antioxidant and Anti-inflammatory Apocarotenoids as Neuroprotectors.

Oxid Med Cell Longev 2020 8;2020:4984927. Epub 2020 Dec 8.

Biophytis, Sorbonne Université-BC9, 4 Place Jussieu, 75005 Paris, France.

Age-related macular degeneration (AMD) is the commonest cause of severe visual loss and blindness in developed countries among individuals aged 60 and older. AMD slowly progresses from early AMD to intermediate AMD (iAMD) and ultimately late-stage AMD. Late AMD encompasses either neovascular AMD (nAMD) or geographic atrophy (GA). nAMD is defined by choroidal neovascularization (CNV) and hemorrhage in the subretinal space at the level of the macula. This induces a rapid visual impairment caused by the death of photoreceptor cells. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) antibodies is the standard treatment of nAMD but adds to the burden of patient care. GA is characterized by slowly expanding photoreceptor, and retinal pigment epithelium (RPE) degeneration patches progressively leading to blindness. There is currently no therapy to cure GA. Late AMD continues to be an unmet medical need representing a major health problem with millions of patients worldwide. Oxidative stress and inflammation are recognized as some of the main risk factors to developing late AMD. The antioxidant formulation AREDS (Age-Related Eye Disease Studies), contains -carotene, which has been replaced by lutein and zeaxanthin in AREDS2, are given to patients with iAMD but have a limited effect on the incidence of nAMD and GA. Thus, to avoid or slowdown the development of late stages of AMD (nAMD or GA), new therapies targeting iAMD are needed such as crocetin obtained through hydrolysis of crocin, an important component of saffron (.), and norbixin derived from bixin extracted from seeds. We have shown that these apocarotenoids preserved more effectively RPE cells against apoptosis following blue light exposure in the presence of A2E than lutein and zeaxanthin. In this review, we will discuss the potential use of apocarotenoids to slowdown the progression of iAMD, to reduce the incidence of both forms of late AMD.
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http://dx.doi.org/10.1155/2020/4984927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803142PMC
May 2021

Systemic administration of the di-apocarotenoid norbixin (BIO201) is neuroprotective, preserves photoreceptor function and inhibits A2E and lipofuscin accumulation in animal models of age-related macular degeneration and Stargardt disease.

Aging (Albany NY) 2020 04 7;12(7):6151-6171. Epub 2020 Apr 7.

Biophytis, Sorbonne Université, Campus Pierre and Marie Curie, Paris 75005, France.

Atrophic A\age-related macular degeneration (AMD) and Stargardt disease (STGD) are major blinding diseases affecting millions of patients worldwide, but no treatment is available. In dry AMD and STGD oxidative stress and subretinal accumulation of -retinylidene--retinylethanolamine (A2E), a toxic by-product of the visual cycle, causes retinal pigment epithelium (RPE) and photoreceptor degeneration leading to visual impairment. Acute and chronic retinal degeneration following blue light damage (BLD) in BALB/c mice and aging of mice, respectively, reproduce features of AMD and STGD. Efficacy of systemic administrations of 9'--norbixin (norbixin), a natural di-apocarotenoid, prepared from seeds with anti-oxidative properties, was evaluated during BLD in BALB/c mice, and in mice of different ages, following three experimental designs: "preventive", "early curative" and "late curative" supplementations. Norbixin injected intraperitoneally in BALB/c mice, maintained scotopic and photopic electroretinogram amplitude and was neuroprotective. Norbixin chronic oral administration for 6 months in mice following the "early curative" supplementation showed optimal neuroprotection and maintenance of photoreceptor function and reduced ocular A2E accumulation. Thus, norbixin appears promising as a systemic drug candidate for both AMD and STGD treatment.
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http://dx.doi.org/10.18632/aging.103014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185133PMC
April 2020

Small-scale analysis of phytoecdysteroids in seeds by HPLC-DAD-MS for the identification and quantification of specific analogues, dereplication and chemotaxonomy.

Phytochem Anal 2020 Sep 17;31(5):643-661. Epub 2020 Mar 17.

Biophytis, Sorbonne Université, Paris, France.

Introduction: Phytoecdysteroids are analogues of arthropod steroids occurring in plants. They contribute to invertebrate deterrence. A wide diversity of ecdysteroids occurs in phytoecdysteroid-containing plant species, sometimes in high amounts. Ecdysteroids demonstrate potentially useful pharmaceutical actions in mammals.

Objectives: Establish reversed-phase high-performance liquid chromatography with tandem mass spectrometry (RP-HPLC-MS/MS) and RP-HPLC-DAD-MS (diode array detector mass spectrometry) methods for the separation, identification and quantification of ecdysteroids to screen for species containing significant amounts of 20-hydroxyecdysone (20E) and other useful ecdysteroids.

Materials And Methods: Micro-extracts of seed samples (ca. 30 mg) in 50% ethanol were subjected to RP-SPE (solid-phase extraction) purification prior to analysis by RP-HPLC-MS/MS and RP-HPLC-DAD-MS. The method was initially applied to genera (Amaranthus, Centaurea, Lychnis, Ourisia, Serratula, Silene and Trollius) where high-accumulating species had been previously encountered. Seeds of 160 randomly selected species, many of which have not previously been assessed, were then analysed. HPLC-MS/MS with a short analysis time initially identifies ecdysteroid-positive extracts and quantifies 20E. The positive extracts (20 ng 20E) are then analysed by HPLC-MS/MS with a longer analysis time to identify and quantify 17 common phytoecdysteroids and, finally, HPLC-DAD-MS (0.1-0.25 μg 20E) is used to obtain UV- and MS-spectra to confirm identifications or as a basis for characterisation of partially identified or novel analogues.

Results: Lychnis coronaria, Silene fimbriata and Silene hookeri ecdysteroids are characterised for the first time and those of Cucubalus baccifer and Ipheion uniflorum are more extensively characterised.

Conclusions: The procedure provides a rapid/sensitive method for screening small plant samples for the presence, quantification and identification of ecdysteroids. It permits ready dereplication of samples, identifying extracts containing large amounts or novel analogues.
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http://dx.doi.org/10.1002/pca.2930DOI Listing
September 2020

The effects of curcumin, mangiferin, resveratrol and other natural plant products on aminopeptidase B activity.

Biochem Biophys Res Commun 2019 05 27;512(4):832-837. Epub 2019 Mar 27.

Sorbonne Université, Institut de Biologie Paris Seine (IBPS), Equipe Biogenèse des Signaux Peptidiques (BIOSIPE), 75005, Paris, France.

Aminopeptidase B (Ap-B) is a Zn-aminopeptidase of the M1 family which is implicated, in conjunction with the nardilysin endoprotease, in the generation of miniglucagon, a peptide involved in the maintenance of glucose homeostasis. Other in vivo physiological roles have been established for this vertebrate enzyme, such as the processing of Arg-extended forms of human insulin and cholecystokinin 9 and the degradation of viral epitopes in the cytoplasm. Among M1 family members, Ap-B is phylogenetically close to leukotriene A hydrolase (LTAH), a bi-functional aminopeptidase also able to transform LTA in LTB (a lipid mediator of inflammation). As the activities of LTAH are reported to be inhibited by resveratrol, a polyphenolic molecule from red wine, the effect of this molecule was investigated on the Ap-B activity. Several other active phenolic compounds produced in plants were also tested. Among them, curcumin and mangiferin are the most effective inhibitors. Dixon analysis indicates that curcumin is a non-competitive inhibitor with a Ki value of 46 μmol.L. Dixon and Lineweaver-Burk representations with mangiferin show a mixed non-competitive inhibition with Ki' and Ki values of 194 μmol.L and 105 μmol.L, respectively. At 200 μmol.L, no significant effect was observed with caffeic, chlorogenic, ferulic, salicylic and sinapic acids as well as with resveratrol. Analyses on the 3D-structure of LTAH with resveratrol (pdb: 3FTS) and the Ap-B 3D-model allow hypothesis to explain theses results.
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http://dx.doi.org/10.1016/j.bbrc.2019.02.143DOI Listing
May 2019

Size matters! Aurora A controls Drosophila larval development.

Dev Biol 2018 08 9;440(2):88-98. Epub 2018 May 9.

CNRS, UMR 6290, Équipe labellisée Ligue contre le Cancer 2014-2016, 35000 Rennes, France; Univ Rennes, IGDR (Institut de Génétique et Développement de Rennes), F-35000 Rennes, France. Electronic address:

In metazoans, organisms arising from a fertilized egg, the embryo will develop through multiple series of cell divisions, both symmetric and asymmetric, leading to differentiation. Aurora A is a serine threonine kinase highly involved in such divisions. While intensively studied at the cell biology level, its function in the development of a whole organism has been neglected. Here we investigated the pleiotropic effect of Aurora A loss-of-function in Drosophila larval early development. We report that Aurora A is required for proper larval development timing control through direct and indirect means. In larval tissues, Aurora A is required for proper symmetric division rate and eventually development speed as we observed in central brain, wing disc and ring gland. Moreover, Aurora A inactivation induces a reduction of ecdysteroids levels and a pupariation delay as an indirect consequence of ring gland development deceleration. Finally, although central brain development is initially restricted, we confirmed that brain lobe size eventually increases due to additive phenotypes: delayed pupariation and over-proliferation of cells with an intermediate cell-identity between neuroblast and ganglion mother cell resulting from defective asymmetric neuroblast cell division.
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http://dx.doi.org/10.1016/j.ydbio.2018.05.005DOI Listing
August 2018

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, .

Front Neurosci 2017 15;11:752. Epub 2018 Jan 15.

School of Biological Sciences, Brambell Laboratories, Bangor University, Bangor, United Kingdom.

Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, . Using aequorin luminescence to measure Ca mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.
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http://dx.doi.org/10.3389/fnins.2017.00752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775280PMC
January 2018

Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo.

PLoS One 2016 16;11(12):e0167793. Epub 2016 Dec 16.

Biophytis, Parc BIOCITECH, 102 Avenue Gaston Roussel, Romainville, France.

The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9'-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9'-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167793PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161507PMC
July 2017

20-Hydroxyecdysone protects wheat seedlings (Triticum aestivum L.) against lead stress.

Plant Physiol Biochem 2016 Jan 19;98:64-71. Epub 2015 Nov 19.

Université Hassan I, Faculté Polydisciplinaire de Khouribga, BP 145, Khouribga, Morocco.

20-Hydroxyecdysone (20E) is the most abundant phytoecdysteroid (PE) produced by plants, where it represents a protective molecule against insect attack. The aim of the present study was to assess the effects of 20E on the growth, metabolic enzymes and antioxidant systems of wheat seedlings under lead stress induced by Pb(NO3)2 (3 mM). Pre-treatment concentrations (0, 1, 3 and 5 μM) of 20E were applied for two days, then wheat seedlings were transferred for 5 days to a treatment solution containing lead nitrate. When plants were grown without pre-treatment, germination and growth were impaired, while signs of oxidative stress were observed. 20E pre-treatment (3 or 5 μM) was able to efficiently protect seedlings from Pb toxicity by reducing Pb uptake and Pb-induced oxidative stress, as well by enhancing Pb excretion. Thus, 20E pre-treatment maintains normal germination, elongation, biomass, and metabolism in spite of the presence of Pb. Our results show that 20E protects oxidative balance and enhances the anti-oxidative systems, and more specifically the ascorbate-glutathione system. To our knowledge, this study shows for the first time that 20E treatment induces metabolic changes that allow the plants to resist heavy metal stress.
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http://dx.doi.org/10.1016/j.plaphy.2015.11.006DOI Listing
January 2016

The ecdysteroidome of Drosophila: influence of diet and development.

Development 2015 Nov 22;142(21):3758-68. Epub 2015 Sep 22.

Max Planck Institute for Cell Biology and Genetics, Pfotenhauerstraße 108, Dresden 01307, Germany

Ecdysteroids are the hormones regulating development, physiology and fertility in arthropods, which synthesize them exclusively from dietary sterols. But how dietary sterol diversity influences the ecdysteroid profile, how animals ensure the production of desired hormones and whether there are functional differences between different ecdysteroids produced in vivo remains unknown. This is because currently there is no analytical technology for unbiased, comprehensive and quantitative assessment of the full complement of endogenous ecdysteroids. We developed a new LC-MS/MS method to screen the entire chemical space of ecdysteroid-related structures and to quantify known and newly discovered hormones and their catabolites. We quantified the ecdysteroidome in Drosophila melanogaster and investigated how the ecdysteroid profile varies with diet and development. We show that Drosophila can produce four different classes of ecdysteroids, which are obligatorily derived from four types of dietary sterol precursors. Drosophila makes makisterone A from plant sterols and epi-makisterone A from ergosterol, the major yeast sterol. However, they prefer to selectively utilize scarce ergosterol precursors to make a novel hormone 24,28-dehydromakisterone A and trace cholesterol to synthesize 20-hydroxyecdysone. Interestingly, epi-makisterone A supports only larval development, whereas all other ecdysteroids allow full adult development. We suggest that evolutionary pressure against producing epi-C-24 ecdysteroids might explain selective utilization of ergosterol precursors and the puzzling preference for cholesterol.
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http://dx.doi.org/10.1242/dev.124982DOI Listing
November 2015

The minor ecdysteroids from Ajuga turkestanica.

Phytochem Anal 2015 Sep-Oct;26(5):293-300. Epub 2015 May 8.

Sorbonne Universités, UPMC Univ Paris 06, IBPS-BIOSIPE, CNRS FR 3631, 7 Quai Saint Bernard, Case Courrier 29, 75252, Paris Cedex 05, France.

Introduction: Ajuga turkestanica is a plant used in traditional medicine for its high ecdysteroid content, including the presence of the particularly active turkesterone, which possess efficient anabolic activity.

Objectives: To isolate and identify minor ecdysteroids present in a semi-purified plant fraction containing ca. 70% turkesterone.

Material And Methods: Multi-step preparative HPLC (combining RP- and NP-HPLC systems) was used to purify the different components present in the turkesterone fraction. Isolated compounds were identified by high-resolution mass spectrometry and 2D-NMR.

Results: Fourteen ecdysteroids (including turkesterone and 20-hydroxyecdysone) were isolated. Seven of these, all bearing an 11α-hydroxy group, were previously unreported.

Conclusion: Ajuga turkestanica ecdysteroids are characterised by the abundance of 11α-hydroxylated compounds and by the simultaneous presence of 24C, 27C, 28C and 29C ecdysteroids. It is expected that even more ecdysteroids are to be found in this plant since the starting material for this study lacked the less polar ecdysteroids. The simultaneous presence of 20-hydroxyecdysone and turkesterone (its 11α-hydroxy analogue) as the two major ecdysteroids suggests that every ecdysteroid is probably present in both 11α-hydroxy and 11-deoxy forms.
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http://dx.doi.org/10.1002/pca.2563DOI Listing
May 2016

Ecdysteroid profiles of two Ajuga species, A. iva and A. remota.

Nat Prod Commun 2014 Aug;9(8):1069-74

Phytoecdysteroids are plant analogues of insect moulting hormones and are used by plants to repel or disturb phytophagous insects. They are also active on mammals and present in many plants used in traditional medicine. The Ajuga genus contains several such species, which occur in various pharmacopoeias. We report the isolation and identification of major and minor ecdysteroids present in two Ajuga species, A. iva and A. remota, both of which are used as medicinal plants in Africa. Three minor ecdysteroids (abutasterone, ponasterone A and sidisterone) have been found for the first time in the Ajuga genus.
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August 2014

Quinoa extract enriched in 20-hydroxyecdysone affects energy homeostasis and intestinal fat absorption in mice fed a high-fat diet.

Physiol Behav 2014 Apr 15;128:226-31. Epub 2014 Feb 15.

INRA, U914, Nutrition Physiology and Ingestive Behavior, F-75005 Paris, France; AgroParisTech, U914, Nutrition Physiology and Ingestive Behavior, F-75005 Paris, France. Electronic address:

In a previous study, we have demonstrated that a supplementation of a high-fat diet with a quinoa extract enriched in 20-hydroxyecdysone (QE) or pure 20-hydroxyecdysone (20E) could prevent the development of obesity. In line with the anti-obesity effect of QE, we used indirect calorimetry to examine the effect of dietary QE and 20E in high-fat fed mice on different components of energy metabolism. Mice were fed a high-fat (HF) diet with or without supplementation by QE or pure 20E for 3 weeks. As compared to mice maintained on a low-fat diet, HF feeding resulted in a marked physiological shift in energy homeostasis, associating a decrease in global energy expenditure (EE) and an increase in lipid utilization as assessed by the lower respiratory quotient (RQ). Supplementation with 20E increased energy expenditure while food intake and activity were not affected. Furthermore QE and 20E promoted a higher rate of glucose oxidation leading to an increased RQ value. In QE and 20E-treated HFD fed mice, there was an increase in fecal lipid excretion without any change in stool amount. Our study indicates that anti-obesity effect of QE can be explained by a global increase in energy expenditure, a shift in glucose metabolism towards oxidation to the detriment of lipogenesis and a decrease in dietary lipid absorption leading to reduced dietary lipid storage in adipose tissue.
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http://dx.doi.org/10.1016/j.physbeh.2014.02.002DOI Listing
April 2014

Effect of lead stress on mineral content and growth of wheat (Triticum aestivum) and spinach (Spinacia oleracea) seedlings.

Saudi J Biol Sci 2013 Jan 8;20(1):29-36. Epub 2012 Sep 8.

PER-Centre des Etudes Environnementales Méditerranéennes, Equipe de recherche Biotechnologies et Génie des Biomolécules, Université Abdelmalek Essaadi, Faculté des Sciences et Techniques, BP 416, Tangier, Morocco.

Lead (Pb) is the most common heavy metal contaminant in the environment. Pb is not an essential element for plants, but they absorb it when it is present in their environment, especially in rural areas when the soil is polluted by automotive exhaust and in fields contaminated with fertilizers containing heavy metal impurities. To investigate lead effects on nutrient uptake and metabolism, two plant species, spinach (Spinacia oleracea) and wheat (Triticum aestivum), were grown under hydroponic conditions and stressed with lead nitrate, Pb(NO3)2, at three concentrations (1.5, 3, and 15 mM). Lead is accumulated in a dose-dependent manner in both plant species, which results in reduced growth and lower uptake of all mineral ions tested. Total amounts and concentrations of most mineral ions (Na, K, Ca, P, Mg, Fe, Cu and Zn) are reduced, although Mn concentrations are increased, as its uptake is reduced less relative to the whole plant's growth. The deficiency of mineral nutrients correlates in a strong decrease in the contents of chlorophylls a and b and proline in both species, but these effects are less pronounced in spinach than in wheat. By contrast, the effects of lead on soluble proteins differ between species; they are reduced in wheat at all lead concentrations, whereas they are increased in spinach, where their value peaks at 3 mM Pb. The relative lead uptake by spinach and wheat, and the different susceptibility of these two species to lead treatment are discussed.
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http://dx.doi.org/10.1016/j.sjbs.2012.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730938PMC
January 2013

Protective role of a methanolic extract of spinach (Spinacia oleracea L.) against Pb toxicity in wheat (Triticum aestivum L.) seedlings: beneficial effects for a plant of a nutraceutical used with animals.

Environ Sci Pollut Res Int 2013 Oct 5;20(10):7377-85. Epub 2013 May 5.

PER-Centre des Etudes Environnementales Méditerranéennes, Equipe de recherche Biotechnologies et Génie des Biomolécules, Faculté des Sciences et Techniques, Université Abdelmalek Essaadi, BP 416, Tangier, Morocco.

Spinach extracts contain powerful natural antioxidants and have been used to improve the response of animal cells to various stress factors. The aim of the present study was to assess the effects of a methanolic extract of spinach (SE) used at two concentrations (21.7 and 217 ppm) on the growth, certain enzymes and antioxidant systems in wheat seedlings under lead stress. When wheat seedlings were grown for 7 days in a solution containing Pb(NO3)2 (3 mM), germination and growth were impaired, while signs of oxidative stress were observed. SE (217 ppm) pretreatment was able to protect seedlings from Pb toxicity by both reducing Pb uptake and Pb-induced oxidative stress. As a consequence, almost normal germination, elongation, biomass and α-amylase activity were restored by SE (217 ppm) pretreatment of wheat seedlings, in spite of the presence of Pb. Our results support the protective role and the antioxidant effect of SE against Pb. These results show an amazing similarity to the effects of SE in animals, which suggests that providing "nutraceuticals" to plants could improve their "health" status.
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http://dx.doi.org/10.1007/s11356-013-1755-1DOI Listing
October 2013

E- and Z-isomers of new phytoecdysteroid conjugates from French Polynesian Microsorum membranifolium (Polypodiaceae) fronds.

Molecules 2012 Sep 28;17(10):11598-606. Epub 2012 Sep 28.

Laboratoire Biodiversité Terrestre et Marine, Université de la Polynésie Française, B.P. 6570 Faa'a, 98702 FAA'A, Tahiti, French Polynesia.

Phytochemical investigation of the fronds of Microsorum membranifolium resulted in the isolation of a new phytoecdysteroid, E-2-deoxy-20-hydroxyecdysone 3-[4-(1-β-D-glucopyranosyl)]-caffeate (1), together with two known phytoecdysteroids, E-2-deoxy-20-hydroxyecdysone 3-[4-(1-β-D-glucopyranosyl)]-ferulate (2), E-2-deoxyecdysone 3-[4-(1-β-D-glucopyranosyl)]-ferulate (3). Their respective Z-isomers 4-6 were also observed and identified for the first time. The new structures were elucidated on the basis of extensive spectroscopic data analysis (1D, 2D-NMR and HR-MS techniques).
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http://dx.doi.org/10.3390/molecules171011598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6268452PMC
September 2012

Mutations in the neverland gene turned Drosophila pachea into an obligate specialist species.

Science 2012 Sep;337(6102):1658-61

CNRS UMR7592, Université Paris Diderot, Sorbonne Paris Cité, Institut Jacques Monod, Paris, France.

Most living species exploit a limited range of resources. However, little is known about how tight associations build up during evolution between such specialist species and the hosts they use. We examined the dependence of Drosophila pachea on its single host, the senita cactus. Several amino acid changes in the Neverland oxygenase rendered D. pachea unable to transform cholesterol into 7-dehydrocholesterol (the first reaction in the steroid hormone biosynthetic pathway in insects) and thus made D. pachea dependent on the uncommon sterols of its host plant. The neverland mutations increase survival on the cactus's unusual sterols and are in a genomic region that faced recent positive selection. This study illustrates how relatively few genetic changes in a single gene may restrict the ecological niche of a species.
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http://dx.doi.org/10.1126/science.1224829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729188PMC
September 2012

Quinoa extract enriched in 20-hydroxyecdysone protects mice from diet-induced obesity and modulates adipokines expression.

Obesity (Silver Spring) 2012 Feb 25;20(2):270-7. Epub 2011 Aug 25.

INRA, U914, Nutrition Physiology and Ingestive Behavior, Paris, France.

Besides their well-known effect in the molting control in insects, ecdysteroids are steroid hormones that display potential pharmacologic and metabolic properties in mammals. The most common ecdysteroid, 20-hydroxyecdysone (20E) is found in many plants such as quinoa. The aim of the present study was to investigate the ability of quinoa extract (Q) enriched in 20E supplementation to prevent the onset of diet-induced obesity and to regulate the expression of adipocyte-specific genes in mice. Mice were fed a standard low-fat (LF) or a high-fat (HF) diet with or without supplementation by 20E-enriched Q or pure 20E for 3 weeks. Supplementation with Q reduced adipose tissue development in HF mice without modification of their body weight gain. This adipose tissue-specific effect was mainly associated with a reduced adipocyte size and a decrease in the expression of several genes involved in lipid storage, including lipoprotein lipase and phosphoenolpyruvate carboxykinase. Furthermore, Q-treated mice exhibited marked attenuation of mRNA levels of several inflammation markers (monocyte chemotactic protein-1, CD68) and insulin resistance (osteopontin, plasminogen activator inhibitor-1 (PAI-1)) as compared to HF mice. Q supplementation also reversed the effects of HF-induced downregulation of the uncoupling protein(s) (UCP(s)) mRNA levels in muscle. Similar results were obtained in mice fed a HF diet supplemented with similar amounts of pure 20E, suggesting that the latter accounted for most of the Q effects. Our study indicates that Q has an antiobesity activity in vivo and could be used as a nutritional supplement for the prevention and treatment of obesity and obesity-associated disorders.
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http://dx.doi.org/10.1038/oby.2011.257DOI Listing
February 2012

Lead phytotoxicity on wheat (Triticum aestivum L.) seed germination and seedlings growth.

C R Biol 2011 Feb 12;334(2):118-26. Epub 2011 Jan 12.

Équipe de recherche biotechnologies et génie des biomolécules, PER - centre des études environnementales méditerranéennes, faculté des sciences et techniques, université Abdelmalek Essaadi, Tangier, Morocco.

Lead (Pb) is an environmental pollutant extremely toxic to plants and other living organisms including humans. To assess Pb phytotoxicity, experiments focusing on germination of wheat seeds were germinated in a solution containing Pb (NO(3))(2) (0.05; 0.1; 0.5; 1g/L) during 6 days. Lead accumulation in seedlings was positively correlated with the external concentrations, and negatively correlated with morphological parameters of plant growth. Lead increased lipid peroxidation, enhanced soluble protein concentrations and induced a significant accumulation of proline in roots. Esterase activity was enhanced in the presence of lead, whereas α-amylase activity was significantly inhibited. Antioxidant enzymes activities, such as, ascorbate peroxidase, peroxidase, superoxide dismutase, catalase and glutathione S-transferase were generally significantly increased in the presence of lead in a dose-dependent manner. The present results thus provide a model system to screen for natural compounds able to counteract the deleterious effects of lead.
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http://dx.doi.org/10.1016/j.crvi.2010.12.006DOI Listing
February 2011

Two minor phytoecdysteroids of the plant Silene viridiflora.

Nat Prod Commun 2010 Oct;5(10):1579-82

Institute of the Chemistry of Plant Substances, Mirzo Ulugbek Str 77, Tashkent 100170, Uzbekistan.

Chemical investigations of Silene viridiflora (L.) yielded a new ecdysteroid, 20-hydroxyecdysone 20,22-monoacetonide-25-acetate (1), and a known ecdysteroid, 2-deoxypolypodine B-3-beta-D-glucoside (2). The elucidation of the chemical structures was established by 1D and 2D NMR experiments.
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October 2010

CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis.

Dev Biol 2011 Jan 7;349(1):35-45. Epub 2010 Oct 7.

Equipe Biogenèse des Signaux hormonaux, Laboratoire Biologie du Développement, UMR7622 CNRS, UPMC, 7 Quai St Bernard, F-75005 Paris, France.

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.
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http://dx.doi.org/10.1016/j.ydbio.2010.09.023DOI Listing
January 2011

Dietary effects of four phytoecdysteroids on growth and development of the Indian meal moth, Plodia interpunctella.

J Insect Sci 2010 ;10:13

PER-Centre des Etudes Environnementales Méditerranéennes, Laboratoire de Biologie Appliquée & Sciences de l'Environnement, Université Abdelmalek Essaadi, Faculté des Sciences et Techniques, BP 416, Tangier, Morocco.

Using pure phytoecdysteroids isolated from Ajuga iva (L.) Schreber (Lamiales: Lamiaceae) and Silene nutans L. (Caryophyllales: Caryophyllaceae), plants known for their high ecdysteroid content, a study was carried out on the effects of ingestion of four different phytoecdysteroids (20-hydroxyecdysone, polypodine B, ponasterone A and makisterone A) on the growth and development of the Indian meal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae) larvae when added at a concentration of 200 ppm in their diet. The experiments clearly showed the susceptibility of P. interpunctella to phytoecdysteroid ingestion. The toxicity of phytoecdysteroids manifested itself by a decrease in larval weight, induction of cannibalism and an increase of mortality, together with disruption of development. The severity of the phytoecdysteroid effect on P. interpunctella depended on the structure of the molecule. The results demonstrate that the minimal structural differences existing between these four phytoecdysteroids significantly affected their toxicity toward P. interpunctella. Makisterone A was the most toxic of the four compounds towards P. interpunctella larvae. In conclusion, phytoecdysteroids ingestion evokes disruptive growth effects on P. interpunctella. This work supports a role for phytoecdysteroids in plant defence against phytophagous insects.
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http://dx.doi.org/10.1673/031.010.1301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014659PMC
October 2010

Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2010 Apr 23;878(13-14):925-32. Epub 2010 Feb 23.

UPMC Univ Paris 06, UMR CNRS 7622, Equipe Biogenèse des signaux hormonaux, Case 29, 7 Quai Saint Bernard, F-75005 Paris, France.

Ecdysteroids are polyhydroxylated steroids that function as molting hormones in insects. 20-Hydroxyecdysone (a 27C-ecdysteroid) is classically considered as the major steroid hormone of Drosophilamelanogaster, but this insect also contains 28C-ecdysteroids. This arises from both the use of several dietary sterols as precursors for the synthesis of its steroid hormones, and its inability to dealkylate the 28C-phytosterols to produce cholesterol. The nature of Drosophila ecdysteroids has been re-investigated using both high-performance liquid chromatography coupled to enzyme immunoassay and a particularly sensitive nano-liquid chromatography-mass spectrometry methodology, while taking advantage of recently available ecdysteroid standards isolated from plants. In vitro incubations of the larval steroidogenic organ, the ring-gland, reveals the synthesis of ecdysone, 20-deoxy-makisterone A and a third less polar compound identified as the 24-epimer of the latter, while wandering larvae contain the three corresponding 20-hydroxylated ecdysteroids. This pattern results from the simultaneous use of higher plant sterols (from maize) and fungal sterols (from yeast). The physiological relevance of all these ecdysteroids, which display different affinities to the ecdysteroid receptors, is still a matter of debate.
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http://dx.doi.org/10.1016/j.jchromb.2010.02.018DOI Listing
April 2010

Ecdysteroids from Cyanotis longifolia Benth. (Commelinaceae).

Arch Insect Biochem Physiol 2009 Dec;72(4):194-209

Laboratoire de Biochimie Structurale et Fonctionnelle des Protéines, CNRS FRE 2852, Université Pierre et Marie Curie, 7 Quai Saint Bernard, Paris Cedex 05, France.

Cyanotis longifolia Benth. (Commelinaceae) contains ecdysteroids, which are highly concentrated in the roots and flowers, whereas leaves contain only very low amounts and stems intermediate amounts. 20-Hydroxyecdysone is the major component found in all tissues, but roots also contain large amounts of 20-hydroxyecdysone 3-acetate and ajugasterone C. A preparative experiment has shown that roots contain a complex ecdysteroid mixture, and the analysis of minor components has allowed the isolation of several already known ecdysteroids (polypodine B, 2-deoxy-20,26-dihydroxyecdysone, isovitexirone, poststerone) together with five new (ajugasterone C 3-acetate, 5beta-hydroxy-poststerone, poststerone 2-acetate, 14(15)-dehydro-poststerone 2-acetate, 24-epi-atrotosterone A [=24-methyl-ajugasterone C]) ecdysteroids that have been fully characterized. A preliminary investigation of 55 species belonging to 15 different genera of the Commelinaceae has shown that several of them contain significant concentrations of ecdysteroids, among which some previously uninvestigated ones appear to be very promising sources of ecdysteroids.
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http://dx.doi.org/10.1002/arch.20329DOI Listing
December 2009

Phytoecdysteroid C2-hydroxylase is microsomal in spinach, Spinacia oleracea L.

Arch Insect Biochem Physiol 2009 Dec;72(4):210-9

UPMC, Université Paris VI, Laboratoire de Biochimie Structurale et Fonctionnelle des Protéines, CNRS FRE 2852, Paris, France.

An enzyme involved in the biosynthesis of phytoecdysteroids, the C2-hydroxylase, has been investigated in spinach, Spinacia oleracea. This enzyme is microsomal and its K(m) has been determined using 2-deoxy-20-hydroxyecdysone as substrate (K(m)=3.72 microM). It is much more efficient with 2-deoxy-20-hydroxyecdysone than with 2-deoxyecdysone and, conversely, the C20-hydroxylase is more active on 2-deoxyecdysone than on ecdysone. These data support the conclusion that C20-hydroxylation precedes C2-hydroxylation. The C2-hydroxylase is inhibited by high concentrations of 20E. Substrate specificity and subcellular localization of C2-hydroxylase differ between plants and insects, and these data, as well as those previously reported on other biosynthetic steps, show the great difference between plant and insect ecdysteroid biosynthetic pathways and suggest an independent origin for the pathways in both kingdoms.
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http://dx.doi.org/10.1002/arch.20330DOI Listing
December 2009
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