Life & Health Sciences

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Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle


ABSTRACT

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis.


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Grape Cane Extracts as Multifunctional Rejuvenating Cosmetic Ingredient: Evaluation of Sirtuin Activity, Tyrosinase Inhibition and Bioavailability Potential


ABSTRACT

Grape canes are waste biomass of viticulture containing bioactive polyphenols valuable in cosmetics. Whereas several studies reported the cosmetic activities of E-resveratrol, only few described the potential of E-ε-viniferin, the second major constituent of grape cane extracts (GCE), and none of them investigated GCE as a natural blend of polyphenols for cosmetic applications. In this study, we considered the potential of GCE from polyphenol-rich grape varieties as multifunctional cosmetic ingredients. HPLC analysis was performed to quantify major polyphenols in GCE i.e., catechin, epicatechin, E-resveratrol, E-piceatannol, ampelopsin A, E-ε-viniferin, hopeaphenol, isohopeaphenol, E-miyabenol C and E-vitisin B from selected cultivars. Skin whitening potential through tyrosinase inhibition assay and the activation capacity of cell longevity protein (SIRT1) of GCE were compared to pure E-resveratrol and E-ε-viniferin. Drug-likeness of GCE polyphenols were calculated, allowing the prediction of skin permeability and bioavailability. Finally, the present data enabled the consideration of GCE from polyphenol-rich varieties as multifunctional cosmetic ingredients in accordance with green chemistry practices.


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Thermal restraint of a bacterial exopolysaccharide of shallow vent origin


ABSTRACT

To dynamically characterize the thermal properties of the fructose-rich exopolysaccharide (EPS1-T14), produced by the marine thermophilic Bacillus licheniformis T14, the Attenuated Total Reflectance Fourier Transform Infra-Red spectroscopy was coupled to variable temperature ranging from ambient to 80 °C.

The spectra were analyzed by the following innovative mathematical tools: i) non-ideal spectral deviation, ii) OH-stretching band frequency center shift, iii) spectral distance, and iv) wavelet cross-correlation analysis.

The thermal restraint analysis revealed that the whole EPS1-T14 system possessed high stability until 80 °C, and suggested that fucose was mainly involved in the EPS1-T14 thermal stability, whereas glucose was responsible for its molecular flexibility.

Our results provide novel insights into the thermal stability properties of the whole EPS1-T14 and into the role of its main monosaccharidic units. As a new biopolymer, the thermostable EPS1-T14 could be used in traditional biotechnology fields and in new biomedical areas, as nanocarriers, requiring high temperature processes.


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Access to Unprotected β-Fluoroalkyl β-Amino Acids and Their α-Hydroxy Derivatives


ABSTRACT

Unprotected β-(het)aryl-β-fluoroalkyl β-amino acids and their α-hydroxy derivatives can be readily obtained using a decarboxylative Mannich-type reaction without protection/deprotection steps. This protocol utilizes lithium hexamethyldisilazide and (het)arylfluoroalkyl ketones to generate NH-ketimine intermediates. The mild reaction conditions allow the preparation of original fluorinated β-amino acids as useful building blocks in a practical and scalable manner.


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GnRH Antagonists Produce Differential Modulation of the Signaling Pathways Mediated by GnRH Receptors


ABSTRACT

Commercial gonadotropin-releasing hormone (GnRH) antagonists differ by 1–2 amino acids and are used to inhibit gonadotropin production during assisted reproduction technologies (ART). In this study, potencies of three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in inhibiting GnRH-mediated intracellular signaling, were compared in vitro. GnRH receptor (GnRHR)-transfected HEK293 and neuroblastoma-derived SH-SY5Y cell lines, as well as mouse pituitary LβT2 cells endogenously expressing the murine GnRHR, were treated with GnRH in the presence or absence of the antagonist. We evaluated intracellular calcium (Ca2+) and cAMP increases, cAMP-responsive element binding-protein (CREB) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation, β-catenin activation and mouse luteinizing-hormone β-encoding gene (Lhb) transcription by bioluminescence resonance energy transfer (BRET), Western blotting, immunostaining and real-time PCR as appropriate. The kinetics of GnRH-induced Ca2+ rapid increase revealed dose-response accumulation with potency (EC50) of 23 nM in transfected HEK293 cells, transfected SH-SY5Y and LβT2 cells. Cetrorelix inhibited the 3 × EC50 GnRH-activated calcium signaling at concentrations of 1 nM–1 µM, demonstrating higher potency than Ganirelix and Teverelix, whose inhibitory doses fell within the 100 nM–1 µM range in both transfected HEK293 and SH-SY5Y cells in vitro. In transfected SH-SY5Y, Cetrorelix was also significantly more potent than other antagonists in reducing GnRH-mediated cAMP accumulation. All antagonists inhibited pERK1/2 and pCREB activation at similar doses, in LβT2 and transfected HEK293 cells treated with 100 nM GnRH. Although immunostainings suggested that Teverelix could be less effective than Cetrorelix and Ganirelix in inhibiting 1 µM GnRH-induced β-catenin activation, Lhb gene expression increase occurring upon LβT2 cell treatment by 1 µM GnRH was similarly inhibited by all antagonists. To conclude, this study has demonstrated Cetrorelix-, Ganirelix- and Teverelix-specific biased effects at the intracellular level, not affecting the efficacy of antagonists in inhibiting Lhb gene transcription.


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Follicle-Stimulating Hormone: Fertility and Beyond


ABSTRACT

Editorial on the Research Topic.

Propagating life to the next generation is a hormone-dependent process relying on the individualwish to generate own progeny and resulting in maintenance of species. This Research Topicis dedicated to Follicle–Stimulating Hormone (FSH) and itsreceptor (FSHR) and their rolein reproduction. FSH is a typical example of a drug which entered clinical use in the “pre-evidence-basedmedicine era,” just for its efficacy in stimulating gonadal function and fertility in hypogonadotropichypogonadism. More recently, FSH entered clinical use in controlled ovarian stimulation in orderto obtain multiple follicular growth for assisted reproduction. Given the progressive increase incouple infertility, the demand for assisted reproduction grows steadily and the FSH market isflourishing. Yet, very little was known about the FSH mode of action until a few years ago,and the therapeutic use of FSH is still far from being evidence-based. But great progress in ourunderstanding of FSH action was made in the last two decades and, since not many scientistsaround the world are active in the gonadotropin/FSH research “niche,” we thought it was time tocall them to report to tell us their view on the state-of-the-art. The result is this “Research Topic.”


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Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars


ABSTRACT

Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f®)- with biosimilar preparations (Bemfola® and Ovaleap®)-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f® or biosimilar (1 × 10−3-1 × 103 ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and β-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and β-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 ± 0.9–24 ± 1.7 ng/ml; β-arrestin 2 EC50 range = 140 ± 14.1–313 ± 18.7 ng/ml; Kruskal-Wallis test; p ≥ 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 μg/ml Gonal-f®, while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC50s were demonstrated (Kruskal-Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.


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Prévention de l’AVC ischémique / Stroke prevention


ABSTRACT

L’accident vasculaire cérébral (AVC) est la première cause de handicap acquis de l’adulte, la deuxième cause de démence et la troisième cause de mortalité en France. Parmi les AVC, 89 % sont ischémiques. Le vieillissement de la population laisse présager une augmentation du nombre de patients victimes d’AVC dans les années à venir.

La prévention des infarctus cérébraux est un enjeu important de la prise en charge des AVC compte tenu du risque élevé de récidive. Après un premier infarctus cérébral, le risque de récidive est estimé à 10 % la première année et entre 20 et 30 % à 5 ans. La survenue d’un AVC est favorisée par des facteurs de risque cardiovasculaire connus et accessibles à la prévention. Le contrôle des facteurs de risque comme l’hypertension artérielle (HTA), le diabète, la dyslipidémie et le tabac est indispensable en cas d’AVC d’origine athéromateuse ou de suspicion de pathologie athéroscléreuse sous-jacente. Lorsque la cause d’un infarctus cérébral ou d’un accident ischémique transitoire (AIT) est connue, le traitement spécifique de l’étiologie est un élément indispensable à la prévention secondaire.


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Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay


ABSTRACT

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.


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Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis


ABSTRACT

Tuberculosis is the leading cause of death by an infectious disease worldwide1. However, the involvement of innate lymphoid cells (ILCs) in immune responses to infection with Mycobacterium tuberculosis (Mtb) is unknown. Here we show that circulating subsets of ILCs are depleted from the blood of participants with pulmonary tuberculosis and restored upon treatment. Tuberculosis increased accumulation of ILC subsets in the human lung, coinciding with a robust transcriptional response to infection, including a role in orchestrating the recruitment of immune subsets. Using mouse models, we show that group 3 ILCs (ILC3s) accumulated rapidly in Mtb-infected lungs and coincided with the accumulation of alveolar macrophages. Notably, mice that lacked ILC3s exhibited a reduction in the accumulation of early alveolar macrophages and decreased Mtb control. We show that the C-X-C motif chemokine receptor 5 (CXCR5)–C-X-C motif chemokine ligand 13 (CXCL13) axis is involved in Mtb control, as infection upregulates CXCR5 on circulating ILC3s and increases plasma levels of its ligand, CXCL13, in humans. Moreover, interleukin-23-dependent expansion of ILC3s in mice and production of interleukin-17 and interleukin-22 were found to be critical inducers of lung CXCL13, early innate immunity and the formation of protective lymphoid follicles within granulomas. Thus, we demonstrate an early protective role for ILC3s in immunity to Mtb infection.