Interaction between the GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX families of transcription factors

KNOTTED1-LIKE HOMEOBOX (KNOX) genes are important regulators of meristem function, and a complex network of transcription factors ensures tight control of their expression. Here, we show that members of the GROWTH-REGULATING FACTOR (GRF) family act as players in this network. A yeast (Saccharomyces cerevisiae) one-hybrid screen with the upstream sequence of the KNOX gene Oskn2 from rice (Oryza sativa) resulted in isolation of OsGRF3 and OsGRF10. Specific binding to a region in the untranslated leader sequence of Oskn2 was confirmed by yeast and in vitro binding assays. ProOskn2:β-glucuronidase reporter expression was down-regulated by OsGRF3 and OsGRF10 in vivo, suggesting that these proteins function as transcriptional repressors. Likewise, we found that the GRF protein BGRF1 from barley (Hordeum vulgare) could act as a repressor on an intron sequence in the KNOX gene Hooded/Barley Knotted3 (Bkn3) and that AtGRF4, AtGRF5, and AtGRF6 from Arabidopsis (Arabidopsis thaliana) could repress KNOTTED-LIKE FROM ARABIDOPSIS THALIANA2 (KNAT2) promoter activity. OsGRF overexpression phenotypes in rice were consistent with aberrant meristematic activity, showing reduced formation of tillers and internodes and extensive adventitious root/shoot formation on nodes. These effects were associated with down-regulation of endogenous Oskn2 expression by OsGRF3. Conversely, RNA interference silencing of OsGRF3, OsGRF4, and OsGRF5 resulted in dwarfism, delayed growth and inflorescence formation, and up-regulation of Oskn2. These data demonstrate conserved interactions between the GRF and KNOX families of transcription factors in both monocot and dicot plants

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties

Λειτουργική ανάλυση των πρωτεινών που δεσμεύουν Σελήνιο σε φυτά και ζώα

The family of selenium binding proteins (SBPs) has highly similar members in moststudied prokaryotes and eukaryotes. The first member of this family was discovered fifteenyears ago and it was named after its association with selenium, a metalloid element withcomplex environmental and biological behavior. Although several reports and hypotheseshave been published about possible roles of SBP, the exact function of this protein is notclear. The aim of the study described in this thesis is to give more insight into the biologicalimportance of SBP and its biochemical function in plants and animals. Chapter 1 presents an overview of the current knowledge on SBP. In this introductory chapter the importance and role of selenium for biological systems is discussedtogether with the progress in research on SBP in both plants and animals.In chapter 2 expression studies of SBP are described, primary in the legume Lotusjaponicus but also in the non leguminous plant Arabidopsis thaliana. Isolation andcharacterization of the L. japonicus gene and its pattern of expression at mRNA and proteinlevels in various tissues are studied. This chapter shows a dynamic expression of the sbpgene in root nodule tissues but also a constitutive expression in root, unrelated withsymbiotic processes. In chapter 3, functional analysis of SBP in A. thaliana is described. Transgenic plants that either over-express or have down-regulated levels of the Atsbp1 endogenousgene are studied with respect to growth and development under normal growth conditionsas well as under selenium challenging conditions. These experiments suggest that SBP might be involved in mechanisms controlling plant tolerance to toxic levels of selenium. The extensive sequence conservation and the relation of SBP genes from differentspecies is discussed in chapter 4. Furthermore, the identification of SBP interacting partnersis also described in this chapter. Two hybrid screenings revealed that the proteins, whichpossibly interact with SBP, are mainly related with vesicle trafficking and membranesynthesis or with the control of the oxidative/redox state of the cell. Additionally, thedemonstration of interaction with other selenium associated proteins suggests an implicationof SBP in a possible protein network that depends on or regulates the selenium metabolism in cells. Finally, chapter 5 presents the work in zebrafish, which is used as a modelorganism for studies of SBP in vertebrates and animals in general. Cell lines, embryos and adult fish are employed for expression, localization and functional analyses. Results on SBP’s dynamic expression, cartilage localization and vesicle association are presented and together suggest that SBP might have a fundamental role in embryonic developmentΗ οικογένεια των πρωτεϊνών που δεσμεύουν σελήνιο (SBPs) έχει ομόλογα μέλη στους περισσότερους μελετημένους προκαρυωτικούς και ευκαρυωτικούς οργανισμούς. Το πρώτο μέλος αυτής της οικογένειας ανακαλύφθηκε πριν δεκαπέντε χρόνια και πήρε το όνομά του από τη συσχέτισή του με το σελήνιο, ένα μεταλλοειδές στοιχείο με πολύπλοκη περιβαλλοντική και βιολογική συμπεριφορά. Αν και αρκετές αναφορές και υποθέσεις έχουν δημοσιευτεί σχετικά με τους πιθανούς ρόλους της SBP, η ακριβής λειτουργία αυτής της πρωτεΐνης δεν είναι σαφής. Στόχος της μελέτης που περιγράφεται σε αυτή τη διατριβή, είναι να δώσει περισσότερες πληροφορίες για τη βιολογική σημασία της SBP και τη βιοχημική λειτουργία της σε φυτά και ζώα.Το Κεφάλαιο 1 παρουσιάζει μια επισκόπηση της τρέχουσας γνώσης για το SBP. Σε αυτό το εισαγωγικό κεφάλαιο, συζητείται η σημασία και ο ρόλος του σεληνίου για τα βιολογικά συστήματα μαζί με την πρόοδο της έρευνας για την SBP τόσο στα φυτά όσο και στα ζώα.Στο κεφάλαιο 2 περιγράφονται μελέτες έκφρασης της SBP, στο ψυχανθές Lotus japonicus αλλά και στο μη ψυχανθές φυτό Arabidopsis thaliana. Έγινε απομόνωση και χαρακτηρισμός του sbp γονιδίου από το L. Japonicus και μελετήθηκε η έκφρασή του σε επιπέδο mRNA και πρωτεΐνης σε διάφορους ιστούς. Αυτό το κεφάλαιο δείχνει μια δυναμική έκφραση του sbp γονιδίου στους ιστούς των φυματίων της ρίζας αλλά και μια συστατική έκφραση στη ρίζα, άσχετη με συμβιωτικές διεργασίες.Στο κεφάλαιο 3, περιγράφεται η λειτουργική ανάλυση της SBP στο φυτό A. thaliana. Διαγονιδιακά φυτά που είτε υπερεκφράζουν είτε έχουν μειωμένα επίπεδα του ενδογενούς Atsbp1 γονιδίου μελετώνται σε σχέση με την ανάπτυξη τόσο υπό κανονικές συνθήκες όσο και κάτω από συνθήκες έκθεσης σε σελήνιο. Αυτά τα πειράματα έδειξαν ότι η SBP μπορεί να εμπλέκεται σε μηχανισμούς που ελέγχουν την ανοχή των φυτών σε τοξικά επίπεδα σεληνίου. Η εκτεταμένη συντήρηση της αλληλουχίας των SBP πρωτεϊνών μεταξύ των διαφορετικών εξελικτικών ειδών και η σχέση αυτών των γονιδίων συζητείται στο κεφάλαιο 4. Επιπλέον, ο προσδιορισμός πρωτεϊνών που αλληλεπιδρούν με την SBP, περιγράφεται επίσης σε αυτό το κεφάλαιο. Μελέτες αλληλεπιδράσεων δύο υβριδίων (two hybrid assay) αποκάλυψαν ότι οι πρωτεΐνες, οι οποίες πιθανώς αλληλεπιδρούν με την SBP, σχετίζονται κυρίως με τη διακίνηση κυστιδίων και τη μεμβρανική σύνθεση ή με τον έλεγχο της οξειδωτικής/οξειδοαναγωγικής κατάστασης του κυττάρου. Επιπλέον, η SBP φαίνεται να αλληλεπιδρά με άλλες πρωτεΐνες που σχετίζονται με το σελήνιο, γεγονός που υποδηλώνει την εμπλοκή της SBP σε ένα πιθανό πρωτεϊνικό δίκτυο που εξαρτάται ή ρυθμίζει το μεταβολισμό του σεληνίου σε κύτταρα.Τέλος, στο κεφάλαιο 5 παρουσιάζονται μελέτες στο zebrafish (Danio rerio) , το οποίο χρησιμοποιείται ως οργανισμός μοντέλο για μελέτες SBP σε σπονδυλωτά και ζώα γενικά. Κυτταρικές σειρές, έμβρυα και ενήλικα ψάρια χρησιμοποιούνται για έκφραση, εντοπισμό και λειτουργικές αναλύσεις. Παρουσιάζονται αποτελέσματα για τη δυναμική έκφραση της SBP, τον εντοπισμό στον χόνδρο και την αλληλεπιδραση με κυστίδια και τα αποτελέσματα αυτά μαζί υποδηλώνουν ότι η SBP μπορεί να έχει θεμελιώδη ρόλο στην εμβρυϊκή ανάπτυξη

Identification of Novel Melanin Synthesis Inhibitors From Crataegus pycnoloba Using an in Vivo Zebrafish Phenotypic Assay

Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes

The SAH7 Homologue of the Allergen Ole e 1 Interacts with the Putative Stress Sensor SBP1 (Selenium-Binding Protein 1) in Arabidopsis thaliana

In this study, we focused on a member of the Ole e 1 domain-containing family, AtSAH7, in Arabidopsis thaliana. Our lab reports for the first time on this protein, AtSAH7, that was found to interact with Selenium-binding protein 1 (AtSBP1). We studied by GUS assisted promoter deletion analysis the expression pattern of AtSAH7 and determined that the sequence 1420 bp upstream of the transcription start can act as a minimal promoter inducing expression in vasculature tissues. Moreover, mRNA levels of AtSAH7 were acutely increased under selenite treatment in response to oxidative stress. We confirmed the aforementioned interaction in vivo, in silico and in planta. Following a bimolecular fluorescent complementation approach, we determined that the subcellular localization of the AtSAH7 and the AtSAH7/AtSBP1 interaction occur in the ER. Our results indicate the participation of AtSAH7 in a biochemical network regulated by selenite, possibly associated with responses to ROS production

Assessment of the Acute Toxicity, Uptake and Biotransformation Potential of Benzotriazoles in Zebrafish (<i>Danio rerio</i>) Larvae Combining HILIC- with RPLC-HRMS for High-Throughput Identification

The current study reports on the toxicity, uptake, and biotransformation potential of zebrafish (embryos and larvae) exposed to benzotriazoles (BTs). Acute toxicity assays were conducted. Cardiac function abnormalities (pericardial edema and poor blood circulation) were observed from the phenotypic analysis of early life zebrafish embryos after BTs exposure. For the uptake and biotransformation experiment, extracts of whole body larvae were analyzed using liquid chromatography–high-resolution tandem mass spectrometry (UPLC-Q-TOF-HRMS/MS). The utility of hydrophilic interaction liquid chromatography (HILIC) as complementary technique to reversed phase liquid chromatography (RPLC) in the identification process was investigated. Through HILIC analyses, additional biotransformation products (bio-TPs) were detected, because of the enhanced sensitivity and better separation efficiency of isomers. Therefore, reduction of false negative results was accomplished. Both oxidative (hydroxylation) and conjugative (glucuronidation, sulfation) metabolic reactions were observed, while direct sulfation proved the dominant biotransformation pathway. Overall, 26 bio-TPs were identified through suspect and nontarget screening workflows, 22 of them reported for the first time. 4-Methyl-1-<i>H</i>-benzotriazole (4-MeBT) demonstrated the highest toxicity potential and was more extensively biotransformed, compared to 1-<i>H</i>-benzotriazole (BT) and 5-methyl-1-<i>H</i>-benzotriazole (5-MeBT). The extent of biotransformation proved particularly informative in the current study, to explain and better understand the different toxicity potentials of BTs

Anti-Melanogenic Properties of Greek Plants. A Novel Depigmenting Agent from Morus alba Wood

In therapeutic interventions associated with melanin hyperpigmentation, tyrosinase is regarded as a target enzyme as it catalyzes the rate-limiting steps in mammalian melanogenesis. Since many known agents have been proven to be toxic, there has been increasing impetus to identify alternative tyrosinase inhibitors, especially from natural sources. In this study, we investigated 900 extracts from Greek plants for potential tyrosinase inhibitive properties. Among the five most potent extracts, the methanol extract of Morus alba wood (MAM) demonstrated a significant reduction in intracellular tyrosinase and melanin content in B16F10 melanoma cells. Bioassay-guided isolation led to the acquisition of twelve compounds: oxyresveratrol (1), kuwanon C (2), mulberroside A (3), resorcinol (4), dihydrooxyresveratol (5), trans-dihydromorin (6), 2,4,3′-trihydroxydihydrostilbene (7), kuwanon H (8), 2,4-dihydroxybenzaldehyde (9), morusin (10), moracin M (11) and kuwanon G (12). Among these, 2,4,3′-trihydroxydihydrostilbene (7) is isolated for the first time from Morus alba and constitutes a novel potent tyrosinase inhibitor (IC50 0.8 ± 0.15). We report here for the first time dihydrooxyresveratrol (5) as a potent natural tyrosinase inhibitor (IC50 0.3 ± 0.05). Computational docking analysis indicated the binding modes of six tyrosinase inhibitors with the aminoacids of the active centre of tyrosinase. Finally, we found both MAM extract and compounds 1, 6 and 7 to significantly suppress in vivo melanogenesis during zebrafish embryogenesis

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<p>Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.</p

Image_3.JPEG

<p>Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.</p

Image_2.JPEG

<p>Zebrafish has emerged as a powerful model organism for high throughput drug screening. Several morphological criteria, transgenic lines and in situ expression screens have been developed to identify novel bioactive compounds and their mechanism of action. Here, we used the inhibition of melanogenesis during early zebrafish embryo development to identify natural compounds that block melanogenesis. We identified an extract from the Greek hawthorn Crataegus pycnoloba as a potent inhibitor of melanin synthesis and used activity based subfractionation to identify active subfractions and eventually three single compounds of the same family (dibenzofurans). These compounds show reversible inhibition of melanin synthesis and do not act via inhibition of tyrosinase. We also showed that they do not interfere with neural crest differentiation or migration. We identified via in silico modeling that the compounds can bind to the aryl hydrocarbon receptor (AHR) and verified activation of the Ahr signaling pathway showing the induction of the expression of target genes.</p