Binding of plant isoperoxidases to pectin in the presence of calcium

AbstractSome of the isoperoxidases present in an extract from zucchini hypocotyls - one anionic and two cationic - exhibited a Ca2+-dependent pelletability which resulted from an interaction with pectins. Endogenous pectins could be replaced by polygalacturonic acid or pectin extracted from citrus, but not by highly esterifled pectin. The interaction between the isoperoxidases and the polysaccharides has been studied by centrifugation, by gel filtration, and with pectins attached in wells of a microtitration plate. These various binding tests have shown that the isoperoxidases had an affinity for the pectins in their Ca2+-induced conformation

Two cell wall associated peroxidases from Arabidopsis influence root elongation

Two class III peroxidases from Arabidopsis, AtPrx33 and Atprx34, have been studied in this paper. Their encoding genes are mainly expressed in roots; AtPrx33 transcripts were also found in leaves and stems. Light activates the expression of both genes in seedlings. Transformed seedlings producing AtPrx33-GFP or AtPrx34-GFP fusion proteins under the control of the CaMV 35S promoter exhibit fluorescence in the cell walls of roots, showing that the two peroxidases are localized in the apoplast, which is in line with their affinity for the Ca2+-pectate structure. The role they can play in cell wall was investigated using (1) insertion mutants that have suppressed or reduced expression of AtPrx33 or AtPrx34 genes, respectively, (2) a double mutant with no AtPrx33 and a reduced level of Atprx34 transcripts, (3) a mutant overexpressing AtPrx34 under the control of the CaMV 35S promoter. The major phenotypic consequences of these genetic manipulations were observed on the variation of the length of seedling roots. Seedlings lacking AtPrx33 transcripts have shorter roots than the wild-type controls and roots are still shorter in the double mutant. Seedlings overexpressing AtPrx34 exhibit significantly longer roots. These modifications of root length are accompanied by corresponding changes of cell length. The results suggest that AtPrx33 and Atprx34, two highly homologous Arabidopsis peroxidases, are involved in the reactions that promote cell elongation and that this occurs most likely within cell wall

An anionic class III peroxidase from zucchini may regulate hypocotyl elongation through its auxin oxidase activity

The high number of peroxidase genes explains the description of numerous physiological functions and the fact that the in planta function of a single isoform has never been characterized yet. We analyzed in transgenic Arabidopsis thaliana the localization of a zucchini isoperoxidase (APRX), previously purified thanks to its pectin binding ability. We confirmed that the protein is localized near the cell wall, mainly produced in the elongation area of the hypocotyls and respond to exogenous auxin. In addition, the ectopic overexpression of APRX induced changes in growth pattern and a significant reduction of endogenous indole-3-acetic acid (IAA) level. In agreement with these observations APRX showed an elevated in vitro auxin oxidase activity. We propose that APRX participates in the negative feedback regulation of auxin level and consequently terminates the hypocotyl elongation proces

Off-label use of targeted therapies in osteosarcomas: data from the French registry OUTC'S (Observatoire de l'Utilisation des Thérapies Ciblées dans les Sarcomes):

BACKGROUND: The objective of this study is to explore the off-label use of targeted therapies (TTs) for patients with osteosarcoma registered within the French Sarcoma Group--Bone Tumor Study Group (GSF-GETO) national registry. METHODS: All patients with an osteosarcoma, registered between January 1, 2009 and July 15, 2013 were analyzed. RESULTS: Twenty-nine patients with refractory relapsed osteosarcomas received 33 treatment lines of TTs. The median age at the beginning of treatment was 19 years (range 9-72). The median number of previous lines of chemotherapy was 3 (range 1-8). Before inclusion, 3 patients were in second complete remission, 26 were in progression for metastatic relapse. Twenty-three patients received sirolimus (in combination with cyclophosphamide for 18); 5, sunitinib; 4, sorafenib; and one, pazopanib. Stable disease was observed for 45.5% of patients (95% Confidence Interval (CI) [20-52.8]). The median Progression-Free Survival (PFS) was 3 months (95% CI [2-5.4]) for patients treated by sirolimus and 1.8 months (95% CI [1.3-2.8]) for patients receiving multi-targeted tyrosine kinase inhibitors; 6-month PFS 15%. The median Overall Survival (OS) was 6.8 months (95% CI [4.7-12.1]), and one-year OS was 24%. In a multivariate analysis, PFS was superior for patients receiving sirolimus compared to other TTs (Hazard Ratio (HR) = 2.7, 95% CI [1.05-7.1]). No toxic death was reported. Grade 3 and 4 toxicities were observed in 27 and 6% of cases respectively. CONCLUSION: Off-label TTs, especially sirolimus, reported benefit in the treatment of refractory osteosarcomas with an acceptable toxicity profile, including in pediatric population

Approche analogique et réalités des phytohormones : des retards et des erreurs stratégiques ?

peer reviewedEndocrine glands could not be seen in plants as in animal organisms which, in addition have a blood vascular system. Therefore, the concept of plant hormone emerged later only with researches on photo- and gravi-tropisms. The peculiar perception by the apices and a polar distribution of at least one substance engendering an unilateral growth in the elongation zone of stems or roots. A first active natural migrating substance was identified as 3-indolylacetic acid (IAA) and called auxin. Coumpounds with a similar or higher activity on growth, after external application were rapidly synthesized, but also generalize the term "growth substance" for both natural and synthetic products. Exogenous application of IAA or a synthetic analogue may exert a positive or negative effect on growth, depending on both the concentration used and an apparent different sensitiveness of the treated organ (stem, root), in a S-shaped curve. This led to speak in term of sub- and over-optimal concentrations used. But nobody effectively compared the endogenous contents nor took care of the real penetration of the treated plant material. The auxin-oxidase activity of the treated tissues was also not assessed. Auxin-like compounds were synthesized (for agricultural purposes, for instance), the most powerful appearing to be the seleniated IAA and 2,4-D prepared by chemists from our laboratories. Secondly, it was rapidly observed that natural or synthetic auxin application, normally on cuttings, enhanced or induced adventitious root formation. Thus, the first type of growth substance discovered could in addition also affect a development process, "probably" with associated ingredients into a complex called rhizocaline. Among the subsequent "growth substances" discovered, gibberellin favored particularly flowering, "probably" (again) in a multicomplex denominated florigen. The association of other components with auxin and gibberellin in rhizocaline and in florigen were never identified although synergistic or antagonistic effectors might exert actions. Plant physiologists progressively recalled to themselves that embryogenesis in higher plants is never ended (certainly not with the completion of an embryo in a seed) and that quantitative growth and qualitative development were intimately interconnected. The term PHYTOHORMONES emerged, without similarities with animal hormones. The phenomenons of differentitation and dedifferentiation were rediscussed, taking into account the spatiotemporal phases of growth and development. The conclusion of the most general assumption actually is that phytohormones rarely act alone, but rather in couples, through crosstalkings and signaling pathways, depending on gradients in their concentrations (changing with feeding and environmental factors) along the whole plants, even in connection with primary and secondary metabolisms. In vitro cultures of calli and vegetative multiplication using sequences of (phyto)hormones (natural and synthetic) in cocktails with minerals and other compounds (carbohydrates) induce traumatology symptoms that help to better distinguish tumors from real plant cancers. The latters also necessitated a specific plant physiological approach. Conscientious that the above considerations might take place in a life science history, where the concept of a plant hormone was distorted by animal physiology, it is hoped that the above reminders will help molecular biologists and genetic engineers to reappraise results on phytohormone specificity.Les premiers botanistes anatomistes n'avaient pu décrire de glandes endocrines à l'image de celles observées et fonctionnelles dans des organismes animaux, disposant d'un système sanguin irriguant tous leurs organes. Le concept d'hormone végétale n'émergea qu'avec les recherches sur les tropismes et la perceptibilité particulière des apex (aussi bien caulinaires que racinaires), aussi avec la découverte de la distribution polarisée, et des croissances sous-jacentes engendrées, d'où le terme premier d'hormone (sinon de substance) de croissance. L'agriculture se mit à rechercher des composés synthétiques en quête d'activités contrôlées... et plus efficaces, et en trouva. L'application externe des unes et des autres sur différents organes sous diverses conditions expérimentales aboutit progressivement à plusieurs découvertes et conclusions importantes pour la suite des recherches. Une même substance peut avoir des effets positifs et négatifs sur la croissance différant en importance selon la supposée "sensibilité" des organes traités, mais surtout selon les concentrations utilisées. Dans la partie "inhibition" des courbes dose-réponse caractéristiques, on parla de concentrations "sus-optimales" atteintes de l'hormone en question, mais rares furent ceux qui vérifièrent que la dite hormone appliquée pénétrait bien comme telle sans être modifiée, pour venir grossir le pool endogène; on se rendit notamment compte plus tard que la plante réagit à des applications qualifiées de sus-optimales d'agents extérieurs par une adaptation de ses systèmes cataboliques (les auxine-oxydases sont les premières en cause), sans se poser la question du devenir et des effets des produits générés; d'où l'ingénierie chimique synthétisant des homologues moins "attaquables" par les systèmes naturels de régulation (nos dérivés séléniés entre autres vraisemblablement). Les effets "rhizogènes" d'une auxine naturelle ou synthétique furent rapidement mis en évidence. Mais une hormone de croissance pouvait-elle seule provoquer un type de développement spécifique? D'où le concept de l'hormone de développement (en l'occurrence la rhizocaline) sous forme d'un complexe auxine + un sucre, des phénols, une oxydase, ...). Il en fut de même plus tard avec le "florigène" à plusieurs composants dont la gibbérelline, une autre hormone de croissance découverte entretemps. L'évidence des cytokinines (importantes celles-là pour la division cellulaire, plutôt que pour l'élongation favorisée par les auxines) mais surtout le rôle joué par la "balance" entre ces deux dernières conduisit à des avancées nouvelles et modifia aussi le concept d'hormone végétale de croissance: chaque type d'hormone pouvait être synthétisé dans tout type de cellule vivante; des gradients des différentes hormones, instaurés sous l'effet des conditions environnementales tout le long de la plante, sont vraisemblablement plus déterminants dans le comportement de la plante qu'une seule hormone dans l'orientation de leur croissance et de leur développement. C'est que, même les botanistes avaient un peu oublié que croissance et développement, dans une embryogénèse sans fin chez les végétaux, étaient intimement liés. Les hormones de développement se firent oublier pour laisser place au terme de PHYTOHORMONES. Ce qui posa immédiatement problème dans la question des phases de développement (induction, initiation ou évocation, expression) spatio-temporelles. Où le terme de dédifférenciation vint sur le tapis "objectivement". A notre connaissance, cette dédifférenciation ne fut réellement observée que dans des processus tératologiques (hyperhydricité dans des cals ou des pousses feuillées), qui par ailleurs mirent en évidence la complexité du vrai rôle des hormones (les couples auxines-cytokinines et polyamines-éthylène) reliées et conditionnées par les métabolismes primaires et secondaires et par les stress. Nous ne sommes pas certains que les biologistes moléculaires actuels tiennent compte de l'état physiologique général (déterminant dans un processus organogénétique) sous l'effet d'une hormone qu'ils croient spécifique, ni que les séquences de gènes allumés ou éteints soient les mêmes spatio-temporellement. L'ingénierie génétique en a déjà fait les frais (par exemple les tomates au mûrissement retardé mais sans saveur!), mais elle devra ramener des physiologistes-hormonologistes à d'autres réalités que les premiers concepts pourtant difficultueusement émergés

Pectin binding proteins: characterization of the binding and comparison with heparin

A small number of pectin binding proteins were obtained by passing soluble proteins from Cucurbita pepo L. hypocotyls through a column containing polygalacturonic acid (PGA) entrapped within polyacrylamide gel. These proteins were bound significantly to the gel only in the presence of Ca2+ and could be eluted with NaCL Two of these proteins were apparently immunologically related to human vitronectin. A comparison with heparinagarose showed that the animal glycosaminoglycan retained the same proteins than Ca2+ -PGA/polyacrylamide. In order to understand how these proteins interacted with the anionic polysaccharides, the binding to Ca2+ -PGA of anionic and cationic isoperoxidases (EC 1.11.1.7), belonging to the set of pectin binding proteins, was measured in the presence of various additions. For this purpose, we used a binding test based on the co-sedimentation of peroxidases and Ca2+ -PGA gel upon centrifugation. The linkage between peroxidases and Ca2+ -PGA was broken by increasing the ionic strength with NaCl or by adding various cationic polyamino acids. To be inhibitory, polycations must be long enough and have a defined conformation. Chitosan was ineffective. Active polycations did not break the Ca2+ -induced polymerization of PGA, but competed with the proteins for the binding to PGA molecules in their Ca2+ -mediated conformation. Experiments with the protein modifiers sulfo-NHS-acetate and phenylglyoxal showed that the amino acids lysine and arginine were involved in the binding mechanism, even in the case of the anionic isoperoxidase. It may be envisaged that this binding mechanism could allow some extracellular enzymes or proteins to be properly distributed within the extracellular matrix, in order to exert their catalytic functions on selected sites