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STUDIO SUL RUOLO DELL’ACIDO Δ1-PIRROLIN-5-CARBOSSILICO NEI FENOMENI DI TOSSICITÀ INDOTTA DA PROLINA IN ARABIDOPSIS THALIANA.
No full textA high accumulation of proline due to increased synthesis and decreased degradation under a variety of stress conditions, such as salt, drought and metals, has been documented in many plant species (Kavi Kishor et al., 2005). This accumulation (up to 80% of total free amino acids under stress, compared to 5% under normal conditions) seems to have diverse physiological roles, such as acting as a compatible osmolyte for osmotic adjustment, stabilization of proteins, membranes and subcellular structures, buffering cellular redox potential and protecting cellular functions by scavenging reactive oxygen species (ROS) (Kavi Kishor et al., 2005; Ashraf and Foolad, 2007).
In plants, proline is synthesized from either glutamate or ornithine (Kavi Kishor et al.,2005; Ashraf and Foolad, 2007). The first two steps of proline biosynthesis from glutamate are catalyzed by a single bifunctional enzyme, δ1-pyrroline-5-carboxylate synthetase (P5CS), yielding glutamic-γ-semialdehyde (GSA). GSA spontaneously cyclises into pyrroline-5-carboxylate (P5C), that is reduced in turn by P5C reductase (P5CR) to proline (Zhang et al., 1995; Kavi Kishor et al., 2005). Plants also synthesize proline from ornithine, through the activity of ornithine-δ-aminotransferase (δOAT) that transaminates ornithine directly to GSA, which is subsequently converted to proline via P5C (Kavi Kishore et al., 2005). The onset of stress-induced proline accumulation is correlated with transcriptional activation of the gene encoding P5CS, which is believed to represent the key and rate limiting enzyme in this biosynthetic pathway (Stines et al., 1999; Zhang et al., 2008).
During recent years a thorough understanding of the mechanisms underlying regulation of Pro biosynthesis has been achieved. However, the role of the catabolic pathway it is still unclear, consisting of two steps sequentially catalyzed by a Pro- and a P5C-dehydrogenase (DH). The oxidative pathway is induced by high intracellular levels of the imino acid; such induction is prevented under hyperosmotic stress conditions (Verbruggen et al., 1996).
Besides its obvious role in Pro recycling after the re-establishment of normo-osmotic conditions, no other physiological roles have been up to now identified for the catabolic pathway. Actually, Arabidopsis thaliana knock-out seedlings for either Pro-DH1 or P5C-DH are morphologically undistinguishable from the wild type and show a normal life cycle. The main effect of the impairment of proline catabolism appears to be a hypersensitivity to the amino acid or to its analogues (Deuschle et al., 2004 ; Mani et al., 2002). Indeed, an unexpected result is that an exogenous Pro supply apparently causes phytotoxic effects. Besides necrotic area formation, the production of reactive oxygen species (ROS) and DNA laddering have been reported (Deuschle et al., 2004 ), suggesting the onset of programmed cell death (PCD). In animal systems it is well known that the PRODH gene is early induced by the tumor suppressor protein p53 (Maxwell et al., 2000), that may inhibit growth and trigger PCD; exogenously-supplied P5C may also cause apoptosis. P5CDH was also reported to represent a p53 target, but it seems able to protect cells from oxidative stress and inhibit cell death (Yoon et al., 2004). Human Pro-DH appears to catalyse ROS production directly, depending to substrate availability (Donald et al.,2001), but Pro-DH has never been purified and fully characterized to date from any plant species.
On the other hand, an early induction of the gene for P5C-DH has been found in crops undergoing a fungal attack, a response typically observed in the case of compatible interactions (Roberts et al., 1995; Ayliffe et al., 2002). Moreover, Pro accumulation through activation of the biosynthetic pathway has been recently shown in Arabidopsis during incompatible plant-pathogen interactions (Fabro et al., 2004). Thus the possibility exists that Pro metabolism is involved in processes leading to PCD during the hypersensitive reaction. Proline effects have been proposed to be mediated by P5C (Phang et al.,1985; Deuschle et al.,2004 ). However, this seems inconsistent with the severe toxicity that occurs in prod1- mutants and plants expressing antisense ProDH constructs (Mani et al., 2002; Nanjo et al., 2003 ).
P5C is the common metabolic intermediate in proline synthesis and catabolism. It is also produced by δOAT, which was recently demonstrated to be localized in the mitochondria (Funck et al., 2008). It is still a matter of debate whether the P5C produced by Arg/Orn catabolism enters proline biosynthesis directly, or it is metabolized to glutamate by mitochondrial P5CDH. Funck and coworkers showed experimental evidence supporting the latter hypothesis. Consistently, the occurrence of a specific P5C transporter has never been reported. This notwithstanding, some authors postulated that P5C can pass the mitochondrial membrane, and that a P5C/Pro cycle mediated by ProDH and P5CR activity can occur (Hu et al., 2007; Miller et al., 2009). Since P5C/Pro cycling is believed to cause ROS production, P5C oxidation by P5CDH would be essential for limiting cycle activity and avoiding ROS-induced damage (Miller et al., 2009).
In Arabidopsis thaliana a single gene is present for both enzymes utilizing P5C as a substrate, namely P5CR and P5CDH. Until now, neither gene product has been purified in this species. Moreover, no obvious correlations between mRNA and protein levels and a RNA silencing-based regulatory mechanism were described for P5CR and P5CDH, respectively. The P5CR mRNA contains a non-coding region in the 5’ untranslated region (UTR) that at the same time mediates transcript stabilization and translation inhibition during salt and heat stress (Hua et al., 2001). Arabidopsis P5CDH has been found to be associated with another gene, which is partially overlapping on the complementary strand. The transcription of this gene is up-regulated under salt stress conditions, with a consequent production and processing of dsRNA. Short interfering RNA being able to downregulate P5CDH are generated from this process (Borsani et al., 2005).
Although many physiological studies suggested that proline may be implicated in multiple stress protection mechanisms, it is also evident that proline accumulation does not represent a response conferring tolerance per se. Accordingly, several salt- and cold-hypersensitive Arabidopsis mutants accumulate proline at high levels without any apparent beneficial effect on stress tolerance (Liu and Zhu, 1997). Therefore, a proper understanding of how proline accumulation influences particular regulatory pathways in complex stress responses still requires many efforts (Maggio et al., 2002).
Aiming at a better comprehension of the role of proline in plant stress response, Arabidopsis was selected as the experimental system. In this weed, P5CDH and P5CR are present as single genes, a feature that may simplify the analysis of expression patterns. Moreover, the availability of a lot of molecular data as well as the fully sequenced genome provide useful tools for the investigation. However, proper evaluations would be hampered in planta by the small size of Arabidopsis seedlings, a drawback that most likely explains why the proteins involved in proline metabolism have not been purified to date. On the other side, in differentiated plants inter-tissue transport, exclusion mechanisms and cell compartmentalization of proline do occur. As a consequence, it is difficult to know the exact conditions (i.e., ionic vs osmotic, extra- and intracellular concentrations, pressure values) to which a single cell is exposed. The availability of suitable experimental systems to quantify resistance to realistic and reproducible low water potential, or salt and freezing stress conditions is well recognized as a crucial point in genetic studies (Verslues et al., 2006). Suspension cultured cells may represent one of these systems. The possibility of using p5cdh seeds, kindly provided by Dr Dietmar Funck (Konstanz, Germany), was a further stimulus to establish cell suspension cultures from A. thaliana seedlings. Two well dispersed cultures were obtained, and used to evaluate cell responses to various experimental conditions, mainly focusing on the metabolic role of the possible effector of Pro toxicity, P5C
Coenzyme preference of Streptococcus pyogenes δ1-pyrroline-5-carboxylate reductase: evidence supporting NADPH as the physiological electron donor
No full textThe streptococcal enzyme that catalyzes the last step in proline biosynthesis was heterologously expressed and the recombinant protein was purified to electrophoretic homogeneity and characterized thoroughly. As for delta-1-pyrroline-5-carboxylate reductases from other sources, it was able to use either NADH or NADPH as the electron donor in vitro. However, with NADH the activity was markedly inhibited by physiological levels of NADP+.
Results also strengthen the possibility that an unusual ordered substrate binding occurs, in which the dinucleotide binds last
Plant P5C reductase as a new target for aminomethylenebisphosphonates.
No full textA series of N-substituted derivatives of aminomethylenebisphosphonic acid were evaluated as potential inhibitors of -pyrroline-5-carboxylate reductase (EC 1.5.1.2), the enzyme that catalyzes the last step in proline biosynthesis, partially purified from Arabidopsis thaliana suspension cultured cells. At millimolar concentrations, three compounds out of 26 were found to interfere with the catalytic mechanism. One of them, namely, 3,5-dichloropyridyl-aminomethylenebisphosphonic acid, retained such inhibitory activity in the micromolar range. Kinetic analyses ruled out the possibility that the inhibition could simply rely upon the chelating properties of bisphosphonates and showed mechanisms of a noncompetitive type against NADH and an uncompetitive type against pyrroline-5-carboxylic acid, with K
I values of 199 +/- 6 and 10.3 +/- 1.5 uM, respectively. A computer-aided docking analysis, performed on the basis of the crystal structure of the enzyme from Streptococcus pyogenes, suggested that this phosphonate may interact with amino acid residues near the binding site of pyrroline-5-carboxylic acid, thus blocking the substrate in a pocket and preventing its interaction with NADH. Because in higher plants the step catalyzed by pyrroline-5-carboxylate reductase is shared by all pathways leading to proline synthesis, such a compound may represent a lead structure to be exploited for the design of new substances endowed with herbicidal activity
δ 1-Pyrroline-5-carboxylate reductase as a new target for therapeutics: Inhibition of the enzyme from Streptococcus pyogenes and effects in vivo
No full textCompounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growth. In both prokaryotic and eukaryotic microorganisms, unless an ornithine cyclodeaminase is present, the activity of delta-1-pyrroline-5-carboxylate (P5C) reductase is mandatory to proline production, and the enzyme inhibition should result in amino acid starvation, blocking in turn protein synthesis. The ability of some substituted derivatives of aminomethylenebisphosphonic acid and its analogues to interfere
with the activity of the enzyme from the human pathogen Streptococcus pyogenes was investigated. Several compounds
were able to suppress activity in the micromolar range of concentrations, with a mechanism of uncompetitive type with respect to the substrate P5C and non-competitive with respect to the electron donor NAD(P)H. The actual occurrence of enzyme inhibition in vivo was supported by the effects of the most active derivatives upon bacterial growth and free amino acid content
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Tailoring nostoclide structure to target the chloroplastic electron transport chain
No full textAiming to improve their effectiveness, three modifications were introduced into the structure of the natural phytotoxins nostoclides, leading to the synthesis of novel 3-benzyl-4-isopropyl-5- (arylmethylene)furan-2(5H)-ones, 3-benzyl-5-(furan-2-ylmethylene)furan-2(5H)-ones, and 3,4 dihalo-5- arylidenefuran-2(5H)-ones. All compounds were characterized by IR, 1H and 13C NMR, NOEDIF, COSY, HETCOR and MS spectrometry. Increasing the length of the molecule was found to reduce the ability to interfere with ferricyanide reduction by isolated chloroplasts. The addition of an isopropyl moiety into the heterocyclic ring, as in naturally-occurring nostoclides, did not influence the inhibitory potential. Also the replacement of the electron-drawing phenyl substituent with two halogen residues did not improve the resulting activity. However, both latter modifications influenced the interaction with the photosynthetic machinery. These analogues could therefore represent novel leads to be explored toward the development of new herbicides targeting the chloroplastic electron transport chain. © ARKAT-USA, Inc
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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