Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

<p>Abstract</p> <p>Background</p> <p>Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts.</p> <p>Results</p> <p>A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way.</p> <p>Conclusions</p> <p>We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.</p

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data

The Molloy Student Literary Magazine Volume 10

The Molloy Student Literary Magazine, sponsored by Molloy College’s Office of Student Affairs, is devoted to publishing the best previously unpublished works of prose, poetry, drama, literary review, criticism, and other literary genres, that the Molloy student community has to offer. The journal welcomes submissions, for possible publication, from currently enrolled Molloy students at all levels. All submitted work will undergo a review process initiated by the Managing Editor prior to a decision being made regarding publication of said work. Given sufficient content, The Molloy Student Literary Magazine is published twice annually in Spring and Fall. Interested contributors from the currently enrolled Molloy student community should send work via e-mail attachment and brief cover letter (including a two-sentence biographical statement) to: Dr. Damian Ward Hey, Managing Editor, The Molloy Student Literary Magazine: [email protected]. Enrolled students who are interested in becoming members of The Molloy Student Literary Magazine staff may e-mail letters of inquiry. Excelsior!https://digitalcommons.molloy.edu/eng_litmag/1002/thumbnail.jp

Isolation of tomato fruit chromoplasts and determination of ATP levels

It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays.Peer reviewe

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts

Background Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. Results A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. Conclusions We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data

Validazione Linguistica e Adattamento Culturale della Scala Infermieristica sul Ragionamento Clinico

 Introduzione: Il ragionamento clinico è un processo nel corso del quale gli infermieri raccolgono spunti, elaborano le informazioni arrivano a comprendere il problema o la situazione del paziente pianificando e attuando gli interventi, valutando i risultati. Disegno dello studio: Obiettivo di questo studio è effettuare la validazione linguistica e culturale per il contesto Italiano della Nurse Clinical Reasoning Scale (NCRS).Metodi: L'adattamento transculturale per sviluppare la versione italiana del questionario è stato effettuato seguendo le linee guida proposte da Beaton e collaboratori nel 2000.Risultati: I risultati hanno evidenziato che la versione italiana dello strumento risulta affidabile sia dimostrando una buona coerenza interna (Alpha di Cronbach = 0.90), che una buona stabilití  (ICC= 0.90; CI = 0.87-0.92; p= &lt;0.001) e può essere proposta come un interessante mezzo di valutazione degli studenti infermieri nel corso dei tirocini clinici ed infermieri nella loro pratica clinica quotidiana.Conclusioni: Avere a disposizione strumenti in lingua italiana favorisce la realizzazione di nuovi progetti di ricerca in vari ambiti, come quello sanitario, al fine di garantire la centralití  del paziente nel processo assistenziale. Parole chiave: ragionamento clinico, competenze, validazione linguistica, infermieristica, studenti infermieri Linguistic validation and cultural adaptation of the Nurse Clinical Reasoning ScaleIntroduction: The clinical reasoning is a process in which nurses collect ideas, process information, they come to understand the problem or the patient's situation by planning and implementing interventions, evaluating the results. Study design: The aim of this study is to perform linguistic and cultural validation for the Italian context of the Nurse Clinical Reasoning Scale (NCRS). Methods: Transcultural adaptation to develop the Italian version of the questionnaire was carried out following the guidelines proposed by Beaton and collaborators in 2000. Results: The results showed that the Italian version of the instrument is reliable both showing a good internal consistency (Cronbach's Alpha = 0.90), and a good stability (ICC = 0.90; CI = 0.87-0.92; p = &lt;0.001) and can be proposed as an interesting means of evaluating nursing students in the course of clinical placements and nurses in their daily clinical practice. Conclusions: Having tools available in Italian promotes the implementation of new research projects in various areas, such as health, in order to ensure the centrality of the patient in the care process. Key words: clinical reasoning, skills, linguistic validation, nursing, nursing student