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1.
Plants (Basel) ; 11(3)2022 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-35161327

RESUMO

The French National Research Institute for Agriculture, Food and the Environment (INRAE) conserves and distributes five vegetable collections as seeds: the aubergine* (in this article the word aubergine refers to eggplant), pepper, tomato, melon and lettuce collections, together with their wild or cultivated relatives, are conserved in Avignon, France. Accessions from the collections have geographically diverse origins, are generally well-described and fixed for traits of agronomic or scientific interest and have available passport data. In addition to currently conserving over 10,000 accessions (between 900 and 3000 accessions per crop), the centre maintains scientific collections such as core collections and bi- or multi-parental populations, which have also been genotyped with SNP markers. Each collection has its own merits and highlights, which are discussed in this review: the aubergine collection is a rich source of crop wild relatives of Solanum; the pepper, melon and lettuce collections have been screened for resistance to plant pathogens, including viruses, fungi, oomycetes and insects; and the tomato collection has been at the heart of genome-wide association studies for fruit quality traits and environmental stress tolerance.

2.
Clin J Sport Med ; 32(2): 114-121, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33605602

RESUMO

OBJECTIVE: To describe the collaborative findings across a broad array of subspecialties in children and adolescents with postconcussion syndrome (PCS) in a pediatric multidisciplinary concussion clinic (MDCC) setting. DESIGN: Retrospective analysis. SETTING: Multidisciplinary concussion clinic at a pediatric tertiary-level hospital. PATIENTS: Fifty-seven patients seen in MDCC for evaluation and management of PCS between June 2014 and January 2016. INTERVENTIONS: Clinical evaluation by neurology, sports medicine, otolaryngology, optometry, ophthalmology, physical therapy, and psychology. MAIN OUTCOME MEASURES: Specialty-specific clinical findings and specific, treatable diagnoses relevant to PCS symptoms. RESULTS: A wide variety of treatable, specialty-specific diagnoses were identified as potential contributing factors to patients' postconcussion symptoms. The most common treatable diagnoses included binocular vision dysfunction (76%), anxiety, (57.7%), depression (44.2%), new or change in refractive error (21.7%), myofascial pain syndrome (19.2%), and benign paroxysmal positional vertigo (17.5%). CONCLUSIONS: Patients seen in a MDCC setting receive a high number of treatable diagnoses that are potentially related to patients' PCS symptoms. The MDCC approach may (1) increase access to interventions for PCS-related impairments, such as visual rehabilitation, physical therapy, and psychological counseling; (2) provide patients with coordinated medical care across specialties; and (3) hasten recovery from PCS.


Assuntos
Traumatismos em Atletas , Concussão Encefálica , Síndrome Pós-Concussão , Adolescente , Traumatismos em Atletas/diagnóstico , Vertigem Posicional Paroxística Benigna/terapia , Concussão Encefálica/complicações , Concussão Encefálica/diagnóstico , Concussão Encefálica/terapia , Criança , Humanos , Síndrome Pós-Concussão/diagnóstico , Síndrome Pós-Concussão/psicologia , Síndrome Pós-Concussão/terapia , Estudos Retrospectivos
3.
Front Plant Sci ; 10: 1091, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31620143

RESUMO

Plant central metabolism generates reactive oxygen species (ROS), which are key regulators that mediate signalling pathways involved in developmental processes and plant responses to environmental fluctuations. These highly reactive metabolites can lead to cellular damage when the reduction-oxidation (redox) homeostasis becomes unbalanced. Whilst decades of research have studied redox homeostasis in leaves, fundamental knowledge in fruit biology is still fragmentary. This is even more surprising when considering the natural profusion of fruit antioxidants that can process ROS and benefit human health. In this review, we explore redox biology in fruit and provide an overview of fruit antioxidants with recent examples. We further examine the central role of the redox hub in signalling during development and stress, with particular emphasis on ascorbate, also referred to as vitamin C. Progress in understanding the molecular mechanisms involved in the redox regulations that are linked to central metabolism and stress pathways will help to define novel strategies for optimising fruit nutritional quality, fruit production and storage.

4.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31028095

RESUMO

TRIM E3 ubiquitin ligases regulate multiple cellular processes, and their dysfunction is linked to disease. They are characterised by a conserved N-terminal tripartite motif comprising a RING, B-box domains, and a coiled-coil region, with C-terminal domains often mediating substrate recruitment. TRIM proteins are grouped into 11 classes based on C-terminal domain identity. Class VI TRIMs, TRIM24, TRIM33, and TRIM28, have been described as transcriptional regulators, a function linked to their C-terminal plant homeodomain and bromodomain, and independent of their ubiquitination activity. It is unclear whether E3 ligase activity is regulated in family members where the C-terminal domains function independently. Here, we provide a detailed biochemical characterisation of the RING domains of class VI TRIMs and describe the solution structure of the TRIM28 RING. Our study reveals a lack of activity of the isolated RING domains, which may be linked to the absence of self-association. We propose that class VI TRIMs exist in an inactive state and require additional regulatory events to stimulate E3 ligase activity, ensuring that associated chromatin-remodelling factors are not injudiciously degraded.


Assuntos
Domínios e Motivos de Interação entre Proteínas , Proteínas com Motivo Tripartido/química , Proteínas com Motivo Tripartido/metabolismo , Sequência de Aminoácidos , Catálise , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade , Proteína 28 com Motivo Tripartido/química , Proteína 28 com Motivo Tripartido/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo
5.
Acta Biol Hung ; 69(4): 464-480, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30587018

RESUMO

We aimed to examine the response of three tomato introgression lines (IL925.3, IL925.5 and IL925.6) to NaCl stress. These lines originated from a cross between M82 (Solanum lycopersicum) and the wild salttolerant tomato Solanum pennellii, each line containing a different fragment of the S.pennellii genome. Salt-sensitive phenotypes related to plant growth and physiology, and the response of antioxidants, pigments and antioxidant enzymes were measured. In general, salt stress decreased the fresh weight of leaves, leaf area and leaf number and an increase of Na+ accumulation in aerial parts was observed, which caused a reduction in the absorption of K+ and Ca2+. Salt stress also induced a decrease in chlorophyll, carotenoids and lipid peroxidation (MDA) and an increase in anthocyanins and reduced ascorbate, although some differences were seen between the lines, for example for carotenoid levels. Guaiacol peroxidase, catalase and glutathione reductase activity enhanced in aerial parts of the lines, but again some differences were seen between the three lines. It is concluded that IL925.5 might be the most sensitive line to salt stress as its dry weight loss was the greatest in response to salt and this line showed the highest Na+ ion accumulation in leaves.


Assuntos
Lycopersicon esculentum/efeitos dos fármacos , Plantas Geneticamente Modificadas/efeitos dos fármacos , Tolerância ao Sal , Cloreto de Sódio/farmacologia , Antocianinas/metabolismo , Ácido Ascórbico/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Cruzamentos Genéticos , Enzimas/metabolismo , Genótipo , Peroxidação de Lipídeos/efeitos dos fármacos , Lycopersicon esculentum/genética , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Tolerância ao Sal/genética , Cloreto de Sódio/metabolismo
6.
Nat Commun ; 9(1): 1820, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29739942

RESUMO

RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1.


Assuntos
Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/imunologia , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/imunologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/imunologia , Sequência de Aminoácidos , Células Cultivadas , Proteína DEAD-box 58/imunologia , Células HEK293 , Humanos , Interferons/biossíntese , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA Viral/imunologia , Receptores Imunológicos , Transdução de Sinais , Fatores de Transcrição/química , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/química , Ubiquitinação
7.
Front Plant Sci ; 9: 137, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29491875

RESUMO

Changing the balance between ascorbate, monodehydroascorbate, and dehydroascorbate in plant cells by manipulating the activity of enzymes involved in ascorbate synthesis or recycling of oxidized and reduced forms leads to multiple phenotypes. A systems biology approach including network analysis of the transcriptome, proteome and metabolites of RNAi lines for ascorbate oxidase, monodehydroascorbate reductase and galactonolactone dehydrogenase has been carried out in orange fruit pericarp of tomato (Solanum lycopersicum). The transcriptome of the RNAi ascorbate oxidase lines is inversed compared to the monodehydroascorbate reductase and galactonolactone dehydrogenase lines. Differentially expressed genes are involved in ribosome biogenesis and translation. This transcriptome inversion is also seen in response to different stresses in Arabidopsis. The transcriptome response is not well correlated with the proteome which, with the metabolites, are correlated to the activity of the ascorbate redox enzymes-ascorbate oxidase and monodehydroascorbate reductase. Differentially accumulated proteins include metacaspase, protein disulphide isomerase, chaperone DnaK and carbonic anhydrase and the metabolites chlorogenic acid, dehydroascorbate and alanine. The hub genes identified from the network analysis are involved in signaling, the heat-shock response and ribosome biogenesis. The results from this study therefore reveal one or several putative signals from the ascorbate pool which modify the transcriptional response and elements downstream.

8.
Med Sci Sports Exerc ; 50(4): 739-746, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29140897

RESUMO

PURPOSE: Few studies have examined the psychological and psychophysiological effects of recuperative music after exhaustive exercise. The main purpose of the present study was to examine the effects of two music conditions compared with a no-music control on psychological and psychophysiological recovery processes after exercise. METHODS: A randomized, fully counterbalanced, crossover design was used. Core affect, salivary cortisol, heart rate, and blood pressure were measured before exhaustive exercise, immediately after, and in 10-, 20-, and 30-min intervals during passive recovery (21 women and 21 men; 20.9 ± 1.7 yr) over three separate trials (slow, sedative music; fast, stimulative music; no-music control). The exercise task entailed incremental cycle ergometry performed at 75 rpm with an increase in intensity of 22.5 W·min at the end of each minute until exhaustion. Data were analyzed using mixed-model 3 (condition) × 4 (time) × 2 (gender) MANOVA/ANCOVA. RESULTS: The largest decline in affective arousal between active and passive recovery phases was evident in the slow, sedative condition (ηp = 0.50). Women had a more pronounced reduction in arousal than did men in the slow, sedative music condition. Heart rate measures showed that fast, stimulative music inhibited the return of heart rate toward resting levels (ηp = 0.06). Similarly, salivary cortisol levels tended to be lower in response to slow, sedative music (ηp = 0.11). There was a main effect of condition for affective valence indicating that the slow, sedative condition elicited more positive affective responses compared with the control and fast, stimulative conditions (ηp = 0.12). CONCLUSIONS: The present findings support the notion that slow, sedative music can expedite the recovery process immediately after strenuous exercise.


Assuntos
Exercício Físico/fisiologia , Exercício Físico/psicologia , Música , Afeto , Nível de Alerta , Pressão Sanguínea , Estudos Cross-Over , Feminino , Frequência Cardíaca , Humanos , Hidrocortisona/análise , Masculino , Psicofisiologia , Descanso , Saliva/química , Adulto Jovem
9.
J Plant Physiol ; 222: 1-8, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29287283

RESUMO

Ascorbate redox metabolism and growth have been shown to be linked and related to the activity of enzymes that produce or remove the radical monodehydroascorbate, the semi-oxidized form of ascorbate (ascorbate oxidase or peroxidase and monodehydroascorbate reductase respectively). Previous work in cherry tomato has revealed correlations between monodehydroascorbate reductase and ascorbate oxidase activity and fruit yield: decreased whole plant MDHAR activity decreases yield while decreased whole plant ascorbate oxidase activity increases yield under unfavourable environmental conditions. We aimed to investigate if similar effects on yield are obtained in a large-fruited variety of tomato, Moneymaker. Furthermore we wished to establish whether previously observed effects on yield in cherry tomato following changes in whole plant enzyme activity could be reproduced by reducing MDHAR activity in fruit only by using a fruit-specific promoter in cherry tomato (West Virginia 106). In Moneymaker, RNAi lines for monodehydroascorbate reductase did not show significant yield decrease compared to control lines when plants were grown under optimal or non-optimal conditions of carbon stress generated by mature leaf removal. In addition, we show that a decrease in monodehydroascorbate reductase activity in fruit of cherry tomato had no effect on yield compared to a reduction in whole-plant monodehydroascorbate reductase activity: we therefore show that whole plant MDHAR activity is necessary to maintain yield in cherry tomato, suggesting that the carbon source in autotrophic tissue is more important than fruit sink activity. The present data also revealed differences between cherry and large fruited tomato that could be linked to a source of genetic variability in the response to monodehydroascorbate metabolism in tomato: maybe the domestication of tomato towards large-fruited lines could have affected the importance of MDHAR in yield maintenance.


Assuntos
Ácido Desidroascórbico/análogos & derivados , Frutas/crescimento & desenvolvimento , Lycopersicon esculentum/genética , NADH NADPH Oxirredutases/genética , Proteínas de Plantas/genética , Ácido Desidroascórbico/metabolismo , Frutas/genética , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo
10.
Plant J ; 89(5): 996-1008, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27888536

RESUMO

Ascorbate content in plants is controlled by its synthesis from carbohydrates, recycling of the oxidized forms and degradation. Of these pathways, ascorbate degradation is the least studied and represents a lack of knowledge that could impair improvement of ascorbate content in fruits and vegetables as degradation is non-reversible and leads to a depletion of the ascorbate pool. The present study revealed the nature of degradation products using [14 C]ascorbate labelling in tomato, a model plant for fleshy fruits; oxalate and threonate are accumulated in leaves, as is oxalyl threonate. Carboxypentonates coming from diketogulonate degradation were detected in relatively insoluble (cell wall-rich) leaf material. No [14 C]tartaric acid was found in tomato leaves. Ascorbate degradation was stimulated by darkness, and the degradation rate was evaluated at 63% of the ascorbate pool per day, a percentage that was constant and independent of the initial ascorbate or dehydroascorbic acid concentration over periods of 24 h or more. Furthermore, degradation could be partially affected by the ascorbate recycling pathway, as lines under-expressing monodehydroascorbate reductase showed a slight decrease in degradation product accumulation.


Assuntos
Ácido Ascórbico/metabolismo , Butiratos/metabolismo , Lycopersicon esculentum/metabolismo , Oxalatos/metabolismo , Frutas/metabolismo , Frutas/efeitos da radiação , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Lycopersicon esculentum/genética , Lycopersicon esculentum/efeitos da radiação , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredução/efeitos da radiação , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação
11.
J Arthroplasty ; 32(2): 601-609, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27597431

RESUMO

BACKGROUND: Short-stemmed femoral components facilitate reduced exposure surgical techniques while preserving native bone. A clinically successful stem should ideally reduce risk for stress shielding while maintaining adequate primary stability for biological fixation. We asked (1) how stem-length changes cortical strain distribution in the proximal femur in a fit-and-fill geometry and (2) if short-stemmed components exhibit primary stability on par with clinically successful designs. METHODS: Cortical strain was assessed via digital image correlation in composite femurs implanted with long, medium, and short metaphyseal fit-and-fill stem designs in a single-leg stance loading model. Strain was compared to a loaded, unimplanted femur. Bone-implant micromotion was then compared with reduced lateral shoulder short stem and short tapered-wedge designs in cyclic axial and torsional testing. RESULTS: Femurs implanted with short-stemmed components exhibited cortical strain response most closely matching that of the intact femur model, theoretically reducing the potential for proximal stress shielding. In micromotion testing, no difference in primary stability was observed as a function of reduced stem length within the same component design. CONCLUSION: Our findings demonstrate that within this fit-and-fill stem design, reduction in stem length improved proximal cortical strain distribution and maintained axial and torsional stability on par with other stem designs in a composite femur model. Short-stemmed implants may accommodate less invasive surgical techniques while facilitating more physiological femoral loading without sacrificing primary implant stability.


Assuntos
Fêmur/cirurgia , Prótese de Quadril , Desenho de Prótese , Fêmur/fisiologia , Humanos , Estresse Mecânico
12.
J Exp Bot ; 67(15): 4767-77, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27382114

RESUMO

GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues.


Assuntos
Ácido Ascórbico/biossíntese , Carboidratos Epimerases/metabolismo , Parede Celular/metabolismo , Lycopersicon esculentum/enzimologia , Carboidratos Epimerases/fisiologia , Parede Celular/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Germinação/fisiologia , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , Pólen/metabolismo
13.
Adv Ther ; 33(2): 199-213, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26833303

RESUMO

INTRODUCTION: The purpose of the present study was to determine whether the abatacept autoinjector can be used by the intended population without patterns of preventable use errors, and is acceptable when assessed against key user needs. METHODS: Two independently conducted simulated-use studies, with no active drug administered, quantified use errors and evaluated the abatacept autoinjector and competitor devices on key attributes (comfort, control, ease of use, confidence of dose) and overall acceptability. Autoinjector preference was also assessed. Participants were patients with rheumatoid arthritis, caregivers, and healthcare professionals (HCPs). Participants were informed that a new rheumatoid arthritis autoinjector was being tested but were blinded to the intended drug and sponsor identity. RESULTS: In the formative (pre-validation) study (n = 54), two high-priority use errors occurred, both of which resulted from protocol non-compliance rather than mental confusion or physical limitations. In the summative (validation) study (n = 99), one high-priority use error occurred; this was deemed a simulated-use study artifact as participant behavior was guided by actual experience associated with the feel of drug delivery into the skin rather than by protocol, so no mitigation steps were considered necessary. Across user groups, average scores were consistently high for the pre-defined key attributes. Overall acceptability scores (7-point scale) were significantly higher for the abatacept versus competitor autoinjectors-formative study: patients 6.7 vs 5.2 (P = 0.0001), caregivers 7.0 vs 4.6 (P = 0.0093), HCPs 6.8 vs 5.1 (P = 0.0020); summative study: patients 6.5 vs 5.9 (P = 0.0404), caregivers 6.8 vs 5.8 (P = 0.0047), HCPs 6.8 vs 5.1 (P = 0.0002). The abatacept autoinjector was preferred to competitor devices: patients 85.7% vs 14.3% (P = 0.00002), caregivers 84.2% vs 15.8% (P = 0.00443), HCPs 95.0% vs 5.0% (P = 0.00004). Positive experiences with the abatacept autoinjector were attributed to the rubberized grip, device size, visualization of dose progression, button ergonomics, and ease of use. CONCLUSION: The abatacept autoinjector demonstrated usability without patterns of preventable use errors, and with high acceptability ratings across all key attributes assessed. Preference over competitor autoinjectors was due to device ergonomics, visualization of dose progression, confidence of dose delivery, and overall ease of use. FUNDING: Bristol-Myers Squibb.


Assuntos
Abatacepte/administração & dosagem , Antirreumáticos/administração & dosagem , Artrite Reumatoide/tratamento farmacológico , Sistemas de Liberação de Medicamentos/instrumentação , Preferência do Paciente , Abatacepte/uso terapêutico , Adolescente , Adulto , Idoso , Antirreumáticos/uso terapêutico , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agulhas , Resultado do Tratamento , Adulto Jovem
14.
Plant Cell Environ ; 39(6): 1279-92, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26510400

RESUMO

Ascorbate is oxidized into the radical monodehydroascorbate (MDHA) through ascorbate oxidase or peroxidase activity or non-enzymatically by reactive oxygen species. Regeneration of ascorbate from MDHA is ensured by the enzyme MDHA reductase (MDHAR). Previous work has shown that growth processes and yield can be altered by modifying the activity of enzymes that recycle ascorbate; therefore, we have studied similar processes in cherry tomato (Solanum lycopersium L.) under- or overexpressing MDHAR. Physiological and metabolic characterization of these lines was carried out under different light conditions or by manipulating the source-sink ratio. Independently of the light regime, slower early growth of all organs was observed in MDHAR silenced lines, decreasing final fruit yield. Photosynthesis was altered as was the accumulation of hexoses and sucrose in a light-dependent manner in plantlets. Sucrose accumulation was also repressed in young fruits and final yield of MDHAR silenced lines showed a stronger decrease under carbon limitation, and the phenotype was partially restored by reducing fruit load. Ascorbate and MDHA appear to be involved in control of growth and sugar metabolism in cherry tomato and the associated enzymes could be potential targets for yield improvement.


Assuntos
Lycopersicon esculentum/fisiologia , NADH NADPH Oxirredutases/metabolismo , Ácido Ascórbico/metabolismo , Metabolismo dos Carboidratos , Clorofila/metabolismo , Ácido Desidroascórbico/análogos & derivados , Ácido Desidroascórbico/metabolismo , Luz , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/crescimento & desenvolvimento , NADH NADPH Oxirredutases/antagonistas & inibidores , Fotossíntese , Transpiração Vegetal
15.
Mol Microbiol ; 99(4): 749-66, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26538516

RESUMO

Flagellar type III secretion systems (T3SS) contain an essential cytoplasmic-ring (C-ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non-flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF-T3SS C-ring component in Shigella flexneri is alternatively translated to produce both full-length (Spa33-FL) and a short variant (Spa33-C), with both required for secretion. They associate in a 1:2 complex (Spa33-FL/C2) that further oligomerises into elongated arrays in vitro. The structure of Spa33-C2 and identification of an unexpected intramolecular pseudodimer in Spa33-FL reveal a molecular model for their higher order assembly within NF-T3SS. Spa33-FL and Spa33-C are identified as functional counterparts of a FliM-FliN fusion and free FliN respectively. Furthermore, we show that Thermotoga maritima FliM and FliN form a 1:3 complex structurally equivalent to Spa33-FL/C2 , allowing us to propose a unified model for C-ring assembly by NF-T3SS and flagellar-T3SS.


Assuntos
Proteínas de Bactérias/metabolismo , Shigella flexneri/genética , Thermotoga maritima/fisiologia , Sistemas de Secreção Tipo III/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalização , Cristalografia por Raios X , Flagelos/fisiologia , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Shigella flexneri/fisiologia
16.
Angew Chem Int Ed Engl ; 54(51): 15374-8, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-26537742

RESUMO

Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1-antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site-specific (15) N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond-nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small-amplitude overall anisotropic motion sampling the interaction interface in the complex.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Ligação Proteica
17.
Angew Chem Weinheim Bergstr Ger ; 127(51): 15594-15598, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-27478273

RESUMO

Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1-antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site-specific 15N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond-nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small-amplitude overall anisotropic motion sampling the interaction interface in the complex.

18.
Plant Physiol ; 165(3): 1120-1132, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24894148

RESUMO

Genome-wide association studies have been successful in identifying genes involved in polygenic traits and are valuable for crop improvement. Tomato (Solanum lycopersicum) is a major crop and is highly appreciated worldwide for its health value. We used a core collection of 163 tomato accessions composed of S. lycopersicum, S. lycopersicum var cerasiforme, and Solanum pimpinellifolium to map loci controlling variation in fruit metabolites. Fruits were phenotyped for a broad range of metabolites, including amino acids, sugars, and ascorbate. In parallel, the accessions were genotyped with 5,995 single-nucleotide polymorphism markers spread over the whole genome. Genome-wide association analysis was conducted on a large set of metabolic traits that were stable over 2 years using a multilocus mixed model as a general method for mapping complex traits in structured populations and applied to tomato. We detected a total of 44 loci that were significantly associated with a total of 19 traits, including sucrose, ascorbate, malate, and citrate levels. These results not only provide a list of candidate loci to be functionally validated but also a powerful analytical approach for finding genetic variants that can be directly used for crop improvement and deciphering the genetic architecture of complex traits.

19.
Angiogenesis ; 17(3): 553-62, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24154861

RESUMO

Hypoxia-inducible factor-1 (HIF-1) plays an important role in retinal and subretinal neovascularization (NV). Increased levels of HIF-1 cause increased expression of vascular endothelial growth factor (VEGF-A) and current therapies for ocular NV focus on neutralizing VEGF-A, but there is mounting evidence that other HIF-1-responsive gene products may also participate. In this study, we tested the effect of a designed ankyrin repeat protein (DARPin) that selectively binds and antagonizes the hypoxia-regulated gene product PDGF-BB in three models of subretinal NV (relevant to neovascular age-related macular degeneration) and compared its effects to a DARPin that selectively antagonizes VEGF-A. Daily intraperitoneal injections of 10 mg/kg of the anti-PDGF-BB DARPin or 1 mg/kg of the anti-VEGF DARPin significantly suppressed subretinal NV from laser-induced rupture of Bruch's membrane. Injections of 1 mg/kg/day of the anti-PDGF-BB DARPin had no significant effect, but when combined with 1 mg/kg/day of the anti-VEGF-A DARPin there was greater suppression than injection of the anti-VEGF-A DARPin alone. In Vldlr (-/-) mice which spontaneously develop subretinal NV, intraocular injection of 1.85 µg of anti-PDGF-BB or anti-VEGF-A DARPin caused significant suppression of NV and when combined there was greater suppression than with either alone. The two DARPins also showed an additive effect in Tet/opsin/VEGF double transgenic mice, a particularly severe model of subretinal NV and exudative retinal detachment. In addition, intraocular injection of 1.85 µg of anti-PDGF-BB DARPin strongly suppressed ischemia-induced retinal NV, which is relevant to proliferative diabetic retinopathy and retinopathy of prematurity. These data demonstrate that PDGF-BB is another hypoxia-regulated gene product that along with VEGF-A contributes to ocular NV and suppression of both provides an additive effect.


Assuntos
Proteínas Proto-Oncogênicas c-sis/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Neovascularização Retiniana/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Becaplermina , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/patologia , Injeções Intraoculares , Isquemia/complicações , Isquemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Opsinas/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-sis/metabolismo , Ratos , Receptores de LDL/deficiência , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Descolamento Retiniano/tratamento farmacológico , Descolamento Retiniano/patologia , Descolamento Retiniano/prevenção & controle , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
PLoS One ; 8(12): e84474, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24367665

RESUMO

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31 °C) and irradiance regimes (darkness or 150 µmol m(-2) s(-1)). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27 °C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12 °C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31 °C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12 °C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.


Assuntos
Ácido Ascórbico/biossíntese , Ácido Ascórbico/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Temperatura Alta , Luz , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Ácido Ascórbico/efeitos da radiação , Frutas/genética , Frutas/metabolismo , Frutas/efeitos da radiação , Regulação da Expressão Gênica de Plantas/genética , Glutationa/metabolismo , Lycopersicon esculentum/efeitos da radiação , Oxirredução/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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