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1.
Mol Cell Proteomics ; 23(11): 100845, 2024 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-39321874

RESUMEN

Protein acetylation is a key co- and post-translational modification. However, how different types of acetylation respond to environmental stress is still unknown. To address this, we investigated the role of a member of the newly discovered family of plastid acetyltransferases (GNAT2), which features both lysine- and N-terminal acetyltransferase activities. Our study aimed to provide a holistic multi-omics acetylation-dependent view of plant acclimation to short-term light changes. We found that both the yield and coverage of the N-terminal acetylome remained unchanged in WT and gnat2-KO backgrounds after 2 h of exposure to high light or darkness. Similarly, no differences in transcriptome or adenylate energy charge were observed between the genotypes under the tested light conditions. In contrast, the lysine acetylome proved to be sensitive to the changes in light conditions, especially in the gnat2 background. This suggests unique strategies of plant acclimation for quick responses to environmental changes involving lysine, but not N-terminal, GNAT2-mediated acetylation activity.

2.
Plant J ; 2024 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-39115024

RESUMEN

Pea is a grain legume crop with a high potential to accelerate the food transition due to its high seed protein content and relatively well-balanced amino acid composition. The critical role of external sulfur (S) supply in determining seed yield and seed quality in pea makes it essential to understand the impact of whole plant S management on the trade-off between these two traits. Here, we investigated the physiological relevance of vacuolar sulfate remobilization by targeting PsSULTR4, the only pea sulfate transporter showing substantial similarity to the vacuolar sulfate exporter AtSULTR4;1. Five mutations in PsSULTR4 were identified by TILLING (Targeting Induced Local Lesions IN Genomes), two of which, a loss of function (W78*) and a missense (E568K), significantly decreased seed yield under S deprivation. We demonstrate that PsSULTR4 triggers S distribution from source tissues, especially lower leaves, to reproductive organs to maintain seed yield under S deficiency. Under sufficient S supply, sultr4 seeds display lower levels of the S-rich storage protein PA1 at maturity. They also overaccumulate sulfate in the endosperm at the onset of seed filling. These findings uncover a role of PsSULTR4 in the remobilization of vacuolar sulfate during embryo development, allowing the efficient synthesis of S-rich proteins. Our study uncovers that PsSULTR4 functions (i) in source tissues to remobilize stored vacuolar sulfate for seed production under low S availability and (ii) in developing seeds well supplied with S to fine-tune sulfate remobilization from the endosperm as a critical control point for storage activities in the embryo.

3.
Plant Physiol ; 195(4): 3097-3118, 2024 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-38588051

RESUMEN

In humans and plants, 40% of the proteome is cotranslationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex is comprised of the catalytic subunit Nα-acetyltransferase 10 (NAA10) and the ribosome-anchoring subunit NAA15. The regulatory subunit Huntingtin Yeast Partner K (HYPK) and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis (Arabidopsis thaliana), only AtHYPK is known to interact with AtNatA. Here we uncover the AtNAA50 interactome and provide evidence for the association of AtNAA50 with NatA at ribosomes. In agreement with the latter, a split-luciferase approach demonstrated close proximity of AtNAA50 and AtNatA in planta. Despite their interaction, AtNatA/HYPK and AtNAA50 exerted different functions in vivo. Unlike NatA/HYPK, AtNAA50 did not modulate drought tolerance or promote protein stability. Instead, transcriptome and proteome analyses of a novel AtNAA50-depleted mutant (amiNAA50) implied that AtNAA50 negatively regulates plant immunity. Indeed, amiNAA50 plants exhibited enhanced resistance to oomycetes and bacterial pathogens. In contrast to what was observed in NatA-depleted mutants, this resistance was independent of an accumulation of salicylic acid prior to pathogen exposure. Our study dissects the in vivo function of the NatA interactors HYPK and NAA50 and uncovers NatA-independent roles for NAA50 in plants.


Asunto(s)
Acetiltransferasas , Proteínas de Arabidopsis , Arabidopsis , Acetiltransferasa E N-Terminal , Inmunidad de la Planta , Acetiltransferasas/metabolismo , Acetiltransferasas/genética , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/microbiología , Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa A N-Terminal/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/genética , Inmunidad de la Planta/genética , Pseudomonas syringae/fisiología , Pseudomonas syringae/patogenicidad , Ácido Salicílico/metabolismo , Acetiltransferasa E N-Terminal/genética , Acetiltransferasa E N-Terminal/metabolismo
4.
Plant Cell ; 34(10): 3814-3829, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-35792878

RESUMEN

Plants, unlike animals, respond to environmental challenges with comprehensive developmental transitions that allow them to cope with these stresses. Here we discovered that antagonistic activation of the Target of Rapamycin (TOR) kinase in Arabidopsis thaliana roots and shoots is essential for the nutrient deprivation-induced increase in the root-to-shoot ratio to improve foraging for mineral ions. We demonstrate that sulfate limitation-induced downregulation of TOR in shoots activates autophagy, resulting in enhanced carbon allocation to the root. The allocation of carbon to the roots is facilitated by the specific upregulation of the sucrose-transporter genes SWEET11/12 in shoots. SWEET11/12 activation is indispensable for enabling sucrose to act as a carbon source for growth and as a signal for tuning root apical meristem activity via glucose-TOR signaling. The sugar-stimulated TOR activity in the root suppresses autophagy and maintains root apical meristem activity to support root growth to enhance mining for new sulfate resources in the soil. We provide direct evidence that the organ-specific regulation of autophagy is essential for the increased root-to-shoot ratio in response to sulfur limitation. These findings uncover how sulfur limitation controls the central sensor kinase TOR to enable nutrient recycling for stress-induced morphological adaptation of the plant body.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autofagia/genética , Carbono , Regulación de la Expresión Génica de las Plantas/genética , Glucosa , Proteínas de Transporte de Membrana , Meristema/metabolismo , Nutrientes , Fosfatidilinositol 3-Quinasas , Raíces de Plantas/metabolismo , Sirolimus , Suelo , Sacarosa , Sulfatos , Azufre , Serina-Treonina Quinasas TOR/metabolismo
5.
Plant Cell ; 34(11): 4428-4452, 2022 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-35938694

RESUMEN

Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling.


Asunto(s)
Arabidopsis , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Mamíferos/metabolismo
6.
New Phytol ; 2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-39351644

RESUMEN

Rice grains typically contain relatively high levels of toxic arsenic (As) but low levels of essential micronutrients. Biofortification of essential micronutrients while decreasing As accumulation in rice would benefit human nutrition and health. We generated transgenic rice expressing a gain-of-function mutant allele astol1 driven by the OsGPX1 promoter. astol1 encodes a plastid-localized O-acetylserine (thiol) lyase (OAS-TL) with Ser189Asn substitution (OsASTOL1S189N), which enhances cysteine biosynthesis by forming an indissociable cysteine synthase complex with its partner serine acetyltransferase (SAT). The effects on growth, As tolerance, and nutrient and As accumulation in rice grain were evaluated in hydroponic, pot and field experiments. The expression of OsASTOL1S189N in pOsGPX1::astol1 transgenic lines enhanced SAT activity, sulphate uptake, biosynthesis of cysteine, glutathione, phytochelatins and nicotianamine, and enhanced tolerance to As. The expression of OsASTOL1S189N decreased As accumulation while increased the accumulation of multiple macronutrients (especially sulphur, nitrogen and potassium) and micronutrients (especially zinc and selenium) in rice grain in a pot experiment and two field experiments, and had little effect on plant growth and grain yield. Our study provides a new strategy to genetically engineer rice to biofortify multiple essential nutrients, reducing As accumulation in rice grain and enhancing As tolerance simultaneously.

7.
Plant Physiol ; 193(3): 2086-2104, 2023 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-37427787

RESUMEN

The acetylation-dependent (Ac/)N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3 ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multiomics approaches to characterize potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3 ligases in the Nt-acetylation-(NTA)-dependent turnover of proteins at global- and protein-specific scales. Arabidopsis has two endoplasmic reticulum (ER)-localized DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wild type, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localized SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.


Asunto(s)
Arabidopsis , Proteínas de Saccharomyces cerevisiae , Animales , Acetilación , Arabidopsis/genética , Arabidopsis/metabolismo , Mamíferos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Escualeno-Monooxigenasa/metabolismo , Esteroles , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
8.
BMC Public Health ; 24(1): 2950, 2024 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-39448994

RESUMEN

BACKGROUND: Allergic diseases are among the most common chronic diseases in childhood. Early childhood allergy prevention (ECAP) behaviors of those caring for the infant during pregnancy and the first months of life may influence the risk of allergy development over the life course. Motivation and intention to use appropriate primary ECAP measures are thus of critical importance. AIMS: To characterize parental ECAP motivation, (a) valid indicators will be developed and (b) typical parental characteristics will be identified. (c) According to socio-cognitive models, the predictive value of parental risk perception, control belief and self-efficacy for parental ECAP motivation shall be determined. METHOD: A sample of N = 343 (expectant) mothers of infants completed a questionnaire on self-reported ECAP motivation, risk perception, control belief, and self-efficacy. The cross-sectional data were analyzed using latent class analysis and structural equation modelling including nominal regression models. RESULTS: Four typical maternal response profiles (motivated to a customary degree, 70%; motivated to use primary preventive measures, 17.8%; reluctant towards new prevention measures, 6.4%; highly motivated to apply preventive measures in case of an existing allergy, 5.8%) could be identified for the items on ECAP motivation. After splitting the model variables "risk perception" (allergy vs. allergy-associated general health problems) and "self-efficacy" (trust vs. insecurity) a satisfactory model-fit was achieved (CFI = .939; RMSEA = .064). Particularly, increased "risk perception-allergy" (OR = 1.655) and "self-efficacy-insecurity" (OR = 2.013) as well as lower "risk perception-general health" (OR = 0.555) and "control belief" (OR = 0.217), respectively, are associated with higher ECAP motivation. CONCLUSION: The use of ECAP-measures by parents to protect their newborns from allergies is important, but there are deficiencies in their implementation. Based on a social cognitive model approach, predictive characteristics could be identified, which are associated with increased motivation to implement ECAP-measures. For public health our findings provide a promising basis for conception of behavioral and environmental ECAP prevention measures and their motivated implementation by parents.


Asunto(s)
Hipersensibilidad , Análisis de Clases Latentes , Madres , Motivación , Humanos , Femenino , Madres/psicología , Madres/estadística & datos numéricos , Lactante , Hipersensibilidad/prevención & control , Adulto , Estudios Transversales , Autoeficacia , Encuestas y Cuestionarios , Masculino
9.
Gesundheitswesen ; 86(S 04): S259-S266, 2024 Sep.
Artículo en Inglés, Alemán | MEDLINE | ID: mdl-39038484

RESUMEN

BACKGROUND: Recent analyses have shown that in health services research in Germany, healthcare organisations are often considered primarily as a study setting, without fully taking their complex organisational nature into account, neither theoretically nor methodologically. Therefore, an initiative was launched to analyse the state of Organisational Health Services Research (OHSR) in Germany and to develop a strategic framework and road map to guide future efforts in the field. This paper summarizes positions that have been jointly developed by consulting experts from the interdisciplinary and international scientific community. METHODS: In July 2023, a scoping workshop over the course of three days was held with 32 (inter)national experts from different research fields centred around OHSR topics using interactive workshop methods. Participants discussed their perspectives on OHSR, analysed current challenges in OHSR in Germany and developed key positions for the field's development. RESULTS: The seven agreed-upon key positions addressed conceptual and strategic aspects. There was consensus that the field required the development of a research agenda that can guide future efforts. On a conceptual level, the need to address challenges in terms of interdisciplinarity, terminology, organisation(s) as research subjects, international comparative research and utilisation of organisational theory was recognized. On a strategic level, requirements with regard to teaching, promotion of interdisciplinary and international collaboration, suitable funding opportunities and participatory research were identified. CONCLUSIONS: This position paper seeks to serve as a framework to support further development of OHSR in Germany and as a guide for researchers and funding organisations on how to move OHSR forward. Some of the challenges discussed for German OHSR are equally present in other countries. Thus, this position paper can be used to initiate fruitful discussions in other countries.


Asunto(s)
Predicción , Investigación sobre Servicios de Salud , Alemania , Investigación sobre Servicios de Salud/tendencias , Objetivos Organizacionales
10.
J Biol Chem ; 298(4): 101749, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35189141

RESUMEN

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain-containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain-containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD-Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3-STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Azufre , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Liasas de Carbono-Azufre , Cisteína/metabolismo , Citosol/metabolismo , Dominios Proteicos , Azufre/metabolismo , Sulfurtransferasas/metabolismo , Tiosulfato Azufretransferasa/genética , Tiosulfato Azufretransferasa/metabolismo
11.
New Phytol ; 238(1): 96-112, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36464787

RESUMEN

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , NAD/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismo , Fotosíntesis , Oxidorreductasas/metabolismo , Hipoxia/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo
12.
J Exp Bot ; 74(11): 3379-3394, 2023 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-36919245

RESUMEN

Cysteine biosynthesis is essential for translation and represents the entry point of reduced sulfur into plant metabolism. The two consecutively acting enzymes serine acetyltransferase (SAT) and O-acetylserine-thiol-lyase catalyse cysteine production and form the cysteine synthase complex, in which SAT is activated. Here we show that tobacco (Nicotiana tabacum) expressing active SAT in plastids (referred to as PSA lines) shows substantial cysteine accumulation in plastids. Remarkably, enhanced cysteine production in plastids entirely abolished granal stack formation, impaired photosynthesis capacity, and decreased the number of chloroplasts in mesophyll cells of the PSA lines. A transgenic tobacco line expressing active SAT in the cytosol accumulated comparable amounts of thiols but displayed no phenotype. To dissect the consequences of cysteine synthase complex formation from enhanced SAT activity in tobacco plastids, we expressed an enzymatically inactive SAT that can still form the cysteine synthase complex in tobacco plastids (PSI lines). The PSI lines were indistinguishable from the PSA lines, although the PSI lines displayed no increase in plastid-localized SAT activity. Neither PSA lines nor PSI lines suffered from an oxidized redox environment in plastids that could have been causative for the disturbed photosynthesis. From these findings, we infer that the association of the plastid cysteine synthase complex itself triggers a signaling cascade controlling sulfur assimilation and photosynthetic capacity in leaves.


Asunto(s)
Cisteína , Nicotiana , Masculino , Humanos , Cisteína/metabolismo , Nicotiana/metabolismo , Cisteína Sintasa/genética , Cisteína Sintasa/metabolismo , Tilacoides/metabolismo , Antígeno Prostático Específico/metabolismo , Plastidios/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Serina O-Acetiltransferasa/genética , Serina O-Acetiltransferasa/metabolismo , Fotosíntesis , Azufre/metabolismo
13.
BMC Med Res Methodol ; 23(1): 17, 2023 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-36647023

RESUMEN

BACKGROUND: Ensuring motivated and successful study participation is a key challenge in the design and conduct of health research studies. Previously, recruitment barriers and facilitators have been identified mainly from experience, and rarely based on theoretical approaches. We developed a framework of intentional and actional components of engaged participation in public health research studies (INTACT-RS), informed by psychological behavioral models. We aimed a) to identify precise indicators for each framework component and b) to better understand which components and decision processes are essential for study participants. METHODS: Within a multicenter research network, we applied various approaches to recruit parents of newborns, pediatricians, and midwives. All recruitment processes were documented from the perspective of both participants and researchers. We used different qualitative and quantitative data material, which we applied in a multistage process according to the basic principles of qualitative content analysis. RESULTS: INTACT-RS encompasses pre-intentional, intentional and actional phases with a total of n = 15 components covering all aspects of an individual's involvement with a research study. During intention formation, an understanding of efforts and benefits, why participation is valuable beyond contributing to research, and how others perceive the study, were particularly important to (potential) participants. Subsequently (intentional phase), participants consider how and when participation is compatible with their own resources, ability and availability, and hence seek for close communication with, and flexibility and support from the research team. During and after (initial) participation (actional phase), participants' assessment of whether expectations and interests have been met impact crucial further steps, especially the willingness to continue and to recommend participation to others. A strong topic-wise and or supportive participation interest as well as active, continuous exchange with the researchers appeared to be central determinants of study completion and data validity. CONCLUSIONS: A theoretical framework is now available to plan and conduct recruitment of different target groups, which accounts for essential motivational and volitional decision-making processes. Based on empirically specified constructs, possible barriers can be addressed even before the initial recruitment process. Therefore, recommendations for scientific practice have been formulated.


Asunto(s)
Comunicación , Salud Pública , Recién Nacido , Humanos , Motivación , Investigación Cualitativa
14.
Nucleic Acids Res ; 49(1): 400-415, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33330923

RESUMEN

In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.


Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Ribosomas/metabolismo , Espectrometría de Masas en Tándem/métodos , Acetilación , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Fraccionamiento Celular/métodos , Chlamydomonas reinhardtii/genética , Regulación de la Expresión Génica de las Plantas , Separación Inmunomagnética , Espectrometría de Masas , Modelos Moleculares , Acetiltransferasas N-Terminal/aislamiento & purificación , Acetiltransferasas N-Terminal/metabolismo , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Procesamiento Proteico-Postraduccional , Subunidades Ribosómicas Grandes/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(1): 741-751, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31871212

RESUMEN

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.


Asunto(s)
Arabidopsis/fisiología , Ciclo del Ácido Cítrico/fisiología , Germinación/fisiología , Mitocondrias/metabolismo , Semillas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Plantas Modificadas Genéticamente , Proteómica/métodos , Semillas/citología , Semillas/crecimiento & desarrollo , Tiorredoxina h/genética , Tiorredoxina h/metabolismo , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo
16.
Plant Physiol ; 186(3): 1507-1525, 2021 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-33856472

RESUMEN

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol, and mitochondria. A single monothiol glutaredoxin (GRX) is involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homolog GRXS15 has only partially been characterized. Arabidopsis (Arabidopsis thaliana) grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to the knockdown line GRXS15amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain, and aconitase in the tricarboxylic acid (TCA) cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine, and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain α-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs. In wild-type plants, pyruvate, glycine, and BCKAs are all metabolized through decarboxylation by mitochondrial lipoyl cofactor (LC)-dependent dehydrogenase complexes. These enzyme complexes are very abundant, comprising a major sink for LC. Because biosynthesis of LC depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why LC-dependent processes are most sensitive to restricted Fe-S supply in grxs15 mutants.


Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Dihidrolipoamida Deshidrogenasa/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/metabolismo , Dihidrolipoamida Deshidrogenasa/genética , Genes de Plantas , Variación Genética , Genotipo , Proteínas Hierro-Azufre/genética
17.
Physiol Plant ; 174(5): e13786, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-36169530

RESUMEN

Plants in nature commonly encounter combined stress scenarios. The response to combined stressors is often unpredictable from the response to single stresses. To address stress interference in roots, we applied salinity, heat, and high light to hydroponically grown sugar beet. Two main patterns of metabolomic acclimation were apparent. High salt of 300 mM NaCl considerably lowered metabolite amounts, for example, those of most amino acids, γ-amino butyric acid (GABA), and glucose. Very few metabolites revealed the opposite trend with increased contents at high salts, mostly organic acids such as citric acid and isocitric acid, but also tryptophan, tyrosine, and the compatible solute proline. High temperature (31°C vs. 21°C) also frequently lowered root metabolite pools. The individual effects of salinity and heat were superimposed under combined stress. Under high light and high salt conditions, there was a significant decline in root chloride, mannitol, ribulose 5-P, cysteine, and l-aspartate contents. The results reveal the complex interaction pattern of environmental parameters and urge researchers to elaborate in much more detail and width on combinatorial stress effects to bridge work under controlled growth conditions to growth in nature, and also to better understand acclimation to the consequences of climate change.


Asunto(s)
Beta vulgaris , Beta vulgaris/metabolismo , Cloruro de Sodio/farmacología , Regulación de la Expresión Génica de las Plantas , Calor , Cloruros/metabolismo , Cisteína/metabolismo , Triptófano , Ácido Aspártico , Sales (Química)/metabolismo , Sales (Química)/farmacología , Salinidad , Prolina/metabolismo , Redes y Vías Metabólicas , Ácido gamma-Aminobutírico/farmacología , Manitol/farmacología , Ácido Cítrico/metabolismo , Glucosa/metabolismo , Tirosina/metabolismo , Tirosina/farmacología , Azúcares/metabolismo , Raíces de Plantas/metabolismo , Estrés Fisiológico
18.
BMC Pregnancy Childbirth ; 22(1): 717, 2022 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-36127645

RESUMEN

BACKGROUND: For quality-oriented evaluation of prenatal and obstetric care, it is important to systematically consider the perspective of the women receiving care in order to comprehensively assess and optimize quality in a woman-centered manner. Empathy and Shared Decision Making (SDM) are essential components of woman-centered midwifery care. The aim of the study was to analyze measurement invariance of the items of the Consultation and Relational Empathy (CARE) and Shared Decision Making-Questionnaire (SDM-Q-9) scales depending on the prenatal versus obstetric care setting. METHODS: One hundred fifty women retrospectively assessed aspects of woman-centered midwifery care in both prenatal and obstetric care setting. The birth of the child was a maximum of 12 months ago. A structural equation modelling approach was adopted to separate true effects from response shift (RS) effects depending on care setting. The latter were analyzed in terms of recalibration (changing women's internal measurement standards), Reprioritization (changing associations of items and construct) as well as Reconceptualization (redefining the target construct). RESULTS: A response shift model was identified for both assessments (pregnancy/birth: CFI = .96/.96; SRMR = .046/.051). At birth, both scales indicated lower quality of care compared with prenatal care (SDM-Q-9-M/CARE-8-M:|d| = 0.190/0.392). Although no reconceptualization is required for the items of both scales, RS effects are evident for individual items. Due to recalibration and reprioritization effects, the true differences in the items are partly underestimated (SDM-Q-9-M/CARE-8-M: 3/2 items) or overestimated (4/2 items). CONCLUSION: The structure of the constructs SDM and Empathy, indicating woman-centered midwifery care, are moderated by the care settings. To validly assess midwives' empathy and shared decision making from women's perspective, setting-dependent response shift effects have to be considered. The proven item-specific response effects contribute to a better understanding of construct characteristics in woman-centered care by midwives during pregnancy and childbirth.


Asunto(s)
Toma de Decisiones Conjunta , Partería , Niño , Toma de Decisiones , Empatía , Femenino , Humanos , Recién Nacido , Participación del Paciente , Embarazo , Estudios Retrospectivos , Vitaminas
19.
BMC Health Serv Res ; 22(1): 588, 2022 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-35501793

RESUMEN

BACKGROUND: Based on the theoretical model of medication adherence (WHO, 2003), the aims of the study were (1) to develop and test a theory-based multidimensional model for the predictive power of barriers to and facilitators of medication adherence and (2) to identify the mediating effects of barriers to medication adherence on drug-related patient outcomes (barrier "MedAd- ": forget; facilitator "MedAd + ": regular intake). METHODS: Within a cross-sectional study entitled "Increasing medication adherence to improve patient safety in cardiological rehabilitation (PaSiMed)", the model was evaluated in structural analytical terms based on data collected online of N = 225 patients with cardiometabolic diseases. The revised "Freiburg questionnaire on medication adherence (FF-MedAd-R)" was used to measure the latent constructs (e.g., facilitator: communication; barrier: reservations)." RESULTS: The structural equation model proved to exhibit an appropriate data fit (RMSEA: .05; CFI: .92). For all first-order facilitators of medication adherence, a high proportion of variance (62-94%) could be explained by the second-order factor "Physician-patient relationship (PPR)". All paths from "PPR" to the constructs depicting barriers to medication adherence showed significant negative effects. Facilitators ("MedAd + ") and barriers ("MedAd-") accounted for 20% and 12% of the variance, respectively, in global items of medication adherence. Whereas "Carelessness" showed a full mediation for "MedAd-", ''Reservations'' showed a partial mediation for "MedAd + ". CONCLUSIONS: "PPR" is an important predictor of patient medication adherence. The results underline the importance of a trustful physician-patient relationship in reducing barriers and enhancing medication adherence.


Asunto(s)
Enfermedades Cardiovasculares , Cumplimiento de la Medicación , Enfermedades Cardiovasculares/tratamiento farmacológico , Estudios Transversales , Humanos , Análisis de Clases Latentes , Encuestas y Cuestionarios
20.
BMC Health Serv Res ; 22(1): 1549, 2022 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-36536337

RESUMEN

BACKGROUND: People with disabilities and without natural speech often rely on care provided by informal caregivers. The caregiving situation of these informal caregivers has been poorly researched. The objectives of the study are 1) to identify stressors, resources, and coping strategies among informal caregivers of people without natural speech and 2) to examine whether a complex intervention in augmentative and alternative communication (AAC) that is not primarily tailored to the needs of informal caregivers can reduce care-related burden. METHODS: The main components of the AAC intervention were (1) initial counselling session, (2) 4 AAC training sessions, (3) 20 AAC therapy sessions and (4) accompanying case management. The control group received only the initial counselling session. Within a quasi-experimental intervention study, survey data on self-perceived burden (Burden Scale for Family Caregivers, BSFC-s) from n = 154 informal caregivers of people without natural speech were collected at three time points between June 2018 and April 2021 from a postal survey. Qualitative interviews with n = 16 informal caregivers were conducted. RESULTS: Caregivers reported various stressors such as limited communication with the cared-for person and concerns about the living situation in adulthood. Diverse resources and effective coping strategies, which the caregivers refer to when dealing with stressors, could also be identified. Burden was significantly reduced in the intervention group compared to the control group. According to the results of the qualitative study, AAC use led to better communication skills and a reduction in behavioural problems and thus a decreased burden. CONCLUSIONS: The AAC intervention seems to have a positive impact on self-perceived burden. Linkages between intervention components and burden reduction as well as stressors and coping strategies could be identified and provide an evidence-based foundation for developing future holistic interventions for families with individuals without natural speech. TRIAL REGISTRATION: German Clinical Trials Register (DRKS); ID: DRKS00013628 (registered on 05/02/2018).


Asunto(s)
Cuidadores , Habla , Adulto , Humanos , Adaptación Psicológica , Manejo de Caso , Consejo , Calidad de Vida
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