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1.
Mol Cell ; 79(3): 376-389.e8, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32640193

RESUMO

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isoenzimas/genética , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Xenoenxertos , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Mutação , Neuroglia/metabolismo , Neuroglia/patologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
2.
PLoS One ; 15(7): e0235556, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614916

RESUMO

To gain a better insight into the selenium nanoparticle (nSe) benefits/toxicity, this experiment was carried out to address the behavior of bitter melon seedlings to nSe (0, 1, 4, 10, 30, and 50 mgL-1) or bulk form (selenate). Low doses of nSe increased biomass accumulation, while concentrations of 10 mgL-1 and above were associated with stem bending, impaired root meristem, and severe toxicity. Responses to nSe were distinct from that of bulk in that the nano-type exhibited a higher efficiency to stimulate growth and organogenesis than the bulk. The bulk form displayed higher phytotoxicity than the nano-type counterpart. According to the MSAP-based analysis, nSe mediated substantial variation in DNA cytosine methylation, reflecting the epigenetic modification. By increasing the concentration of nSe, the expression of the WRKY1 transcription factor linearly up-regulated (mean = 7.9-fold). Transcriptions of phenylalanine ammonia-lyase (PAL) and 4-Coumarate: CoA-ligase (4CL) genes were also induced. The nSe treatments at low concentrations enhanced the activity of leaf nitrate reductase (mean = 52%) in contrast with the treatment at toxic concentrations. The toxic concentration of nSe increased leaf proline concentration by 80%. The nSe supplement also stimulated the activities of peroxidase (mean = 35%) and catalase (mean = 10%) enzymes. The nSe-treated seedlings exhibited higher PAL activity (mean = 39%) and soluble phenols (mean = 50%). The nSe toxicity was associated with a disrupted differentiation of xylem conducting tissue. The callus formation and performance of the explants originated from the nSe-treated seedlings had a different trend than that of the control. This experiment provides new insights into the nSe-associated advantage/ cytotoxicity and further highlights the necessity of designing convincing studies to introduce novel methods for plant cell/tissue cultures and agriculture.


Assuntos
Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Momordica charantia/metabolismo , Nanopartículas/toxicidade , Selênio/química , Citosina/metabolismo , Momordica charantia/efeitos dos fármacos , Momordica charantia/crescimento & desenvolvimento , Nanopartículas/química , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Fenóis/metabolismo , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Prolina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos
3.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 1023-1028, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701248

RESUMO

OBJECTIVE: To investigate the effects of stachydrine (STA) on apoptosis of Aß25-35-induced PC12 cells mimicking Alzheimer's disease and explore the mechanisms. METHODS: The differential genes of STA were analyzed based on GSE85871 data, and the target genes of STA were identified using STITCH database. PC12 cells were treated with Aß25-35 to establish a cell model of Alzheimer's disease, and the changes in cell viability and cell cycle in response to STA treatment were assessed using MTT assay and flow cytometry, respectively. RT-PCR and Western blotting were used to detect the relevant gene or protein expressions in the treated cells. RESULTS: GSE85871 data showed 37 up-regulated genes and 48 down-regulated genes in cells following treatment with STA. Analysis of the data from the STITCH database indicated that RPS8 and EED were the target genes of STA. Treatment of PC12 cells with Aß25-35 significantly lowered the cell viability (P < 0.05) and the expressions of RPS8 and EED at both the mRNA and protein levels (P < 0.05), and obviously inhibited the expression of apoptosis-related proteins Bcl-2 and p53 (P < 0.05). STA treatment of the cells significantly reversed the effect of Aß25-35 and induced cell cycle arrest in G2/M phase, causing also significantly increases in the expression levels of RPS8, EED, Bcl-2 and p53 (P < 0.05). CONCLUSIONS: STA plays an important role in inhibiting the apoptosis of PC12 cells induced by Aß25-35 possibly by regulating RPS8 and EED expression to promote the expressions of Bcl-2 and p53.


Assuntos
Doença de Alzheimer , Apoptose , Prolina/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Células PC12 , Prolina/farmacologia , Ratos
4.
Cell Res ; 30(8): 678-692, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32541865

RESUMO

A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.


Assuntos
Antivirais/farmacologia , Betacoronavirus/enzimologia , Infecções por Coronavirus/metabolismo , Glicoproteínas/farmacologia , Pneumonia Viral/metabolismo , Prolina/análogos & derivados , Pirrolidinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Células CACO-2 , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Descoberta de Drogas/métodos , Humanos , Concentração Inibidora 50 , Cinética , Pandemias , Pneumonia Viral/virologia , Prolina/farmacologia , Conformação Proteica , Pirrolidinas/química , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
5.
Ecotoxicol Environ Saf ; 202: 110890, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32593096

RESUMO

The presence of tetracycline is ubiquitous and has adverse effects on aquatic systems. A hydroponic experiment was conducted to investigate the ecological sensitivity of Hydrocharis dubia (Bl.) Backer and Trapa bispinosa Roxb. Exposed to different concentrations of tetracycline (0, 0.1, 1, 10, 30 and 50 mg/L) for one day (1D) and 14 days (14D). The results showed that after 1D of tetracycline exposure, the physiological indices of H. dubia had no remarkable change except for proline which was significantly stimulated under 0.1 mg/L tetracycline. For T. bispinosa, guaiacol peroxidase (POD), polyphenol oxidase (PPO) and ascorbate peroxidase (APX) activity and protein and proline content were notably promoted under different concentrations of tetracycline, but PPO activity was significantly decreased in 50 mg/L. After 14D, tetracycline caused no harm to the growth and protein content of H. dubia, but negatively influenced lipid peroxidation product and chlorophyll content in H. dubia under high tetracycline concentrations. Superoxide dismutase (SOD) and POD activity of H. dubia significantly increased at high tetracycline concentrations, while catalase (CAT) and PPO activity significantly decreased. APX activity in H. dubia increased with tetracycline concentrations at low tetracycline concentrations. For T. bispinosa, high concentrations of tetracycline application significantly inhibited its growth and the content of protein and chlorophyll. SOD, POD, CAT, and PPO activity of T. bispinosa were induced under different concentrations of tetracycline and no lipid peroxidation was observed. APX activity in T. bispinosa was significantly inhibited at high tetracycline concentrations. The results suggest that tetracycline can cause oxidative damage in H. dubia but harm the metabolism process of T. bispinosa without inducing oxidative damage. Overall, the sensitivity of T. bispinosa exposed to tetracycline exposure is higher than that of H. dubia.


Assuntos
Antibacterianos/toxicidade , Hydrocharitaceae/fisiologia , Tetraciclina/toxicidade , Antioxidantes/metabolismo , Ascorbato Peroxidases/metabolismo , Catalase/metabolismo , Clorofila/metabolismo , Hydrocharitaceae/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Lythraceae , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Peroxidase , Prolina/metabolismo , Superóxido Dismutase/metabolismo
6.
Food Chem ; 329: 126775, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512387

RESUMO

Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.


Assuntos
Colágeno Tipo I/química , Hidroxiprolina/química , Prolina/química , Serina/química , Animais , Dicroísmo Circular , Peixes , Desnaturação Proteica , Temperatura
7.
Proc Natl Acad Sci U S A ; 117(25): 14512-14521, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513714

RESUMO

Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Domínios Proteicos/genética , Regulação Alostérica , Animais , Cátions Bivalentes/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/ultraestrutura , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oócitos , Técnicas de Patch-Clamp , Prolina/genética , Conformação Proteica em alfa-Hélice/genética , Relação Estrutura-Atividade , Xenopus laevis
8.
Proc Natl Acad Sci U S A ; 117(25): 14158-14167, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513738

RESUMO

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 µM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 µM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.


Assuntos
Prolina/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte Vesicular/química , Humanos , Modelos Moleculares , Prolina/metabolismo , Complexo de Endopeptidases do Proteassoma , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
9.
PLoS Biol ; 18(6): e3000741, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520929

RESUMO

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.


Assuntos
Metabolômica , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcriptoma/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
10.
Ecotoxicol Environ Saf ; 201: 110869, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585490

RESUMO

Chromium (Cr) contamination is a potential threat to the agricultural soil. Arbuscular mycorrhizal (AM) fungi have potential to remediate the heavy metal polluted soils. It was hypothesized that Cr phytoremediation potentiality of AM fungi could be enhanced in combination with saprophytic filamentous fungi and soil amendment. Tomato plants were raised in Cr polluted technosol amended with compost, inoculated with mixed-culture of AM fungi and Aspergillus terreus. It was found that, triple treatment (soil amendment with compost along with AM fungi and A. terreus inoculation) enhanced biomass production (up to 315%), fruit setting (up to 49%), photosynthetic pigments (up to 214%) and carbohydrate content (up to 400%) whereas reduced the proline (up to 76.5%), catalase (up to 34.2%), peroxidase (up to 58.9%) and root membrane permeability (up to 74.2%). The effect of AM fungi with compost amendment was additive, while it was synergistic with A. terreus. AM fungi enhanced the extraction of Cr from the substrate, but retained in the mycorrhizal root, thereby reduced the translocation into shoot and in fruit, Cr translocation was undetectable. At the end of experiment Cr content in the substrate was significantly decreased (up to 37.9%). Soil amendment with compost along with AM fungi and A. terreus inoculation can be used for reclamation of Cr polluted soils at field scale.


Assuntos
Aspergillus/fisiologia , Cromo/farmacocinética , Lycopersicon esculentum/metabolismo , Micorrizas/fisiologia , Poluentes do Solo/farmacocinética , Biodegradação Ambiental , Biomassa , Metabolismo dos Carboidratos , Catalase/metabolismo , Compostagem , Lycopersicon esculentum/enzimologia , Peroxidase/metabolismo , Raízes de Plantas/metabolismo , Prolina/metabolismo
11.
Food Chem ; 327: 127057, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32464461

RESUMO

Chilling injury (CI) restricts the quality and shelf life of bell pepper fruits; reducing these CI-induced detrimental effects is therefore of high economic and agricultural relevance. Here, we investigated the effects of trisodium phosphate (TSP), salicylic acid (SA), and TSP + SA treatments on pepper fruits under cold stress at 4 °C for 25 d. Combined TSP + SA treatment performed an optimal effect. Specifically, TSP + SA treatment enhanced fatty-acid desaturation efficiency, as indicated by the increased expression of key fatty acid desaturase genes, and higher content of unsaturated fatty acids. Meanwhile, TSP + SA treatment inhibited the CI-induced membrane damage, manifested as lower electrolyte leakage and malondialdehyde content. Furthermore, low field-nuclear magnetic resonance and proline content also revealed that TSP + SA treatment mitigated CI through enhancing water retention in pepper fruits. Collectively, our results may shed new light on optimizing the low-temperature storage conditions of post-harvest peppers.


Assuntos
Capsicum/química , Ácidos Graxos/química , Fosfatos/química , Ácido Salicílico/química , Água/química , Capsicum/efeitos dos fármacos , Capsicum/metabolismo , Parede Celular/efeitos dos fármacos , Temperatura Baixa , Ácidos Graxos/metabolismo , Frutas/química , Frutas/efeitos dos fármacos , Frutas/metabolismo , Malondialdeído/química , Malondialdeído/metabolismo , Permeabilidade/efeitos dos fármacos , Fosfatos/farmacologia , Prolina/química , Ácido Salicílico/farmacologia
12.
J Anim Sci ; 98(6)2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32415842

RESUMO

This study determined whether extracellular citrulline is degraded by ruminal bacteria of sheep. In the first experiment, whole rumen fluid (3 mL) from six adult Suffolk sheep was incubated at 37 °C with 5 mM l-glutamine (Gln), l-glutamate (Glu), l-arginine (Arg), or l-citrulline (Cit) for 0, 0.5, 1, and 2 h or with 0, 0.5, 2, or 5 mM Gln, Glu, Arg, or Cit for 2 h. An aliquot (50 µL) of the incubation solution was collected at the predetermined time points for amino acids (AA) analyses. Results showed extensive hydrolysis of Gln into Glu and ammonia, of Arg into l-ornithine and l-proline, but little or no degradation of extracellular Cit or Glu by ruminal microbes. In the second experiment, six adult Suffolk sheep were individually fed each of three separate supplements (8 g Gln , Cit, or urea) on three separate days along with regular feed (800 g/animal). Blood (2 mL) was sampled from the jugular vein prior to feeding (time 0) and at 0.5, 1, 2, and 4 h after consuming the supplement. Plasma was analyzed for AA, glucose, ammonia, and urea. The concentrations of Cit in the plasma of sheep consuming this AA increased (P < 0.001) by 117% at 4 h and those of Arg increased by 23% at 4 h, compared with the baseline values. Urea or Gln feeding did not affect (P > 0.05) the concentrations of Cit or Arg in plasma. These results indicate that Cit is not metabolized by ruminal microbes of sheep and is, therefore, absorbed as such by the small intestine and used for the synthesis of Arg by extrahepatic tissues.


Assuntos
Bactérias/metabolismo , Citrulina/metabolismo , Rúmen/microbiologia , Ovinos/microbiologia , Amônia/metabolismo , Ração Animal/análise , Animais , Arginina/metabolismo , Dieta/veterinária , Suplementos Nutricionais , Feminino , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ornitina/sangue , Prolina/metabolismo , Ureia
13.
PLoS Pathog ; 16(5): e1008244, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365082

RESUMO

Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antivirais/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Proteínas de Ligação a DNA/imunologia , Epitopos/imunologia , Epitopos de Linfócito T/imunologia , Genes RAG-1/imunologia , Ligantes , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/metabolismo , Prolina/metabolismo , Ligação Proteica , Linfócitos T Citotóxicos/imunologia , Vacinação/métodos
14.
Environ Sci Pollut Res Int ; 27(23): 29130-29142, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32430722

RESUMO

The continuous increase of the human population worldwide has led to an increase of pharmaceuticals' consumption, such as diclofenac (DCF), a widely used non-steroidal anti-inflammatory drug (NSAID), that is not removed by wastewater treatment processes. Although there is some research regarding the effects of DCF on animals and aquatic invertebrates, information concerning its influence on plants' metabolism is still scarce. Through an integrated approach, using combined biochemical and molecular biology techniques, this work aimed to evaluate the phytotoxicity of DCF in Solanum lycopersicum L., focusing on the primary plant processes: nitrogen (N) assimilation and photosynthesis. The exposure of tomato plants to increasing concentrations of DCF (0, 0.5, and 5 mg L-1) revealed that glutamine synthetase (GS) was differentially affected, in an organ-dependent manner, by this contaminant at the gene expression, protein, and activity levels, with an increased activity of 0.2-fold in shoots of plants treated with the lowest concentration of DCF although a general decrease was registered for the SlGS gene family expression, revealing that post-translational regulation was in order, since GS2 polypeptide content did not change. Glutamate dehydrogenase (GDH) activity was generally enhanced, accompanied by increases of 0.4- to 1.9-fold in proline levels, revealing GDH as an important compensatory route for both N assimilation and proline production under stressful conditions. No alterations in most photosynthetic endpoints were noticed after DCF treatments, but small decreases of 0.1- to 0.8-fold in the accumulation of RuBisCO-encoding transcripts were observed, along with a reduction in starch content. Some alterations in the soluble polypeptide profile were also detected in response to DCF, evidencing the participation of some stress-related proteins in the plant's response to DCF.


Assuntos
Lycopersicon esculentum , Carbono , Diclofenaco , Glutamato Desidrogenase , Glutamato-Amônia Ligase , Nitrogênio , Prolina
15.
Ecotoxicol Environ Saf ; 200: 110721, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32464438

RESUMO

Glutaredoxins (Grxs) are small (10-15 kDa) glutathione (GSH) - dependent redox proteins. The role of Grxs are well documented in tolerance to heavy metal stress in prokaryotic and mammalian systems and a few plant genera, but is poorly understood in plants against drought. In the present study, two rice glutaredoxin (Osgrx) genes (LOC_Os02g40500 and LOC_Os01g27140) responsible for tolerance against heavy metal stress have been studied for investigating their role against drought. Each glutaredoxin gene was over-expressed in Arabidopsis thaliana to reveal their role in drought stress. The relative expression of both Osgrx genes was higher in the transgenic lines. Transgenic lines of both Osgrxs showed longer roots, higher seed germination, and survival efficiency during drought stress. The physiological parameters (PN, gs, E, WUE, qP, NPQ and ETR), antioxidant enzymes (GRX, GR, GPX, GST, APX, POD, SOD, CAT, DHAR, and MDHAR), antioxidant molecules (ascorbate and GSH) and stress-responsive amino acids (cysteine and proline) levels were additionally increased in transgenic lines of both Osgrxs to provide drought tolerance. The outcomes from this study strongly determined that each Osgrx gene participated in the moderation of drought and might be utilized in biological engineering strategies to overcome drought conditions in different crops.


Assuntos
Glutarredoxinas/genética , Oryza/enzimologia , Estresse Fisiológico , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Ácido Ascórbico/metabolismo , Cisteína/metabolismo , Secas , Genes de Plantas , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Prolina/metabolismo , Estresse Fisiológico/genética
16.
Ecotoxicol Environ Saf ; 200: 110732, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32460049

RESUMO

This paper reports the role of exogenous glycine betaine (25 and 50 mM GB at a rate of 50 mL per plant) in enhancing NaCl-stress tolerance in common bean (Phaseolus vulgaris L.). Irrigating plants by simulated saline water, containing 0, 50 and 100 mM sodium chloride (NaCl), significantly reduced the growth dynamics, photosynthetic pigments (i.e., Chl a, Chl b, and carotenoids), membrane stability index (MSI), relative water content (RWC), and pod yield. While, malondialdehyde (MDA), endogenous proline, and glutathione contents, electrolyte leakage (EL), antioxidant defense system, and Na+ accumulation markedly increased upon exposure to NaCl-stress. However, the application of exogenous GB significantly improved salt tolerance of common bean as it increased the antioxidant defense including both enzymatic (i.e., peroxidase, superoxide dismutase, and catalase) and nonenzymatic (i.e., proline and glutathione) agents. Consequently, MSI, RWC, EL, and photosynthetic pigments have been improved recording significantly higher values than the control. Moreover, the pod yield increased by 29.8 and 59.4% when plants grown under 50 and 100 mM NaCl, respectively, were sprayed with 25 mM GB. Our results show that GB-induced slat tolerance in common bean plants mainly depends on the osmoregulation effect of GB and to a lesser extent on its antioxidant capacity. Foliar application of GB significantly reduced the accumulation of Na+ and at the same time induced K+ uptake maintaining a higher K+/Na+ ratio. Despite some changes in the activities of antioxidant enzymes induced by the application of GB, no consistent contribution in the salt tolerance could be cited in this study. Therefore, we suggest that salt tolerance is largely unrelated to the antioxidant defense ability of GB in common bean. While the potential role of GB in ameliorating salt tolerance is mainly due to the adjustment of ions uptake through limiting Na+ uptake and alternatively increasing K+ accumulation in plant tissues.


Assuntos
Betaína/farmacologia , Phaseolus/efeitos dos fármacos , Potássio/metabolismo , Tolerância ao Sal , Sódio/metabolismo , Antioxidantes/metabolismo , Transporte Biológico/efeitos dos fármacos , Catalase/metabolismo , Cátions , Glutationa/metabolismo , Malondialdeído/análise , Osmorregulação/efeitos dos fármacos , Peroxidase/metabolismo , Phaseolus/química , Phaseolus/enzimologia , Phaseolus/metabolismo , Fotossíntese/efeitos dos fármacos , Potássio/análise , Prolina/metabolismo , Sódio/análise , Superóxido Dismutase/metabolismo
17.
Chem Biol Interact ; 326: 109136, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32417162

RESUMO

Osteoarthritis (OA) is a common degenerative joint disease that is closely associated with inflammation. Stachydrine (STA) is a bioactive alkaloid with anti-inflammatory activity. However, the role of STA in OA remains unknown. This study aimed to explore the effects of STA on OA chondrocytes in the presence of IL-1ß. Primary human OA chondrocytes were pretreated with various concentrations of STA for 2 h and then stimulated with IL-1ß for 24 h. Inflammatory mediators and cytokines including NO, PGE2, TNF-α and IL-6 in chondrocytes were detected to reflect inflammation status. Production of extracellular matrix (ECM) degrading enzymes including MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in chondrocytes was measured using ELISA. The expression levels of iNOS, COX-2, p65, p-p65, p-IκBα, and IκBα were detected by Western blot analysis. Our results showed that STA significantly suppressed IL-1ß-induced inflammation with decreased levels of inflammatory mediators and cytokines including NO, PGE2, iNOS, COX-2, TNF-α and IL-6. Treatment with STA suppressed the production of ECM degrading enzymes including MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 in IL-1ß-induced chondrocytes. Furthermore, STA blocked the IL-1ß-mediated potentiation of NF-κB pathway in chondrocytes. In conclusion, these findings demonstrated that STA protected chondrocytes from IL-1ß-induced inflammation through the NF-κB signaling pathway.


Assuntos
Condrócitos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Osteoartrite/tratamento farmacológico , Prolina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Linhagem Celular , Condrócitos/metabolismo , Humanos , Inflamação/metabolismo , Osteoartrite/metabolismo , Prolina/farmacologia
18.
PLoS One ; 15(4): e0231348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298338

RESUMO

Salt stress is one of the devastating factors that hampers growth and productivity of soybean. Use of Pseudomonas pseudoalcaligenes to improve salt tolerance in soybean has not been thoroughly explored yet. Therefore, we observed the response of hydroponically grown soybean plants, inoculated with halotolerant P. pseudoalcaligenes (SRM-16) and Bacillus subtilis (SRM-3) under salt stress. In vitro testing of 44 bacterial isolates revealed that four isolates showed high salt tolerance. Among them, B. subtilis and P. pseudoalcaligenes showed ACC deaminase activity, siderophore and indole acetic acid (IAA) production and were selected for the current study. We determined that 106 cells/mL of B. subtilis and P. pseudoalcaligenes was sufficient to induce tolerance in soybean against salinity stress (100 mM NaCl) in hydroponics by enhancing plant biomass, relative water content and osmolytes. Upon exposure of salinity stress, P. pseudoalcaligenes inoculated soybean plants showed tolerance by the increased activities of defense related system such as ion transport, antioxidant enzymes, proline and MDA content in shoots and roots. The Na+ concentration in the soybean plants was increased in the salt stress; while, bacterial priming significantly reduced the Na+ concentration in the salt stressed soybean plants. However, the antagonistic results were observed for K+ concentration. Additionally, soybean primed with P. pseudoalcaligenes and exposed to 100 mM NaCl showed a new protein band of 28 kDa suggesting that P. pseudoalcaligenes effectively reduced salt stress. Our results showed that salinity tolerance was more pronounced in P. pseudoalcaligenes as compared to B. subtilis. However, a detailed study at molecular level to interpret the mechanism by which P. pseudoalcaligenes alleviates salt stress in soybean plants need to be explored.


Assuntos
Bacillus subtilis/patogenicidade , Pseudomonas pseudoalcaligenes/patogenicidade , Tolerância ao Sal , Soja/metabolismo , Bacillus subtilis/metabolismo , Transporte de Íons , Malondialdeído/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Pseudomonas pseudoalcaligenes/metabolismo , Espécies Reativas de Oxigênio , Soja/microbiologia
19.
Ecotoxicol Environ Saf ; 196: 110525, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32224370

RESUMO

Fragrant rice is a high-valued quality rice type which is gaining much popularity over the globe due to its better cooking qualities and special aromatic characteristics. Selenium (Se) and silicon (Si) could improve the growth and yield of rice; however, the combine effects of Se and Si (Se-Si treatments) on rice grain quality, aroma and lodging in fragrant rice were rarely investigated. The pot and field experiments were conducted with two fragrant rice cultivars i.e., Xiangyaxiangzhan and Yuxiangyouzhan, grown under three Se levels i.e., 0, 120, and 240 mg kg-1 of soil (for pot experiment) and 0, 300, and 600 kg ha-1 (for field experiment) regarded as LSe, MSe and HSe, respectively and two Si levels i.e., 0 and 60 mg kg-1 of soil (for pot experiment) and 0 and 150 kg ha-1 (for field experiment) regarded as -Si and +Si, respectively. Results depicted that the Se-Si treatments regulated head rice yield, grain yield and yield related traits and the HSe+Si treatment sustainably improved the grain yield and head rice yield by regulating plant growth, antioxidant response and malondialdehyde (MDA) contents in fragrant rice. The Se-Si treatments also improved the grain 2AP contents owing to regulation in the proline, pyrroline-5-carboxylate (P5C) and γ-aminobutyric acid (GABA) contents. Besides, Se-Si treatments also regulated the grain quality attributes and influenced the plant Se contents. Moreover, the Si mitigated Se-induced lodging resulted from changes in the lodging parameters i.e., lodging index, fresh weight per tiller, pushing resistance force, plant height and bending moment. Overall, the Se and Si application improved the grain yield and regulated the dry weight accumulation, antioxidant attributes and quality attributes. Meanwhile, the Si application mitigated the negative effect of Se-induced lodging in fragrant rice.


Assuntos
Grão Comestível/efeitos dos fármacos , Odorantes/análise , Oryza/efeitos dos fármacos , Selênio/farmacologia , Silício/farmacologia , Antioxidantes/análise , Relação Dose-Resposta a Droga , Grão Comestível/química , Grão Comestível/crescimento & desenvolvimento , Malondialdeído/análise , Oryza/química , Oryza/crescimento & desenvolvimento , Prolina/análise , Pirróis/análise , Solo/química
20.
Chem Biol Interact ; 324: 109092, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32278739

RESUMO

Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. Km and kcat values for butyrylthiocholine were independent of subunit assembly status. The Km for all forms of MaBChE was about 70 µM, compared to 13 µM for HuBChE. The kcat was about 100,000 min-1 for MaBChE and 30,000 min-1 for HuBChE. The reversible inhibitor ethopropazine had similar Ki values of 0.05 µM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5 × 107 M-1 min-1 for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4 M-1 min-1, but was 380 fold faster for DFP-inhibited HuBChE (kr 531 M-1 min-1). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8-fold (kr 92 M-1 min-1) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2 M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.


Assuntos
Butirilcolinesterase/química , Reativadores da Colinesterase/química , Compostos de Pralidoxima/química , Prolina/química , Sequência de Aminoácidos , Animais , Inibidores da Colinesterase/farmacologia , Humanos , Cinética , Macaca , Macaca mulatta , Fenotiazinas/farmacologia , Alinhamento de Sequência
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