RESUMO
In plants, heat stress induces changes in alternative splicing, including intron retention; these events can rapidly alter proteins or downregulate protein activity, producing nonfunctional isoforms or inducing nonsense-mediated decay of messenger RNA (mRNA). Nuclear cyclophilins (CYPs) are accessory proteins in the spliceosome complexes of multicellular eukaryotes. However, whether plant CYPs are involved in pre-mRNA splicing remain unknown. Here, we found that Arabidopsis thaliana CYP18-1 is necessary for the efficient removal of introns that are retained in response to heat stress during germination. CYP18-1 interacts with Step II splicing factors (PRP18a, PRP22, and SWELLMAP1) and associates with the U2 and U5 small nuclear RNAs in response to heat stress. CYP18-1 binds to phospho-PRP18a, and increasing concentrations of CYP18-1 are associated with increasing dephosphorylation of PRP18a. Furthermore, interaction and protoplast transfection assays revealed that CYP18-1 and the PP2A-type phosphatase PP2A B'η co-regulate PRP18a dephosphorylation. RNA-seq and RT-qPCR analysis confirmed that CYP18-1 is essential for splicing introns that are retained under heat stress. Overall, we reveal the mechanism of action by which CYP18-1 activates the dephosphorylation of PRP18 and show that CYP18-1 is crucial for the efficient splicing of retained introns and rapid responses to heat stress in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Processamento Alternativo/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclofilinas/genética , Ciclofilinas/metabolismo , Resposta ao Choque Térmico/genética , Íntrons/genética , Splicing de RNA , RNA Mensageiro/genéticaRESUMO
Alternative splicing (AS) is a critical means by which plants respond to changes in the environment, but few splicing factors contributing to AS have been reported and functionally characterized in rice (Oryza sativa L.). Here, we explored the function and molecular mechanism of the spliceosome-associated protein OsFKBP20-1b during AS. We determined the AS landscape of wild-type and osfkbp20-1b knockout plants upon abscisic acid (ABA) treatment by transcriptome deep sequencing. To capture the dynamics of translating intron-containing mRNAs, we blocked transcription with cordycepin and performed polysome profiling. We also analyzed whether OsFKBP20-1b and the splicing factors OsSR34 and OsSR45 function together in AS using protoplast transfection assays. We show that OsFKBP20-1b interacts with OsSR34 and regulates its stability, suggesting a role as a chaperone-like protein in the spliceosome. OsFKBP20-1b facilitates the splicing of mRNAs with retained introns after ABA treatment; some of these mRNAs are translatable and encode functional transcriptional regulators of stress-responsive genes. In addition, interacting proteins, OsSR34 and OsSR45, regulate the splicing of the same retained introns as OsFKBP20-1b after ABA treatment. Our findings reveal that spliceosome-associated immunophilin functions in alternative RNA splicing in rice by positively regulating the splicing of retained introns to limit ABA response.
Assuntos
Oryza , Íntrons/genética , Oryza/genética , Oryza/metabolismo , Ácido Abscísico/farmacologia , Ácido Abscísico/metabolismo , Splicing de RNA/genética , Processamento Alternativo/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plantas/metabolismo , Fatores de Processamento de RNA/genéticaRESUMO
Peptidyl-prolyl isomerase-like 1 (PPIL1) is associated with the human spliceosome complex. However, its function in pre-mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18-2 (AtCYP18-2), a PPIL1 homolog, plays an essential role in heat tolerance by regulating pre-mRNA splicing. Under heat stress conditions, AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP18-2 interacts with several spliceosome complex BACT components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress. The AtCYP18-2 loss-of-function allele cyp18-2 engineered by CRISPR/Cas9-mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type. Moreover, global transcriptome profiling showed that the cyp18-2 mutation affects alternative splicing of heat stress-responsive genes under heat stress conditions, particularly intron retention (IR). The abundance of most intron-containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18-2 compared to the wild type. Furthermore, the intron-containing transcripts of two heat stress-related genes, HEAT SHOCK PROTEIN 101 (HSP101) and HEAT SHOCK FACTOR A2 (HSFA2), produced functional proteins. HSP101-IR-GFP localization was responsive to heat stress, and HSFA2-III-IR interacted with HSF1 and HSP90.1 in plant cells. Our findings reveal that CYP18-2 functions as a splicing factor within the BACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Resposta ao Choque Térmico , Humanos , Processamento Alternativo/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Resposta ao Choque Térmico/genética , Íntrons/genética , Precursores de RNA/genéticaRESUMO
Sessile plants have evolved distinct mechanisms to respond and adapt to adverse environmental conditions through diverse mechanisms including RNA processing. While the role of RNA processing in the stress response is well understood for Arabidopsis thaliana, limited information is available for rice (Oryza sativa). Here, we show that OsFKBP20-1b, belonging to the immunophilin family, interacts with the splicing factor OsSR45 in both nuclear speckles and cytoplasmic foci, and plays an essential role in post-transcriptional regulation of abiotic stress response. The expression of OsFKBP20-1b was highly upregulated under various abiotic stresses. Moreover genetic analysis revealed that OsFKBP20-1b positively affected transcription and pre-mRNA splicing of stress-responsive genes under abiotic stress conditions. In osfkbp20-1b loss-of-function mutants, the expression of stress-responsive genes was downregulated, while that of their splicing variants was increased. Conversely, in plants overexpressing OsFKBP20-1b, the expression of the same stress-responsive genes was strikingly upregulated under abiotic stress. In vivo experiments demonstrated that OsFKBP20-1b directly maintains protein stability of OsSR45 splicing factor. Furthermore, we found that the plant-specific OsFKBP20-1b gene has uniquely evolved as a paralogue only in some Poaceae species. Together, our findings suggest that OsFKBP20-1b-mediated RNA processing contributes to stress adaptation in rice.
Assuntos
Oryza/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Processamento de RNA/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/genética , Proteínas de Plantas/genética , Ligação Proteica , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/fisiologia , Fatores de Processamento de RNA/genética , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologiaRESUMO
Precise flowering timing is critical for the plant life cycle. Here, we examined the molecular mechanisms and regulatory network associated with flowering in Chinese cabbage (Brassica rapa L.) by comparative transcriptome profiling of two Chinese cabbage inbred lines, "4004" (early bolting) and "50" (late bolting). RNA-Seq and quantitative reverse transcription PCR (qPCR) analyses showed that two positive nitric oxide (NO) signaling regulator genes, nitrite reductase (BrNIR) and nitrate reductase (BrNIA), were up-regulated in line "50" with or without vernalization. In agreement with the transcription analysis, the shoots in line "50" had substantially higher nitrogen levels than those in "4004". Upon vernalization, the flowering repressor gene Circadian 1 (BrCIR1) was significantly up-regulated in line "50", whereas the flowering enhancer genes named SUPPRESSOR OF OVEREXPRESSION OF CONSTANCE 1 homologs (BrSOC1s) were substantially up-regulated in line "4004". CRISPR/Cas9-mediated mutagenesis in Chinese cabbage demonstrated that the BrSOC1-1/1-2/1-3 genes were involved in late flowering, and their expression was mutually exclusive with that of the nitrogen signaling genes. Thus, we identified two flowering mechanisms in Chinese cabbage: a reciprocal negative feedback loop between nitrogen signaling genes (BrNIA1 and BrNIR1) and BrSOC1s to control flowering time and positive feedback control of the expression of BrSOC1s.
Assuntos
Brassica rapa/fisiologia , Flores/fisiologia , Proteínas de Domínio MADS/fisiologia , Nitrogênio/metabolismo , Proteínas de Plantas/fisiologia , Sistemas CRISPR-Cas , Retroalimentação Fisiológica , Redes Reguladoras de Genes , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Análise de Sequência de RNA , TranscriptomaRESUMO
OsFKBP20-1b, a plant-specific cyclophilin protein, has been implicated to regulate pre-mRNA splicing under stress conditions in rice. Here, we demonstrated that OsFKBP20-1b is SUMOylated in a reconstituted SUMOylation system in E.coli and in planta, and that the SUMOylation-coupled regulation was associated with enhanced protein stability using a less SUMOylated OsFKBP20-1b mutant (5KR_OsFKBP20-1b). Furthermore, OsFKBP20-1b directly interacted with OsSUMO1 and OsSUMO2 in the nucleus and cytoplasm, whereas the less SUMOylated 5KR_OsFKBP20-1b mutant had an impaired interaction with OsSUMO1 and 2 in the cytoplasm but not in the nucleus. Under heat stress, the abundance of an OsFKBP20-1b-GFP fusion protein was substantially increased in the nuclear speckles and cytoplasmic foci, whereas the heat-responsiveness was remarkably diminished in the presence of the less SUMOylated 5KR_OsFKBP20-1b-GFP mutant. The accumulation of endogenous SUMOylated OsFKBP20-1b was enhanced by heat stress in planta. Moreover, 5KR_OsFKBP20-1b was not sufficiently associated with the U snRNAs in the nucleus as a spliceosome component. A protoplast transfection assay indicated that the low SUMOylation level of 5KR_OsFKBP20-1b led to inaccurate alternative splicing and transcription under heat stress. Thus, our results suggest that OsFKBP20-1b is post-translationally regulated by SUMOylation, and the modification is crucial for proper RNA processing in response to heat stress in rice.
Assuntos
Resposta ao Choque Térmico , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Splicing de RNA , Sumoilação , Escherichia coliRESUMO
KEY MESSAGE: Plant possesses particular Golgi-resident cyclophilin 21 proteins (CYP21s) and the catalytic isomerase activities have a negative effect on ABA signalling gene expression during early seedling development. Cyclophilins (CYPs) are essential for diverse cellular process, as these catalyse a rate-limiting step in protein folding. Although Golgi proteomics in Arabidopsis thaliana suggests the existence of several CYPs in the Golgi apparatus, only one putative Golgi-resident CYP protein has been reported in rice (Oryza sativa L.; OsCYP21-4). Here, we identified the Golgi-resident CYP21 family genes and analysed their molecular characteristics in Arabidopsis and rice. The CYP family genes (CYP21-1, CYP21-2, CYP21-3, and CYP21-4) are plant-specific, and their appearance and copy numbers differ among plant species. CYP21-1 and CYP21-4 are common to all angiosperms, whereas CYP21-2 and CYP21-3 evolved in the Malvidae subclass. Furthermore, all CYP21 proteins localize to cis-Golgi, trans-Golgi or both cis- and trans-Golgi membranes in plant cells. Additionally, based on the structure, enzymatic function, and topological orientation in Golgi membranes, CYP21 proteins are divided into two groups. Genetic analysis revealed that Group I proteins (CYP21-1 and CYP21-2) exhibit peptidyl prolyl cis-trans isomerase (PPIase) activity and regulate seed germination and seedling growth and development by affecting the expression levels of abscisic acid signalling genes. Thus, we identified the Golgi-resident CYPs and demonstrated that their PPIase activities are required for early seedling growth and development in higher plants.
Assuntos
Ciclofilinas/genética , Ciclofilinas/metabolismo , Complexo de Golgi/metabolismo , Desenvolvimento Vegetal , Plântula/metabolismo , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclofilinas/classificação , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Oryza/genética , Oryza/metabolismo , Peptidilprolil Isomerase/metabolismo , Filogenia , Desenvolvimento Vegetal/genética , Desenvolvimento Vegetal/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ProteômicaRESUMO
The crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway, and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of strains with single and double deletions of ΔtreY and ΔtreT in S. acidocaldarius revealed that in addition to these two pathways, a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family, establishing a new function within this group of enzymes. This novel GT4-like TPS was found to be present mainly in the Sulfolobales The ΔtreY ΔtreT Δtps triple mutant of S. acidocaldarius, which lacks the ability to synthesize trehalose, showed no altered phenotype under standard conditions or heat stress but was unable to grow under salt stress. Accordingly, in the wild-type strain, a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress-mediated induction of all three trehalose-synthesizing pathways. This demonstrates that in Archaea, trehalose plays an essential role for growth under high-salt conditions.IMPORTANCE The metabolism and function of trehalose as a compatible solute in Archaea was not well understood. This combined genetic and enzymatic approach at the interface of microbiology, physiology, and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea Here, we unraveled the complexity of trehalose metabolism, and we present a comprehensive study on trehalose function in stress response in S. acidocaldarius This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea, involving many novel biocatalysts, such as glycosyltransferases, with great potential in biotechnology.
Assuntos
Proteínas Arqueais/genética , Estresse Salino/genética , Sulfolobus acidocaldarius/enzimologia , Trealose/metabolismo , Proteínas Arqueais/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Redes e Vias Metabólicas , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate target gene expression by binding to sequences in messenger RNA processing. Inflammation is a protective reaction from harmful stimuli. MiRNAs can be biomarkers of diseases related to inflammation and are widely expressed in serum. However, overall changes in serum miRNA levels during inflammation have yet to be observed. Here, we selected studies published until 20 January 2020 that examined miRNAs in mouse models of inflammation. Serum microRNA, inflammation, inflammatory and mouse were used as search terms to select articles from PubMed and MEDLINE. Among the articles, sepsis and 18 related miRNAs were mainly examined. Eleven miRNAs were related to brain disease and 10 with fibrosis. Seventeen injury-induced inflammatory disease studies were included, as well as other inflammatory diseases, such as metabolic disease, vascular disease, arthritis, asthma, autoimmune disease, inflammatory bowel disease, and thyroiditis. The data described miRNA-associated downstream pathways associated with inflammation as well as mitochondrial responses, oxidative responses, apoptosis, cell signalling, and cell differentiation. We expect that the data will inform future animal inflammation-related miRNA studies.
Assuntos
Biomarcadores/sangue , Inflamação/sangue , MicroRNAs/sangue , Sepse/sangue , Animais , Modelos Animais de Doenças , Humanos , Inflamação/patologia , Camundongos , MicroRNAs/genética , Sepse/patologiaRESUMO
BACKGROUND: Despite recent advances in studies on the gastric microbiome, the role of the non-Helicobacter pylori gastric microbiome in gastric carcinogenesis remains unclear. We evaluated the characteristics of the gastric microbiome and metagenomic functions in patients with IM. METHODS: Participants were classified into six groups according to disease status (chronic superficial gastritis [CSG], intestinal metaplasia [IM], and cancer) and H. pylori- infection status (H. pylori-positive and H. pylori-negative). The gastric microbiome was analyzed in mucosal tissues at the gastric antrum by 16S rRNA gene sequencing. Moreover, we assessed the metagenome including the type IV secretion system (T4SS) gene, as T4SS proteins are essential for transferring CagA from H. pylori- into the human gastric epithelium. RESULTS: Among the 138 included patients, 48, 9, 23, 14, 12, and 32 were classified into the H. pylori-negative CSG, H. pylori-negative IM, H. pylori-negative cancer, H. pylori-positive CSG, H. pylori-positive IM, and H. pylori-positive cancer groups, respectively. Cyanobacteria were predominant in the H. pylori-negative CSG group compared to in the H. pylori-negative IM and H. pylori-negative cancer groups (H. pylori-negative CSG vs H. pylori-negative IM vs H. pylori-negative cancer: 14.0% vs 4.2% vs 0.04%, P < 0.001). In contrast, Rhizobiales were commonly observed in the H. pylori-negative IM group (H. pylori-negative CSG vs H. pylori-negative IM vs H. pylori-negative cancer: 1.9% vs 15.4% vs 2.8%, P < 0.001). The relative abundance of Rhizobiales increased as H. pylori-infected stomachs progressed from gastritis to IM. In the H. pylori-negative IM group, genes encoding T4SS were prevalent among the metagenome. Additionally, after H. pylori- eradication therapy, the gastric microbiome was similar to the microbiome observed after spontaneous clearance of H. pylori-. CONCLUSIONS: The relative abundance of Rhizobiales was higher in patients with H. pylori-negative IM than in those with H. pylori-negative CSG or cancer. Additionally, T4SS genes were highly observed in the metagenome of patients with IM. Highly abundant T4SS proteins in these patients may promote gastric carcinogenesis.
Assuntos
Microbioma Gastrointestinal/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Intestinos/microbiologia , Intestinos/patologia , Metaplasia/microbiologia , Adulto , Idoso , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Progressão da Doença , Feminino , Gastrite/microbiologia , Gastrite/patologia , Gastrite Atrófica/tratamento farmacológico , Gastrite Atrófica/microbiologia , Gastrite Atrófica/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Masculino , Metagenômica , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Sistemas de Secreção Tipo IV/genética , Adulto JovemRESUMO
In this study, the energy and exergy of an automobile refrigeration system using R134a and R134a/R1234yf were analyzed experimentally with respect to outdoor air temperature and compressor speed. As outdoor air temperature increased from 32.5 °C to 37.5 °C, the coefficient of performance (COP) and total exergy destruction rate of the refrigeration system using Mix30 decreased by 5.19% and 25.8% on average, compared to that of the system using R134a. The exergy efficiency of the Mix30 refrigeration system was on average 21.8% higher than that of the R134a system. As the compressor rotating speed increased from 1000 to 2000 rpm, the cooling capacity of the refrigeration system using R134a and R134a/R1234yf increased, while the COP decreased. The COP and total exergy destruction rate of the refrigeration system using Mix30 decreased by 4.82% and 19.5%, compared to that of the system using R134a. The exergy efficiency of the Mix30 refrigeration system increased on average by 20.7%, compared to that of the R134a system. The total exergy destruction rate of the automobile refrigeration system using R134a/R1234yf decreased with increase in R1234yf, while exergy efficiency increased. In addition, the exergy destruction rate of the automobile refrigeration system decreased as the amount of R1234yf in the R134a/R1234yf automobile refrigeration system increased.
RESUMO
BACKGROUND: Smartphone addiction has recently been highlighted as a major health issue among adolescents. In this study, we assessed the degree of agreement between adolescents' and parents' ratings of adolescents' smartphone addiction. Additionally, we evaluated the psychosocial factors associated with adolescents' and parents' ratings of adolescents' smartphone addiction. METHODS: In total, 158 adolescents aged 12-19 years and their parents participated in this study. The adolescents completed the Smartphone Addiction Scale (SAS) and the Isolated Peer Relationship Inventory (IPRI). Their parents also completed the SAS (about their adolescents), SAS-Short Version (SAS-SV; about themselves), Generalized Anxiety Disorder-7 (GAD-7), and Patient Health Questionnaire-9 (PHQ-9). We used the paired t-test, McNemar test, and Pearson's correlation analyses. RESULTS: Percentage of risk users was higher in parents' ratings of adolescents' smartphone addiction than ratings of adolescents themselves. There was disagreement between the SAS and SAS-parent report total scores and subscale scores on positive anticipation, withdrawal, and cyberspace-oriented relationship. SAS scores were positively associated with average minutes of weekday/holiday smartphone use and scores on the IPRI and father's GAD-7 and PHQ-9 scores. Additionally, SAS-parent report scores showed positive associations with average minutes of weekday/holiday smartphone use and each parent's SAS-SV, GAD-7, and PHQ-9 scores. CONCLUSION: The results suggest that clinicians need to consider both adolescents' and parents' reports when assessing adolescents' smartphone addiction, and be aware of the possibility of under- or overestimation. Our results cannot only be a reference in assessing adolescents' smartphone addiction, but also provide inspiration for future studies.
Assuntos
Comportamento Aditivo/patologia , Pais/psicologia , Smartphone , Adolescente , Criança , Feminino , Humanos , Internet , Masculino , Psicologia do Adolescente , Inquéritos e Questionários , Adulto JovemRESUMO
Protein kinases are deeply involved in immune-related diseases and various cancers. They are a potential target for structure-based drug discovery, since the general structure and characteristics of kinase domains are relatively well-known. However, the ATP binding sites in protein kinases, which serve as target sites, are highly conserved, and thus it is difficult to develop selective kinase inhibitors. To resolve this problem, we performed molecular dynamics simulations on 26 kinases in the aqueous solution, and analyzed topological water networks (TWNs) in their ATP binding sites. Repositioning of a known kinase inhibitor in the ATP binding sites of kinases that exhibited a TWN similar to interleukin-1 receptor-associated kinase 4 (IRAK4) allowed us to identify a hit molecule. Another hit molecule was obtained from a commercial chemical library using pharmacophore-based virtual screening and molecular docking approaches. Pharmacophoric features of the hit molecules were hybridized to design a novel compound that inhibited IRAK4 at low nanomolar levels in the in vitro assay.
Assuntos
Desenho de Fármacos , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Água/química , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Estaurosporina/química , Estaurosporina/farmacologiaRESUMO
Ultra-performance convergence chromatography, which integrates the advantages of supercritical fluid chromatography and ultra high performance liquid chromatography technologies, is an environmentally friendly analytical method that uses dramatically reduced amounts of organic solvents. An ultra-performance convergence chromatography method was developed and validated for the quantification of decursinol angelate and decursin in Angelica gigas using a CSH Fluoro-Phenyl column (2.1 mm × 150 mm, 1.7 µm) with a run time of 4 min. The method had an improved resolution and a shorter analysis time in comparison to the conventional high-performance liquid chromatography method. This method was validated in terms of linearity, precision, and accuracy. The limits of detection were 0.005 and 0.004 µg/mL for decursinol angelate and decursin, respectively, while the limits of quantitation were 0.014 and 0.012 µg/mL, respectively. The two components showed good regression (correlation coefficient (r2 ) > 0.999), excellent precision (RSD < 2.28%), and acceptable recoveries (99.75-102.62%). The proposed method can be used to efficiently separate, characterize, and quantify decursinol angelate and decursin in Angelica gigas and its related medicinal materials or preparations, with the advantages of a shorter analysis time, greater sensitivity, and better environmental compatibility.
Assuntos
Angelica/química , Cromatografia Líquida de Alta Pressão , Cromatografia com Fluido Supercrítico , Extratos Vegetais/análise , Controle de QualidadeRESUMO
Alternative splicing (AS) is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1) transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER) targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H) and bimolecular fluoresence complementation (BiFC assays), although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1.
Assuntos
Processamento Alternativo/genética , Aromatase/metabolismo , Resposta ao Choque Frio/genética , Oryza/genética , Peptidilprolil Isomerase/metabolismo , Isoformas de Proteínas/genética , Aromatase/genética , Sequência de Bases , Retículo Endoplasmático/metabolismo , Immunoblotting , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Peptidilprolil Isomerase/genética , Mapas de Interação de Proteínas , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVE: Aberrant regulation of the proliferation, survival, and migration of endothelial cells (ECs) is closely related to the abnormal angiogenesis that occurs in hypoxia-induced pathological situations, such as cancer and vascular retinopathy. Hypoxic conditions and the subsequent upregulation of hypoxia-inducible factor-1α and target genes are important for the angiogenic functions of ECs. Phospholipase D2 (PLD2) is a crucial signaling mediator that stimulates the production of the second messenger phosphatidic acid. PLD2 is involved in various cellular functions; however, its specific roles in ECs under hypoxia and in vivo angiogenesis remain unclear. In the present study, we investigated the potential roles of PLD2 in ECs under hypoxia and in hypoxia-induced pathological angiogenesis in vivo. APPROACH AND RESULTS: Pld2 knockout ECs exhibited decreased hypoxia-induced cellular responses in survival, migration, and thus vessel sprouting. Analysis of hypoxia-induced gene expression revealed that PLD2 deficiency disrupted the upregulation of hypoxia-inducible factor-1α target genes, including VEGF, PFKFB3, HMOX-1, and NTRK2. Consistent with this, PLD2 contributed to hypoxia-induced hypoxia-inducible factor-1α expression at the translational level. The roles of PLD2 in hypoxia-induced in vivo pathological angiogenesis were assessed using oxygen-induced retinopathy and tumor implantation models in endothelial-specific Pld2 knockout mice. Pld2 endothelial-specific knockout retinae showed decreased neovascular tuft formation, despite a larger avascular region. Tumor growth and tumor blood vessel formation were also reduced in Pld2 endothelial-specific knockout mice. CONCLUSIONS: Our findings demonstrate a novel role for endothelial PLD2 in the survival and migration of ECs under hypoxia via the expression of hypoxia-inducible factor-1α and in pathological retinal angiogenesis and tumor angiogenesis in vivo.
Assuntos
Carcinoma Pulmonar de Lewis/irrigação sanguínea , Células Endoteliais/enzimologia , Hipóxia/complicações , Neovascularização Patológica , Fosfolipase D/deficiência , Neovascularização Retiniana/enzimologia , Vasos Retinianos/enzimologia , Animais , Animais Recém-Nascidos , Hipóxia Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipase D/genética , Interferência de RNA , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Vasos Retinianos/patologia , Fatores de Tempo , Técnicas de Cultura de Tecidos , TransfecçãoRESUMO
AIMS/HYPOTHESIS: Obesity-induced inflammation is initiated by the recruitment of macrophages into adipose tissue. The recruited macrophages, called adipose tissue macrophages, secrete several proinflammatory cytokines that cause low-grade systemic inflammation and insulin resistance. The aim of this study was to find macrophage-recruiting factors that are thought to provide a crucial connection between obesity and insulin resistance. METHODS: We used chemotaxis assay, reverse phase HPLC and tandem MS analysis to find chemotactic factors from adipocytes. The expression of chemokines and macrophage markers was evaluated by quantitative RT-PCR, immunohistochemistry and FACS analysis. RESULTS: We report our finding that the chemokine (C-X-C motif) ligand 12 (CXCL12, also known as stromal cell-derived factor 1), identified from 3T3-L1 adipocyte conditioned medium, induces monocyte migration via its receptor chemokine (C-X-C motif) receptor 4 (CXCR4). Diet-induced obese mice demonstrated a robust increase of CXCL12 expression in white adipose tissue (WAT). Treatment of obese mice with a CXCR4 antagonist reduced macrophage accumulation and production of proinflammatory cytokines in WAT, and improved systemic insulin sensitivity. CONCLUSIONS/INTERPRETATION: In this study we found that CXCL12 is an adipocyte-derived chemotactic factor that recruits macrophages, and that it is a required factor for the establishment of obesity-induced adipose tissue inflammation and systemic insulin resistance.
Assuntos
Tecido Adiposo/metabolismo , Quimiocina CXCL12/metabolismo , Resistência à Insulina/fisiologia , Macrófagos/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Linhagem Celular , Quimiotaxia/fisiologia , Camundongos , Obesidade/metabolismoRESUMO
AMP-activated protein kinase has been described as a key signaling protein that can regulate energy homeostasis. Here, we aimed to characterize novel AMP-activated kinase (AMPK)-activating compounds that have a much lower effective concentration than metformin. As a result, emodin, a natural anthraquinone derivative, was shown to stimulate AMPK activity in skeletal muscle and liver cells. Emodin enhanced GLUT4 translocation and [(14)C]glucose uptake into the myotube in an AMPK-dependent manner. Also, emodin inhibited glucose production by suppressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species and Ca(2+)/calmodulin-dependent protein kinase kinase activity. Finally, we confirmed that a single dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection of emodin for 8 days using an insulin tolerance test and insulin-stimulated PI3K phosphorylation in wild type and high fat diet-induced diabetic mouse models. Our study suggests that emodin regulates glucose homeostasis in vivo by AMPK activation and that this may represent a novel therapeutic principle in the treatment of type 2 diabetic models.