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Ligularia fischeri, a leafy edible plant found in damp shady regions, has been used as an herbal medicine and is also consumed as a horticultural crop. In this study, we investigated the physiological and transcriptomic changes, especially those involved in phenylpropanoid biosynthesis, induced by severe drought stress in L. fischeri plants. A distinguishing characteristic of L. fischeri is a color change from green to purple due to anthocyanin biosynthesis. We chromatographically isolated and identified two anthocyanins and two flavones upregulated by drought stress using liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses in this plant for the first time. In contrast, all types of caffeoylquinic acids (CQAs) and flavonol contents were decreased under drought stress. Further, we performed RNA sequencing to examine the molecular changes in these phenolic compounds at the transcriptome level. In an overview of drought-inducible responses, we identified 2,105 hits for 516 distinct transcripts as drought-responsive genes. Moreover, differentially expressed genes (DEGs) associated with phenylpropanoid biosynthesis accounted for the greatest number of both up- and downregulated DEGs by Kyoto Encyclopedia of Genes and Genomes enrichment analysis. We identified 24 meaningful DEGs based on the regulation of phenylpropanoid biosynthetic genes. Potential drought-responsive genes included upregulated flavone synthase (LfFNS, TRINITY DN31661 c0 g1 i1) and anthocyanin 5-O-glucosyltransferase (LfA5GT1, TRINITY DN782 c0 g1 i1), which could contribute to the high levels of flavones and anthocyanins under drought stress in L. fischeri. In addition, the downregulated shikimate O-hydroxycinnamolytransferase (LfHCT, TRINITY DN31661 c0 g1 i1) and hydroxycinnamoyl-CoA quinate/shikimate transferase (LfHQT4, TRINITY DN15180 c0 g1 i1) genes led to a reduction in CQAs. Only one or two BLASTP hits for LfHCT were obtained for six different Asteraceae species. It is possible that the HCT gene plays a crucial role in CQAs biosynthesis in these species. These findings expand our knowledge of the response mechanisms to drought stress, particularly regarding the regulation of key phenylpropanoid biosynthetic genes in L. fischeri.
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BACKGROUND: Chaperon-mediated autophagy (CMA) has taken on a new emphasis in cancer biology. However, the roles of CMA in hypoxic tumours are poorly understood. We investigated the anti-tumour effects of the natural product ManA through the activation of CMA in tumour progression under hypoxia. METHODS: The effect of ManA on CMA activation was assessed in mouse xenograft models and cells. The gene expressions of HIF-1α, HSP90AA1, and transcription factor EB (TFEB) were analysed using The Cancer Genome Atlas (TCGA) datasets to assess the clinical relevance of CMA. RESULTS: ManA activates photoswitchable CMA reporter activity and inhibits Hsp90 chaperone function by disrupting the Hsp90/F1F0-ATP synthase complex. Hsp90 inhibition enhances the interaction between CMA substrates and LAMP-2A and TFEB nuclear localisation, suggesting CMA activation by ManA. ManA-activated CMA retards tumour growth and displays cooperative anti-tumour activity with anti-PD-1 antibody. TCGA datasets show that a combined expression of HSP90AA1High/HIF1AHigh or TFEBLow/HIF1AHigh is strongly correlated with poor prognosis in patients with lung cancer. CONCLUSIONS: ManA-induced CMA activation by modulating Hsp90 under hypoxia induces HIF-1α degradation and reduces tumour growth. Thus, inducing CMA activity by targeting Hsp90 may be a promising therapeutic strategy against hypoxic tumours.
Assuntos
Autofagia Mediada por Chaperonas , Neoplasias Pulmonares , Camundongos , Animais , Humanos , Hipóxia , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares , Autofagia/genéticaRESUMO
Antibiotic resistance is one of the most challenging global health issues and presents an urgent need for the development of new antibiotics. In this regard, phospho-MurNAc-pentapeptide translocase (MraY), an essential enzyme in the early stages of peptidoglycan biosynthesis, has emerged as a promising new antibiotic target. We recently reported the crystal structures of MraY in complex with representative members of naturally occurring nucleoside antibiotics, including muraymycin D2. However, these nucleoside antibiotics are synthetically challenging targets, which limits the scope of medicinal chemistry efforts on this class of compounds. To gain access to active muraymycin analogs with reduced structural complexity and improved synthetic tractability, we prepared and evaluated cyclopentane-based muraymycin analogs for targeting MraY. For the installation of the 1,2-syn-amino alcohol group of analogs, the diastereoselective isocyanoacetate aldol reaction was explored. The structure-activity relationship analysis of the synthesized analogs suggested that a lipophilic side chain is essential for MraY inhibition. Importantly, the analog 20 (JH-MR-23) showed antibacterial efficacy against Staphylococcus aureus. These findings provide insights into designing new muraymycin-based MraY inhibitors with improved chemical tractability.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Ciclopentanos/farmacologia , Transferases/antagonistas & inibidores , Uridina/análogos & derivados , Uridina/farmacologia , Antibacterianos/síntese química , Arginina/análogos & derivados , Arginina/farmacologia , Ciclopentanos/síntese química , Ensaios Enzimáticos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
PURPOSE: Osteoporosis is a metabolic skeletal disease characterized by bone loss and an increased risk of fractures. This study aimed to investigate the therapeutic effect of Agastache rugosa on postmenopausal osteoporosis and elucidate its mechanisms in modulating the bone status. METHODS AND RESULTS: In the osteoblast differentiation process with MC3T3-E1 pre-osteoblasts, ethanol extract of Agastache rugosa (EEAR) and its compounds increased the expression of the proteins and genes of the osteoblast differentiation-related markers such as Runt-related transcription factor 2 (RUNX2) and ß-catenin along with the elevation of calcium deposits. An ovariectomized mouse model was utilized to determine the impact of EEAR extract on postmenopausal osteoporosis. Twelve weeks of AR treatment suppressed the loss of bone strength, which was observed through micro-computed tomography. AR elevated osteogenic markers in the bone marrow cells, and collagen type 1 alpha 1 in the distal femoral bone. The results of the 16S rRNA gene sequencing analysis of cecal gut microbiomes demonstrated that AR reversed the ovariectomy-induced changes in the gut microbiomes. CONCLUSION: Ethanol extract of Agastache rugosa has a therapeutic effect on postmenopausal osteoporosis via bone morphogenic protein, transforming growth factor ß, and Wnt signaling pathway. It also increases the diversity of gut microbiota. Therefore, these data suggest that EEAR could be a potential candidate to treat postmenopausal osteoporosis.
Assuntos
Agastache/química , Microbioma Gastrointestinal/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Etanol/química , Feminino , Microbioma Gastrointestinal/genética , Camundongos Endogâmicos C57BL , Osteoblastos/fisiologia , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Osteoporose/microbiologia , Ovariectomia , Extratos Vegetais/química , RNA Ribossômico 16S , Via de Sinalização Wnt/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
Rosmarinic acid (RA) is an active constituent of Ocimum basilicum. It has been shown that hairy root production (measured as dry weight) improves when green basil (O. basilicum "Cinnamon") is cultured under the light. In contrast, purple basil (O. basilicum "Purpurascens") shows greater hairy root production when cultured under dark conditions. The level of gene expression was highest in hairy roots of green basil under dark conditions for up to 1 week. Transcript levels were highest in hairy roots of purple basil under both dark and light conditions after 2 weeks of culturing. After 3 weeks of culture under light conditions, green basil had accumulated 1.9-fold higher RA content than that of purple basil, which in turn was fivefold higher than that of the natural roots (42.86 µg/mg). Tyrosine aminotransferase showed a higher transcript level when compared to the other phenylpropanoid pathway genes (phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and coenzyme-A ligase) in both dark and light conditions and in all-time regimens. RA accumulation was higher in the cultured hairy roots of green basil than those of purple basil under both light and dark conditions.
Assuntos
Antioxidantes/metabolismo , Cinamatos/metabolismo , Depsídeos/metabolismo , Expressão Gênica , Ocimum basilicum/genética , Ocimum basilicum/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Expressão Gênica/efeitos da radiação , Luz , Ocimum basilicum/classificação , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Transdução de Sinais/efeitos da radiação , Transcrição Gênica/efeitos da radiação , Tirosina Transaminase/genética , Ácido RosmarínicoRESUMO
Tumors adapt to hypoxia by regulating angiogenesis, metastatic potential, and metabolism. These adaptations mediated by hypoxia-inducible factor 1 (HIF-1) make tumors more aggressive and resistant to chemotherapy and radiation. Therefore, HIF-1 is a validated therapeutic target for cancer. In order to develop new HIF-1 inhibitors for cancer chemotherapy by harnessing the potential of the natural product manassantin A, we synthesized and evaluated manassantin A analogues with modifications in the tetrahydrofuran core region of manassantin A. Our structure-activity relationship study indicated that the α,α'-trans-configuration of the central ring of manassantin A is critical to HIF-1 inhibition. We also demonstrated that a combination of manassantin A with an epidermal growth factor receptor inhibitor shows cooperative antitumor activity (â¼80% inhibition for combination vs â¼30% inhibition for monotherapy). Our findings will provide important frameworks for the future therapeutic development of manassantin A-derived chemotherapeutic agents.
Assuntos
Receptores ErbB/antagonistas & inibidores , Furanos/química , Lignanas/química , Lignanas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Células HEK293 , HumanosRESUMO
PURPOSE: The aim of this study was to evaluate the effectiveness of evidence-based guidelines for fever management of critically ill adult patients with brain injury. METHODS: We used a pretest-posttest design with 48 patients 19 years or older admitted to an intensive care unit after surgery for brain injury. We applied evidence-based guidelines only to an experimental group of 24 patients and compared with 24 control patients who did not receive evidence-based guidelines. Experimental and control groups were matched 1:1 using the Acute Physiology and Chronic Health Evaluation II score. Data included the proportion of patients with reduced fever and time to normalized temperature. RESULTS: The proportion of patients whose temperature fell to normal after fever was 4.5 times higher in the experimental group than in the control group. The time it took the patients' highest fever to fall to normal during their intensive care unit stay was 4.84 times faster in the experimental group than in the control group (hazard ratio, 4.84; 95% confidence interval, 1.79-13.11; P = .002). CONCLUSION: Evidence-based guidelines for fever management in patients with a brain injury can be used in nursing practice with rapid response, improving healthcare efficiency and contributing to better outcomes for critically ill patients.
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Lesões Encefálicas/complicações , Estado Terminal , Medicina Baseada em Evidências , Febre , Guias como Assunto , Lesões Encefálicas/cirurgia , Feminino , Febre/enfermagem , Febre/terapia , Mortalidade Hospitalar , Hospitalização , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Novel antibacterial agents are needed to address the emergence of global antibiotic resistance. MraY is a promising candidate for antibiotic development because it is the target of five classes of naturally occurring nucleoside inhibitors with potent antibacterial activity. Although these natural products share a common uridine moiety, their core structures vary substantially and they exhibit different activity profiles. An incomplete understanding of the structural and mechanistic basis of MraY inhibition has hindered the translation of these compounds to the clinic. Here we present crystal structures of MraY in complex with representative members of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors. Our structures reveal cryptic druggable hot spots in the shallow inhibitor binding site of MraY that were not previously appreciated. Structural analyses of nucleoside inhibitor binding provide insights into the chemical logic of MraY inhibition, which can guide novel approaches to MraY-targeted antibiotic design.
Assuntos
Antibacterianos/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Produtos Biológicos/química , Inibidores Enzimáticos/química , Nucleosídeos/antagonistas & inibidores , Transferases/química , Aminoglicosídeos/química , Arginina/análogos & derivados , Arginina/química , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Transferases/antagonistas & inibidores , Transferases/genética , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
After a difficult brain tumor surgery, refractory intracranial hypertension (RICH) may occur due to residual tumor or post-operative complications such as hemorrhage, infarction, and aggravated brain edema. We investigated which predictors are associated with prognosis when using barbiturate coma therapy (BCT) as a second-tier therapy to control RICH after brain tumor surgery. The study included adult patients who underwent BCT after brain tumor surgery between January 2010 and December 2016. The primary outcome was neurological status upon hospital discharge, which was assessed using the Glasgow Outcome Scale (GOS). In the study period, 4,296 patients underwent brain tumor surgery in total. Of these patients, BCT was performed in 73 patients (1.7%). Among these 73 patients, 56 (76.7%) survived to discharge and 25 (34.2%) showed favorable neurological outcomes (GOS scores of 4 and 5). Invasive monitoring of intracranial pressure (ICP) was performed in 60 (82.2%) patients, and revealed that the maximal ICP within 6 h after BCT was significantly lower in patients with favorable neurological outcome as well as in survivors (p = 0.008 and p = 0.028, respectively). Uncontrolled RICH (ICP ≥ 22 mm Hg within 6 h of BCT) was an important predictor of mortality after BCT (adjusted hazard ratio 12.91, 95% confidence interval [CI] 2.788-59.749), and in particular, ICP ≥ 15 mm Hg within 6 h of BCT was associated with poor neurological outcome (adjusted odds ratio 9.36, 95% CI 1.664-52.614). Therefore, early-controlled ICP after BCT was associated with clinical prognosis. There were no significant differences in the complications associated with BCT between the two neurological outcome groups. No BCT-induced death was observed. The active and timely control of RICH may be beneficial for clinical outcomes in patients with RICH after brain tumor surgery.
Assuntos
Barbitúricos/administração & dosagem , Edema Encefálico , Neoplasias Encefálicas , Coma , Pressão Intracraniana/efeitos dos fármacos , Complicações Pós-Operatórias , Adulto , Edema Encefálico/etiologia , Edema Encefálico/mortalidade , Edema Encefálico/terapia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/fisiopatologia , Neoplasias Encefálicas/cirurgia , Coma/induzido quimicamente , Coma/mortalidade , Coma/fisiopatologia , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/mortalidade , Complicações Pós-Operatórias/fisiopatologia , Complicações Pós-Operatórias/terapia , Taxa de SobrevidaRESUMO
We have aimed to investigate the expression of genes related to rosmarinic acid (RA) synthesis and rosmarinic acid content in 2 Ocimum basilicum cultivars, green (cinnamon) and purple (red rubin) basil. Specifically, genes related to rosmarinic acid biosynthesis were cloned and characterized for O. basilicum. We obtained partial cDNAs of tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate reductase (HPPR), which were of 323 bp and 616 bp in size, respectively. The transcription levels of most genes related to rosmarinic acid synthesis were higher in green basil compared to purple basil, except for ObPAL and Ob4CL in the root. The highest expression was obtained in the leaves of green basil for all genes and the roots of purple basil for all genes, except for TAT. The highest rosmarinic acid content was obtained in the leaves of both cultivars, with higher RA accumulating in green basil compared to purple basil. The leaves had the highest RA content out of all plant organs, with the RA accumulation in the leaves of green basil being 1.64 times higher compared to purple basil. Further study is required to investigate whether a similar trend is observed across O. basilicum cultivars of different color types.
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The UDP-2,3-diacylglucosamine pyrophosphatase LpxH in the Raetz pathway of lipid A biosynthesis is an essential enzyme in the vast majority of Gram-negative pathogens and an excellent novel antibiotic target. The 32P-radioautographic thin-layer chromatography assay has been widely used for analysis of LpxH activity, but it is inconvenient for evaluation of a large number of LpxH inhibitors over an extended time period. Here, we report a coupled, nonradioactive LpxH assay that utilizes the recently discovered Aquifex aeolicus lipid A 1-phosphatase LpxE for quantitative removal of the 1-phosphate from lipid X, the product of the LpxH catalysis; the released inorganic phosphate is subsequently quantified by the colorimetric malachite green assay, allowing the monitoring of the LpxH catalysis. Using such a coupled enzymatic assay, we report the biochemical characterization of a series of sulfonyl piperazine LpxH inhibitors. Our analysis establishes a preliminary structure-activity relationship for this class of compounds and reveals a pharmacophore of two aromatic rings, two hydrophobic groups, and one hydrogen-bond acceptor. We expect that our findings will facilitate the development of more effective LpxH inhibitors as potential antibacterial agents.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Fosfatos/química , Piperazina/química , Pirofosfatases/química , Corantes de Rosanilina/química , Antibacterianos/química , Antibacterianos/farmacologia , Aquifex , Bactérias/química , Proteínas de Bactérias/antagonistas & inibidores , Biocatálise , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Glicolipídeos/química , Piperazina/farmacologia , Pirofosfatases/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
NF00659B1 is a novel α-pyrone diterpenoid natural product with potent anti-colon cancer activity. A stereoselective approach to the 2,2-dimethyl oxepanol core of NF00659B1 is described enlisting a sequence of olefinic ester ring-closing metathesis, epoxidation, and Grignard addition. This strategy paves the way to a total synthesis of NF00659B1 for further biological studies.
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Alcenos/síntese química , Produtos Biológicos/síntese química , Alcenos/química , Produtos Biológicos/química , Conformação Molecular , EstereoisomerismoRESUMO
Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A's mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein-ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action.
Assuntos
Lignanas/metabolismo , Dobramento de Proteína , Estabilidade Proteica , Antineoplásicos/metabolismo , Produtos Biológicos , Células Cultivadas , Filaminas/metabolismo , Humanos , Marcação por Isótopo , Ligantes , Oxirredução , Fator 1 de Elongação de Peptídeos/metabolismo , Ligação Proteica , Saururaceae/químicaRESUMO
To cope with hypoxia, tumor cells have developed a number of adaptive mechanisms mediated by hypoxia-inducible factor 1 (HIF-1) to promote angiogenesis and cell survival. Due to significant roles of HIF-1 in the initiation, progression, metastasis, and resistance to treatment of most solid tumors, a considerable amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer. Isolated from Saururus cernuus, manassantins A (1) and B (2) are potent inhibitors of HIF-1 activity. To define the structural requirements of manassantins for HIF-1 inhibition, we prepared and evaluated a series of manassantin analogues. Our SAR studies examined key regions of manassantin's structure in order to understand the impact of these regions on biological activity and to define modifications that can lead to improved performance and drug-like properties. Our efforts identified several manassantin analogues with reduced structural complexity as potential lead compounds for further development. Analogues MA04, MA07, and MA11 down-regulated hypoxia-induced expression of the HIF-1α protein and reduced the levels of HIF-1 target genes, including cyclin-dependent kinase 6 (Cdk6) and vascular endothelial growth factor (VEGF). These findings provide an important framework to design potent and selective HIF-1α inhibitors, which is necessary to aid translation of manassantin-derived natural products to the clinic as novel therapeutics for cancers.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Lignanas/química , Lignanas/farmacologia , Técnicas de Química Sintética , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/química , Avaliação Pré-Clínica de Medicamentos/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Concentração Inibidora 50 , Lignanas/síntese química , Estrutura MolecularRESUMO
Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Proteínas de Transporte de Nucleosídeos/química , Uridina/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/metabolismo , Feminino , Expressão Gênica , Humanos , Cinética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Acoplamento Molecular , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribavirina/química , Ribavirina/metabolismo , Especificidade por Substrato , Termodinâmica , Uridina/química , Vibrio cholerae/química , Vibrio cholerae/metabolismo , Xenopus laevis , GencitabinaRESUMO
To improve the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1) using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA.
Assuntos
Proteínas de Arabidopsis/metabolismo , Ácido Clorogênico/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Platycodon/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Platycodon/genética , Fatores de Transcrição/genéticaRESUMO
Gamma-aminobutyric acid (GABA), synthesized by glutamate decarboxylase (GAD), plays an important role in plants. To study the molecular mechanism of GAD regulation and to examine the levels of GABA in Scutellaria baicalensis, we isolated cDNA clones (SbGAD1 and 2) encoding GAD from S. baicalensis. The open reading frames of SbGADI and 2 were 1,503 and 1,494 bp long and had 450 and 497 amino acid residues, respectively. Quantitative real-time RT-PCR analysis was performed to show the variation of transcript levels among different organs of S. baicalensis. Transcript levels of SbGAD1 and 2 were highest in the root and flower, respectively. The GABA content of different parts (ranked in descending order) was as follows: leaf > flower > stem > root. We concluded that the expression pattern of SbGAD1 and 2 did not match the accumulation pattern of GABA in different organs. We presume that GABA biosynthesis might be more controlled by SbGAD2 than SbGADI. These data will aid in future studies that seek to understand the mechanisms underlying GABA biosynthesis, an important amino acid that is synthesized by the GAD enzyme. To explain adequately the GABA biosynthesis mechanisms in S. baicalensis, the enzyme activities of SbGAD1 and 2 should be determined in the near future.
Assuntos
Glutamato Descarboxilase/genética , Scutellaria baicalensis/genética , Ácido gama-Aminobutírico/biossíntese , Sequência de Bases , Cálcio/metabolismo , Calmodulina/metabolismo , DNA Complementar/química , Glutamato Descarboxilase/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Scutellaria baicalensis/enzimologia , Análise de Sequência de DNARESUMO
MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg(2+) within the active site, and provide a structural basis of catalysis for this class of enzyme.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Parede Celular/química , Proteínas de Membrana/química , Transferases/química , Proteínas de Bactérias/genética , Domínio Catalítico , Parede Celular/enzimologia , Cristalografia por Raios X , Citoplasma/enzimologia , Proteínas de Membrana/genética , Periplasma/enzimologia , Conformação Proteica , Estrutura Secundária de Proteína , Transferases/genética , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
In the present study, carotenoids, anthocyanins, and phenolic acids of cauliflowers ( Brassica oleracea L. ssp. botrytis) with various colored florets (white, yellow, green, and purple) were characterized to determine their phytochemical diversity. Additionally, 48 metabolites comprising amino acids, organic acids, sugars, and sugar alcohols were identified using gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). Carotenoid content was considerably higher in green cauliflower; anthocyanins were detected only in purple cauliflower. Phenolic acids were higher in both green and purple cauliflower. Results of partial least-squares discriminant, Pearson correlation, and hierarchical clustering analyses showed that green cauliflower is distinct on the basis of the high levels of amino acids and clusters derived from common or closely related biochemical pathways. These results suggest that GC-TOFMS-based metabolite profiling, combined with chemometrics, is a useful tool for determining phenotypic variation and identifying metabolic networks connecting primary and secondary metabolism.
Assuntos
Brassica/metabolismo , Metaboloma , Aminoácidos/análise , Antocianinas/análise , Brassica/classificação , Carboidratos/análise , Ácidos Carboxílicos/análise , Carotenoides/análise , Cromatografia Gasosa-Espectrometria de Massas , Hidroxibenzoatos/análise , Análise dos Mínimos Quadrados , Pigmentação , Extratos Vegetais/química , Metabolismo Secundário , Especificidade da Espécie , Álcoois Açúcares/análiseRESUMO
The relationship between carotenoid accumulation and expression of carotenoid biosynthesis genes was investigated in the flowers, stems, young leaves, old leaves, and roots of Chinese cabbage (Brassica rapa subsp. pekinensis). Quantitative real-time PCR analysis showed that the mRNA levels of BrPSY, BrPDS, BrZDS, BrLCYB, BrLCYE, BrCHXB, and BrZEP leading to the production of carotenoids were highest in the flowers or the leaves and lowest in the roots of Chinese cabbage. In contrast, the mRNA expression of BrNCED, a gene involved in abscisic acid (ABA) biosynthesis, was highest in the roots. High-performance liquid chromatography revealed that carotenoids, namely, lutein and ß-carotene, were distributed predominantly in the flowers and leaves, with very little in the underground organ, the roots. Specifically, old leaves contained 120.3 µg/g lutein and 103.93 µg/g ß-carotene, which is the most potent dietary precursor of vitamin A. Moreover, we found a relatively large amount of cis isomers of ß-carotene, namely, 9-cis ß-carotene and 13-cis ß-carotene, in Chinese cabbage. These results provide insight into carotenoid biosynthetic mechanisms in Chinese cabbage and may be helpful in the metabolic engineering of carotenoid biosynthesis in plants.