984 resultados para secondary metabolism
Resumo:
The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.
Resumo:
Plant cell cultures have been suggested as a feasible technology for the production of a myriad of plant-derived metabolites. However, commercial application of plant cell culture has met limited success with only a handful of metabolites produced at the pilot- and commercial-scales. To improve the production of secondary metabolites in plant cell cultures, efforts have been devoted predominantly to the optimization of biosynthetic pathways by both process and genetic engineering approaches. Given that secondary metabolism includes-the synthesis. metabolism and catabolism of endogenous compounds by the specialized proteins, this review intends to draw attention to the manipulation and optimization of post-biosynthetic events that follow the formation of core metabolite structures in biosynthetic pathways. These post-biosynthetic events-the chemical and enzymatic modifications, transport, storage/secretion and catabolism/degradation have been largely unexplored in the past. Potential areas are identified where further research is needed to answer fundamental questions that have implications for advanced bioprocess design. Anthocyanin production by plant cell cultures is used as a case study for this discussion, as it presents a good example of compounds for which there are extensive research publications but still no commercial bioprocess. It is perceived that research on post-biosynthetic processes may lead to future opportunities for significant advances in commercial plant cell cultures. (C) 2002 Elsevier Science Inc. All rights reserved.
Resumo:
Aspergillus nidulans contains two functionally distinct fatty acid synthases (FASs): one required for primary fatty acid metabolism (FAS) and the other required for secondary metabolism (sFAS). FAS mutants require long-chain fatty acids for growth, whereas sFAS mutants grow normally but cannot synthesize sterigmatocystin (ST), a carcinogenic secondary metabolite structurally and biosynthetically related to aflatoxin. sFAS mutants regain the ability to synthesize ST when provided with hexanoic acid, supporting the model that the ST polyketide synthase uses this short-chain fatty acid as a starter unit. The characterization of both the polyketide synthase and FAS may provide novel means for modifying secondary metabolites.
Resumo:
The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.
Resumo:
Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome evolution, usually associated with increased fitness and/or changes in behavior, especially if one considers pathogenic vs. non-pathogenic bacterial groups. The genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, were previously inspected for genome islands originating from LGT events, and, in this work, potentially early and late LGT events were identified according to their altered nucleotide composition. The biological role of the islands was also assessed, and pathogenicity, virulence and secondary metabolism pathways were functions highly represented, especially in islands that were found to be recently transferred. However, old islands are composed of a high proportion of genes related to cell primary metabolic functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally inherited by vertical processes.
Resumo:
Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and sourcesink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory.
Resumo:
Myora Springs is one of many groundwater discharge sites on North Stradbroke Island (Queensland, Australia). Here spring waters emerge from wetland forests to join Moreton Bay, mixing with seawater over seagrass meadows dominated by eelgrass, Zostera muelleri. We sought to determine how low pH / high CO2 conditions near the spring affect these plants and their interactions with the black rabbitfish (Siganus fuscescens), a co-occurring grazer. In paired-choice feeding trials S. fuscescens preferentially consumed Z. muelleri shoots collected nearest to Myora Springs. Proximity to the spring did not significantly alter the carbon and nitrogen contents of seagrass tissues but did result in the extraordinary loss of soluble phenolics, including Folin-reactive phenolics, condensed tannins, and phenolic acids by ?87%. Conversely, seagrass lignin contents were, in this and related experiments, unaffected or increased, suggesting a shift in secondary metabolism away from the production of soluble, but not insoluble, (poly)phenolics. We suggest that groundwater discharge sites such as Myora Springs, and other sites characterized by low pH, are likely to be popular feeding grounds for seagrass grazers seeking to reduce their exposure to soluble phenolics.
Resumo:
In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Resumo:
Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.
Resumo:
Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.
Resumo:
The regulation of carotenoid biosynthesis in a high-carotenoid-accumulating Fei group Musa cultivar, Asupina, has been examined and compared to that of a low-carotenoid-accumulating cultivar, Cavendish, to understand the molecular basis underlying carotenogenesis during banana fruit development. Comparisons in the accumulation of carotenoid species, expression of isoprenoid genes, and product sequestration are reported. Key differences between the cultivars include greater carotenoid cleavage dioxygenase 4 (CCD4) expression in Cavendish and the conversion of amyloplasts to chromoplasts during fruit ripening in Asupina. Chromoplast development coincided with a reduction in dry matter content and fruit firmness. Chromoplasts were not observed in Cavendish fruits. Such information should provide important insights for future developments in the biofortification and breeding of banana.
Resumo:
A biodiversidade brasileira abrange plantas de importncia medicinal que podem ser utilizadas na formulao de novos frmacos. Contudo, tem sido reduzida em velocidade alarmante, em funo de diferentes aes antrpicas. A cultura de tecidos vegetais propicia a conservao e uso do germoplasma permitindo a obteno de substncias de importncia medicinal. As leishmanioses so consideradas um problema de sade pblica mundial sendo a espcie Leishmania braziliensis de maior importncia epidemiolgica no Brasil. Recentemente tem-se registrado aumento da resistncia linha de tratamento usual. Do mesmo modo, o uso indiscriminado de antibiticos levou ao aumento de bactrias multirresistentes, que representam srio risco de infeco. A espcie Annona mucosa (Jacq.) possui substncias, como acetogeninas e alcaloides, que apresentam atividades antiparasitria e antimicrobiana. Nesse sentido, o objetivo do trabalho foi avaliar o potencial leishmanicida e antibacteriano de extratos de A. mucosa de material produzido in vitro e in vivo. Foi proposto um protocolo de germinao in vitro, ainda no reportada para a espcie, com vistas obteno de plntulas axnicas. Em meio WPM foram cultivados explantes hipocotiledonares e foliares em meio MS, suplementados com PIC e diferentes concentraes de KIN, BAP ou TDZ. Os calos obtidos foram cultivados em meio lquido de mesma composio para a produo de suspenses celulares. Os materiais foram submetidos extrao metanlica e posterior fracionamento em hexano e diclorometano. Para a avaliao da atividade dos extratos sobre L. braziliensis foi usado o modelo in vitro, com a forma promastigota, e in vivo na forma amastigota, a partir do tratamento de macrfagos peritoneais de camundongos infectados com o parasito. Ambas as formas foram tratadas com os extratos por 96 e 48h, respectivamente. A atividade antimicrobiana foi avaliada por macrodiluio do extrato em Mueller-Hinton, sendo avaliado o crescimento das cepas aps 16h de incubao a 48C. A germinao in vitro da espcie foi alcanada em substrato vermiculita estril umedecido com soluo de sais do meio MS, com taxa mdia de 85%. A maior produo de calos friveis foi obtida em meios contendo KIN, com potencial uso para cultivo em suspenses celulares. Os extratos do material in situ e in vitro apresentaram atividade leishmanicida, apesar da toxicidade para macrfagos. Culturas de clulas em suspenso apresentaram potencial leishmanicida in vitro e reduo da infeco em macrfagos. Os extratos do material avaliado apresentaram atividade antimicrobiana seletiva, com inibio do crescimento de Streptococcus pyogenes e Bacillus thurigiensis em diferentes concentraes avaliadas. Os mtodos biotecnolgicos empregados permitiram a obteno de materiais com propriedades medicinais para as atividades leishmanicida e antibacteriana, assim como o material in vivo, constituindo este estudo o primeiro relato para as atividades propostas em A. mucosa.
Resumo:
Saussurea medusa Maxim3-cDNA 1. cDNA4106pfu/ml1011 pfu/ml98%PCR0.5kb3kb1kb62%chsdfrMybSmPPCR 2. RT-PCRSmchiTD-PCRcDNASmchi cDNA831bp232cDNASmchi DNASmchiSmchi cDNAchiORF84%79.3%Smchi mRNASmCHIChalconeCHI7-8N115M115CHISmchiSmchi50%670%85.1%SmchiSmCHICHICHI 3. Smchi3-Smf3hcDNA1334bp343aacDNASmf3h DNA1630bp43Smf3h mRNA SmF3H2OG-FeII_OxyH6H45%F3H40%SmF3HF3HH6HSmF3HF3HSmf3hSmf3hf3hh6h 4. SmchicDNASmPcDNA969bp256 aacDNASmPDNASmPSmPcDNA mRNAA+T63%A-TSmPR2R3-MybN-Myb DNA-binding DomainMyb1A5JOsMYBThMYB71.3%70.8%;C-NtMYBVlMYBS170S206SmP51%0.5-6SmPSmP92%-100% 5. Erich Grotewold EST50SmANSSmALDHSmANSPCR50SmANScDNA1229bp356aaSmANScDNAANSORF80%mRNA ;ANS82.9%SmANS2OG-FeII_Oxy2OG-FeII_OxyANS94%---711-2OG-FeII_OxyHis84His235His291Asp237 6. ESTSmALDHPCR50SmALDHcDNA 1664bp491aaSmALDHcDNA3/-UTRA+T80%5/-UTRA+TG+C50%ORF52%mRNA5/-UTRSmALDHcDNAORF64.03%63.89%63.72%54.9%54.3%54.0%SmALDHAldedh 1OF7-H---1015-ALDHSmALDH
Resumo:
(Saussurea medusa Maxim), SAYESAYESA620 MSA532 mg/l630 mg/l130%150%SAYEYESA1040 g/mlYE741 mg/l303 mg/l2.82.5 20 M100 M SASACHSCHI24hPAL7.548 hSACHSCHIPAL cDNASmCHScDNAcDNA1313bp38988%SmCHSCHSlikeCHSCHSSmCHSSmCHSSmCHSchs
Resumo:
JAMeJA -AlCl3-NaClF-CHATUInd-IleMe70 % Ind-IleMecoronalonMeJAHPLC15%20%coronalon 1 mol/LInd-IleMe10 mol/LMeJA100 mol/L MeJASODMDAPALMeJA VOCsSPMEGC-MSSPME