ABSTRACT
The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.
Subject(s)
Aldehyde Reductase/metabolism , Carbohydrate Epimerases/metabolism , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Plant , Morphine/biosynthesis , Papaver/metabolism , Plant Proteins/metabolism , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Alkaloids/biosynthesis , Alkaloids/chemistry , Base Sequence , Benzylisoquinolines/chemistry , Benzylisoquinolines/metabolism , Bromoviridae/genetics , Bromoviridae/metabolism , Carbohydrate Epimerases/antagonists & inhibitors , Carbohydrate Epimerases/genetics , Cytochrome P-450 Enzyme System/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Exons , Gene Fusion , Introns , Ligases/genetics , Ligases/metabolism , Molecular Sequence Data , Morphinans/chemistry , Morphinans/metabolism , Morphine/chemistry , Open Reading Frames , Opium/chemistry , Opium/metabolism , Oxidation-Reduction , Papaver/genetics , Plant Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , StereoisomerismABSTRACT
BACKGROUND: Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. RESULTS: A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. CONCLUSIONS: The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.
Subject(s)
Alkaloids/metabolism , Gene Expression Profiling , Plant Proteins/analysis , Proteome/analysis , Alkaloids/chemistry , Benzophenanthridines/chemistry , Benzophenanthridines/metabolism , Benzylisoquinolines/chemistry , Benzylisoquinolines/metabolism , Biological Factors/pharmacology , Biosynthetic Pathways/drug effects , Botrytis/chemistry , Cells, Cultured , Chromatography, High Pressure Liquid , Cluster Analysis , Electrophoresis, Polyacrylamide Gel , High-Throughput Nucleotide Sequencing , Isoquinolines/chemistry , Isoquinolines/metabolism , Mass Spectrometry , Molecular Sequence Data , Molecular Structure , Morphine/chemistry , Morphine/metabolism , Opium/chemistry , Opium/metabolism , Papaver/cytology , Papaver/genetics , Papaver/metabolism , Proteomics , Tyrosine/chemistry , Tyrosine/metabolismSubject(s)
Agriculture/legislation & jurisprudence , Agriculture/trends , Analgesics, Opioid/isolation & purification , Drug and Narcotic Control/legislation & jurisprudence , Papaver/chemistry , Papaver/growth & development , Afghanistan , Agriculture/economics , Analgesics, Opioid/chemical synthesis , Analgesics, Opioid/chemistry , Analgesics, Opioid/economics , Codeine/chemical synthesis , Codeine/economics , Codeine/isolation & purification , Heroin/chemical synthesis , Heroin/economics , Heroin/isolation & purification , Humans , Morphine/chemistry , Morphine/economics , Morphine/isolation & purification , Opium/chemistry , Opium/economics , Opium/isolation & purificationABSTRACT
BACKGROUND: The measurement of morphine in biological samples has become a routine in many clinical and forensic toxicology laboratories. A high-performance liquid chromatography (HPLC) method was developed for the determination of morphine in plasma. METHODS: Samples were extracted using Zeolite Y column followed by reversed phase HPLC with fluorescence detection. This method was based on an ex-calibration procedure and was linear between 20 and 200 ng/ml of morphine. Blood from 10 male opiate addicts were obtained from Rosbeh Hospital. All of the male smoked opiate (heroin, opium) and cigarettes. RESULTS: The mean total level 5 h after the last abuse was 152.4 ng/ml and 37.6 ng/ml at 10-15 h. The method was reliable for morphine determination in blood even after 5 half-lives after the last abuse. CONCLUSIONS: This method is simple and rapid and may be useful for routine monitoring of plasma morphine concentration.
Subject(s)
Chromatography, High Pressure Liquid/methods , Morphine/blood , Zeolites/chemistry , Adult , Aged , Heroin Dependence/blood , Humans , Male , Morphine/chemistry , Morphine/isolation & purification , Opium , Reproducibility of Results , Substance-Related Disorders/bloodABSTRACT
This year is the 300th anniversary of the publication of one of the first books written about opiates and their subjective effects. Since that time the influence of opiates in Western society has grown enormously, as has our knowledge of the mechanisms by which these drugs produce their effects. Wars have been fought over the use of opiates and the economies of several countries depend on their production. In this article, some aspects of the history and effects of opiates on the arts in particular are explored.
Subject(s)
Opium/history , History, 17th Century , History, 18th Century , History, 19th Century , History, 20th Century , Medicine in the Arts , Morphine/chemistryABSTRACT
To investigate morphine degradation and optimize turning frequency in opium poppy processing waste composting, a pilot scale windrow composting trial was run for 55 days. Four treatments were designed as without turning (A1), every 5 days turning (A2), every 10 days turning (A3) and every 15 days turning (A4). During composting, a range of physicochemical parameters including the residual morphine degradation, temperature, pH, and the contents of total C, total N, total P and total K were investigated. For all treatments, the residual morphine content decreased below the detection limit and reached the safety standards after day 30 of composting, the longest duration of high temperature (⩾50 °C) was observed in A3, pH increased 16.9-17.54%, total carbon content decreased 15.5-22.5%, C/N ratio reduced from 46 to 26, and the content of total phosphorus and total potassium increased slightly. The final compost obtained by a mixture of all four piles was up to 55.3% of organic matter, 3.3% of total nutrient (N, P2O5 and K2O) and 7.6 of pH. A turning frequency of every ten days for a windrow composting of opium poppy processing waste is recommended to produce homogenous compost.