Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures.

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.

Anesthesiologist suicide with atracurium.

Atracurium is a nondepolarizing skeletal muscle relaxant used to facilitate endotracheal intubation and to induce skeletal muscle relaxation during surgery or mechanical ventilation. The drug undergoes a spontaneous non-enzymatic biotransformation, yielding laudanosine and an acrylate moiety. This report documents the case of a 45-year-old anesthesiologist who was found dead at the hospital where he worked. The victim was known to be depressed and undergoing treatment with venlafaxine. An empty syringe was found near the body. Toxicological analysis revealed the presence of laudanosine in the syringe, 0.6 mg/L of laudanosine in heart blood, 0.3 mg/L in urine, and 0.02 mg/L in vitreous humor. Meanwhile, concentrations of venlafaxine and O-desmethyl-venlafaxine, its active metabolite, were 0.7 and 1.1 mg/L in heart blood, 1.7 and 5.2 mg/L in urine, 0.5 and 0.7 mg/L in vitreous humor, and 400 and 20 mg in gastric content, respectively. All drugs and metabolites involved in the case were detected using gas chromatography with nitrogen-phosphorus detection (GC-NPD) and confirmed using GC-mass spectrometry in full scan mode after solid-phase extraction using Bond-Elut Certify columns. Additional high-performance liquid chromatography coupled to diode-array detection screening also obtained the same results. Quantitation of laudanosine and venlafaxine together with its metabolite was carried out using GC-NPD. No other drugs, including ethanol, were detected. Recoveries for laudanosine and venlafaxine were 89% and 86%, respectively, at 0.5 mg/L; intraday and interday precisions were 2% and 6%, and 3% and 7%, respectively; and limits of detection and quantitation were 6 and 20 ng/mL and 18 and 59 ng/mL, respectively. The linearity of the blood calibration curves was excellent for both drugs with r(2) values of > 0.999 (range 0.1-2.0 mg/L). Based on the autopsy findings, case history, and toxicology results, the forensic pathologists ruled that the cause of death was an overdose of atracurium, and the manner of death was suicide.

Concentrations of atracurium and laudanosine in cerebrospinal fluid and plasma during intracranial surgery.

Concentrations of atracurium and laudanosine in cerebrospinal fluid (CSF) and plasma were assayed in nine patients receiving atracurium infusions of 111-251 min duration to maintain neuromuscular block during intracranial surgery. The total dose of atracurium was 1.57-2.60 mg kg-1 and the plasma concentration of atracurium was 1.27-5.44 micrograms ml-1. Concentrations of laudanosine in CSF and plasma increased during the infusion period, and after 125-140 min reached means of 202.5 ng ml-1 and 1448.7 ng ml-1, respectively. The highest recorded concentration of laudanosine in CSF was 570 ng ml-1, in one of two CSF samples found to contain atracurium. After operation, two patients had fits, but these were not thought to be related to laudanosine. It is concluded that during infusion of atracurium, laudanosine accumulates in both plasma and CSF.

Concentrations of atracurium and laudanosine in cerebrospinal fluid and plasma in three intensive care patients.

We have measured concentrations of atracurium and laudanosine in cerebrospinal fluid (CSF) and plasma in three intensive care patients receiving atracurium infusions of 22.5-106 h duration to maintain neuromuscular block. Two patients had suffered severe closed head injuries and the third patient had developed respiratory failure following the clipping of two intracranial aneurysms. The total dose of atracurium given was 14.3-136.6 mg kg-1; rate of infusion was 0.6-1.38 mg kg-1 h-1. Plasma concentrations of atracurium and laudanosine were 0.73-3.11 micrograms ml-1 and 0.48-8.65 micrograms ml-1, respectively; CSF concentration of laudanosine was 70-440 ng ml-1. No adverse effects attributable to these concentrations of laudanosine were observed.

[Atracurium: neuromuscular blockade in repeated administration]. / Atracurium: Die neuromuskuläre Blockade bei wiederholter Gabe.

The neuromuscular blocking action of repeated injections of atracurium and vecuronium was studied in 74 surgical patients during balanced anaesthesia (methohexitone or etomidate, intubation after suxamethonium, fentanyl, droperidol, N2O). The initial bolus dose (ID) of atracurium was 0.25 mg/kg and of vecuronium 0.05 mg/kg followed by repeated increments (RD) of atracurium 0.1 mg/kg and vecuronium 0.0125 mg/kg when neuromuscular function (EMG) had recovered to about 30% of pre-relaxant control. Dose-response relationships revealed atracurium to be about 1/5 as potent as vecuronium; the ED50 of atracurium was 0.13 +/- 0.03 mg/kg and of vecuronium 0.023 +/- 0.007 mg/kg. The ID of both relaxants produced a neuromuscular blockade of about 90% within 4 min. The duration from the time of injection to 30% recovery was slightly longer in atracurium 26 +/- 9 min. In all patients the RD produced within 3.5 min satisfactory muscle relaxation with a neuromuscular block of about 85%. The mean duration of atracurium (18 min) was 5-10 min longer than of vecuronium (12 min). To maintain good surgical relaxation (more than 70% blockade) atracurium 0.32 mg/kg X h and vecuronium 0.056 mg/kg X h were required. No cumulation could be measured after repeated injections. The recovery time of atracurium and vecuronium at the end of anaesthesia was 10-12 min. Neither cardiovascular side-effects nor signs of histamine release were observed after both relaxants in our particular dose range. It is concluded, that atracurium is a favourable blocker for anaesthetic practice: The time of onset is approximately the same compared with vecuronium. The duration of action, however, is slightly longer but still truly intermediate long.(ABSTRACT TRUNCATED AT 250 WORDS)

New muscle relaxants.

There are many new neuromuscular blocking drugs now going through the clinical trials required before new drugs can be used clinically on a routine basis. It is safe to say that within the next five years the muscle relaxants now in common use will be replaced by new and more "ideal" muscle relaxants. This review has attempted to define for the practicing anesthesiologist the goals of muscle relaxant research and the drugs that have been developed from that research. It is hoped that some familiarity with these drugs before they become widely available may hasten their extensive use by practitioner in the interest of better anesthetic patient care.

Clinical pharmacology of vecuronium and atracurium.

Vecuronium and atracurium provide addition flexibility to the clinician using neuromuscular blocking drugs. The shorter duration of action, lack of significant cardiovascular effects, and the lack of dependence on the kidney for elimination provide clinical advantages over, or alternatives to, currently available nondepolarizing neuromuscular blocking drugs.