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Abstract Background Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly. Results Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C10:C10). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/gglucose corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation. Conclusions A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.forsciint.2008.10.022 Byline: Torsten Arndt (a), Birgit Gierten (a), Brunhilde Gussregen (a), Annika Werle (a), Joachim Gruner (b) Keywords: Alcohol; Chloral hydrate; Ethyl glucuronide; False-positive; Enzyme immunoassay; LC-MS Abbreviations: EtG, ethyl glucuronide; EtS, ethyl sulfate Abstract: Urine-ethyl glucuronide (EtG) concentrations are considered as a specific marker of recent alcohol consumption. We describe false-positive EtG screening results by the DRI.sup.[R] ethyl glucuronide enzyme immunoassay caused by chloral hydrate intake. Author Affiliation: (a) Bioscientia, Institut fur Medizinische Diagnostik GmbH, D-55218 Ingelheim, Germany (b) Private Practice, Frankfurt, Germany Article History: Received 28 August 2008; Revised 27 October 2008; Accepted 30 October 2008

Amantadine-sulfate has been used for several decades to treat acute influenza A, Parkinson's disease (PD), and acute or chronic drug-induced dyskinesia. Several mechanisms of actions detected in vivo/in vitro including N-methyl-D-aspartate (NMDA)-receptor antagonism, blockage of potassium channels, dopamine receptor agonism, enhancement of noradrenergic release, and anticholinergic effects have been described. We used transcranial magnetic stimulation (TMS) to evaluate the effect of single doses of amantadine on human motor cortex excitability in normal subjects. Using a double-blind, placebo-controlled, crossover study design, motor thresholds, recruitment curves, cortical stimulation-induced silent period (CSP), short intracortical inhibition (ICI), intracortical facilitation (ICF), and late inhibition (L-ICI) in 14 healthy subjects were investigated after oral doses of 50 and 100 mg amantadine with single and paired pulse TMS paradigms. Spinal cord excitability was investigated by distal latencies and M-amplitudes of the abductor digiti minimi muscle. After intake of amantadine, a significant dose-dependent decrease of ICF was noticed as well as a significant increase of L-ICI as compared to placebo. The effect on ICF and L-ICI significantly correlated with amantadine serum levels. ICI was slightly increased after amantadine intake, but the effect failed to be significant. Furthermore, amantadine had no significant effects on motor thresholds, MEP recruitment curves, CSP, or peripheral excitability. In conclusion, a low dose of amantadine is sufficient in modulating human motor cortex excitability. The decrease of ICF and increase of L-ICI may reflect glutamatergic modulation or a polysynaptic interaction of glutamatergic and GABA-ergic circuits. Although amantadine has several mechanisms of action, the NMDA-receptor antagonism seems to be the most relevant effect on cortical excitability. As L-ICI can be influenced by this type of drug, it may be an interesting parameter for studies of motor learning and use-dependent plasticity.Neuropsychopharmacology (2006) 31, 2758–2766. doi:10.1038/sj.npp.1301122; published online 14 June 2006 [ABSTRACT FROM AUTHOR]

Abstract: Background: Amantadine (1-adamantylamine) is used for treatment of influenza, hepatitis C, parkinsonism, and multiple sclerosis. Current amantadine analysis by HPLC or gas chromatography (GC) requires a laborious sample pretreatment with extraction and/or derivatization steps. We established an LC-MS/MS method without protein precipitation, centrifugation, extraction and derivatization steps. Material and methods: 50 μl sample+50 μl of 0.4 mg/l 1-(1-adamantyl)pyridinium bromide as internal standard+1000 μl water (96-well plate). Of this 25 μl+500 μl water (96-well plate; final serum dilution 1:462). LC-MS/MS: Surveyor MS pump, Autosampler, triple-quadrupole TSQ Quantum mass spectrometer (Thermo Electron). Autosampling: 2 μl of each sample. Chromatography: isocratic water/acetonitrile (60/40 v/v) with 5 g/l formic acid, flow rate 0.2 ml/min, run time 3 min, Phenomenex Luna C8(2) (100×2.0 mm (i.d.); 3-μm bead size) column. Mass spectrometry: electrospray atmospheric pressure ionization, positive ion and selective reaction monitoring mode, ion transitions m/z 152.0→135.1 (at 22 eV amantadine) and 214.1→135.1 (at 26 eV internal standard). Results: Calibration curves were constructed with spiked serum samples (amantadine 50–1000 μg/l, r >0.99). No carry over (5000 μg/l). No ion suppression with retention times similar to those of amantadine (1.8 min) and the internal standard (2.1 min). Detection limit 20 mg/l, linearity 20–5000 mg/l, intra-assay/inter-assay CV<6%/<8%, recovery 99–101%. Method comparison: LC-MS/MS=1.23×GC-45 (Passing-Bablok regression). No significant bias between GC and LC-MS/MS (Bland-Altman plot). Conclusion: We consider the sample pretreatment without deproteination, derivatization and centrifugation steps and the specificity of the tandem mass spectrometry as the most important points of our amantadine analysis method. [Copyright &y& Elsevier]