Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525     

Gradient reconstitution of membrane proteins for solid-state NMR studies

We here adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. We established conditions under which quantitative reconstitution of active protein at low lipid-to-protein ratios can be obtained, and also how to upscale these conditions in order to produce adequate amounts for NMR. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution, and shall represent a valuable alternative in solid-state NMR membrane protein sample preparation.     

Criteria to distinguish between natural situations and illegal use of boldenone,boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap?). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L^?1 and 0.4 μg L^?1 on triple quadrupole and 0.1 μg L^?1 and 0.2 μg L^?1 on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.     

Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes

The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [(32)P]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.     

Cocaine Is Low on the Value Ladder of Rats: Possible Evidence for Resilience to Addiction

Background

Assessing the relative value of cocaine and how it changes with chronic drug use represents a long-standing goal in addiction research. Surprisingly, recent experiments in rats – by far the most frequently used animal model in this field – suggest that the value of cocaine is lower than previously thought.

Methodology/Principal Findings

Here we report a series of choice experiments that better define the relative position of cocaine on the value ladder of rats (i.e., preference rank-ordering of different rewards). Rats were allowed to choose either taking cocaine or drinking water sweetened with saccharin – a nondrug alternative that is not biologically essential. By systematically varying the cost and concentration of sweet water, we found that cocaine is low on the value ladder of the large majority of rats, near the lowest concentrations of sweet water. In addition, a retrospective analysis of all experiments over the past 5 years revealed that no matter how heavy was past cocaine use most rats readily give up cocaine use in favor of the nondrug alternative. Only a minority, fewer than 15% at the heaviest level of past cocaine use, continued to take cocaine, even when hungry and offered a natural sugar that could relieve their need of calories.

Conclusions/Significance

This pattern of results (cocaine abstinence in most rats; cocaine preference in few rats) maps well onto the epidemiology of human cocaine addiction and suggests that only a minority of rats would be vulnerable to cocaine addiction while the large majority would be resilient despite extensive drug use. Resilience to drug addiction has long been suspected in humans but could not be firmly established, mostly because it is difficult to control retrospectively for differences in drug self-exposure and/or availability in human drug users. This conclusion has important implications for preclinical research on the neurobiology of cocaine addiction and for future medication development.     

Elimination kinetic of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate ester metabolites in calves' urine

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C(18) SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CCalpha) for steroids were below 0.1 microg L(-1), except for 19-norandrosterone (CCalpha=0.7 microg L(-1)) and estrone (CCalpha=0.3 microg L(-1)). Kinetics of elimination of the administered 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate were established by monitoring 17beta-estradiol, 17alpha-estradiol, estrone and 17beta-nandrolone, 17alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17alpha-estradiol and 17alpha-nandrolone (>20 and 2 mg L(-1), respectively after 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate administration) whereas 17beta-estradiol, estrone, 17beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the microg L(-1) level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17beta form.