Determination of yohimbine and its two hydroxylated metabolites in humans by high-performance liquid chromatography and mass spectral analysis

The existence of at least two metabolites of yohimbine (YO) in humans is demonstrated. Combined high-performance liquid chromatographic (HPLC), NMR and mass spectral analyses permitted them to be identified as hydroxylated metabolites at the C-10 and C-11 positions. A normal-phase HPLC method allowing the simultaneous determination of YO and its main metabolite, 11-hydroxyyohimbine (11-OHYO), in biological samples is described. This assay was performed using a LiChrosorb Si 60 column and a mobile phase consisting of 0.02 M sodium acetate (pH 5)—methanol (5:95, v/v) at a flow-rate of 1 ml/min. Detection was achieved by a fluorimetric method (excitation at 280 nm and emission at 320 nm). The extraction yields of YO, 10-OHYO and 11-OHYO from plasma were 91.8, 45.3 and 17.8%, respectively, and their respective within-day reproducibilities were 3.8, 1.4 and 5.9%. The between-day reproducibility for YO at the concentrations of 1 and 10 ng/ml were 8.9 and 6.4%, respectively. The accuracy of the method for YO at concentrations of 1 and 10 ng/ml were 5.1 and 2.3%, respectively. The limits of determination of YO, 10-OHYO and 11-OHYO were 0.1, 0.5 and 1 ng/ml, respectively. The method was used in bioavailability study of YO following oral and intravenous administration in humans.     

Genetic variation at allozyme and RAPD loci in sessile oak Quercus petraea (Matt.) Liebl.: the role of history and geography

The nuclear genetic variation within and among 21 populations of sessile oak was estimated at 31 RAPD loci in conjunction with previous estimates of variation at eight allozyme loci. The aim of the study was to assess the relative role of isolation-by-distance and postglacial history on patterns of nuclear variation. Because of its small effective population size and maternal transmission, the chloroplast genome is a good marker of population history. Both kinds of nuclear variation (RAPD and allozyme) were therefore compared, first, to the geographical distances among populations and, secondly, to chloroplast DNA restriction polymorphism in the same populations. Multiple Mantel tests were used for this purpose. Although RAPDs revealed less genetic diversity than allozymes, levels of genetic differentiation ( G _ST) were identical. The standard genetic distance calculated at all RAPD loci was correlated with geographical distances but not with the genetic distance calculated from chloroplast DNA data. Conversely, allozyme variation was correlated with chloroplast DNA variation, but not with geography. Possibly, divergent selection at two allozyme loci during the glacial period could explain this pattern. Because of its greater number of loci assayed, RAPDs probably provided a less biased picture of the relative role of geography and history.     

Host–microbe interactions as a driver of acclimation to salinity gradients in brown algal cultures

Like most eukaryotes, brown algae live in association with bacterial communities that frequently have beneficial effects on their development. Ectocarpus is a genus of small filamentous brown algae, which comprises a strain that has recently colonized freshwater, a rare transition in this lineage. We generated an inventory of bacteria in Ectocarpus cultures and examined the effect they have on acclimation to an environmental change, that is, the transition from seawater to freshwater medium. Our results demonstrate that Ectocarpus depends on bacteria for this transition: cultures that have been deprived of their associated microbiome do not survive a transfer to freshwater, but restoring their microflora also restores the capacity to acclimate to this change. Furthermore, the transition between the two culture media strongly affects the bacterial community composition. Examining a range of other closely related algal strains, we observed that the presence of two bacterial operational taxonomic units correlated significantly with an increase in low salinity tolerance of the algal culture. Despite differences in the community composition, no indications were found for functional differences in the bacterial metagenomes predicted to be associated with algae in the salinities tested, suggesting functional redundancy in the associated bacterial community. Our study provides an example of how microbial communities may impact the acclimation and physiological response of algae to different environments, and thus possibly act as facilitators of speciation. It paves the way for functional examinations of the underlying host–microbe interactions, both in controlled laboratory and natural conditions.     

Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.

The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.     

Effect of training on the resting heart rate of rats

Adult male rats were progressively trained 5 days/week on a motor-driven treadmill. The training period lasted 12 weeks and consisted of 60 min/day of wind-sprints and endurance work. No significant difference in resting heart rates was observed between the control and exercise groups during week 1 (394±7 vs. 388±5). However, at week 12 the exercise group had a lower resting heart rate (359±6 vs. 331±4). Heart rates observed following saline, propranolol, atropine, and propranolol plus atropine injections were lower in the exercise group in all cases. The difference in heart rates between the control and exercise groups was 19 beats/min following propranolol plus atropine which was less than the 28 beats/min difference observed under control conditions. With atropine and then with propranolol the differences were 33 and 27 beats/min. These heart rate differences were observed without the presence of cardiac hypertrophy as assessed from ventricle weights.Our data indicate that the bradycardia resulting from exercise training is due primarily to changes other than neural influences on the heart.     

Proteinase activity of dairy Propionibacterium

A proteolytic system was found in all the species of dairy Propionibacterium. Two types of activities could act on [¹⁴C] \gb-casein or [¹⁴C] \ga_s1-casein. The first was the highest at the beginning of the exponential growth phase, tightly linked to the cells and hydrolysed preferably \gb-casein. The second was released at the end of growth and acted similarly on both substrates tested. This activity was located in the cell membrane of these cheese-ripening bacteria. The enzyme could be gently extracted from the cell by incubation in Ca^2\s+ \t- or Mg Mg^2\s+-free buffer.     

Impacts and management of unowned and owned cats at a seabird colony on Reunion Island (Western Indian Ocean)

Biological Invasions - Cats (Felis catus) introduced on islands have strong impacts on biodiversity, and the main conservation actions to protect native fauna is cat eradication or control (i.e.,...     

Aryl hydrocarbon receptor- and calcium-dependent induction of the chemokine CCL1 by the environmental contaminant benzo[a]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1beta and tumor necrosis factor-alpha. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner.     

Demography and conservation of the White-tailed Tropicbird Phaethon lepturus on Aride Island, Western Indian Ocean

The White-tailed Tropicbird Phaethon lepturus breeding population of Aride Island, Seychelles, has experienced a strong decline in the recent past. To predict the future trends of the White-tailed Tropicbird population on Aride Island and understand the urgency of applying management procedures, we developed a population matrix model. We estimated White-tailed Tropicbird adult survival rate over a 12-year period and investigated whether oceanographic conditions (El Ni?o Southern Oscillation, ENSO, and inter-annual climate variability in the Indian Ocean, Indian Ocean Dipole, IOD) or local factors (Pisonia grandis fruiting events) potentially affect adult survival. Annual adult survival could not be linked to inter-annual variability in oceanographic conditions, but may be more influenced by Pisonia fruiting events, which, overall, accounts for 23.2% of the adult mortality. The growth rate of the population, primarily influenced by annual adult survival, was estimated to be 0.946, which indicates an annual population decline of 5.4%. At the current rate, the probability of extinction of this population would be 99% in 170 years. Management scenarios studied through Monte Carlo simulations, assuming small increments in adult survival and/or breeding success, dramatically reduce the risk of extinction of White-tailed Tropicbirds. Habitat management, in order to maintain an open canopy and little understorey vegetation, appears to be crucial for the conservation of White-tailed Tropicbirds on Aride Island, as has already been shown for other ground-nesting tropical seabirds nesting on forested islands.