Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective, sensitive and reproducible liquid chromatographic method with tandem mass spectrometric detection has been developed and validated for the analysis of a new specific bradycardic agent, ivabradine (S 16257) and six potentially active metabolites in human plasma. Isolation of these compounds and of the internal standard was performed by an automated solid-phase extraction system using Oasis cartridges. Separation and detection of ivabradine and its metabolites were achieved using a C₁₈ column and a MS–MS detector with a positive electrospray ionization source. Ivabradine and its metabolites gave a linear response ranging from 0.1 or 0.2 to 20 ng/ml and the limits of quantitation ranged from 0.1 to 0.2 ng/ml using a 0.5 ml plasma sample size. A complete validation demonstrated the method to be accurate, precise and specific for the simultaneous quantification of ivabradine and its metabolites in human plasma. The method was subsequently applied to the quantitative determination of ivabradine and its metabolites in human plasma samples from healthy volunteers participating in a clinical study to provide pharmacokinetic data.     

Acute Moderate Exercise Does Not Further Alter the Autonomic Nervous System Activity in Patients with Sickle Cell Anemia

A decreased global autonomic nervous system (ANS) activity and increased sympathetic activation in patients with sickle cell anemia (SCA) seem to worsen the clinical severity and could play a role in the pathophysiology of the disease, notably by triggering vaso-occlusive crises. Because exercise challenges the ANS activity in the general population, we sought to determine whether a short (<15 min) and progressive moderate exercise session conducted until the first ventilatory threshold had an effect on the ANS activity of a group of SCA patients and a group of healthy individuals (CONT group). Temporal and spectral analyses of the nocturnal heart rate variability were performed before and on the 3 nights following the exercise session. Standard deviation of all normal RR intervals (SDNN), total power, low frequencies (LF) and high frequencies powers (HF) were lower but LF/HF was higher in SCA patients than in the CONT group. Moderate exercise did not modify ANS activity in both groups. In addition, no adverse clinical events occurred during the entire protocol. These results imply that this kind of short and moderate exercise is not detrimental for SCA patients.     

Reversible Alterations of Cerebral γ-Aminobutyric Acid in Pyrithiamine-Treated Rats: Implications for the Pathogenesis of Wernicke''s Encephalopathy

Treatment of rats with the central thiamine antagonist, pyrithiamine, results in severe neurological symptoms such as loss of righting reflex. Measurement of gamma-aminobutyric acid (GABA) content of brain tissue from symptomatic pyrithiamine-treated (PT) rats revealed significant reductions in thalamus, cerebellum, and pons. GABA content of cerebral cortex, however, was unaltered. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha KGDH) were reduced in parallel with the GABA changes. On the other hand, activities of the GABA-synthetic enzyme glutamic acid decarboxylase (GAD) remained within normal limits, with the exception of a small but significant decrease in thalamus of symptomatic PT rats. Affinities and densities of high-affinity [3H]muscimol binding sites on crude cerebral membrane preparations from symptomatic PT rats were unchanged. Thiamine administration to symptomatic animals resulted in correction of abnormal righting reflexes and in normalization of decreased GABA levels and reduced alpha KGDH activities in cerebellum and pons. Thalamic GABA levels and alpha KGDH activities, on the other hand, remained significantly lower than normal. These results suggest that the reversible symptoms of pyrithiamine treatment may result from imparied GABA synthesis in cerebellum and pons of these animals. Similar mechanisms may play a role in the pathogenesis of the reversible symptoms of Wernicke's encephalopathy in man.     

Steady state peripheral blood provides cells with functional and metabolic characteristics of real hematopoietic stem cells

Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non‐SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non‐SP cells. From a cell regulation point of view, we showed that SP activity depended on O₂ concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia‐induced factors HIF‐1α and HIF‐2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non‐SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy.     

Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.

A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.     

Inhibition of δ8 → δ7-sterol isomerase and of cycloeucalenol—obtusifoliol isomerase by N-benzyl-8-aza-4α, 10-dimethyl-trans-decal-3β-ol,an analogue of a carbocationic high energy intermediate

An enzymatic assay for the δ⁸ → δ⁷-sterol isomerase, an enzyme involved in sterol biosynthesis, has been developed in higher plants. This assay has been used in the study of various inhibitors. N-Benzyl-8-aza-4α, 10-dimethyl- trans-decal-3β-ol was designed to mimic the C-8 and the C-9 carbocationic high energy intermediates occurring during the reactions catalysed by the δ⁸ → δ⁷-sterol isomerase and the cycloeucalenol obtusifoliol isomerase, respectively. In accordance with the ‘transition state analogues’ theory, this analogue of a high energy intermediate was found to be a very potent and specific inhibitor of the two enzymatic reactions both in vitro and in vivo.     

Ethical challenges at the science-policy interface: an ethical risk assessment and proposition of an ethical infrastructure

Developing an interface between knowledge holders, stakeholders and decision makers on biodiversity issues, just as any science-policy interface, will face many challenges. In the crucial endeavour to tackle all those challenges, determining an ethical course of actions will be essential to the prestige and credibility of such an interface. The paper identifies and assesses potential ethical risks that may arise in interactions between science, society and policy and uses the Network of Knowledge (NoK) process as an example to show how an ethical infrastructure could be developed for minimizing the ethical risks and their potential consequences. Indeed, when various actors from different spheres (politics, academia, lobbyism, media, etc.) are called upon to interact within one process as complex as the NoK, the integrity and credibility of the latter are at high risk of being compromised if the ethical risks are not adequately addressed. In order to limit those risks, which science-policy interfaces such as IPCC and IPBES have already encountered, we propose to set up an ethical governance infrastructure that will guide (and regulate) interactions among internal actors of the NoK (knowledge coordination body, secretariat, expert working groups, etc.) as well as with external actors (requesters, stakeholders, etc.). A thorough evaluation of the interaction between the actors for every step of the process is carried out and potential ethical risks are identified. Suggestions as to how the risks can be handled and prevented are presented and integrated as part of an ethical infrastructure. The main objective of the paper is to address how a science-policy interface and the scientific community as a whole would benefit from implementing ethical measures and instruments to help prevent sensitive issues and undesired consequences undermining credibility and legitimacy.     

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP‐galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.