Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma

In order to study the effect of a diet on metabolites found in body fluids such as plasma, we have developed and validated a UPLC/MS method. While methods using NMR have been well established to analyse different biological tissues, recent studies have described robust untargeted UPLC-MS methods for plasma analysis. One major concern when profiling plasma is the presence of an important quantity of proteins which have to be precipitated without any loss of metabolites prior to LC/MS analysis. The utilization of untargeted approaches in nutritional metabolomics still suffers from the lack of identification of specific biomarkers. We therefore suggest an alternative method still using a global approach but focusing at the same time on metabolites previously described in human plasma in order to detect biomarkers of metabolic dysregulations. Thus, to fulfil our objectives, analytical parameters were tested (i) the anticoagulant type for sample collection, (ii) the protein precipitation method and (iii) UPLC/MS analytical conditions. Three protein precipitation methods and two anticoagulants were tested and compared. The method utilizing blood collection on heparin and methanol precipitation was chosen for giving the most reproducible results while keeping the complexity of the sample. Finally, a validation was proposed to evaluate the stability of this analytical method applied to a large batch of samples for nutritional metabolomic studies.     

QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

Background

Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes.

Results

The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue.

Conclusions

A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.     

Biochemical alterations in human cells irradiated with alpha particles delivered by macro- or microbeams

Irradiation of individual cell nuclei with charged-particle microbeams requires accurate identification and localization of cells using Hoechst staining and UV illumination before computer-monitored localization of each cell. Using Fourier-transform infrared microspectroscopy (FT-IRM), we investigated whether the experimental conditions used for cell recognition induce cellular changes prior to irradiation and compared biochemical changes and DNA damage after targeted and nontargeted irradiation with alpha particles delivered by macro- or microbeams, using gamma radiation as a reference. Molecular damage in single HaCaT cells was studied by means of FT-IRM and comet assay (Gault et al., Int. J. Radiat. Biol. 81, 767-779, 2005). Hoechst 33342-stained HaCaT cells were exposed to single doses of 2 Gy (239)Pu alpha particles from a broad-beam irradiator, five impacted alpha particles from a microbeam irradiator, or 6 Gy gamma rays from (137)Cs, each of which resulted in about 5% clonogenic survival. FT-IRM of control cells indicated that Hoechst binding to nuclear DNA induced subtle changes in DNA conformation, and its excitation under UV illumination induced a dramatic shift of the DNA conformation from A to B as well as major DNA damage as measured by the comet assay. Comparison of the FT-IRM spectra of cells exposed to gamma rays or alpha particles specifically targeted to the nucleus, alpha particles from a broad-beam irradiator revealed spectral changes corresponding to all changes in constitutive bases in nucleic acids, suggesting oxidative damage in these bases, as well as structural damage in the deoxyribose-phosphate backbone of DNA and the osidic structure of nucleic acids. Concomitantly, spectral changes specific to protein suggested structural modifications. Striking differences in IR spectra between targeted microbeam- and nontargeted macrobeam-irradiated cells indicated greater residual unrepaired or misrepaired damage after microbeam irradiation. This was confirmed by the comet assay data. These results show that FT-IRM, together with the comet assay, is useful for assessing direct radiation-induced damage to nucleic acids and proteins in single cells and for investigating the effects of radiation quality. Significantly, FT-IRM revealed that Hoechst 33342 binding to DNA and exposure to UV light induce a dramatic change in DNA conformation as well as DNA damage. These findings suggest that fluorochrome staining should be avoided in studies of ionizing radiation-induced bystander effects based on charged-particle microbeam irradiation. An alternative cell nucleus recognition system that avoids nuclear matrix damage and its possible contribution to propagation of biological effects from irradiated cells to neighboring nontargeted cells needs to be developed.     

Opposite regulation of endothelial NO synthase by HSP90 and caveolin in liver and lungs of rats with hepatopulmonary syndrome

The hepatopulmonary syndrome is a complication of cirrhosis that associates an overproduction of nitric oxide (NO) in lungs and a NO defect in the liver. Because endothelial NO synthase (eNOS) is regulated by caveolin that decreases and heat shock protein 90 (HSP90) that increases NO production, we hypothesized that an opposite regulation of eNOS by caveolin and HSP90 might explain the opposite NO production in both organs. Cirrhosis was induced by a chronic bile duct ligation (CBDL) performed 15, 30, and 60 days before sample collection and pharmacological tests. eNOS, caveolin, and HSP90 expression were measured in hepatic and lung tissues. Pharmacological tests to assess NO released by shear stress and by acetylcholine were performed in livers (n = 28) and lungs (n = 28) isolated from normal and CBDL rats. In lungs from CBDL rats, indirect evidence of high NO production induced by shear stress was associated with a high binding of HSP90 and a low binding of caveolin to eNOS. Opposite results were observed in livers from CBDL rats. Our study shows an opposite posttranslational regulation of eNOS by HSP90 and caveolin in lungs and liver from rats with CBDL. Such opposite posttranslational regulation of eNOS by regulatory proteins may explain in part the pulmonary overproduction of NO and the hepatic NO defect in rats with hepatopulmonary syndrome.     

Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50

Nicotinic acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels that mediate fast synaptic transmission at the neuromuscular junction (NMJ). After assembly in the endoplasmic reticulum (ER), AChRs must be transported to the plasma membrane through the secretory apparatus. Little is known about specific molecules that mediate this transport. Here we identify a gene that is required for subtype-specific trafficking of assembled nicotinic AChRs in Caenorhabditis elegans. unc-50 encodes an evolutionarily conserved integral membrane protein that localizes to the Golgi apparatus. In the absence of UNC-50, a subset of AChRs present in body-wall muscle are sorted to the lysosomal system and degraded. However, the trafficking of a second AChR type and of GABA ionotropic receptors expressed in the same muscle cells is not affected in unc-50 mutants. These results suggest that, in addition to ER quality control, assembled AChRs are sorted within the Golgi system by a mechanism that controls the amount of cell-surface AChRs in a subtype-specific way.     

Messinian palaeoenvironments and hydrology in Sicily (Italy): The dinoflagellate cyst record

Hydrological conditions prevailing before, during and after the Messinian salinity crisis in Sicily have been approached using dinoflagellate cyst records. The synthetic sequence considered is based on five classical sections from the Caltanissetta Basin. Our interpretations are based on the recognition of autochthonous, allochthonous and reworked population among the dinocyst assemblages. For the first time, sea-surface temperatures and seasonal salinity contrasts were tentatively reconstructed using a “Mutual Climatic Range Method”. Sicilian late Tortonian deposits correspond to marine environment with significant terrestrial inputs favourable to eutrophic dinocyst species. Immediately after the beginning of the Messinian Stage, euryhaline assemblages took place, followed by meso-hyperhaline taxa, within a general trend to shallowing. At the end of the Tripoli diatomitic Formation, environment appears confined, with regular oceanic inflows. Such marine inflows remain persistent during the deposition of the salt Member, witnessing the probable persistence of nearby normal marine sea-surface water conditions in the Mediterranean Sea but with possible reduced hydrological circulation and/or low nutrient component. Such inflows are slightly decreasing up to the top of the Sicilian Upper Evaporites. At the same time, river inputs appear weak during the salt deposition, as the consequence of a rather dry climatic context. As shown by reworking activity, terrestrial inputs increase progressively from the base of the Upper Evaporites. During the Lago Mare period, while climate remains rather dry (absence of freshwater algae inputs, very low amount in trees requiring humid conditions), local deposition environment is confined with mesohaline to hypohaline sea-surface waters, in a context with relatively high seasonal sea-surface salinity contrast (up to 6‰). The very high reworking observed in the Arenazzolo silts, in addition to slightly increasing water depth, led us to consider the presence of a discontinuity between the Lago Mare and the Arenazzolo Fms. We consider the Arenazzolo Formation as a transgressive facies following a deep downcutting period. The Arenazzolo Formation presents a two-step development. The first one, correlative with the presence of a G. etrusca (a species with Paratethyan affinities), corresponds to a relative high-stand sea-level with oceanic influxes, low seasonal sea-surface salinity contrast and probable more humid context, as revealed by the important freshwater algal inputs. During the second step, salinity becomes much more variable, with a clear increase of seasonal sea-surface salinity contrast, a possible slight mean sea-surface temperature increase (only few degrees) and a clear weakening of the river inputs. Taking the proposed Zanclean position of the Arenazzolo Fm. into consideration, we state that mean sea-surface temperature did not change significantly from the base of the Messinian to the earliest Zanclean. At 5.33 Ma, the suddenly achieved flooding restored a fair, deep oceanic environment characterized, at the beginning, by a clear mean sea-surface temperature cooling (up to 6-7 °C) and a nutrient depletion, associated with the basins starvation. Sea-surface salinities were normal, with very low seasonal contrast. Hydrodynamics then nutrient supply became then quite normal from c.a. 5.08 Ma. The status of the Sicilian Caltanissetta Basin as a marginal basin although fastly deepening and the stratigraphical location of the Messinian discontinuity at the base of the Arenazzolo is the scenario that best matches our dinocyst record.     

Carbonic anhydrase inhibitors: the X-ray crystal structure of the adduct of N-hydroxysulfamide with isozyme II explains why this new zinc binding function is effective in the design of potent inhibitors

N-Hydroxysulfamide is a 2000-fold more potent inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) as compared to sulfamide. It also inhibits other physiologically relevant isoforms, such as the tumor-associated CA IX and XII (K(I)s in the range of 0.865-1.34microM). In order to understand the binding of this inhibitor to the enzyme active site, the X-ray crystal structure of the human hCA II-N-hydroxysulfamide adduct was resolved. The inhibitor coordinates to the active site zinc ion by the ionized primary amino group, participating in an extended network of hydrogen bonds with amino acid residues Thr199, Thr200 and two water molecules. The additional two hydrogen bonds in which N-hydroxysulfamide bound to hCA II is involved as compared to the corresponding adduct of sulfamide may explain its higher affinity for the enzyme, also providing hints for the design of tight-binding CA inhibitors possessing an organic moiety substituting the NH group in the N-hydroxysulfamide structure.     

Carbonic anhydrase inhibitors. Inhibition of isoforms I, II, IV, VA, VII, IX, and XIV with sulfonamides incorporating fructopyranose-thioureido tails

A series of aromatic/heterocyclic sulfonamides incorporating 2,3:4,5-bis-O-(isopropylidene)-beta-d-fructopyranosyl-thioureido moieties has been synthesized and assayed for the inhibition of seven human isoforms of the zinc enzyme carbonic anhydrase (hCA, EC 4.2.1.1). The new derivatives behaved as weak hCA I inhibitors (K(I)s of 9.4 -13.3microM), were efficient hCA II inhibitors (K(I)s of 6-750nM), and slightly inhibited isoforms hCA IV and hCA VA. Only the sulfanilamide derivative showed efficient and selective inhibition of hCA IV (K(I) of 10nM). These derivatives also showed excellent hCA VII inhibitory activity (K(I)s of 10-79nM), being less efficient as inhibitors of the transmembrane isoforms hCA IX (K(I)s of 10-4500nM) and hCA XIV (K(I)s of 21-3500nM). Two of the new compounds showed anticonvulsant action in a maximal electroshock seizure test in mice, with the fluorosulfanilamide derivative being a more efficient anticonvulsant than the antiepileptic drug topiramate.