Differential distribution of 4-hydroxynonenal adducts to sulfur and nitrogen residues in blood proteins as revealed using Raney nickel and gas chromatography–mass spectrometry

Quantification of 4-hydroxy-2-nonenal (HNE) bound to circulating proteins may prove to be useful in evaluating the role of this bioactive lipoperoxidation by-product in the pathogenesis of various diseases. Recently, we developed a quantitative gas chromatography–mass spectrometry (GCMS) assay of total protein-bound HNE (HNE-P) in blood after reduction with NaB²H₄ and cleavage with Raney nickel. Whereas it has been assumed that Raney nickel cleaves only Michael adducts of HNE to cysteine via a thioether bond (HNE-SP), results from this study demonstrate that our GCMS method also detects with precision picomoles of HNE adducts via nitrogen residues (HNE-NP). Specifically, evidence was obtained using various study models, including polyamino acids consisting of cysteine, lysine, and histidine and a biologically relevant molecule, albumin. Furthermore, we show that dinitrophenylhydrazine treatment before Raney nickel treatment can be used to discriminate and quantify the various HNE-P molecular species in plasma and blood samples from normal rats, which range between 0.15 and 3 pmol/mg protein or 10 to 600 nM. However, whereas HNE-SP predominated in whole blood, we detected HNE-NP only in plasma. We also identified another significant MS signal, which we attribute to protein-bound 1,4-dihydroxynonane (DHN-P) presumably formed from the enzymatic reduction of HNE-P. The distribution profile of all these species in plasma differed from that observed when physiologically relevant concentrations of albumin and HNE were incubated in vitro. Furthermore, interestingly, hypercholesterolemic rabbits showed higher plasma levels of HNE-NP, but not of DHN-P. Beyond documenting the presence of various types of HNE-P in circulating proteins, our results emphasize the importance of enzymatic mechanisms in situ as a factor determining their distribution in the various blood compartments under various conditions.     

Plasma Leptin and Acute Serotoninergic Stimulation of the Corticotropic Axis in Women Who Are Normal Weight or Obese

OPPERT, JEAN-MICHEL, NAJIBA LAHLOU, BLANDINE LAFERRÈRE, MARC ROGER, ARNAUD BASDEVANT, BERNARD GUY-GRAND. Plasma leptin and acute serotoninergic stimulation of the corticotropic axis in women who are normal weight or obese. In some recent studies, glucocorticoid treatment was associated with rapid induction of obese (ob) gene expression in adipose tissue of normal rats and in isolated adipocytes. We studied the effect of acute stimulation of the corticotropic axis on plasma leptin, the ob gene product, in 7 women of normal weight and 12 women with obesity. Under double-blind, placebo-controlled conditions, a single 12.5-mg dose of clomipramine, a serotonin uptake inhibitor, was administered intravenously in 15 minutes. Mean basal plasma leptin was increased more than 3-fold in subjects with obesity compared with subjects of normal weight (35.1 ± 4.9 ng/mL vs. 8.9 ± 1.4 ng/mL, p=0.001). Whereas corticotropin (ACTH) and Cortisol responses were increased in women who were obese compared with women who were lean, no significant effect of clomipramine infusion was found on plasma leptin concentrations measured during the following 150 minutes in both groups. There was a strong positive correlation between basal plasma leptin concentrations and body mass index (r=0.92, p<0.0001). In six subjects with obesity studied after a moderate weight loss, mean basal plasma leptin was significantly decreased (43.7 ± 6.4 ng/mL before vs. 28.0 ± 8.1 ng/mL after, p=0.04), but the hormonal response pattern to clomipramine administration was unchanged. We conclude that, at least in the short term, an acute stimulation of the corticotropic axis does not seem to increase leptin secretion in humans, as shown by the response to the serotoninergic agent clomipramine.     

Korean and Caucasian overweight premenopausal women have different relationship of body mass index to percent body fat with age.

The aim of the study was to investigate in premenopausal women whether the relationship between percentage body fat (PBF) and body mass index (BMI; in kg/m2) differs between Korean Asians (Ko-As) living in Seoul, South Korea, and Caucasians (Ca) living in New York City. Healthy premenopausal women (50 Ko-As; 38 Ca), ages 22-50 yr, were studied. Weight, height, and PBF by dual-energy X-ray absorptiometry were measured. Total body dual-energy X-ray absorptiometry data were collected using GE-Lunar systems (Prodigy-Korea and DPXL-New York), and all scan analyses were performed by one technician in New York. Similar soft tissue phantoms were used for daily instrument calibrations at both sites. The relationship between PBF and BMI was assessed by multiple regression analysis with race, age, reciprocal of BMI (1/BMI), and a race-by-age interaction as the final independent variables. Race (P = 0.003) and 1/BMI (P < 0.001) were significantly related to PBF in this model. A significant race-by-age interaction (P = 0.039) indicated that the slope of the lines for PBF vs. age differed between Ko-As and Ca. This study demonstrates in a Ko-As sample that the BMI-fat relationship differs significantly from that in a comparable group of Caucasian women. Investigators who use BMI as an index of fatness should be aware of the well documented differences in the relationship of BMI and fatness across race/ethnic groups.     

Low but contrasting neutral genetic differentiation shaped by winter temperature in European great tits

Gene flow is usually thought to reduce genetic divergence and impede local adaptation by homogenising gene pools between populations. However, evidence for local adaptation and phenotypic differentiation in highly mobile species, experiencing high levels of gene flow, is emerging. Assessing population genetic structure at different spatial scales is thus a crucial step towards understanding mechanisms underlying intraspecific differentiation and diversification. Here, we studied the population genetic structure of a highly mobile species – the great tit Parus major – at different spatial scales. We analysed 884 individuals from 30 sites across Europe including 10 close‐by sites (< 50 km), using 22 microsatellite markers. Overall we found a low but significant genetic differentiation among sites (F_ST = 0.008). Genetic differentiation was higher, and genetic diversity lower, in south‐western Europe. These regional differences were statistically best explained by winter temperature. Overall, our results suggest that great tits form a single patchy metapopulation across Europe, in which genetic differentiation is independent of geographical distance and gene flow may be regulated by environmental factors via movements related to winter severity. This might have important implications for the evolutionary trajectories of sub‐populations, especially in the context of climate change, and calls for future investigations of local differences in costs and benefits of philopatry at large scales.     

β cell membrane remodelling and procoagulant events occur in inflammation‐driven insulin impairment: a GLP‐1 receptor dependent and independent control

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell‐derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon‐like peptide (GLP)‐1 analogue, is known to promote insulin secretion and β‐cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin‐m5f β‐cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross‐talk model. Methyl‐β‐cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N‐éthylmaleimide‐sensitive‐factor Attachment protein Receptor (SNARE)‐dependent exocytosis. Cytokines induced a two‐fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two‐fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD‐treated cells showed similar patterns. Cells pre‐treated by saturating concentration of the GLP‐1r antagonist exendin (9‐39), showed a partial abolishment of the liraglutide‐driven insulin secretion and liraglutide‐decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP‐1r‐dependent and ‐independent pathways. Our results confirm an integrative β‐cell response to GLP‐1 that targets receptor‐mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation‐driven procoagulant events.     

Comparative Study of 35 Bacteriophages of Lactobacillus helveticus: Morphology and Host Range

This survey included 23 phages isolated from cheese whey and 12 temperate phages induced with mitomycin from their lysogenic host strains. All of the phages had an isometric head and a tail with a contractile sheath. In addition, short-tailed (160-nm-long) and long-tailed (260-nm-long) phages were distinguished. Short-tailed phages were by far the most widespread in French cheese factories (32 of the 35 phages studied). The study of phage relationships enabled two large groups of strains to be distinguished: those not or slightly sensitive to phages and those very sensitive to phages. There was an obvious relationship in the first group between phage sensitivity (or resistance) and the geographic origin of the strains. The second group contained primarily strains from large international collections and those isolated from commercial starters. The relationships among short-tailed phages, either temperate or isolated as lytic, suggest that lysogenic strains could be the major source of phages in French cheese factories.     

Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as 'Hit and Run' and 'vector recycling' which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.