A genetic screen for modifiers of the delta1-dependent notch signaling function in the mouse

The Notch signaling pathway is an evolutionarily conserved transduction pathway involved in embryonic patterning and regulation of cell fates during development. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, which are also involved in distinct human diseases. Delta1 is one of the known ligands of the Notch receptors. Mice homozygous for a loss-of-function allele of the Delta1 gene Dll1(lacZ/lacZ) die during embryonic development. Here, we present the results of two phenotype-driven modifier screens. Heterozygous Dll1(lacZ) knockout animals were crossed with ENU-mutagenized mice and screened for dysmorphological, clinical chemical, and immunological variants that are dependent on the Delta1 loss-of-function allele. First, we show that mutagenized heterozygous Dll1(lacZ) offspring have reduced body weight and altered specific clinical chemical parameters, including changes in metabolites and electrolytes relevant for kidney function. In our mutagenesis screen we have successfully generated 35 new mutant lines. Of major interest are 7 mutant lines that exhibit a Dll1(lacZ/+)-dependent phenotype. These mutant mouse lines provide excellent in vivo tools for studying the role of Notch signaling in kidney and liver function, cholesterol and iron metabolism, cell-fate decisions, and during maturation of T cells in the immune system.     

Cellular recognition of trimyristoylated peptide or enterobacterial lipopolysaccharide via both TLR2 and TLR4

Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.     

Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants

Background

Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity.

Methodology/Principal Findings

a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency ≥10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13×10⁻⁷, corrected p = 0.0494; odds ratio (OR)_CT 1.67, 95% confidence interval (CI) 1.22–2.27; OR_TT 2.76, 95% CI 1.88–4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium.

Conclusions/Significance

Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings.     

Secular trends of obesity and cardiovascular risk factors in a Mediterranean population

Objective: To evaluate time trends of obesity, abdominal obesity, and cardiovascular risk factors (CRFs) according to BMI and waist circumference (WC) categories in a Mediterranean population. Research Methods and Procedures: Subjects were Spanish men (n = 2383) and women (n = 2525) 25 to 74 years old, examined in 1994 to 1995 and 1999 to 2000 in two independent population‐based cross‐sectional surveys in the northeast of Spain. Lifestyle measures, CRFs, and anthropometric variables were analyzed. Results: Over the 5 years of the study, mean age‐standardized BMI increased by 1.0 units in men and by 0.8 units in women. At the same time the prevalence of obesity increased from 15.4% to 21.9% in men and from 15.4% to 21.4% in women. An upward trend was observed for WC and abdominal obesity (WC > 102 cm in men and WC > 88 cm in women) only in men. The proportion of men and women with hypercholesterolemia, diabetes, and low high‐density lipoprotein‐cholesterol plasma concentration remained stable within BMI and WC categories. The proportion of hypertension and smoking in obese men significantly increased from 1995 to 2000. Discussion: The 5‐year increase in BMI and WC is of considerable magnitude in the present population, although several CRFs remained stable within BMI and WC categories.     

Methane production in cattle calculated by the nutrient composition of the diet

In this study data originating from complete metabolic trials with cattle of both sexes, fed 337 rations at feed intake levels between one to three times maintenance energy requirement were used to regress the total CH4 emission to the level of DM intake and to the nutrient composition, respectively. A major component of the measured CH4 emission cannot be explained by DM intake but is rather due to differences in dietary nutrient composition. The amount of digestible nutrients especially of the carbohydrate fraction (starch, sugar, N-free residuals) are reliable to estimate CH4 release with high precision (r2 = 0.885). Its production rate increased to 1.9-fold higher levels (range 1.8-2.1) per g of N-free residuals compared to that induced per g of protein, starch or sugar, respectively. Furthermore, diets rich in fat reduced CH4 formation in the rumen. The regression equations fit a wide range of diets and diet compositions, and more importantly, they are applicable to various types of production systems.     

High Performance Proteomics: 7th HUPO Brain Proteome Project Workshop March 7-9, 2007 Wellcome Trust Conference Centre, Hinxton, UK

The Wellcome Trust Conference Centre at Hinxton, UK, was the meeting place of the 7th HUPO Brain Proteome Project Workshop entitled "High Performance Proteomics". It started on Wednesday, March 7, 2007 with a steering committee meeting followed by a two days series of talks dealing with the standardization and handling of tissues, body fluids as well as of proteomics data. The presentation and accompanying vivid discussions created a picture of actual strategies and standards in recent proteomics.     

Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii

The basic question addressed in this study is how energy metabolism is adjusted to cope with iron deficiency in Chlamydomonas reinhardtii. To investigate the impact of iron deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Although photosynthetic electron transfer is largely compromised under iron deficiency, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased. Concomitantly, stress-related chloroplast polypeptides, like 2-cys peroxiredoxin and a stress-inducible light-harvesting protein, LhcSR3, as well as a novel light-harvesting protein and several proteins of unknown function were induced under iron-deprivation. Respiratory oxygen consumption did not decrease and accordingly, polypeptides of respiratory complexes, harboring numerous iron-sulfur clusters, were only slightly diminished or even increased under low iron. Consequently, iron-deprivation induces a transition from photoheterotrophic to primarily heterotrophic metabolism, indicating that a hierarchy for iron allocations within organelles of a single cell exists that is closely linked with the metabolic state of the cell.     

Impact of Surface Active Compounds on Iron Catalyzed Oxidation of Methyl Linolenate in AOT–Water–Hexadecane Systems

Edible oils contain minor surface active components that form micro-heterogeneous environments, such as reverse micelles, which can alter the rate and direction of chemical reactions. However, little is known about the role of these micro-heterogeneous environments on lipid oxidation of bulk oil. Our objective was to evaluate the ability of water, cumene hydroperoxide, oleic acid, and phosphatidylcholine to influence the structure of reverse micelles in a model oil system: sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT; AOT) in n-hexadecane. The influence of reverse micelle structure on iron catalyzed lipid oxidation was determined using methyl linolenate as an oxidizable substrate. The size and shape of the reverse micelle were investigated by small-angle x-ray scattering, and water contents was determined by Karl Fischer titrations. Lipid hydroperoxides and thiobarbituric acid reactive substances were used to follow lipid oxidation. Our results showed that AOT formed spherical reverse micelles in hexadecane. The size of the reverse micelles increased with increased water or phosphatidylcholine concentration, but decreased upon addition of cumene hydroperoxide or oleic acid. Iron catalyzed oxidation of methyl linolenate in the reverse micelle system decreased with increasing water concentration. Addition of phosphatidylcholine into the reverse micelle systems decreased methyl linolenate oxidation compared to control and reverse micelles with added oleic acid. These results indicate that water, cumene hydroperoxide, oleic acid, and phosphatidylcholine can alter reverse micelle size and lipid oxidation rates. Understanding how these compounds influence reverse micelle structure and lipid oxidation rates could provide information on how to modify bulk oil systems to increase oxidative stability.     

Proteomic analysis of peripheral blood mononuclear cells: selective protein processing observed in patients with rheumatoid arthritis

In a comparative proteome analysis of peripheral blood mononuclear cells (PBMCs), we analyzed 130 two-dimensional gels obtained from 33 healthy control individuals and 32 patients diagnosed with rheumatoid arthritis (RA). We found 16 protein spots that are deregulated in patients with RA and, using peptide mass fingerprinting and Western blot analyses, identified these spots as belonging to 9 distinct proteins. A hierarchical clustering procedure organizes the study subjects into two main clusters based on the expression of these 16 protein spots, one that contains mostly healthy control individuals and the other mostly RA patients. The majority of the proteins differentially expressed in RA patients when compared with healthy controls can be detected as protein fragments in PBMCs obtained from RA patients. This set of deregulated proteins includes several factors that have been shown to be autoantigens in autoimmune diseases.