Genome-wide association meta-analysis of cortical bone mineral density unravels allelic heterogeneity at the RANKL locus and potential pleiotropic effects on bone

Previous genome-wide association (GWA) studies have identified SNPs associated with areal bone mineral density (aBMD). However, this measure is influenced by several different skeletal parameters, such as periosteal expansion, cortical bone mineral density (BMD_C) cortical thickness, trabecular number, and trabecular thickness, which may be under distinct biological and genetic control. We have carried out a GWA and replication study of BMD_C, as measured by peripheral quantitative computed tomography (pQCT), a more homogenous and valid measure of actual volumetric bone density. After initial GWA meta-analysis of two cohorts (ALSPAC n = 999, aged ∼15 years and GOOD n = 935, aged ∼19 years), we attempted to replicate the BMD_C associations that had p<1×10⁻⁵ in an independent sample of ALSPAC children (n = 2803) and in a cohort of elderly men (MrOS Sweden, n = 1052). The rs1021188 SNP (near RANKL) was associated with BMD_C in all cohorts (overall p = 2×10⁻¹⁴, n = 5739). Each minor allele was associated with a decrease in BMD_C of ∼0.14SD. There was also evidence for an interaction between this variant and sex (p = 0.01), with a stronger effect in males than females (at age 15, males −6.77mg/cm³ per C allele, p = 2×10⁻⁶; females −2.79 mg/cm³ per C allele, p = 0.004). Furthermore, in a preliminary analysis, the rs1021188 minor C allele was associated with higher circulating levels of sRANKL (p<0.005). We show this variant to be independent from the previously aBMD associated SNP (rs9594738) and possibly from a third variant in the same RANKL region, which demonstrates important allelic heterogeneity at this locus. Associations with skeletal parameters reflecting bone dimensions were either not found or were much less pronounced. This finding implicates RANKL as a locus containing variation associated with volumetric bone density and provides further insight into the mechanism by which the RANK/RANKL/OPG pathway may be involved in skeletal development.     

HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity

Background

Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure.

Methodology/Principal Findings

We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA^all-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles.

Conclusions/Significance

The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.     

Alterations of mitochondrial DNA in CEM cells selected for resistance toward ddC toxicity

2 ',3 '-dideoxycytidine (ddC) is a nucleoside analog that has been shown to produce a delayed toxicity which may be due to the depletion of mitochondrial DNA (mtDNA). In order to gain further understanding of the events involved in mitochondrial toxicity, two different CEM cell lines were selected for resistance to the delayed ddC toxicity.     

Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK).     

Physiochemical characterization of the Alzheimer's disease-related peptides A beta 1-42Arctic and A beta 1-42wt

The amyloid beta peptide (A beta) is crucial for the pathogenesis of Alzheimer's disease. Aggregation of monomeric A beta into insoluble amyloid fibrils proceeds through several soluble A beta intermediates, including protofibrils, which are believed to be central in the disease process. The main reason for this is their implication in familial Alzheimer's disease with the Arctic amyloid precursor protein mutation (E693G). This mutation gives rise to early onset Alzheimer's disease, and synthetic A beta 1-40Arctic displays an enhanced rate of protofibril formation in vitro[Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J & Lannfelt L. (2001) Nat Neurosci4, 887-893]. To increase our understanding of the mechanisms involved in A beta aggregation, especially A beta monomer oligomerization into protofibrils and protofibril fibrillization into fibrils, the kinetics of A beta 1-42wt and A beta 1-42Arctic aggregation were examined under different physiochemical conditions, such as concentration, temperature, ionic strength and pH. We used size exclusion chromatography for this purpose, where monomers are separated from protofibrils, and fibrils are separated from protofibrils in a centrifugation step. The Arctic mutation significantly accelerated both A beta 1-42wt protofibril formation and protofibril fibrillization. In addition, we demonstrated that two distinct chemical processes - monomer oligomerization and protofibril fibrillization - were affected differently by changes in the micro-environment and that the Arctic mutation alters the peptide response to such changes.     

Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes; segregation of specific proteins and functions

Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm. They constitute sites of entry into the cell as well as platforms for cell signalling. We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy. We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells. These caveolae contained caveolin-1 and caveolin-2. Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4. This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol. A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4. Small amounts of these proteins were also detected in the high-density caveolae. In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae. The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae. There was no correlation between the caveolar contents of caveolin and cholesterol. The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake/metabolism. These findings demonstrate a segregation of functions in caveolae subclasses.     

DNA oxidative damage and strand breaks in young healthy individuals: a gender difference and the role of life style factors

The aim of this study was to analyze background levels of DNA damage in young (19-31 years) non-smoking individuals and to correlate damage to gender and life style. DNA single strand breaks (SSB) and alkali labile sites (ALS) were measured in 99 subjects living in Stockholm, Sweden. Further, oxidative DNA damage was analyzed using the DNA repair glycosylase FPG as well as HPLC-ECD for specific analysis of 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). We found that males had higher (P < 0.001) levels of SSB + ALS than females, but no difference was seen for oxidative lesions. There was no correlation between FPG sites and 8-oxodG. For females, there was a positive correlation between FPG levels and body mass index and a negative correlation between SSB + ALS and fruit intake. We conclude that the background level of oxidative DNA damage, analyzed with improved methods, is low and that gender, fruit intake and BMI can affect DNA damage.     

Nutrition and theory of mind--The role of polyunsaturated fatty acids (PUFA) in the development of theory of mind

Breast-milk provides nutrients required for the development of the brain. n-6 and n-3 long-chain polyunsaturated fatty acids (LCPUFAs) have been suggested to be particularly involved. In this study levels of fatty acids in breast-milk were examined in relation to theory of mind (ToM) (n = 13) and WISC-III (n = 22) in six-year-old children. ToM tasks comprised four illustrated stories with questions about emotional (sad) events. Single polyunsaturated fatty acids (PUFA) were estimated as well as ratios between different fatty acids in order to describe putative associations between PUFA and psychological measures. Results show correlations between both ToM and WISC-III with single n-6 PUFA and the ratios DHA/AA and DHA/DPA. The correlations remained when socio-demographic factors were statistically controlled for. The positive findings related to the n-6 and n-3 LCPUFAs corroborate previous findings related to child cognitive development.     

Negative feedback predominates over cross-regulation to control ERK MAPK activity in response to FGF signalling in embryos

Expression of the gene encoding the MKP-3/Pyst1 protein phosphatase, which inactivates ERK MAPK, is induced by FGF. However, which intracellular signalling pathway mediates this expression is unclear, with essential roles proposed for both ERK and PI(3)K in chick embryonic limb. Here, we report that MKP-3/Pyst1 expression is sensitive to inhibition of ERK or MAPKK, that endogenous MKP-3/Pyst1 co-localizes with activated ERK, and expression of MKP-3/Pyst1 in mice lacking PDK1, an essential mediator of PI(3)K signalling. We conclude that MKP-3/Pyst1 expression is mediated by ERK activation and that negative feedback control predominates in limiting the extent of FGF-induced ERK activity.     

Characterization of the outer membrane protein profile from disease-related Helicobacter pylori isolates by subcellular fractionation and nano-LC FT-ICR MS analysis

Because of the important role of membrane proteins in adhesion, invasion, and intracellular survival of pathogens in the host, membrane proteins are of potential interest in the search for drug targets or biomarkers. We have established a mass spectrometry-based method that allows characterization of the outer membrane protein (OMP) profile of clinical isolates from of the human gastric pathogen Helicobacter pylori. Subcellular fractionation and one-dimensional gel electrophoresis (1D-GE) analysis was combined with nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) and tandem mass spectrometry (MS/MS) analysis of fifteen H. pylori strains associated either with duodenal ulcers, gastric cancer, or isolated from asymptomatic H. pylori infected carriers. Over 60 unique membrane or membrane-associated proteins, including 30 of the 33 theoretically predicted OMPs, were identified from the strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. In the search for clinical markers we found that Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. In addition, presence of Omp30 and absence of Omp6 seemed to be associated with H. pylori strains causing duodenal ulcer.