Genetic determinants of serum testosterone concentrations in men

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterone''s high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10⁻⁴¹ and rs6258, p = 2.3×10⁻²²). Subjects with ≥3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10⁻¹⁶). The rs6258 polymorphism in exon 4 of SHBG affected SHBG''s affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.     

Heterozygosity for a loss-of-function mutation in GALNT2 improves plasma triglyceride clearance in man

Genome-wide association studies have identified GALNT2 as a candidate gene in lipid metabolism, but it is not known how the encoded enzyme ppGalNAc-T2, which contributes to the initiation of mucin-type O-linked glycosylation, mediates this effect. In two probands with elevated plasma high-density lipoprotein cholesterol and reduced triglycerides, we identified a mutation in GALNT2. It is shown that carriers have improved postprandial triglyceride clearance, which is likely attributable to attenuated glycosylation of apolipoprotein (apo) C-III, as observed in their plasma. This protein inhibits lipoprotein lipase (LPL), which hydrolyses plasma triglycerides. We show that an apoC-III-based peptide is a substrate for ppGalNAc-T2 while its glycosylation by the mutant enzyme is impaired. In addition, neuraminidase treatment of apoC-III which removes the sialic acids from its glycan chain decreases its potential to inhibit LPL. Combined, these data suggest that ppGalNAc-T2 can affect lipid metabolism through apoC-III glycosylation, thereby establishing GALNT2 as a lipid-modifying gene.     

Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression: a proteomic approach

Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study, we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress to specific receptors on the cell surface. We showed that mechanical stimuli promote an increase in FAK (focal adhesion kinase) phosphorylation. In order to further shed light in the process of myoblast-induced differentiation by mechanical stimuli, we performed a proteomic analysis. Thirteen proteins were found to be affected by mechanical stimulation including galectin-1, annexin III and RhoGDI (Rho guanine-nucleotide-dissociation inhibitor). In this study, we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of galectin-1 in mechanically stimulated myoblasts. A potential involvement of galectin-1 in myoblast differentiation is presented.     

RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.     

UniCarb-DB: a database resource for glycomic discovery

Glycosylation is one of the most important post-translational modifications of proteins, known to be involved in pathogen recognition, innate immune response and protection of epithelial membranes. However, when compared to the tools and databases available for the processing of high-throughput proteomic data, the glycomic domain is severely lacking. While tools to assist the analysis of mass spectrometry (MS) and HPLC are continuously improving, there are few resources available to support liquid chromatography (LC)-MS/MS techniques for glycan structure profiling. Here, we present a platform for presenting oligosaccharide structures and fragment data characterized by LC-MS/MS strategies. The database is annotated with high-quality datasets and is designed to extend and reinforce those standards and ontologies developed by existing glycomics databases. AVAILABILITY: http://www.unicarb-db.org     

Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24?h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1?h, but lowest at 24?h, whereas the slow pH decline group showed the reverse case. The same pattern was also observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p<0.05). Protein identification revealed that most of the phosphoproteins were glycometabolism-related enzymes, and the others were involved in stress response, phosphocreatine metabolism, and other functions. The phosphorylation of pyruvate kinase and triosephosphate isomerase-1 showed to be related to PM muscle pH decline rate. Our work sheds light on the potential role of protein phosphorylation on regulating meat quality development.     

Multilocus genotyping of human Giardia isolates suggests limited zoonotic transmission and association between assemblage B and flatulence in children

Background

Giardia intestinalis is one of the most common diarrhea-related parasites in humans, where infection ranges from asymptomatic to acute or chronic disease. G. intestinalis consists of eight genetically distinct genotypes or assemblages, designated A–H, and assemblages A and B can infect humans. Giardiasis has been classified as a possible zoonotic disease but the role of animals in human disease transmission still needs to be proven. We tried to link different assemblages and sub-assemblages of G. intestinalis isolates from Swedish human patients to clinical symptoms and zoonotic transmission.

Methodology/Principal Findings

Multilocus sequence-based genotyping of 207 human Giardia isolates using three gene loci: ß-giardin, glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) was combined with assemblage-specific tpi PCRs. This analysis identified 73 patients infected with assemblage A, 128 with assemblage B, and six with mixed assemblages A+B. Multilocus genotypes (MLGs) were easily determined for the assemblage A isolates, and most patients with this genotype had apparently been infected through anthroponotic transmission. However, we also found evidence of limited zoonotic transmission of Giardia in Sweden, since a few domestic human infections involved the same assemblage A MLGs previously reported in Swedish cats and ruminants. Assemblage B was detected more frequently than assemblage A and it was also more common in patients with suspected treatment failure. However, a large genetic variability made determination of assemblage B MLGs problematic. Correlation between symptoms and assemblages was found only for flatulence, which was significantly more common in children less than six years of age infected with assemblage B.

Conclusions/Significance

This study shows that certain assemblage A subtypes are potentially zoonotic and that flatulence is connected to assemblage B infections in young children. Determination of MLGs from assemblages A and B can be a valuable tool in outbreak situations and to help identify possible zoonotic transmission.