Elements in autopsy liver tissue samples from Greenlandic inuit and Danes. V. Selenium measured by X-ray fluorescence spectrometry

The content of selenium in normal liver tissue samples from Greenlandic Inuit was measured and the results compared with those obtained in normal liver tissue samples from Danes. Normal liver tissue samples were obtained at autopsy from 50 Greenlandic Inuit (27 men, 23 women) with a median age of 61 years (range 23–83) and from 74 Danes (44 men, 30 women) with a median age of 60 years (range 15–87). Total liver selenium content was measured by X-ray fluorescence spectrometry. The content of selenium (median) was in Inuit 26.6 mol/kg dry liver (5–95 percentile: 15.2–49.4) and in Danes 17.7 mol/kg dry liver (5–95 percentile: < 3.8–36.5) (p < 0.0001). Liver selenium content displayed no significant gender difference, either in Inuit or Danes. In Inuit men, there was a negative correlation between liver selenium content and age (r_s = −0.39, p < 0.05), whereas Danish men displayed a positive correlation between liver selenium content and age (r_s = 0.37, p = 0.02). There was no correlation in Inuit or Danish women. In Inuit, the median hepatic selenium index (liver selenium content divided by age) was 0.48 and in Danes 0.33 (p = 0.001). There was an inverse correlation between hepatic selenium index and age both in Inuit (r_s = −0.77, p < 0.0001) and in Danes (r_s = −0.47, p < 0.0001). In conclusion, Inuit had a higher liver content of selenium and a higher hepatic selenium index compared with Danes. The more favourable selenium status is due to a higher nutritional selenium intake with fish and meat from sea mammals.     

The Genome-wide Patterns of Variation Expose Significant Substructure in a Founder Population

Although high-density SNP genotyping platforms generate a momentum for detailed genome-wide association (GWA) studies, an offshoot is a new insight into population genetics. Here, we present an example in one of the best-known founder populations by scrutinizing ten distinct Finnish early- and late-settlement subpopulations. By determining genetic distances, homozygosity, and patterns of linkage disequilibrium, we demonstrate that population substructure, and even individual ancestry, is detectable at a very high resolution and supports the concept of multiple historical bottlenecks resulting from consecutive founder effects. Given that genetic studies are currently aiming at identifying smaller and smaller genetic effects, recognizing and controlling for population substructure even at this fine level becomes imperative to avoid confounding and spurious associations. This study provides an example of the power of GWA data sets to demonstrate stratification caused by population history even within a seemingly homogeneous population, like the Finns. Further, the results provide interesting lessons concerning the impact of population history on the genome landscape of humans, as well as approaches to identify rare variants enriched in these subpopulations.     

Directed evolution of barnase stability using proteolytic selection

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability.Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.     

Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells

Recent evidence implicates the volume-regulated anion current (VRAC) and other anion currents in control or modulation of cell cycle progression; however, the precise involvement of anion channels in this process is unclear. Here, Cl- currents in Ehrlich Lettre Ascites (ELA) cells were monitored during cell cycle progression, under three conditions: (i) after osmotic swelling (i.e., VRAC), (ii) after an increase in the free intracellular Ca2+ concentration (i.e., the Ca2+-activated Cl- current, CaCC), and (iii) under steady-state isotonic conditions. The maximal swelling-activated VRAC current decreased in G1 and increased in early S phase, compared to that in G0. The isotonic steady-state current, which seems to be predominantly VRAC, also decreased in G1, and increased again in early S phase, to a level similar to that in G0. In contrast, the maximal CaCC current (500 nM free Ca2+ in the pipette), was unaltered from G0 to G1, but decreased in early S phase. A novel high-affinity anion channel inhibitor, the acidic di-aryl-urea NS3728, which inhibited both VRAC and CaCC, attenuated ELA cell growth, suggesting a possible mechanistic link between cell cycle progression and cell cycle-dependent changes in the capacity for conductive Cl- transport. It is suggested that in ELA cells, entrance into the S phase requires an increase in VRAC activity and/or an increased potential for regulatory volume decrease (RVD), and at the same time a decrease in CaCC magnitude.     

The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1

We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.     

Occurrence and Identification of Yeast Species in Fermented Liquid Feed for Piglets

The major objective of the present study was to investigate the occurrence and identity of yeast species in fermented liquid feed (FLF) used for feeding piglets. In total, 40 different Danish farms were included in the analysis. The preparation and composition of FLF was found to be very heterogeneous with high variations in both yeast counts and yeast species composition. The yeast population varied between 6.0?×?10³ and 4.2?×?10⁷?cfug^?1 with an average yeast count of 8.7?×?10⁶?±?1.1?×?10⁷?cfug^?1. A total of 766 yeasts were isolated and identified by conventional and/or molecular typing techniques. The predominant yeast species in the FLF samples were found to be Candida milleri (58.4%), Kazachstania exigua (17.5%), Candida pararugosa (6.40%) and Kazachstania bulderi (5.09%). No clear separation between isolates of C. milleri and Candida humilis could be obtained based on sequencing of the D1/D2 region of the 26S rRNA gene. The combined use of ITS-RFLP analysis and phenotypic criteria did meanwhile suggest a closer relationship with C. milleri than C. humilis.     

Crystal structure of the sulfotransferase domain of human heparan sulfate N-deacetylase/ N-sulfotransferase 1

Heparan sulfate N-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single alpha/beta fold with a central five-stranded parallel beta-sheet and a three-stranded anti-parallel beta-sheet bearing an interstrand disulfide bond. The structural regions alpha1, alpha6, beta1, beta7, 5'-phosphosulfate binding loop (between beta1 and alpha1), and a random coil (between beta8 and alpha13) constitute the PAP binding site of NST1. The alpha6 and random coil (between beta2 and alpha2), which form an open cleft near the 5'-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5'-phosphate.     

Na+,K+-pump stimulation improves contractility in isolated muscles of mice with hyperkalemic periodic paralysis

In patients with hyperkalemic periodic paralysis (HyperKPP), attacks of muscle weakness or paralysis are triggered by K(+) ingestion or rest after exercise. Force can be restored by muscle work or treatment with β(2)-adrenoceptor agonists. A missense substitution corresponding to a mutation in the skeletal muscle voltage-gated Na(+) channel (Na(v)1.4, Met1592Val) causing human HyperKPP was targeted into the mouse SCN4A gene (mutants). In soleus muscles prepared from these mutant mice, twitch, tetanic force, and endurance were markedly reduced compared with soleus from wild type (WT), reflecting impaired excitability. In mutant soleus, contractility was considerably more sensitive than WT soleus to inhibition by elevated [K(+)](o). In resting mutant soleus, tetrodotoxin (TTX)-suppressible (22)Na uptake and [Na(+)](i) were increased by 470 and 58%, respectively, and membrane potential was depolarized (by 16 mV, P < 0.0001) and repolarized by TTX. Na(+),K(+) pump-mediated (86)Rb uptake was 83% larger than in WT. Salbutamol stimulated (86)Rb uptake and reduced [Na(+)](i) both in mutant and WT soleus. Stimulating Na(+),K(+) pumps with salbutamol restored force in mutant soleus and extensor digitorum longus (EDL). Increasing [Na(+)](i) with monensin also restored force in soleus. In soleus, EDL, and tibialis anterior muscles of mutant mice, the content of Na(+),K(+) pumps was 28, 62, and 33% higher than in WT, respectively, possibly reflecting the stimulating effect of elevated [Na(+)](i) on the synthesis of Na(+),K(+) pumps. The results confirm that the functional disorders of skeletal muscles in HyperKPP are secondary to increased Na(+) influx and show that contractility can be restored by acute stimulation of the Na(+),K(+) pumps. Calcitonin gene-related peptide (CGRP) restored force in mutant soleus but caused no detectable increase in (86)Rb uptake. Repeated excitation and capsaicin also restored contractility, possibly because of the release of endogenous CGRP from nerve endings in the isolated muscles. These observations may explain how mild exercise helps locally to prevent severe weakness during an attack of HyperKPP.