Developmentally regulated thyroid hormone distributor proteins in marsupials, a reptile, and fish

Thyroid hormones are essential for vertebrate development. There is a characteristic rise in thyroid hormone levels in blood during critical periods of thyroid hormone-regulated development. Thyroid hormones are lipophilic compounds, which readily partition from an aqueous environment into a lipid environment. Thyroid hormone distributor proteins are required to ensure adequate distribution of thyroid hormones, throughout the aqueous environment of the blood, and to counteract the avid partitioning of thyroid hormones into the lipid environment of cell membranes. In human blood, these proteins are albumin, transthyretin and thyroxine-binding globulin. We analyzed the developmental profile of thyroid hormone distributor proteins in serum from a representative of each order of marsupials (M. eugenii; S.crassicaudata), a reptile (C. porosus), in two species of salmonoid fishes (S. salar; O. tshawytsch), and throughout a calendar year for sea bream (S. aurata). We demonstrated that during development, these animals have a thyroid hormone distributor protein present in their blood which is not present in the adult blood. At least in mammals, this additional protein has higher affinity for thyroid hormones than the thyroid hormone distributor proteins in the blood of the adult. In fish, reptile and polyprotodont marsupial, this protein was transthyretin. In a diprotodont marsupial, it was thyroxine-binding globulin. We propose an hypothesis that an augmented thyroid hormone distributor protein network contributes to the rise in total thyroid hormone levels in the blood during development.     

Use of bioluminescence for monitoring the viability of individual Pseudomonas putida KT2442 cells

Exponential phase cells of Pseudomonas putida KT2442 rapidly lost viability when incubated at 0°C without entering a viable but non-culturable state. The majority of dead cells retained their cellular integrity and contained DNA. However, their cellular rRNA content was substantially reduced. By employing a luciferase-marked derivative of P. putida KT2442 in combination with a highly sensitive low-light imaging system, live and dead cells could be distinguished.     

Structure and dynamics of small soluble Aβ(1-40) oligomers studied by top-down hydrogen exchange mass spectrometry

Aβ peptides can assemble into amyloid fibrils, which represent one of the hallmarks of Alzheimer's disease. Recent studies, however, have focused on the behavior of small soluble Aβ oligomers that possess a much greater neurotoxicity than mature fibrils. The structural characterization of these oligomers remains difficult because of their highly dynamic and polymorphic nature. This work explores the behavior of Aβ(1-40) in a slightly basic solution (pH 9.3) at a low salt concentration (10 mM ammonium acetate). These conditions lead to the formation of small oligomers, without any signs of fibrillation for several hours. The structure and dynamics of these oligomers were characterized by circular dichroism spectroscopy, size exclusion chromatography, and millisecond time-resolved hydrogen exchange mass spectrometry (MS). Our results reveal rapid interconversion between Aβ(1-40) oligomers and monomers. The mole fraction of monomeric molecules is on the order of 40%. Oligomers consist of ~4 Aβ(1-40) molecules on average, and the resulting assemblies have a predominantly β-sheet secondary structure. Hydrogen exchange proceeds in the EX1 regime. This feature allows the application of conformer-specific top-down MS. Electron capture dissociation is used for interrogating the deuteration behavior of the Aβ(1-40) oligomers. This approach provides a spatial resolution of ~2 residues. The backbone amide deuteration pattern uncovered in this way is consistent with a β-turn-β motif for L17-M35. The N-terminus is involved in hydrogen bonding, as well, whereas protection gradually tapers off for C-terminal residues 35-40. Our data are consistent with earlier proposals, according to which Aβ(1-40) oligomers adopt a β-barrel structure. In general terms, this study demonstrates how top-down MS with precursor ion selection can be employed for structural studies of specific protein conformers within a heterogeneous mix.     

Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase II alpha

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.     

Complete genome sequence of Desulfocapsa sulfexigens,a marine deltaproteobacterium specialized in disproportionating inorganic sulfur compounds

Desulfocapsa sulfexigens SB164P1 (DSM 10523) belongs to the deltaproteobacterial family Desulfobulbaceae and is one of two validly described members of its genus. This strain was selected for genome sequencing, because it is the first marine bacterium reported to thrive on the disproportionation of elemental sulfur, a process with a unresolved enzymatic pathway in which elemental sulfur serves both as electron donor and electron acceptor. Furthermore, in contrast to its phylogenetically closest relatives, which are dissimilatory sulfate-reducers, D. sulfexigens is unable to grow by sulfate reduction and appears metabolically specialized in growing by disproportionating elemental sulfur, sulfite or thiosulfate with CO₂ as the sole carbon source. The genome of D. sulfexigens contains the set of genes that is required for nitrogen fixation. In an acetylene assay it could be shown that the strain reduces acetylene to ethylene, which is indicative for N-fixation. The circular chromosome of D. sulfexigens SB164P1 comprises 3,986,761 bp and harbors 3,551 protein-coding genes of which 78% have a predicted function based on auto-annotation. The chromosome furthermore encodes 46 tRNA genes and 3 rRNA operons.     

Patterns of time since last meal revealed by sparse PCA in an observational LC–MS based metabolomics study

In metabolomics studies, liquid chromatography mass spectrometry (LC–MS) provides comprehensive information on biological samples. However, extraction of few relevant metabolites from this large and complex data is cumbersome. To resolve this issue, we have employed sparse principal component analysis (SPCA) to capture the underlying patterns and select relevant metabolites from LC–MS plasma profiles. The study involves a small pilot cohort with 270 subjects where each subject’s time since last meal (TSLM) has been recorded prior to plasma sampling. Our results have demonstrated that both PCA and SPCA can capture the TSLM patterns. Nevertheless, SPCA provides more easily interpretable loadings in terms of selection of relevant metabolites, which are identified as amino acids and lyso-lipids. This study demonstrates the utility of SPCA as a pattern recognition and variable selection tool in metabolomics. Furthermore, amino acids and lyso-lipids are determined as dominating compounds in response to TSLM.     

Effects on metabolic markers are modified by PPARG2 and COX2 polymorphisms in infants randomized to fish oil

Long-chain n-3 fatty acids (n-3 LCPUFA) improve blood pressure (BP) and lipid profile in adults and improve insulin sensitivity in rodents. We have previously shown that n-3 LCPUFA reduces BP and plasma triacylglycerol (TAG) in infants. Few studies have found effects on glucose homeostasis in humans. We explored possible effect modification by FADS, PPARG2, and COX2 genotypes to support potential effects of n-3 LCPUFA on metabolic markers in infants. Danish infants (133) were randomly allocated to daily supplementation with a teaspoon (~5 mL/day) of fish oil (FO) or sunflower oil (SO) from 9 to 18 months of age. Before and after the intervention, we assessed BP, erythrocyte n-3 LCPUFA, plasma lipid profile, insulin, and glucose in addition to functional single nucleotide polymorphisms in FADS, PPARG2, and COX2. At 18 months, plasma TAG was lower in the FO compared with SO group (p = 0.014). This effect was modified by PPARG2-Pro12Ala, as TAG only decreased among heterozygotes. FO supplemented PPARG2 Pro12Ala heterozygotes also had decreased plasma glucose compared with the SO group (p = 0.043). The effect of FO on mean arterial BP at 18 months was gender dependent (p = 0.020) and reduced in boys only (p = 0.028). Diastolic BP was, however, lower among all FO supplemented homozygous COX2-T8473C variant allele carriers compared with the SO group (p = 0.001). In conclusion, our results confirm that FO supplementation in late infancy reduces TAG and BP and indicates that the effects are mediated via peroxisome proliferator-activated receptor-γ and cyclooxygenase-2. Furthermore, FO reduced plasma glucose only in PPARG2 heterozygotes.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0396-4) contains supplementary material, which is available to authorized users.     

A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.     

Characterization of the interaction of interleukin-8 with hyaluronan, chondroitin sulfate, dermatan sulfate and their sulfated derivatives by spectroscopy and molecular modeling

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG-protein systems.     

A longitudinal study to assess the persistence of vancomycin-resistant Enterococcus faecium (VREF) on an intensive broiler farm in the United Kingdom

Seven years after the ban of avoparcin, VREF could still be isolated within sectors of the UK broiler industry. The aim of this study was to assess whether there is a carryover of VREF between consecutive flocks of birds, to conduct a preliminary investigation of possible routes of entry of VREF into broiler houses and to follow the dynamics of VREF shed by growing birds. A series of nine visits were made to two of six houses on a conventional broiler farm. A total of 343 vanA VREF were recovered from environmental (95/843) and faecal (248/416) samples. Significant differences were observed in the carryover of VREF between pre- and postcohort postcleaning and disinfection visits (RR 0.57, P=0.006). Ninety-nine percent of the VREF isolates were resistant to more than five antimicrobials, with 42 isolates (n=49) positive for erm(B) and 32 (n=40) for vat(E). Pulsed field gel electrophoresis (PFGE) typing identified 50 PFGE types within 15 different PFGE clusters of 90% similarity, demonstrating a high level of genetic diversity within VREF populations from epidemiologically related broiler flocks and broiler houses. Further characterization of Tn1546 from different clones showed a low diversity of Tn-types, suggesting horizontal transfer of resistance determinants between different genetic clones. Thus, this study does not only show the persistence of VREF but also of multi-drug resistant lineages of VREF.