176 research outputs found
Strain-Specific Liver Metabolite Profiles in Medaka
The relationship between genetic variation and phenotypic traits is often poorly understood since specific genotypes do not always easily translate into associated phenotypes, especially for complex disorders. The genetic background has been shown to affect metabolic pathways and thus contribute to variations in the metabolome. Here, we tested the suitability of NMR metabolomics for comparative analysis of fish lines as a first step towards phenotype-genotype association studies. The Japanese rice fish, medaka (Oryzias latipes), is a widely used genetic vertebrate model with several isogenic inbred laboratory strains. We used liver extracts of medaka iCab and HO5 strains as a paradigm to test the feasibility of distinguishing the metabolome of two different inbred strains. Fifteen metabolites could be detected in uni- and multivariate analyses that showed strain-specific levels. Differences could be assigned to specific metabolic pathways. Our results show that NMR spectroscopy is a suitable method to detect variance of the metabolome caused by subtle genetic differences. Thus, it has the potential to address genotypeâphenotype associations in medaka, providing an additional level of phenotypic analysis
Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by Îł-Secretase
FâTag Induced Acyl Shift in the Photochemical Cyclization of o âAlkynylated N âAlkylâ N âacylamides to Indoles
A photochemical cyclization of F-tagged, o-alkynylated N-alkylamides to indoles catalyzed by [Ir(dF(CF)ppy)(dtbpy)]PF is presented. This straightforward and efficient reaction involves an intramolecular rearrangement due to the presence of an electron withdrawing group in the acyl moiety and is the first example of photochemically induced 1,3-acyl shift in the cyclization towards 3-acylindoles. A four-step reaction sequence including the photoreaction as a key step to the desired indoles has been developed and optimized. The compatibility of differently substituted F-tagged precursors with the photocyclization step was investigated and the robustness of this step towards modifications could be shown. In total, 16 so far unknown derivatives with diverse modifications in positions N1 and C2, bearing a pentadecafluorooctanoyl moiety as F-tag, were synthesized in very good yields and fully characterized
Lipase-Catalyzed Production of Sorbitol Laurate in a â2-in-1â Deep Eutectic System: Factors Affecting the Synthesis and Scalability
Surfactants, such as glycolipids, are specialty compounds that can be encountered daily in cleaning agents, pharmaceuticals or even in food. Due to their wide range of applications and, more notably, their presence in hygiene products, the demand is continuously increasing worldwide. The established chemical synthesis of glycolipids presents several disadvantages, such as lack of specificity and selectivity. Moreover, the solubility of polyols, such as sugars or sugar alcohols, in organic solvents is rather low. The enzymatic synthesis of these compounds is, however, possible in nearly water-free media using inexpensive and renewable building blocks. Using lipases, ester formation can be achieved under mild conditions. We propose, herein, a â2-in-1â system that overcomes solubility problems, as a Deep Eutectic System (DES) made of sorbitol and choline chloride replaces either a purely organic or aqueous medium. For the first time, 16 commercially available lipase formulations were compared, and the factors affecting the conversion were investigated to optimize this process, owing to a newly developed High-Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) method for quantification. Thus, using 50 g/L of lipase formulation Novozym 435Âź at 50 °C, the optimized synthesis of sorbitol laurate (SL) allowed to achieve 28% molar conversion of 0.5 M of vinyl laurate to its sugar alcohol monoester when the DES contained 5 wt.% water. After 48h, the de novo synthesized glycolipid was separated from the media by liquidâliquid extraction, purified by flash-chromatography and characterized thoroughly by one- and two-dimensional Nuclear Magnetic Resonance (NMR) experiments combined to Mass Spectrometry (MS). In completion, we provide initial proof of scalability for this process. Using a 2.5 L stirred tank reactor (STR) allowed a batch production reaching 25 g/L in a highly viscous two-phase system
Urinary NMR Profiling in Pediatric Acute Kidney InjuryâA Pilot Study
Acute kidney injury (AKI) in critically ill children and adults is associated with significant short- and long-term morbidity and mortality. As serum creatinine- and urine output-based definitions of AKI have relevant limitations, there is a persistent need for better diagnostics of AKI. Nuclear magnetic resonance (NMR) spectroscopy allows for analysis of metabolic profiles without extensive sample manipulations. In the study reported here, we examined the diagnostic accuracy of NMR urine metabolite patterns for the diagnosis of neonatal and pediatric AKI according to the Kidney Disease: Improving Global Outcomes (KDIGO) definition. A cohort of 65 neonatal and pediatric patients (0â18 years) with established AKI of heterogeneous etiology was compared to both a group of apparently healthy children (n = 53) and a group of critically ill children without AKI (n = 31). Multivariate analysis identified a panel of four metabolites that allowed diagnosis of AKI with an area under the receiver operating characteristics curve (AUC-ROC) of 0.95 (95% confidence interval 0.86â1.00). Especially urinary citrate levels were significantly reduced whereas leucine and valine levels were elevated. Metabolomic differentiation of AKI causes appeared promising but these results need to be validated in larger studies. In conclusion, this study shows that NMR spectroscopy yields high diagnostic accuracy for AKI in pediatric patients
Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase
Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin
The strictly conserved Cys76 plays a crucial role in the conformational stability of reduced Px III
Macrocyclic tetramersâstructural investigation of peptide-peptoid hybrids
Outstanding affinity and specificity are the main characteristics of peptides, rendering them interesting compounds for basic and medicinal research. However, their biological applicability is limited due to fast proteolytic degradation. The use of mimetic peptoids overcomes this disadvantage, though they lack stereochemical information at the α-carbon. Hybrids composed of amino acids and peptoid monomers combine the unique properties of both parent classes. Rigidification of the backbone increases the affinity towards various targets. However, only little is known about the spatial structure of such constrained hybrids. The determination of the three-dimensional structure is a key step for the identification of new targets as well as the rational design of bioactive compounds. Herein, we report the synthesis and the structural elucidation of novel tetrameric macrocycles. Measurements were taken in solid and solution states with the help of X-ray scattering and NMR spectroscopy. The investigations made will help to find diverse applications for this new, promising compound class
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