DFT and MP2 studies on the C2C2α bond cleavage in thiamin catalysis

Thiamin diphosphate (ThDP), the biologically active derivative of vitamin B₁, is an important cofactor of several enzymes that catalyze the oxidative and non-oxidative conversion of α-keto acids. The final step of non-oxidative decarboxylation of pyruvate by pyruvate decarboxylase – the liberation of acetaldehyde – requires deprotonation of the α-hydroxyl group and cleavage of the C2–C2α bond of the transitory 2-(1-hydroxyethyl)-ThDP intermediate. It has been proposed that the cofactor 4′-amino/imino function is essentially involved in the deprotonation of the α-hydroxyl group. Proton transfer and C2–C2α cleavage may occur in a stepwise manner, or, alternatively in a concerted mechanism. Here, density functional theory (DFT) calculations as well as second order Møller–Plesset perturbation theory (MP2) studies were performed on a simple model for the enzyme using the program package Gaussian 03. Calculations favor a stepwise mechanism with initial formation of the C2α alkoxide, followed by C2–C2α bond cleavage.     

Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses

Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.     

A Pilot Study to Assess the Feasibility of Transcutaneous Glomerular Filtration Rate Measurement Using Fluorescence-Labelled Sinistrin in Dogs and Cats

In dogs and cats an assessment of renal function is often needed, however, existing methods including urine and plasma clearances are invasive, cumbersome and time consuming. This pilot study evaluated the feasibility of a transcutaneous glomerular filtration rate (GFR) measurement in dogs and cats. Additionally the optimal dose and location for the transcutaneous measurement device were investigated. Renal elimination of fluorescein-isothiocyanate-labelled sinistrin (FITC-S) was measured transcutaneously for 4 hours. The procedures were performed in awake, freely moving animals using escalating doses of FITC-S (10 mg/kg, 30 mg/kg, 50 mg/kg) with a wash-out period of at least 24 h in between. Multiple devices were placed on each animal. The resulting FITC-S disappearance curves were visually assessed to determine the most suitable location and the appropriate dose to reach an adequate transcutaneous peak signal for kinetic analysis. In both species 30 mg/kg were adequate for kinetic calculation. The most suitable place for the device was the lateral thoracic wall in dogs and the ventral abdominal wall in cats, respectively. Transcutaneous FITC-S clearance was then repeated using the optimal dose and location and in parallel with an additional plasma sinistrin clearance. Plasma elimination half-lives [min] were 26, 31 and 35, and corresponding transcutaneous elimination half-lives [min] were 26, 34 and 55, respectively in the dogs. Plasma elimination half-lives [min] were 51, 60 and 61, and corresponding transcutaneous elimination half-lives [min] were 75, 96 and 83, respectively in the cats. In conclusion, transcutaneous FITC-S clearance is a feasible method for the assessment of GFR in awake dogs and cats. It is noninvasive, well tolerated and easy to perform even in a clinical setting with results being readily available. A dose of 30 mg/kg of FITC-S seems adequate for kinetic assessment. Further studies are now needed to establish reference values and evaluate transcutaneous renal clearance in various conditions.     

Complete assignment of Ala,Ile, Leu,Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl–methyl NOESY

Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70 kDa homodimeric proteins. Assignments were obtained from methyl–methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.     

Toxic gain of function from mutant FUS protein is crucial to trigger cell autonomous motor neuron loss

FUS is an RNA‐binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS‐containing aggregates are often associated with concomitant loss of nuclear FUS. Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell‐specific CRE‐mediated expression of wild‐type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.