An Automated Coupled-Column HPLC-System for the Direct Analysis of Ribonucleosides and Ribonucleotides in Biological Fluids

Abstract

We developed a coupled dual column HPLC-system for on-line sample processing, trace enrichment and analysis of ribonucleosides and ribonucleotides. The fully automated HPLC-analyzer tolerates the direct and repetitive injection of biological fluids such as serum or urine by use of an unique bonded-phase precolumn material which allows the simultaneous performance of covalent affinity and size-exclusion chromatography.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Myogenic differentiation of mesenchymal stem cells co‐cultured with primary myoblasts

TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co‐cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)‐transduced rat MSC co‐cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC—myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α‐sarcomeric actin, and MEF2 expression was up‐regulated upon co‐cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co‐cultures. In summary, we have determined optimal co‐culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell‐based therapies.     

Body mass variations in disturbed mallards Anas platyrhynchos fit to the mass‐dependent starvation‐predation risk trade‐off

For passerines the starvation‐predation risk theory predicts that birds should decrease their body mass to improve escape flight performance, when predation pressure increases. To investigate whether this theory may apply to large birds, which manage body reserves differently from small passerines, we experimentally increased the predation risk in mallards Anas platyrhynchos. Two groups were disturbed at different frequencies during experimental sessions lasting one week, while a control group was left undisturbed. We found that body mass loss and final wing loading were similar in both disturbed groups and significantly differed from the control group. Food intake in disturbed groups was reduced up to day four of the disturbance session and was lower than in the control group. Altogether our results suggest that disturbed mallards may adjust their body mass to reach a more favorable wing loading, supposedly to improve escape flight performance. Nevertheless, body mass loss in our mallards was double than what has been observed in passerines. This greater mass decrease might be explained by different strategies concerning energy storage. Furthermore, in large birds the predation component of the starvation‐predation trade‐off might be of greater importance. Hence, the observed relevance of this trade‐off over a large size range suggests that the starvation‐predation risk theory is of major ecological significance for many animal species.     

Evidence for effect of random genetic drift on G+C content after lateral transfer of fucose pathway genes to Escherichia coli K-12

The cps cluster of Escherichia coli K-12 comprises genes involved in synthesis of capsular polysaccharide colanic acid. Part of the E. coli K-12 cps region has been cloned and sequenced and compared to its Salmonella enterica LT2 counterpart. The cps genes from the two organisms are homologous; in the case of the LT2 genes, with G+C content of 0.61 and codons characteristic of high G+C species, it seems clear that they have been acquired relatively recently by lateral transfer from a high G+C species. The K-12 form of these cps genes is closely related to those of LT2 so must derive from the same high G+C species, but it appears to have transferred much earlier such that random genetic drift has brought P3 (the corrected G+C content of codon base 3) down from 0.77 to 0.64, more than halfway to the E. coli average of 0.57. We estimate, using an equation developed by Sueoka, that the lateral transfer to E. coli took place approximately 45 million years ago. This is the first report we are aware of demonstrating the expected adjustment of P3 after lateral transfer between species with different G+C content DNA.      

Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator.

Glycerol transport is commonly cited as the only example of facilitated diffusion across the Escherichia coli cytoplasmic membrane. Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. We found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation are different, depending on the presence or absence of the facilitator protein. We conclude that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria.     

Aberrations in SMAD family of genes among HNSCC patients

Head and neck cancer is a debilitating disease with several etiological factors. One of the main etiologies to be noticed is the alteration, which is either caused by genetic or environmental factors. Therefore, it is of interest to assess the effect of genetic alterations, especially the non-synonymous mutations of the SMAD gene family and its possible association with HNSCC. Data shows a significant novel mutation in the SMAD gene family in association with head and neck squamous cell carcinoma (HNSCC), which would aid in better diagnosis and treatment planning for cancer.     

Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.     

Methionine on the rise: how mitochondria changed their codon usage

The tRNA specific for methionine (tRNA^Met) of human mitochondria contains a formyl‐cytosine at the wobble position of the anticodon to facilitate its binding to AUG, AUA and (in one instance) to AUU. In this issue of The EMBO Journal, Haag et al identify a two‐step enzyme pathway facilitating the modification of the tRNA. Sequential reactions of the methyltransferase NSUN3 and the dioxygenase ALKBH1/ABH1 are important to render the tRNA as able to recognize the non‐canonical methionine codons AUA and AUUs, a property critical for efficient protein synthesis in human mitochondria.