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Gundner A.L.,University of Bonn | Gundner A.L.,Pharma Research and Early Development | Hahn I.,University of Bonn | Sendscheid O.,University of Bonn | And 3 more authors.

Phosphoinositide-3-kinase enhancer (PIKE) proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH) domain, a GTPase-activating (GAP) domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK) and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to third instar larval transition. © 2014 Gündner et al. Source

Rudolph M.G.,Pharma Research and Early Development | Del Toro Duany Y.,University of Munster | Jungblut S.P.,University of Basel | Ganguly A.,University of Munster | Klostermeier D.,University of Munster
Nucleic Acids Research

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presented. © 2012 The Author(s). Source

Miner J.H.,University of Washington | Baigent C.,University of Oxford | Flinter F.,Guys and St Thomas NHS Foundation Trust | Gross O.,University Medicine Goettingen | And 14 more authors.
Kidney International

Alport syndrome, historically referred to as hereditary glomerulonephritis with sensorineural deafness and anterior lenticonus, is a genetic disease of collagen α3α4α5(IV) resulting in renal failure. The collagen α3α4α5(IV) heterotrimer forms a network that is a major component of the kidney glomerular basement membrane (GBM) and basement membranes in the cochlea and eye. Alport syndrome, estimated to affect 1 in 5000-10,000 individuals, is caused by mutations in any one of the three genes that encode the α chain components of the collagen α3α4α5(IV) heterotrimer: COL4A3, COL4A4, and COL4A5. Although angiotensin-converting enzyme inhibition is effective in Alport syndrome patients for slowing progression to end-stage renal disease, it is neither a cure nor an adequate long-term protector. The 2014 International Workshop on Alport Syndrome, held in Oxford, UK, from January 3-5, was organized by individuals and families living with Alport syndrome, in concert with international experts in the clinical, genetic, and basic science aspects of the disease. Stakeholders from diverse communities - patient families, physicians, geneticists, researchers, Pharma, and funding organizations - were brought together so that they could meet and learn from each other and establish strategies and collaborations for the future, with the overall aim of discovering much needed new treatments to prolong kidney function. Source

Marchal J.,CNRS Mechanical Adaptation and Evolution | Dal-Pan A.,CNRS Mechanical Adaptation and Evolution | Epelbaum J.,University of Paris Descartes | Blanc S.,CNRS Hubert Curien Multi-disciplinary Institute | And 4 more authors.
Experimental Gerontology

Oxidative stress is a key factor in the aging process and in the development of age-related diseases. Because nutritional interventions such as caloric restriction (CR) delay the onset of age-related diseases and increase the lifespan of many species, the impact of a moderate CR was tested on male grey mouse lemur (Microcebus murinus), which have a median survival time of 5.7years in captivity. The effects of CR on these lemurs were compared with a potential mimetic, resveratrol (RSV), a polyphenol naturally found in grapes. We hypothesized that both CR and RSV impact oxidative DNA and RNA damage compared to standard-fed control (CTL) animals. Adult (3-4years old) male mouse lemurs were assigned to three dietary groups: a CTL group, a CR group receiving 30% fewer calories than the CTL and a RSV group receiving the CTL diet supplemented with RSV (200mg·day-1·kg-1). Oxidative stress was estimated after 3, 9, 15 and 21months of treatment using the measurement of oxidized nucleosides in urine samples by mass spectrometry. The resting metabolic rate, adjusted for changes in body composition, was also measured to assess the potential relationship between oxygen consumption and oxidative damage markers. This study provides evidence for oxidative stress accumulation with age in grey mouse lemur. Dietary interventions resulted in a short-term increase in oxidative stress levels followed by reduced levels with increasing age. Moreover, in this photoperiod-dependent heterotherm primate, seasonal variations in oxidative stress were observed, which was likely due to a season-dependent, cost-benefit trade-off between torpor use and oxidative stress. © 2013 Elsevier Inc. Source

Schoch A.,Pharma Research and Early Development | Thorey I.S.,Roche Holding AG | Engert J.,Ludwig Maximilians University of Munich | Winter G.,Ludwig Maximilians University of Munich | Emrich T.,Pharma Research and Early Development
Lab Animal

In pharmacokinetic studies, intravenous (i.v.) administration of antibodies to mice is usually done via the lateral tail vein. This approach can cause stress to the mice and has a high rate of failure because it is challenging to perform correctly. Administration via the retro-orbital venous sinus has been suggested as a good alternative to tail vein i.v. administration of antibodies. Evidence is still needed, however, to determine whether the route of administration has an effect on the absorption or the pharmacokinetic activity of the injected antibody. The authors compared serum concentrations and pharmacokinetic parameters of a therapeutic antibody administered via tail vein injection or via retro-orbital injection. The findings suggest that there is no difference in the absorption or pharmacokinetic activity of therapeutic antibodies when administered via the lateral tail vein versus the retro-orbital venous sinus. Source

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