Thomas Beiter

Antisense transcription: a critical look in both directions.

Abstract.The mammalian genome contains a large layer of hidden biological information. High-throughput methods have provided new insights into the regulatory networks that orchestrate the “when, where and how” of gene expression, revealing a complex interplay between proteins, regulatory RNAs, and chemical and structural alterations of the genome itself. Naturally occurring antisense transcription has been considered as an important feature in creating transcriptional and hence cellular and organismal complexity. Here, we review the current understanding of the extent, functions and significance of antisense transcription. We critically discuss results from genome-wide studies and documented examples of individual antisense transcripts. So far, the regulatory potential of gene overlaps has been demonstrated only in a few selected cases of experimentally characterized antisense transcripts. Facing the large-scale antisense transcription observed in eukaryotic genomes, it still remains an open challenge to distinguish transcriptional noise from biological function of gene overlapping patterns.

COX-3—a virtual pain target in humans?

A long-sought molecule (1) was recently identified as acting independent of COX-1/-2 (2). The potent analgesic and anti-pyretic actions of acetaminophen lacking anti-inflammatory potency suggested the presence of an additional cyclo-oxygenase that could be directly responsible for acetaminophen-sensitive generation of prostanoids in neuronal systems. This could now be understood at least in part as modulation of the recently identified COX-3 (2). In canine, alternative splicing generates four different mRNA variants derived from the COX-1 geneOCOX-3, COX-1, and two partial COX-1 (denoted as PCOX-1 proteins), PCOX-1a and PCOX-1b, encoding novel members of the COX-1 protein family. Among them only canine COX-1 and canine COX-3 possessed glycosylation-dependent cyclooxygenase activity. The discovery by Dan Simmons and colleagues of COX-3 will most surely herald more interest in these pathways and enzymes as recently illustrated by the number, scholarship, and import of several commentaries (3–6). Predicted canine COX-3 is composed of COX-1 and the retained intron 1 (2). Identification of canine COX-3 might have a significant effect on understanding inflammatory messengers, characterizing new routes of prostanoid formation and pain conduction (3–6). These findings may be crucial to the development of new therapeutic agents targeting both COX-2 and COX-3 in order to reduce CNS inflammatory reactions, fever, and pain. However, this may hold true only for the dog. Database analysis of human COX-1 (Fig. 1) showed a frameshift induced by intron 1, possibly revealing COX-3 to be a virtual protein in humans. However, Western blot analysis of human aorta tissue using polyclonal antibodies directed against the first 13 amino acids of the predicted human and dog/mouse COX-3 detected (surprisingly) a 65 kDa protein postulated to be human COX-3 (2). The high sequence similarity of the canine insert to intron 1 of either human or mouse COX-1 genes and the presence of 5 and 3 consensus splice sites indicated the complete retention of intron 1 in this transcript. The predicted retention of a complete intron 1 sequence in human COX-1 (OMIM accession #176805) from a screening of several databases revealed a frameshift due to insertion of intron 1 consisting of 94 nucleotides or 98 nucleotides in the murine COX-1 gene, respectively. The frameshift after the insert (see Fig. 2) results 1) in a TGA stop codon 48 amino acids later and 2) a completely changed protein sequence not related to human COX-1. The compensating molecular events suggested by Chandrasekharan et al. are unlikely. COX-1 polymorphisms or sequencing errors concerning the nucleotides of intron 1 are implausible since recent screening of the human COX-1 gene, including the complete intron 1 region, revealed no polymorphisms or deletions in intron 1 (7). Moreover, compensation by “-1” ribosomal frame shifting has not been reported in eukaryotic mRNAs to date. COX-3 is considered a new and important lead in the generation of anti-inflammatory and analgesic agents. However, a frameshift caused by complete retention of intron 1 in the human sequence questions its relevance to human pathophysiology.

Short-Term Treadmill Running as a Model for Studying Cell-Free DNA Kinetics In Vivo

BACKGROUND Increased plasma concentrations of cell-free DNA (cf-DNA) are considered a hallmark of various clinical conditions. Despite intensive research in this field, limited data are available concerning the time course of release and clearance of cf-DNA in vivo. METHODS We extracted cf-DNA from plasma samples taken before and immediately after a 10-km cross-country run, and from samples taken before, immediately after, and 30 min after exhaustive short-term treadmill exercise. The contribution of nuclear (nDNA) and mitochondrial DNA (mtDNA) was measured by quantitative real-time PCR. The incremental treadmill exercise setup was exploited to delineate the precise sequencing and timing of cf-nDNA, lactate, and high-mobility group box 1 protein (HMGB1) release during the exercise and recovery phases. RESULTS Postexercise plasma cf-nDNA concentrations in cross-country and treadmill runners were significantly increased, by 7.6-fold and 9.9-fold, respectively (P < 0.001). cf-nDNA concentrations were not correlated with age, sex, or body mass index. Plasma concentrations of cf-nDNA and HMGB1 in postexercise samples of treadmill runners were significantly correlated (r = 0.84; P = 0.004). cf-mtDNA concentrations were not affected by treadmill exercise. Time-course analyses demonstrated that cf-nDNA is released within minutes after the onset of exercise and is rapidly cleared from the circulation after the cessation of exercise. Nearly congruent kinetics for cf-nDNA, lactate, and HMGB1 were observed during the exercise phase. CONCLUSIONS A single bout of exhaustive short-term treadmill exercise constitutes a versatile model system suitable for addressing basic questions about cf-DNA biology.

Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma

Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1–183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6–23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

Experimental autoimmune neuritis induces differential microglia activation in the rat spinal cord

The reactive spatial and temporal activation pattern of parenchymal spinal cord microglia was analyzed in rat experimental autoimmune neuritis (EAN). We observed a differential activation of spinal cord microglial cells. A significant increase in ED1(+) microglia predominantly located in the dorsal horn grey matter of lumbar and thoracic spinal cord levels was observed on Day 12. As revealed by morphological criteria and by staining with further activation markers [allograft inflammatory factor 1 (AIF-1), EMAPII, OX6, P2X(4)R], reactive microglia did not reach a macrophage-like state of full activation. On Day 12, a significant proliferative response could be observed, affecting all spinal cord areas and including ED1(+) microglial cells and a wide range of putative progenitor cells. Thus, in rat EAN, a reactive localized and distinct microglial activation correlating with a generalized proliferative response could be observed.

Neutrophils release extracellular DNA traps in response to exercise

Intense exercise evokes a rapid and transient increase in circulating cell-free DNA (cf-DNA), a phenomenon that is commonly observed in a variety of acute and chronic inflammatory conditions. In this study, we aimed to shed new light on the release and clearance mechanisms of cf-DNA in response to exercise. We hypothesized that activated neutrophils may primarily contribute to exercise-evoked cf-DNA levels by releasing neutrophil extracellular traps (NETs). Analysis of plasma and/or serum samples from male athletes at rest and in response to exhaustive treadmill exercise revealed an immediate and transient increase in cf-DNA that was concomitantly counterbalanced by an increase in serum DNase activity. Consistently, rapid release and clearance kinetics for cf-DNA could also be observed in response to intensive cycling exercise, with no significant differences between endurance-trained (V̇o2max >57 ml·min(-1)·kg(-1)) and healthy (V̇o2max <49 ml·min(-1)·kg(-1)) sedentary individuals. In postexercise blood smear samples, we detected seemingly intact neutrophils displaying morphological signs of NET release, as indicated by abnormal swollen nuclei and emanating DNA fibers. In support, we observed a striking correlation of postexercise cf-DNA concentrations with plasma levels of the granule-derived enzyme myeloperoxidase. Our study indicates that intense exercise induces liberation of NETs, which is sufficiently counterbalanced in healthy individuals by a concomitant rise in serum DNase activity. As aberrant release of NETs has been linked to diverse disease states, monitoring of cf-DNA/DNase levels or activities in response to standardized exercise testing could provide a valuable tool to identify people who are at increased risk for cardiac ischemia, thrombosis, autoimmunity, or chronic fatigue.

Basal and exercise induced label-free quantitative protein profiling of m. vastus lateralis in trained and untrained individuals.

UNLABELLED Morphological and metabolic adaptations of the human skeletal muscle to exercise are crucial to improve performance and prevent chronic diseases and metabolic disorders. In this study we investigated human skeletal muscle protein composition in endurance trained (ET) versus untrained individuals (UT) and its modulation by an acute bout of endurance exercise. Participants were recruited based on their VO2max and subjected to a bicycle exercise test. M. vastus lateralis biopsies were taken before and three hours after exercise. Muscle lysates were analyzed using off-gel LC-MS/MS. Relative protein abundances were compared between ET and UT at rest and after exercise. Comparing UT and ET, we identified 92 significantly changed proteins under resting conditions. Specifically, fiber-type-specific and proteins of the oxidative phosphorylation and tricarboxylic acid cycle were increased in ET. In response to acute exercise, 71 proteins in ET and 44 in UT were altered. Here, a decrease of proteins involved in energy metabolism accompanied with alterations of heat shock and proteasomal proteins could be observed. In summary, long-term endurance training increased the basal level of structural and mitochondrial proteins in skeletal muscle. In contrast, acute exercise resulted in a depletion of proteins related to substrate utilization, especially in trained athletes. BIOLOGICAL SIGNIFICANCE The investigation of the human skeletal muscle proteome in response to exercise may provide novel insights into the process of muscular plasticity. It is of importance in the development of exercise-based strategies in the prevention and therapy of many chronic inflammatory and degenerative diseases which are often accompanied by muscular deconditioning. Up to date, proteomic investigations of the human muscle proteome in adaptation to exercise are mainly focused on untrained individuals and often restricted to animal studies. In the present study we compare the protein composition in endurance trained athletes and untrained individuals in the resting muscle and its modulation in response to acute exercise. To our knowledge, we present the first comprehensive analysis of skeletal muscle proteome alterations in response to acute and long-term exercise intervention.

Effects of Acute Endurance Exercise on Plasma Protein Profiles of Endurance-Trained and Untrained Individuals over Time

Acute physical exercise and repeated exercise stimuli affect whole-body metabolic and immunologic homeostasis. The aim of this study was to determine plasma protein profiles of trained (EET, n = 19) and untrained (SED, n = 17) individuals at rest and in response to an acute bout of endurance exercise. Participants completed a bicycle exercise test at an intensity corresponding to 80% of their VO2max. Plasma samples were taken before, directly after, and three hours after exercise and analyzed using multiplex immunoassays. Seventy-eight plasma variables were included in the final analysis. Twenty-nine variables displayed significant acute exercise effects in both groups. Seven proteins differed between groups, without being affected by acute exercise. Among these A2Macro and IL-5 were higher in EET individuals while leptin showed elevated levels in SED individuals. Fifteen variables revealed group and time differences with elevated levels for IL-3, IL-7, IL-10, and TNFR2 in EET individuals. An interaction effect could be observed for nine variables including IL-6, MMP-2, MMP-3, and muscle damage markers. The proteins that differ between groups indicate a long-term exercise effect on plasma protein concentrations. These findings might be of importance in the development of exercise-based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring.

Neutrophil Extracellular Traps: A Walk on the Wild Side of Exercise Immunology

Intense exercise evokes a rapid and transient increase in circulating cell-free DNA (cf-DNA), a phenomenon that is commonly observed in a variety of acute and chronic inflammatory conditions. While the potential value of cf-DNA for the prediction of disease outcome and therapeutic response is well documented, the release mechanisms and biological relevance of cf-DNA have long remained enigmatic. The discovery of neutrophil extracellular traps (NETs) provided a novel mechanistic explanation for increased cf-DNA levels. Now there is increasing evidence that NETs may contribute to cf-DNA in diverse infectious, non-infectious and autoinflammatory conditions, as well as in response to acute exercise. NETs have now been firmly established as a fundamental immune mechanism used by neutrophils to respond to infection and tissue injury. On the other side, aberrant formation of NETs appears to be a driving force in the pathogenesis of autoimmunity and cardiovascular disease. Thus, the emergence of NETs in the ‘exercising vasculature’ raises important questions considering beneficial effects, as well as occasional adverse effects, of exercise on immune homeostasis. This review gives an overview of the current state of research into the mechanisms of how NETs are released, contribute to host defence and participate in inflammatory disorders. We discuss the impact of exercise-induced NETs, considering a potentially beneficial role in the prevention of lifestyle-related diseases, as well as putative detrimental effects that may arise in elite sports. Finally, we propose that exercise-induced cf-DNA responses could be exploited for diagnostic/prognostic purposes to identify individuals who are at increased risk of cardiovascular events or autoimmunity.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.