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The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions. Author Summary We study a crucial virulence factor produced by pathogenic Escherichia coli strains, the Cytotoxic Necrotizing Factor 1 (CNF1). More than 80% of urinary tract infections (UTIs), which are counted among the most common bacterial infections of humans, are caused by Uropathogenic Escherichia coli (UPEC) strains. We and others elucidated the molecular mechanism of the E. coli toxin CNF1. It constitutively activates Rho GTPases by a direct covalent modification. The toxin enters mammalian cells by receptor-mediated endocytosis. Here, we identified the protein receptor for CNF1 by co-precipitation of cell surface molecules with the tagged toxin and subsequent Maldi-TOF analysis. We identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as receptor for CNF1 and located its interaction site to the C-terminal part of the toxin. We performed direct protein-protein interaction analysis and competition studies. Moreover, cells deficient in Lu/BCAM could not bind labeled CNF1. The identification of a toxin's cellular receptor and receptor binding region is an important task for understanding the pathogenic function of the toxin and, moreover, to make the toxin accessible for its use as a cellbiological and pharmacological tool, for example for the generation of immunotoxins.
FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.
Background: Because most human stroke victims are elderly, studies of experimental stroke in the aged rather than the young rat model may be optimal for identifying clinically relevant cellular responses, as well for pinpointing beneficial interventions.
Methodology/Principal Findings: We employed the Affymetrix platform to analyze the whole-gene transcriptome following temporary ligation of the middle cerebral artery in aged and young rats. The correspondence, heat map, and dendrogram analyses independently suggest a differential, age-group-specific behaviour of major gene clusters after stroke. Overall, the pattern of gene expression strongly suggests that the response of the aged rat brain is qualitatively rather than quantitatively different from the young, i.e. the total number of regulated genes is comparable in the two age groups, but the aged rats had great difficulty in mounting a timely response to stroke. Our study indicates that four genes related to neuropathic syndrome, stress, anxiety disorders and depression (Acvr1c, Cort, Htr2b and Pnoc) may have impaired response to stroke in aged rats. New therapeutic options in aged rats may also include Calcrl, Cyp11b1, Prcp, Cebpa, Cfd, Gpnmb, Fcgr2b, Fcgr3a, Tnfrsf26, Adam 17 and Mmp14. An unexpected target is the enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 in aged rats, a key enzyme in the cholesterol synthesis pathway. Post-stroke axonal growth was compromised in both age groups.
Conclusion/Significance: We suggest that a multi-stage, multimodal treatment in aged animals may be more likely to produce positive results. Such a therapeutic approach should be focused on tissue restoration but should also address other aspects of patient post-stroke therapy such as neuropathic syndrome, stress, anxiety disorders, depression, neurotransmission and blood pressure.
Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.
Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells
(2011)
In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e. g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.
Growth factor induced signaling cascades are key regulatory elements in tissue development, maintenance and regeneration. Perturbations of these cascades have severe consequences, leading to developmental disorders and neoplastic diseases. As a major function in signal transduction, activating mutations in RAF family kinases are the cause of human tumorigenesis, where B-RAF V600E has been identified as the prevalent mutant. In order to address the oncogenic function of B-RAF V600E, we have generated transgenic mice expressing the activated oncogene specifically in lung alveolar epithelial type II cells. Constitutive expression of B-RAF V600E caused abnormalities in alveolar epithelium formation that led to airspace enlargements. These lung lesions showed signs of tissue remodeling and were often associated with chronic inflammation and low incidence of lung tumors. The inflammatory cell infiltration did not precede the formation of the lung lesions but was rather accompanied with late tumor development. These data support a model where the continuous regenerative process initiated by oncogenic B-RAF-driven alveolar disruption provides a tumor-promoting environment associated with chronic inflammation.
Background
Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties.
Results
The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0\(\pm\)0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions.
Conclusion
Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the blood-brain-barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemo-lymphatic as well as neurological stages of sleeping sickness.
Background: Hereditary spastic paraplegias (HSPs) are characterised by lower limb spasticity due to degeneration of the corticospinal tract. We set out for an electrophysiological characterisation of motor and sensory tracts in patients with HSP.
Methods: We clinically and electrophysiologically examined a cohort of 128 patients with genetically confirmed or clinically probable HSP. Motor evoked potentials (MEPs) to arms and legs, somato-sensory evoked potentials of median and tibial nerves, and nerve conduction studies of tibial, ulnar, sural, and radial nerves were assessed.
Results: Whereas all patients showed clinical signs of spastic paraparesis, MEPs were normal in 27% of patients and revealed a broad spectrum with axonal or demyelinating features in the others. This heterogeneity can at least in part be explained by different underlying genotypes, hinting for distinct pathomechanisms in HSP subtypes. In the largest subgroup, SPG4, an axonal type of damage was evident. Comprehensive electrophysiological testing disclosed a more widespread affection of long fibre tracts involving peripheral nerves and the sensory system in 40%, respectively. Electrophysiological abnormalities correlated with the severity of clinical symptoms.
Conclusions: Whereas HSP is primarily considered as an upper motoneuron disorder, our data suggest a more widespread affection of motor and sensory tracts in the central and peripheral nervous system as a common finding in HSP. The distribution patterns of electrophysiological abnormalities were associated with distinct HSP genotypes and could reflect different underlying pathomechanisms. Electrophysiological measures are independent of symptomatic treatment and may therefore serve as a reliable biomarker in upcoming HSP trials.
Ziel der vorliegenden Arbeit war es, die Auswirkungen von Aminosäuren (AS) und Proteinen auf die physikalischen und chemischen Eigenschaften von Calciumphosphat-Zement in Hinblick auf ihre klinische Verwendbarkeit zu untersuchen. Im Rahmen der Arbeit wurden ein zweikomponentiger Zement bestehend aus Tetracalciumphosphat (TTCP) und Calciumhydrogenphosphat (Monetit, DCPA), sowie zwei einkomponentige Zemente aus mechanisch aktiviertem alpha-Tricalciumphosphat (alpha-TCP) bzw. beta-Tricalciumphosphat (beta-TCP) verwendet. Die Zemente wurden mit verschiedenen Aminosäuren und Proteinen durch Zusatz zur flüssigen Zementphase modifiziert. Untersuchte Qualitätsparameter waren die Abbindezeit nach Gillmore, die mechanische Stabilität sowie die Phasenzusammensetzung nach Aushärtung und Änderungen der Oberflächenladung und des pH-Werts der Zementpartikel nach Modifikation. Die Abbindezeit wurde mittels der Gillmor-Nadel-Methode untersucht. Hierbei zeigten sich teilweise deutlich verlängerte Abbindephasen wie z.B mit Arginin (ST = 18 min) auf das Vierfache des Zementnormwertes. Eine Abhängigkeit der Abbindezeit von der Konzentration konnte nur für TTCP-DCPA-Zement mit Proteinen nachgewiesen werden. Untersuchungen der Partikelladung der Zementbestandteile über das Zeta-Potential ergaben für Arginin in Verbindung mit allen Zementen die höchsten Potentiale von bis zu -35,1 ± 1,1 mV, was über die verstärkten Abstoßungskräfte der CPC-Partikel die Verlängerung der Abbindezeiten erklärt. Die Bestimmung der pH-Werte der suspendierten Zementpartikel in Aminosäurelösungen ergab für alle Proben basischere pH-Werte als die jeweiligen isoelektrischen Punkte der Aminosäuren. Dies bedingt, dass alle Verbindungen in deprotonierter Form vorliegen. Die Ermittlung der Druck- und Zugfestigkeit der Zemente erfolgte im standardisierten Verfahren nach Verdichtung der Zementpaste. Die Druckfestigkeit (CS) der unmodifizierten Zemente lag bei 64,1 ± 3,0 MPa (alpha-TCP), 51,8 ± 4,1 MPa (beta-TCP) und 83 ± 10 MPa (TTCP-DCPA). Es zeigte sich, dass Albumin und Fibrinogen zu einer Verringerung der Zementstabilität führen. Die Zugabe von Aminosäuren zu alpha-und beta-TCP Zementen erbrachte gleichbleibende bzw. verringerte Festigkeiten. Bei TTCP-DCPA-Zement verursachte die Modifikation mit einigen Aminosäuren höhere Festigkeiten bis 133,4 ± 4,2 MPa (CS) durch 20% Glycin Zusatz, erklärbar durch eine höhere Dichte und damit geringere Porosität der Zementmatrix. Rasterlektronenmikroskopische Untersuchungen der TTCP-DCPA-Zementtextur zeigten zusätzlich eine Veränderung des mikrostrukuturellen Aufbaus der Zementmatrix. Durch infrarotspektrometrische Untersuchung der abgebundenen Zemente konnte gezeigte werden, dass alle Aminosäuren als chemisch nicht gebundene Additive in der Zementmatrix vorliegen und sich mit Wasser auswaschen lassen. Eine Umsetzung der Zementreaktanden zu nanokristallinem Hydroxylapatit konnte durch die röntgendiffraktometrische Untersuchung für alle Formulierungen gezeigt werden. Die Verbesserungen der Zementeigenschaften einiger Proben sind im Bezug auf den klinischen Einsatz interessant, da sich so die Indikationsbreite der verstärkten CPC erweitern ließe, beispielsweise auf gering kraft-belastete Defekte im Bereich der oberen Extremitäten oder der Halswirbelsäule. Weiterführende Untersuchungen müssten sich vor allem mit dem Mechanismus der beobachteten Zementverstärkung beschäftigen. Hierfür müssten oberflächensensitive Verfahren zur Charakterisierung der Wechselwirkung von Zementpartikel und Aminosäure, beispielsweise Festkörper -NMR, zum Einsatz kommen.
DOT1A-dependent H3K76 methylation is required for replication regulation in Trypanosoma brucei
(2012)
Cell-cycle progression requires careful regulation to ensure accurate propagation of genetic material to the daughter cells. Although many cell-cycle regulators are evolutionarily conserved in the protozoan parasite Trypanosoma brucei, novel regulatory mechanisms seem to have evolved. Here, we analyse the function of the histone methyltransferase DOT1A during cell-cycle progression. Over-expression of DOT1A generates a population of cells with aneuploid nuclei as well as enucleated cells. Detailed analysis shows that DOT1A over-expression causes continuous replication of the nuclear DNA. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. As histone H3K76 methylation has never been associated with replication control in eukaryotes before, we have discovered a novel function of DOT1 enzymes, which might not be unique to trypanosomes.