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The adaptive immune system of jawed vertebrates is based on recognition and elimination of cells that are either invaded by intracellular pathogens or malignantly transformed. One essential component of these processes is the cell surface presentation of antigenic peptides via major histocompatibility complex (MHC) class I molecules to cytotoxic T-cells (CTLs). Cells degrade defective ribosomal products and misfolded or unwanted proteins by the ubiquitin-proteasome pathway. The resulting degradation products are recognized and translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER) lumen, where they are loaded onto MHC I molecules. Assembled peptide-MHC complexes are then shuttled by the secretory pathway to the cell surface for antigen presentation to CTLs, leading in the case of viral infection or malignant transformation to lysis and apoptosis of the target cell. Due to the fact that the TAP complex represents a key control point within the antigen presentation pathway, several viruses have evolved sophisticated strategies to evade immune surveillance by interfering with TAP function.
Detailed studies of the TAP mechanism or its viral inhibition have been severely impeded by difficulties in expressing sufficient amounts of functional heterodimeric TAP complex. Thus, the overexpression of TAP in the methylotrophic yeast Pichia pastoris was established for functional analysis of this important ABC complex. Biomass production was scaled up by fermentation using classical batch and feed methods. Extensive screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex. Notably, only the very mild detergent digitonin preserved TAP function. Hereby, the optimal solubilization and purification strategy yielded in 30 mg TAP transporter per liter culture. Remarkably, the protein amount was 50-fold increased compared to previously described expression/purification in cultured insect cells.
The high yield and quality of TAP produced in P. pastoris allowed an extensive analysis of substrate binding and transport kinetics of the transport complex in the membrane, its solubilized and purified state, as well as the reconstituted state. Thereby, a strong and direct effect of the lipid bilayer on ATP hydrolysis and peptide transport was discovered. These important results were extended further by successful functional reconstitution of the antigen translocation machinery in different lipid environments. For the first time, a stimulation of the transport activity by phosphatidylinositol (PI) and phosphatidylethanolamine (PE) was observed, whereas cholesterol was identified as an inhibitor of TAP activity.
Purification of TAP and subsequent thin-layer chromatography (TLC)/liquid chromatography Fourier transform-mass spectrometry (LC FT-MS) fingerprinting of residual lipids exhibited specifically associated glycerophospholipids; mainly PC, PE, and PI species. Strikingly, these lipids not only represent the primary class of phospholipids of the ER but were also shown to be essential for functional reactivation of delipidated, and thus inactive, TAP. The results demonstrate that transport of antigenic peptides by the ABC transporter TAP strictly requires specific glycerophospholipids.
In addition to the biochemical characterization of heterologous produced TAP, the soluble domain of the viral inhibitor US6 from human cytomegalovirus was expressed in E. coli. Optimization of the purification and refolding strategy yielded in functional protein, with a 35-fold increased protein amount compared to previous purification procedures. Protein activity was analyzed by specific inhibition of ATP binding to TAP. Furthermore, high protein yields allowed detailed investigation of TAP-dependent spatial and mechanistic separation of MHC I restricted cross-presentation in professional antigen presenting cells (pAPC).
Stem cells are often referred to as potential candidates for the treatment of different pathologies. Their ability to differentiate into various tissue specific cell types offers the possibility to engineer cell systems or organs for replacement. One of the main questions in stem cell biology is how stemness properties are regulated and to what extend this regulation is intrinsic or conveyed by the direct microenvironment (‘niche’). In order to elucidate such regulatory processes, it is informative to analyze processes or molecules that are shared between different stem cell populations.
One such molecule that is expressed on a wide range of different embryonic and adult as well as tumor stem cells is the ABC transporter Abcg2. ABC transporters in general are transmembrane proteins that actively extrude endo- and exotoxins as well as xenobiotics, thereby protecting cells and organs. Additionally, ABC transporters are responsible for drug resistance in many cancers. A well-described characteristic of stem cells expressing Abcg2 is the formation of the ‘side population’ (SP) phenotype: An active Abcg2 transporter mediates the efflux of a particular fluorescent dye that is taken up by all cells, thus leading to a less brightly stained population. This phenomenon is widely used to characterize and isolate the most primitive stem cell subpopulation from embryonic and adult tissues, including tumors. Besides its role as toxin transporter little is known about the function of Abcg2 in stem cells. This is mainly due to the fact that its physiological substrate in stem cells remains unknown. The identification of such substrates is therefore of high interest because it would directly link the activity of ABC transporters to regulatory mechanisms in stem cell biology.
In the present study we wanted to test the hypothesis that the sphingolipid ceramide is a physiological substrate of the ABC transporter Abcg2. Sphingolipids are potent second messengers and are known to have regulatory functions in stem cells. In particular, the sphingolipid ceramide is described as a mediator of controlled cell death and inducer of differentiation. It is suggested that stem cells need to keep their intracellular ceramide content at low levels in order to prevent apoptosis or differentiation. We propose that Abcg2 and ceramide interact and that this interaction leads to changes in the absolute or relative amounts of ceramide. This in turn influences basic stem cell functions such as self renewal and differentiation.
We show that Abcg2 prevents cells from accumulating fluorescence labeled ceramide. Furthermore, exogenously applied ceramides inhibit the transport activity of Abcg2, measured by a decrease of the side population phenotype. This inhibitory effect is consistent with a competitive inhibition mechanism. Additionally, we show that active Abcg2 can increase the ceramide concentration in cell culture supernatant. Finally we demonstrate that Abcg2 protects from ceramide induced cytotoxicity in human cell lines. In summary, these in vitro results strongly suggest that Abcg2 has the ability to regulate ceramide levels.
Murine hematopoietic stem cells (HSCs) are the best characterized adult stem cell system so far. By using 7-colour fluorescence-activated cell sorting (FACS) we established the purification of the most primitive HSCs, reflected by their high engraftment capability when transplanted to lethally irradiated mice. By using this sorted cell populations it was in addition possible to establish a system to reproducibly manipulate HSCs ex vivo. This experimental system will serve in further elucidating the physiological consequences of Abcg2 mediated changes in ceramide levels on stem cells in vivo.
Taken together, this study shows that Abcg2 has the ability to regulate ceramide levels in cells. This in turn can lead to cellular protection from ceramide induced apoptosis. Additionally, the experimental techniques to further analyze the role of Abcg2 and ceramide in the most primitive hematopoietic stem cells were successfully established, enabling more detailed analysis in the future.
Conclusion: Proteins containing a Jumonji C (JmjC) domain appear in almost all living organisms and catalyze a variety of oxidation reactions. Therefore, they are important regulators in many biological processes such as proliferation and differentiation. They act either as protein hydroxylases, histone demethylases or by regulate mRNA splicing. Given the fact that some of the JmjC domain-containing proteins are shown to be upregulated in response to hypoxia as well as the dependency of JmjC domain catalytic activity on oxygen led to the assumption of an involvement in angiogenesis. For Jmjd6, a member of the JmjC domain-containing protein family, a regulatory involvement in mRNA splicing has been shown. The Jmjd6-/- mouse dies perinatally due to several severe organ malformations, especially in the heart. Despite the pale appearance, the growth retardation and the cardiac defects, it is unclear whether these mice exhibit defects of cells comprising the vasculature. Therefore, the involvement of Jmjd6 in angiogenesis was examined in vitro using angiogenesis assays as well as in vivo using the Jmjd6+/- mouse. An siRNA-mediated knockdown of Jmjd6 in ECs significantly impaired the formation of capillary-like networks in the tube formation assay as well as sprouting in the spheroid assay. Moreover, after siRNA-mediated knockdown of Jmjd6 in ECs cell migration was significantly reduced. These findings were confirmed in the matrigel plug assay in vivo. Implanted matrigel plugs of Jmjd6+/- mice exhibited significantly less perfused vessels compared to wildtype littermates. Furthermore, cultured lung ECs from Jmjd6+/- mice exhibited impaired network forming activity ex vivo compared to cells isolated from wildtype littermates. To elucidate the mechanisms underlying the requirement of Jmjd6 in angiogenesis, an Affymetrix exon-array was performed, which allows detection of changes in gene expression as well as splicing. The siRNA-mediated knockdown of Jmjd6 altered the expression of genes known to play a role in vascular biology. The bioinformatic assessment of alternative splice variants revealed that Jmjd6 silencing affects the splicing of the VEGF receptor 1 (Flt1). Differential splicing of Flt1 was shown to generate a short and soluble form of Flt1 (sFlt1), which sequestrates VEGF and PlGF, and thereby inhibits angiogenesis. In particular, a significant increase in sFlt1 expression was observed. Jmjd6 was recently reported to hydroxylate the splicing factor U2AF65. Therefore, we investigated whether U2AF65 might mediate Flt1 splicing and binds to Flt1 mRNA. Indeed, U2AF65 co-immunoprecipitated with Jmjd6 in ECs, while an interaction of U2AF65 with sFlt1 was demonstrated. Moreover, inhibition of Jmjd6 catalytic function by reduced oxygen concentration altered splicing of Flt1 resulted in an increase of the sFlt1 splice variant. Finally, saturating concentrations of VEGF or PlGF or neutralizing antibodies against sFlt1 significantly reduced the inhibition of sprouting caused by Jmjd6 knockdown in vitro.
Collectively, our results indicate that Jmjd6 has an essential role in the oxygen-dependent regulation of angiogenesis by controlling the splicing of Flt1 mRNA, thereby adjusting the generation of the anti-angiogenic short splice variant sFlt1. Several publications demonstrated a major importance for sFlt1 as a biomarker for many severe human diseases such as preeclampsia, sepsis, cancer, myocardial infarction as well as chronic heart failure. Therefore, the identification of the molecular mechanism behind the generation of sFlt1 might enable the development of new or more precise clinical markers for the diagnosis of the corresponding diseases. Furthermore, the discovery of the enzymes involved in the generation of sFlt1 provides further possibilities to modulate sFlt1 levels and thereby may potentially gives rise to the development of new therapies.
The translocation of nuclear-encoded precursor proteins into chloroplasts is a highly ordered process involving the action of several components to regulate this molecular ensemble. Not only GTP hydrolysis and GDP release but also the phosphorylation of TOC GTPases is a widely discussed mechanism to regulate protein import. The receptor component (Toc34) and its isoform of A. thaliana (atToc33) were found to be regulated by phosphorylation. Although the phosphorylation of Toc33 is already known for several years, several questions regarding the molecular components involved in the regulation of the phosphorylation process, precisely what is the protein kinase and where this kinase is initially localized, so far remained unclear.
This thesis aimed at the defining of the phosphorylation status of TOC GTPases in monomeric and/or dimeric states, the identification of the nature of Toc33-PK (protein kinase), and in the same context it aimed at gaining first insights into the physiological significance of Toc33 phosphorylation. To this end, (I) An in vitro and in vivo system for investigating of TOC GTPases Phosphorylation (in monomeric or dimeric state) was developed. Since no information is available about the phosphorylation status of the Toc159 isoforms, the second receptor of the TOC complex, it was interesting to investigate whether these isoforms undergo phosphorylation or not. The results indicated that atToc159 isoforms are able to be phosphorylated by the kinase activity in purified outer envelope membranes (OEMs) of pea, but not atToc132. Moreover, an artificial dimer of psToc34 based on the interaction of a C-terminally fused leucine zipper was not phosphorylated. This result reflected the inability of the OEM kinase to phosphorylate the dimers of TOC GTPases. Also, In vivo labeling of atToc33 was developed and occurred in a dose-dependent manner. Therefore, this results evidenced that in vitro phosphorylation of atToc33 (both endogenous wild type and recombinant expressed proteins) is not artificial labeling but represents a physiological relevance. CD (circular dichroism) measurements revealed that recombinant GTPase domain of atToc33 is preferentially phosphorylated in its folded state. Therefore, it could be suggested that folding of atToc33rec is a prerequisite for its phosphorylation and the phosphorylation event occurs as a posttranslational modification most likely after insertion of Toc33 (Toc34) into the OE of chloroplasts.
Secondly, (II) Isolation and identification of Toc33-PK from OEMs of chloroplasts was performed. Four independent strategies were developed to identify the Toc33-protein kinase: UV-induced and chemically-based crosslinking, different applied chromatographic techniques, identification of PK-Toc33 interaction by means of HDN-PAGE (histidine- and deoxycholate-based native PAGE), and finally mass spectrometric approaches were performed on fractions including the potential kinase activity. UV-induced crosslinking procedure was developed and resulted in covalent bonding of nine proteins to [a-32P] ATP, while chemically-based one was not significant. The applied chromatographic and HDN-PAGE approaches, including mass spectrometry, have revealed the identification of 13 protein kinases. Of these identified kinases, phototropin2 (Phot2, AT5G58140), leucine-rich repeat PK (LRR-PK, AT4G28650.1), and receptor-like transmembrane PK (RLK, AT5G56040.2) were selected as the most promising candidates (ca. kinase type and one transmembrane helix for membrane localization).
(III) The physiological significance of Toc33 phosphoryation was shown to link this process with the environmental changes (especially, the light conditions). Identification of chloroplast OE-located PKs performed by nLC-MALDI-MS/MS resulted in the detection of Phot2. Furthermore, the subcellular localization of Phot2 in OEM of chloroplasts was confirmed by immunoblotting experiments using a-Phot2 antibody. The kinase activity of Phot2 towards TOC GTPases was characterized and revealed that fused GST-KD (kinase domain) protein able to specifically phosphorylate atToc33rec, but not atToc159rec. Also, endogenous atPhot2 was upregulated and heavily detected in the ppi1-S181A plant line (where serine to alanine exchange was performed to abolish the phosphorylation of atToc33). Hence, we suggested that certain signal cascades may directly or indirectly link Toc33 receptor phosphorylation, protein levels of Phot2 (as promising PK candidate), and irradiation conditions (as an inducing signal of the subsequent phosphorylation events). Light-dependent phosphorylation of Toc33 was shown either after de-etiolation conditions or after high light intensities of blue light was performed. Therefore, phosphorylation of Toc33 might be identified as an external regulatory signal to regulate preproteins import into chloroplasts in response to environmental conditions (e.g. light changes) or as a signal of chloroplast biogenesis.
Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.
The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer.
Clathrin-mediated endocytosis (CME) involves spatially and temporally restricted molecular dynamics.
Although protein kinases and the actin cytoskeleton contribute to the process, whether and how
functions of kinases and actin are integrated remains unknown. Here, we demonstrate that neural
Wiskott-Aldrich syndrome protein (N-WASP) and protein kinase CK2 form a complex and localize on
clathrin-coated vesicles (CCVs). N-WASP binds to and is phosphorylated by CK2, thereby reducing the
kinase activity of CK2. By contrast, N-WASP-promoted actin polymerization is decreased upon both
phosphorylation and binding of CK2. Knockdown of N-WASP and CK2, alone or in combination, results
in impaired endocytosis of epidermal growth factor (EGF) and increased cell-surface levels of EGF
receptor (EGFR). In order to rescue the phenotype of N-WASP-CK2 knockdown cells, both N-WASP and
CK2 activities and abilities to assemble in a complex are required. In summary, this study shows that the
N-WASP-CK2 complex integrates in a single circuit different activities contributing to CME of EGFR and
that the interplay between the two proteins optimizes this process.
NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation
Axonal growth is essential for establishing neuronal circuits during brain development and for regenerative processes in the adult brain. Unfortunately, the extracellular signals controlling axonal growth are poorly understood. Here we report that a reduction in extracellular ATP levels by tissue-nonspecific alkaline phosphatase (TNAP) is essential for the development of neuritic processes by cultured hippocampal neurons. Selective blockade of TNAP activity with levamisole or specific TNAP knockdown with short hairpin RNA interference inhibited the growth and branching of principal axons, whereas addition of alkaline phosphatase (ALP) promoted axonal growth. Neither activation nor inhibition of adenosine receptors affected the axonal growth, excluding the contribution of extracellular adenosine as a potential hydrolysis product of extracellular ATP to the TNAP-mediated effects. TNAP was colocalized at axonal growth cones with ionotropic ATP receptors (P2X7 receptor), whose activation inhibited axonal growth. Additional analyses suggested a close functional interrelation of TNAP and P2X7 receptors whereby TNAP prevents P2X7 receptor activation by hydrolyzing ATP in the immediate environment of the receptor. Furthermore inhibition of P2X7 receptor reduced TNAP expression, whereas addition of ALP enhanced P2X7 receptor expression. Our results demonstrate that TNAP, regulating both ligand availability and protein expression of P2X7 receptor, is essential for axonal development.
The canonical Wnt/β-catenin and the Shh pathway as well as the Notch signaling cascade
are key regulators in stem cell biology and are independently associated with the development
of cancer. Despite the knowledge of a balanced signaling for cellular maintenance, the
fundamental biochemical mechanisms of crosstalk are still poorly understood. This study
demonstrates that the outcome of interaction between Wnt and Shh is cell type specific. A
combined inhibitory mechanism of the Shh and Notch2/Jagged2 pathways on dominant
active β-catenin signaling in the adult tongue epithelium keeps Wnt/β-catenin signaling
restricted to physiological tolerable levels. In the opposite crosstalk the activation of
Wnt/β-catenin signaling in medulloblastoma (MB) of the Shh subtype, in turn inhibits the Hh
pathway.
The inhibitory mechanism of Shh and Notch2/Jagged2 on Wnt/β-catenin signaling is
independent of the degradation complex of β-catenin and takes place inside the nucleus.
Furthermore, the negative feedback on Wnt/β-catenin signaling by the Shh pathway relies
on transcriptional activity of Gli1/2A. Inhibition of Gli1/2A with the specific inhibitor GANT61
abrogated the negative impact of Shh on β-catenin signaling in vitro. Although the negative
feedback loop of Shh is still functional in human SCC25 cells, the inhibitory effect of
Notch2/Jagged2 is lost and contributes to the cancerogenic phenotype of these cells. In the
inverse situation, the activation of β−catenin signaling has a negative feedback on
constantly active Shh signaling and significantly inhibits the Hh pathway. This was shown in
Ptch+/- and Math1-Cre:SmoM2Fl/+ MB tumor spheres in vitro, in which inhibition of sphere
formation and growth was observed and Hh target gene transcription was down-regulated.
This demonstrates for the first time that the activation of canonical Wnt/β-catenin signaling
in primary MB cells with a Hh pathway over-activation has a negative effect on the growth of
these cells in vitro.
In summary the results show that crosstalk of Wnt/β-catenin and Shh signaling has context
specific outcome on pathway activity. Elucidation of the molecular interactions will improve
our understanding of Wnt and Hh associated tumors and contribute to the development of
new therapeutic strategies.
5-lipoxygenase (5-LO) catalyzes the first two steps in leukotriene (LT) biosynthesis. In a two step reaction the enzyme oxygenates arachidonic acid (AA) to form the highly unstable epoxide leukotriene A4 (LTA4) in dehydrating a hydroperoxide intermediate (20). LTA4 can then be further metabolized by two terminal synthases yielding either the potent chemoattractant leukotriene B4 (LTB4) or the cysteinyl leukotrienes (CysLTs). 5-LO enzyme expression is primarily found in mature leukocytes (22) where it can either reside in the cytoplasm or in the nucleus associated with euchromatin (29). Its enzymatic activity is embedded in a complicated network in intact cells regulating LT synthesis by various factors dependent on the cell type and nature of stimulus. Factors such as the amount of free AA released by phospholipase A2 enzymes, levels of enzymes involved, catalytic activity per enzyme molecule and availability of different small molecules influence 5-LO activity (36).
The 5-LO derived LTs are lipid mediators which were shown to primarily mediate inflammatory and allergic reactions and their role in the pathogenesis of asthma is well defined. CysLTs are among the most potent bronchoconstrictors yet studied in man and play an important role in airway remodeling. LTB4 has no bronchoconstrictory effects in healthy and asthmatic humans but displays potent chemoattractant properties on neutrophils and increases leukocyte adhesion to the vessel wall endothelium (22). Therefore, LTB4 enhances the capacity of macrophages and neutrophils to ingest and kill microbes. In concert with LTB4, histamine and prostaglandin E2 (PGE2) CysLTs are thought to maintain the tone of the human airways (82).
Besides their well studied role in asthma, 5-LO derived LTs have also been implicated to play a role in cardiovascular diseases and cancer. In contrast to healthy tissues, LT pathway enzymes and receptors were found to be abundantly expressed in cancer tissues, atherosclerotic lesions in the aorta, heart and carotid artery (86). Pharmacological inhibition of 5-LO potently suppressed tumour cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway (92, 93). In several studies LTs were found to exhibit cardiovascular actions by promotion of plasma leakage in postcapillary venules, coronary artery vasoconstriction and impaired ventricular contraction leading to reduced coronary blood flow and cardiac output (24). Unfortunately, the precise molecular mechanisms through which LTs influence carcinogenesis and cardiovascular diseases are still incompletely understood.
In contrast, an increasing number of studies questions the correlation between 5-LO and cancer (95-97) since extreme LT concentrations were applied to induce proliferative effects in the majority of the publications. A few studies exist which show susceptibility towards 5-LO products in physiological concentrations or achieve anti-proliferation by applying low concentrations of 5-LO inhibitors (98) ...
Regulation of dissimilatory sulfur oxidation in the purple sulfur bacterium Allochromatium vinosum
(2011)
In the purple sulfur bacterium Allochromatium vinosum, thiosulfate oxidation is strictly dependent on the presence of three periplasmic Sox proteins encoded by the soxBXAK and soxYZ genes. It is also well documented that proteins encoded in the dissimilatory sulfite reductase (dsr) operon, dsrABEFHCMKLJOPNRS, are essential for the oxidation of sulfur that is stored intracellularly as an obligatory intermediate during the oxidation of thiosulfate and sulfide. Until recently, detailed knowledge about the regulation of the sox genes was not available. We started to fill this gap and show that these genes are expressed on a low constitutive level in A. vinosum in the absence of reduced sulfur compounds. Thiosulfate and possibly sulfide lead to an induction of sox gene transcription. Additional translational regulation was not apparent. Regulation of soxXAK is probably performed by a two-component system consisting of a multi-sensor histidine kinase and a regulator with proposed di-guanylate cyclase activity. Previous work already provided some information about regulation of the dsr genes encoding the second important sulfur-oxidizing enzyme system in the purple sulfur bacterium. The expression of most dsr genes was found to be at a low basal level in the absence of reduced sulfur compounds and enhanced in the presence of sulfide. In the present work, we focused on the role of DsrS, a protein encoded by the last gene of the dsr locus in A. vinosum. Transcriptional and translational gene fusion experiments suggest a participation of DsrS in the post-transcriptional control of the dsr operon. Characterization of an A. vinosum ΔdsrS mutant showed that the monomeric cytoplasmic 41.1-kDa protein DsrS is important though not essential for the oxidation of sulfur stored in the intracellular sulfur globules.
Forest fragmentation and selective logging are two main drivers of global environmental change and modify biodiversity and environmental conditions in many tropical forests. The consequences of these changes for the functioning of tropical forest ecosystems have rarely been explored in a comprehensive approach. In a Kenyan rainforest, we studied six animal-mediated ecosystem processes and recorded species richness and community composition of all animal taxa involved in these processes. We used linear models and a formal meta-analysis to test whether forest fragmentation and selective logging affected ecosystem processes and biodiversity and used structural equation models to disentangle direct from biodiversity-related indirect effects of human disturbance on multiple ecosystem processes. Fragmentation increased decomposition and reduced antbird predation, while selective logging consistently increased pollination, seed dispersal and army-ant raiding. Fragmentation modified species richness or community composition of five taxa, whereas selective logging did not affect any component of biodiversity. Changes in the abundance of functionally important species were related to lower predation by antbirds and higher decomposition rates in small forest fragments. The positive effects of selective logging on bee pollination, bird seed dispersal and army-ant raiding were direct, i.e. not related to changes in biodiversity, and were probably due to behavioural changes of these highly mobile animal taxa. We conclude that animal-mediated ecosystem processes respond in distinct ways to different types of human disturbance in Kakamega Forest. Our findings suggest that forest fragmentation affects ecosystem processes indirectly by changes in biodiversity, whereas selective logging influences processes directly by modifying local environmental conditions and resource distributions. The positive to neutral effects of selective logging on ecosystem processes show that the functionality of tropical forests can be maintained in moderately disturbed forest fragments. Conservation concepts for tropical forests should thus include not only remaining pristine forests but also functionally viable forest remnants.
Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.
Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.
Acanthocephalans are attractive candidates as model organisms for studying the ecology and co-evolutionary history of parasitic life cycles in the marine ecosystem. Adding to earlier molecular analyses of this taxon, a total of 36 acanthocephalans belonging to the classes Archiacanthocephala (3 species), Eoacanthocephala (3 species), Palaeacanthocephala (29 species), Polyacanthocephala (1 species) and Rotifera as outgroup (3 species) were analyzed by using Bayesian Inference and Maximum Likelihood analyses of nuclear 18S rDNA sequence. This data set included three re-collected and six newly collected taxa, Bolbosoma vasculosum from Lepturacanthus savala, Filisoma rizalinum from Scatophagus argus, Rhadinorhynchus pristis from Gempylus serpens, R. lintoni from Selar crumenophthalmus, Serrasentis sagittifer from Johnius coitor, and Southwellina hispida from Epinephelus coioides, representing 5 new host and 3 new locality records. The resulting trees suggest a paraphyletic arrangement of the Echinorhynchida and Polymorphida inside the Palaeacanthocephala. This questions the placement of the genera Serrasentis and Gorgorhynchoides within the Echinorhynchida and not the Polymorphida, necessitating further insights into the systematic position of these taxa based on morphology.
Background and Objective: The slow delayed rectifier current (IKs) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced IKs and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for IKs modulation is poorly understood and was subject of this study.
Methods: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy.
Results: All KCNE1 constructs physically interacted with Kv7.1. IKs resulting from co-expression of Kv7.1 with non-atrial fibrillation ‘38S’ was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions (‘38G-3xA’) were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct (‘Δ1-38’). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct.
Conclusions: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of IKs. Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.
Membrane proteins (MPs) constitute about 30% of the genome and are essential in many cellular processes. In particular structural characterisation of MPs is challenged by their hydrophobic nature resulting in expression difficulties and structural instability upon extraction from the membrane. Despite these challenges, progress in sample preparation and the techniques to solve MP structures has led to 281 unique MP structures as of January 2011. Through the combination of a cell-free expression system and selective labelling strategies, this thesis aimed to advance the structure determination of α-helical MPs by NMR spectroscopy and resulted in the structure determination of a seven-ransmembrane-helix protein. Results were obtained for the 5-lipoxygenase-activating protein (FLAP) and proteorhodopsin (PR). The detergent-based cell-free expression mode proved most efficient for production of both targets, but optimisation of FLAP and PR followed different routes. The presence of a retinal cofactor in PR greatly facilitated the search for an appropriate hydrophobic environment. For structural studies, NMR spectra of FLAP indicated favourable properties of the lysolipid LPPG. In contrast, PR was stable and homogenous in the short-chain lipid diC7PC. As NMR spectra of α-helical MPs are generally characterised by broad lines and signal overlap, selective labelling strategies were essential in the assignment process of both targets. For the backbone assignment of FLAP the transmembrane segment-enhanced (TMS) labelling was developed, employing the six amino acids AFGILV. These residues cluster predominantly in transmembrane helices and form long stretches allowing a large extent of backbone assignment. Besides that, the combinatorial labelling enables identification of unique pairs in the sequence based on a mixture of 15N and 1-13C-labelled amino acids. To find the optimal labelling pattern for a given primary structure, the UPLABEL algorithm has been made available and successfully applied in the backbone assignment of PR. Both selective labelling approaches greatly benefitted from the use of a cell-free expression system to reduce isotope scrambling. Additionally, the de novo structure of PR was determined with an average backbone rmsd of 1.2 Å based on TALOS-derived backbone torsion angles, intrahelical hydrogen bond restraints and distance restraints from the NOE and paramagnetic relaxation enhancement (PRE). A major bottleneck in the NMR structure determination of MPs concerns the number of long-range distances which are often limited. In PR, side chain assignment was enabled by stereo-array isotope labelling as well as selective labelling which provided 33 long-range NOEs. These NOEs stabilised the symmetry of the seven helix bundle. With a total number of 1031, the majority of long-range distances were derived from PREs. The structure of PR reveals differences to its homologues such as the absence of an anti-parallel β-sheet between helices B and C and allows conclusions towards the mechanism of colour tuning.
The role of small leucine-rich proteoglycans, biglycan and decorin, in podocytopathy and albuminuria
(2011)
Biglycan is a member of the small leucine-rich proteoglycan (SLRP) family and is involved in the assembly of extracellular matrix components. In macrophages soluble biglycan acts as an endogenous ligand of the innate immunity receptors TLR2 and TLR4. Data addressing the role of biglycan in renal pathology are surprisingly limited. In a normal kidney, biglycan is expressed mainly in the tubulointerstitium; however, in the course of various renal diseases its expression may be altered. The biological role and mechanisms of biglycan action in the pathology of renal diseases, especially those affecting glomeruli, remain poorly understood.
Albuminuria is the first detectable clinical abnormality in diabetic nephropathy. In this study we detected increased biglycan mRNA expression in glomeruli of renal biopsies of patients with incipient diabetic nephropathy, with predominant localization in podocytes. This novel finding raised the question about the role and mechanisms of biglycan action in diabetic podocyte injury and whether the mechanisms of biglycan signaling causing podocyte injury and albuminuria could be extrapolated to other glomerular diseases.
To investigate the role of biglycan in the cause of diabetic podocyte injury and albuminuria we used the murine model of STZ-induced diabetic nephropathy and wild type (Bgn+/0) and biglycan deficient (Bgn-/0) mice. We observed that biglycan was expressed on mRNA and protein levels in podocytes of diabetic Bgn+/0 mice and that diabetic Bgn+/0 mice also had significantly higher albuminuria compared to non-diabetic mice 6 and 12 weeks after disease induction. Biglycan deficiency was shown to be an important factor in albuminuria development. Namely, we observed that diabetic Bgn-/0 mice had significantly lower levels of urinary albumin compared to diabetic Bgn+/0 mice. We showed that less severe podocyte loss in the urine of diabetic Bgn-/0 mice was associated with significantly higher nephrin and podocin glomerular expression compared to diabetic Bgn+/0 mice. Our data suggested that biglycan deficiency was protective against podocyte loss into urine and might be beneficial against development of albuminuria in diabetes.
Biglycan contributed to podocyte actin rearrangement due to increased phosphorylation of Rac1 in vitro. Furthermore, biglycan induced caspase-3 activity and production of reactive oxygen species (ROS), thus enhancing apoptosis in cultured podocytes. Biglycan-induced ROS generation was TLR2/TLR4-dependent. Overexpression of soluble biglycan in wild type mice induced albuminuria under normal conditions and significantly increased albuminuria under pathological conditions (murine model of LPS-induced albuminuria). Inhibition of Rac1 activity in vivo decreased the albuminuria induced by biglycan overexpression. In patients with glomerular diseases, biglycan was detected in urine and was associated with nephrin appearance in the urine of these patients and with increased albuminuria. Collectively, our results elucidate a novel mechanism for biglycan-induced TLR2- and TLR4-dependent, Rac1- and ROS-mediated podocytopathy leading to podocyturia, albuminuria development and progression of glomerular diseases. Interfering with biglycan actions and blocking its signaling via TLR2 and TLR4 might be a potential therapeutic strategy against these diseases. To achieve this goal, the specific mechanisms for binding of biglycan to TLR2 and TLR4 must be elucidated and effective ways of preventing this binding must be developed. Nevertheless, biglycan remains the “danger signal” that activates innate immune receptors in non-immune cells and triggers the deleterious mechanisms leading to aggravation of renal injury.
Ubiquitin is a highly conserved protein involved in several cellular processes like protein degradation, endocytosis, signal transduction and DNA repair. The discovery of ubiquitin-like proteins (UBL) and ubiquitin-like domains (ULD) increases the number of regulation pathways where the property of the ubiquitin-fold is profitable.
Autophagy is the catabolic pathway used in cells to deliver cytosolic components and dysfunctional organelles to the lysosome for degradation. MAP1LC3 proteins are ubiquitin-like proteins involved in one hand for the expansion of the autophagosome, which sequesters cytosolic substrates. In the other hand, these proteins (LC3- and GABARAP- subfamilies) bind to autophagic receptors linked to polyubiquitinated proteins aggregates. For this project, the 3D structure of the GABARAPL-1/NBR1-LIR complex was determined and confirmed that GABARAPL-1 belongs to the MAP1LC3 proteins family, structurally characterized by an ubiquitin-fold, consisting of a central beta-sheet formed by four beta-strands and two alpha-helices on one side of the beta-sheet, preceded N terminally by two alpha-helices, resulting in the formation of two hydrophobic pockets, hp1 and hp2. The autophagic receptor NBR1 interacts with GABARAPL-1 through the hp1 and hp2 with its LIR motif taking an extended beta conformation upon binding, forming an intermolecular beta-sheet with the second beta-strand of GABARAPL 1. This LC3- interacting region (LIR) consists of an Theta XX Gamma sequence preceded by acidic amino acids, with Theta and Gamma represented by any aromatic and hydrophobic residues, respectively. Interaction studies of the LIR domains of p62, Nix and NBR1 with different members of the MAP1LC3 proteins family indicate that the presence of a tryptophan in the LIR motif increases the binding affinity. Substitution to other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation, suggesting that effector proteins can interact with a wide variety of different sequences with similar and moderate binding affinities.
Additionally to be present in proteins dealing with protein folding and degradation, ubiquitin-like domain were found protein involved in the regulation of signal transduction like TBK1, a serine/threonine kinase responsible for induction of immune response. In this second project, based on the NMR chemical shifts of the TBK1 domain contained between amino acids 302 and 383, secondary structure prediction programs (TALOS and CSI) confirmed the presence of an Ubiquitin-like domain in TBK1 by identifying one alpha-helix and four beta-strands sequentially aligned like following beta-beta-alpha-beta-beta. This alignment corresponds perfectly with the secondary structure elements of Ubiquitin and proved that TBK1_ULD belongs to the UBL protein superfamily. The similarity to ubiquitin was even bigger by the presence in addition of a small beta-strand and a short helix, which are observed as the beta 5-strand and a 310-helix in Ubiquitin, respectively. The first attempts on the 3D structure determination confirmed the Ub-fold but due to the lack of assignment in TBK1_ULD, only a structure based on ubiquitin as a model was determined. Interaction studies of TBK1_ULD with the IAD-SRR domain of IRF3 showed that both side of the molecule seems involved and that the TBK1/IRF3 interaction is more complex than a one to one binding process. Unfortunately, the instability of TBK1_ULD associated to the difficulty in the purification of IAD-SRR did not allow to further study this interaction more precisely.
Finally, to overcome the difficulty encountered in NMR experiments because of low expression and/or poor solubility, an expression vector using the intrinsic property of ubiquitin was designed. Fused to proteins or peptides targets, this construct produced proteins and peptides in a larger amount than with traditional expression vectors and also with a less cost than chemical synthesis for pure labeled peptides for NMR structural studies. The presence of a hexa histidine tag was useful for the isolation and the purification of the constructs. The existence of a TEV cleavage site was created to keep the possibility of releasing the ubiquitin moiety from the expressed protein or peptide. Moreover, the ubiquitin-tag could also still be attached to the protein/peptide of interest when biophysical methods like NMR, ITC or CD spectroscopy are applied, providing the same results than for the protein/peptide moiety alone.
A Bayesian framework to estimate diversification rates and their variation through time and space
(2011)
Background: Patterns of species diversity are the result of speciation and extinction processes, and molecular phylogenetic data can provide valuable information to derive their variability through time and across clades. Bayesian Markov chain Monte Carlo methods offer a promising framework to incorporate phylogenetic uncertainty when estimating rates of diversification.
Results: We introduce a new approach to estimate diversification rates in a Bayesian framework over a distribution of trees under various constant and variable rate birth-death and pure-birth models, and test it on simulated phylogenies. Furthermore, speciation and extinction rates and their posterior credibility intervals can be estimated while accounting for non-random taxon sampling. The framework is particularly suitable for hypothesis testing using Bayes factors, as we demonstrate analyzing dated phylogenies of Chondrostoma (Cyprinids) and Lupinus (Fabaceae). In addition, we develop a model that extends the rate estimation to a meta-analysis framework in which different data sets are combined in a single analysis to detect general temporal and spatial trends in diversification.
Conclusions: Our approach provides a flexible framework for the estimation of diversification parameters and hypothesis testing while simultaneously accounting for uncertainties in the divergence times and incomplete taxon sampling.
Blood vessel formation is a well orchestrated process where multiple components including different cells types, growth factors as well as extracellular matrix proteins act in synergistic and highly regulated manner to support the growth of new blood vessels. During embryonic development this process is marked as vasculogenesis and entails the differentiation of mesodermal cells into angioblasts and their subsequent fusion into a primitive vascular plexus. Angiogenesis, in contrast, describes the formation of new vessels from the pre-existing vasculature and it occurs in the embryo during remodeling of the primitive plexus into a mature vascular network. Furthermore, in the adult, angiogenic processes play a role in various physiological and pathological conditions. Angiogenesis is governed by a set of factors and molecular mechanisms whose identification has been a major focus of cardiovascular research for the past several decades. Most recently, Epidermal growth factor-like domain 7 (EGFL7) has been described as a novel molecular player in this context. This secreted protein is produced by endothelial cells and has been implicated in vessel development. Studies performed in zebrafish revealed an important role for EGFL7 in lumen formation during vasculogenesis although the underlying molecular mechanism has not been elucidated yet. In contrast, the investigation of EGFL7’s functions during angiogenic sprouting has faced several challenges and the role of EGFL7 in angiogenesis remained elusive. The purpose of this thesis was to identify the functions of EGFL7 during angiogenic mode of vessel formation in a systematic fashion using numerous in vitro as well as in vivo approaches.
Previously it has been suggested that EGFL7 might associate with the extracellular matrix from where it could exert its effects. Indeed, we could show that EGFL7 accumulates on the outer surface of endothelial cells in vivo by demonstrating its co-localization with collagen IV, a major constituent of the basal lamina. Furthermore, after its secretion to the extracellular matrix (ECM), EGFL7 seemed to interact with some components of the extracellular matrix including fibronectin and vitronectin, but not collagens and laminin.
A major group of receptors that mediate the interaction between the cells and the ECM are integrin receptors. Our co-immunoprecipitation studies revealed that EGFL7 associated with integrin αvβ3 which is highly expressed in endothelial cells and known to be important for vessel growth. Importantly, this EGFL7-αvβ3 integrin interaction was dependent on Arg-Gly-Asp (RGD) motif present within the second EGF-like domain of EGFL7 protein. Adhesion assays performed with human umbilical vein endothelial cells (HUVEC) revealed that EGFL7 promoted endothelial cell adhesion compared to BSA used as a negative control, however, adhesion seemed to be less efficient as compared to bona fide ECM proteins such as fibronectin and vitronectin. In addition, cultivation of endothelial cells on EGFL7 was characterized by the absence of mature focal adhesions and stress fibers, but was paralleled by increased phosphorylation of kinases typical for integrin activation signaling cascade such as FAK, Src and Akt. This led us to the hypothesis that EGFL7 creates an environment that supports a motile phenotype of endothelial cells by serving as a modulator of existing interactions between the cells and the surrounding matrix. Indeed, EGFL7 increased random migration of HUVEC on fibronectin in an αvβ3 integrin dependent manner as shown using a live cell imaging platform. Most importantly, this was paralleled by a decrease in endothelial cell adhesion to fibronectin which is consistent with previous reports on secreted proteins that support a medium strength of adhesion and such promote cellular migration. To assess the overall effect of EGFL7 on the process of blood formation several in vitro and in vivo approaches were employed. First, the addition of EGFL7 to Matrigel injected subcutaneously into mice significantly increased the invasion of endothelial cells into the plugs. Second, a spheroid-based sprouting assay in three-dimensional collagen matrix clearly demonstrated the ability of EGFL7 to support angiogenic sprouting in an integrin dependent manner. This is consistent with the observed effects of EGFL7 on endothelial cell migration. Third, using in vivo assays such as the chick chorioallantoic membrane (CAM) assay as well as a zebrafish model system we were able to validate the importance of the EGFL7-integrin interaction for the process of angiogenesis in vivo. Taken together, I identified some of the major cellular functions EGFL7 modulates during angiogenesis. In addition, with integrin αvβ3 I unraveled a novel interaction partner of EGFL7 that delivers a mechanistical explanation for EGFL7’s effects on blood vessel formation. Most importantly, data presented in this PhD thesis contribute substantially to the existing literature on EGFL7 unambiguously assigning a role for this protein in the process of angiogenesis.
Since combinatorial chemistry and high throughput screening have become a common technique in the drug discovery phase the number of compounds being considered has increased frequently. These structures are often characterized by high molecular weight, high lipophilicity and low solubility in aqueous and physiological media. Due to the generally poor bioavailability, new in vitro techniques were needed for screening of pharmacokinetic properties. An important parameter for these screening methods is the implementation at an early state of drug discovery phase, to find potential lead structures, before investment costs become significant. The established in vitro methods for the prediction of membrane interaction are not reliable especially for poorly soluble compounds. A new method that is fast and easy to use, requires only small amounts of NCE and which can provide more reliable predictions is needed. In this study, a new screening technique based on surface activity profiling for the prediction of oral drug absorption was evaluated with special emphasis on the predictability of biological membrane interaction of poorly soluble drug compounds. It was demonstrated that drug absorption through a bilayer membrane can be modeled by the orientation of compounds at the air/water interface. Thus amphilicity of a drug is generally related to both oral absorption and blood brain barrier penetration. In turn, amphiphilicity is influenced by the lipophilicity, size and charge distribution of a drug. Surface activity profiling was determined by analysis of surface pressure profiles using the Gibbs adsorption isotherm. The surface activity measurements were carried out using a multichannel tensiometer Delta 8, which was developed by Kibron to be utilized in conjugation high throughput screening in early drug discovery processes. For this study two test sets were analyzed, one for the prediction of gastrointestinal wall interaction and the second for the prediction of the penetration behavior at the blood brain barrier. Both test sets consist of drug compounds with a wide range of absorption properties and consist mainly of compounds with poor water solubility. Since the drugs characteristics varied, they were classified according to water solubility and surface activity and a sample preparation method for each group was established. For the prediction of oral drug absorption, three different methods were established to model the interaction of compound and gastrointestinal wall. For drug compounds with solubility above 1mmol/L the traditional shake-flask method enabled the determination of the amphiphilic properties of drug compounds in pure aqueous media. Compounds with solubility below 1mmol/L tend to not to exhibit any increase in surface activity. Thus surface tension measurements of compounds, which exhibited a limited surface activity due to poor aqueous solubility, were conducted from stock solutions prepared with various organic solvents. Mainly polar organic solvents were used. A mixture of DMSO and DMF resulted in the best combination of properties: the intensive solubility enhancing effect of DMF and the lower intrinsic surface activity of DMSO. The polar solvent ruptured the water clusters, so that highly lipophilic structures had a higher affinity to the solvent and higher concentrations could be obtained. For these compounds higher maximum surface pressure were generated than was possible in pure aqueous media. The surface pressure data were correlated with the fraction absorbed values in vivo. However it was found that poor water solubility is not the only limiting step to exhibiting any surface activity. Some compounds were showed no surface activity in either solvent system. Therefore a micelle vehicle method was established using short chain phospholipids to mimic the gastrointestinal wall. It could be concluded from the results, that non surface active drugs can interact with the phospholipids micelle vehicle in a way analogous to their interaction with the membrane bilayer. The relative critical micelle concentration was correlated with the fraction absorbed of this test set. A sample preparation schema based on the three types of drugs was established. This schema enabled us to predict the absorbance of slightly soluble and poorly soluble drugs with acceptable reliability for early compound screening. For the prediction of blood brain barrier penetration using surface activity profiling as analyzing method, a test set with very poorly soluble characteristics was chosen. The sample preparation method was based on a strictly aqueous approach using the ‘shake flask’ method. The surface tension measurements enabled correlation of the amphiphilic properties of the very poorly soluble drug compounds with BBB uptake. From the aqueous surface pressure profiles and the determination of physicochemical parameters, it was found that blood brain barrier is more likely when a drug provides a small cross-sectional area, As, at the interface. The cross-sectional area is the only parameter which is independent from the maximal concentration in aqueous media and it is particularly suitable for lower solubility compounds. In summary, it was shown that amphilicity is related to biological membrane interaction in the human body and that surface activity profiling with appropriate sample preparation can be used as a reliable screening tool for the prediction of oral drug absorption of poorly soluble drugs. Furthermore an in vitro screening method of blood-brain-barrier penetration was established.
This article reports on the second Young Environmental Scientists Meeting that was hosted from 28 February to 2 March 2011 by the Institute for Environmental Research at RWTH Aachen University, Germany. This extraordinary meeting was again initiated and organized by the Student Advisory Council under the umbrella of Society of Environmental Toxicology and Chemistry Europe. A movie about the meeting and the abstracts of poster and platform presentations are freely available as supplemental material of this article.
Almost two decades ago, microRNAs were discovered as novel posttranscriptional regulators of gene expression. Since then, research efforts have uncovered their involvement in the control of various cellular processes including migration, proliferation and cell survival. Even more complex events, such as the formation of new blood vessels or organ development, have been shown to be tightly regulated and orchestrated by microRNAs. Due to their crucial regulatory role in tissue homeostasis in vertebrates, it does not come as a big surprise that dysregulated microRNA ex-pression is associated with pathology of diverse diseases. In this regard, the miR-17-92 cluster is a prime example since it has become famous for its amplified expression in tumours and its on-cogenic potential. Our lab demonstrated the expression of the members of the miR-17-92 cluster, namely miR-17, -18a, -19a, -20a, -19b and -92a, in endothelial cells and provided evidence for the anti-angiogenic activity of miR-92a in ECs as well as its important regulatory role in tissue re-covery after ischemia. In this work we addressed the function of the remaining members of the miR-17-92 cluster, i.e. miR-17, miR-18a, miR-19a and miR-20a, in endothelial cells and angiogenesis. Surprisingly, the individual members all displayed anti-angiogenic properties in endothelial cells in vitro, although overexpression of the whole cluster in transformed colonocytes was shown to promote tumour angiogenesis in a mouse model. In this context, we provide evidence that the individual miRs differentially affect the paracrine angiogenic activity of endothelial and tumour cells. Moreover, Antagomir-mediated inhibition of miR-17/20 in a mouse tumour model did not affect tumour angi-ogenesis, although miR-17/20 inhibition profoundly increased vascularization of Matrigel plugs. Thus, our research efforts suggest a differential involvement of the members of the miR-17-92 cluster in physiological and tumour angiogenesis. Additionally, we identified Janus kinase (JAK) 1 as a novel miR-17 target in endothelial cells and demonstrated the involvement of JAK1 in angio-genesis and in the phosphorylation of STAT3 in response to different cytokines in vitro. Overall, inhibition of specific members of the miR-17-92 cluster might represent an attractive therapeutic strategy to enhance angiogenesis in ischemic diseases. In the second part of the present work we investigated the therapeutic value of Antagomir-mediated microRNA inhibition in animal models of pulmonary arterial hypertension. Collectively, inhibition of miR-17 by the respective Antagomir revealed a significant improvement of pulmonary hemodynamics and cardiac function in both the chronic hypoxia mouse model and the mono-crotaline-induced lung injury rat model. Histomorphometric analysis of the lungs of the pulmonary hypertensive mice and rats uncovered a significant reduction of disease associated musculariza-tion of pulmonary arteries in Antagomir-17 treated animals compared to the control animals indicating interference with smooth muscle cell proliferation or survival. Probing of lung tissue of the pulmonary hypertensive rats for selected miR-17 targets uncovered a profound increase in the expression of the cyclin dependent kinase inhibitor p21 in the Antagomir-17 treated rats suggest-ing that inhibition of miR-17 impairs proliferation by impeding cell cycle progression. Analysis of miR-17 function in human smooth muscle cells in vitro corroborated the results from the animal experiments by demonstrating pro-proliferative activity of miR-17 and decreased levels of p21 in these cells. Collectively, our results indicate that Antagomir-17 improves pulmonary hemodyna-mics and cardiac function by interfering with vascular remodelling within the lung. Hence, inhibi-tion of miR-17 might be of therapeutic value to ameliorate the disease pattern in pulmonary arte-rial hypertension. In summary, the present work provides insights into the regulatory functions of members of the miR-17-92 cluster, especially miR-17, in blood vessels and suggests that specific inhibition of members of the miR-17-92 cluster might be a novel option to treat vascular diseases.
Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1tm1a) that reduces mRNA to ~10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1tm1a/tm1a). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1tm1a/tm1a embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.
Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
Recovery of an ecosystem following disturbance can be severely hampered or even shift altogether when a point disturbance exceeds a certain spatial threshold. Such scale-dependent dynamics may be caused by preemptive competition, but may also result from diminished self-facilitation due to weakened ecosystem engineering. Moreover, disturbance can facilitate colonization by engineering species that alter abiotic conditions in ways that exacerbate stress on the original species. Consequently, establishment of such counteracting engineers might reduce the spatial threshold for the disturbance, by effectively slowing recovery and increasing the risk for ecosystem shifts to alternative states. We tested these predictions in an intertidal mudflat characterized by a two-state mosaic of hummocks (humps exposed during low tide) dominated by the sediment-stabilizing seagrass Zostera noltii) and hollows (low-tide waterlogged depressions dominated by the bioturbating lugworm Arenicola marina). In contrast to expectations, seagrass recolonized both natural and experimental clearings via lateral expansion and seemed unaffected by both clearing size and lugworm addition. Near the end of the growth season, however, an additional disturbance (most likely waterfowl grazing and/or strong hydrodynamics) selectively impacted recolonizing seagrass in the largest (1 m2) clearings (regardless of lugworm addition), and in those medium (0.25 m2) clearings where lugworms had been added nearly five months earlier. Further analyses showed that the risk for the disturbance increased with hollow size, with a threshold of 0.24 m2. Hollows of that size were caused by seagrass removal alone in the largest clearings, and by a weaker seagrass removal effect exacerbated by lugworm bioturbation in the medium clearings. Consequently, a sufficiently large disturbance increased the vulnerability of recolonizing seagrass to additional disturbance by weakening seagrass engineering effects (sediment stabilization). Meanwhile, the counteracting ecosystem engineering (lugworm bioturbation) reduced that threshold size. Therefore, scale-dependent interactions between habitat-mediated facilitation, competition and disturbance seem to maintain the spatial two-state mosaic in this ecosystem.
Spherical harmonics coeffcients for ligand-based virtual screening of cyclooxygenase inhibitors
(2011)
Background: Molecular descriptors are essential for many applications in computational chemistry, such as ligand-based similarity searching. Spherical harmonics have previously been suggested as comprehensive descriptors of molecular structure and properties. We investigate a spherical harmonics descriptor for shape-based virtual screening. Methodology/Principal Findings: We introduce and validate a partially rotation-invariant three-dimensional molecular shape descriptor based on the norm of spherical harmonics expansion coefficients. Using this molecular representation, we parameterize molecular surfaces, i.e., isosurfaces of spatial molecular property distributions. We validate the shape descriptor in a comprehensive retrospective virtual screening experiment. In a prospective study, we virtually screen a large compound library for cyclooxygenase inhibitors, using a self-organizing map as a pre-filter and the shape descriptor for candidate prioritization. Conclusions/Significance: 12 compounds were tested in vitro for direct enzyme inhibition and in a whole blood assay. Active compounds containing a triazole scaffold were identified as direct cyclooxygenase-1 inhibitors. This outcome corroborates the usefulness of spherical harmonics for representation of molecular shape in virtual screening of large compound collections. The combination of pharmacophore and shape-based filtering of screening candidates proved to be a straightforward approach to finding novel bioactive chemotypes with minimal experimental effort.
Background: The human pathogen Helicobacter pylori (H. pylori) is a main cause for gastric inflammation and cancer. Increasing bacterial resistance against antibiotics demands for innovative strategies for therapeutic intervention. Methodology/Principal Findings: We present a method for structure-based virtual screening that is based on the comprehensive prediction of ligand binding sites on a protein model and automated construction of a ligand-receptor interaction map. Pharmacophoric features of the map are clustered and transformed in a correlation vector (‘virtual ligand’) for rapid virtual screening of compound databases. This computer-based technique was validated for 18 different targets of pharmaceutical interest in a retrospective screening experiment. Prospective screening for inhibitory agents was performed for the protease HtrA from the human pathogen H. pylori using a homology model of the target protein. Among 22 tested compounds six block E-cadherin cleavage by HtrA in vitro and result in reduced scattering and wound healing of gastric epithelial cells, thereby preventing bacterial infiltration of the epithelium. Conclusions/Significance: This study demonstrates that receptor-based virtual screening with a permissive (‘fuzzy’) pharmacophore model can help identify small bioactive agents for combating bacterial infection.
The tumor suppressor p53 plays a crucial role in cellular growth control inducing a plethora of different response pathways. The molecular mechanisms that discriminate between the distinct p53-responses have remained largely elusive. Here, we have analyzed the p53-regulated pathways induced by Actinomycin D and Etoposide treatment resulting in more growth arrested versus apoptotic cells respectively. We found that the genome-wide p53 DNA-binding patterns are almost identical upon both treatments notwithstanding transcriptional differences that we observed in global transcriptome analysis. To assess the role of post-translational modifications in target gene choice and activation we investigated the genome-wide level of phosphorylation of Serine 46 of p53 bound to DNA (p53-pS46) and of Serine 15 (p53-pS15). Interestingly, the extent of S46 phosphorylation of p53 bound to DNA is considerably higher in cells directed towards apoptosis while the degree of phosphorylation at S15 remains highly similar. Moreover, our data suggest that following different chemotherapeutical treatments, the amount of chromatin-associated p53 phosphorylated at S46 but not at pS15 is higher on certain apoptosis related target genes. Our data provide evidence that cell fate decisions are not made primarily on the level of general p53 DNA-binding and that post-translationally modified p53 can have distinct DNA-binding characteristics.
The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.
Background: Metabolic engineering is an attractive approach in order to improve the microbial production of drugs. Triterpenes is a chemically diverse class of compounds and many among them are of interest from a human health perspective. A systematic experimental or computational survey of all feasible gene modifications to determine the genotype yielding the optimal triterpene production phenotype is a laborious and time-consuming process. Methodology/Principal Findings: Based on the recent genome-wide sequencing of Saccharomyces cerevisiae CEN.PK 113-7D and its phenotypic differences with the S288C strain, we implemented a strategy for the construction of a beta-amyrin production platform. The genes Erg8, Erg9 and HFA1 contained non-silent SNPs that were computationally analyzed to evaluate the changes that cause in the respective protein structures. Subsequently, Erg8, Erg9 and HFA1 were correlated with the increased levels of ergosterol and fatty acids in CEN.PK 113-7D and single, double, and triple gene over-expression strains were constructed. Conclusions: The six out of seven gene over-expression constructs had a considerable impact on both ergosterol and beta-amyrin production. In the case of beta-amyrin formation the triple over-expression construct exhibited a nearly 500% increase over the control strain making our metabolic engineering strategy the most successful design of triterpene microbial producers.
Bacteria are generally assumed to be monoploid (haploid). This assumption is mainly based on generalization of the results obtained with the most intensely studied model bacterium, Escherichia coli (a gamma-proteobacterium), which is monoploid during very slow growth. However, several species of proteobacteria are oligo- or polyploid, respectively. To get a better overview of the distribution of ploidy levels, genome copy numbers were quantified in four species of three different groups of proteobacteria. A recently developed Real Time PCR approach, which had been used to determine the ploidy levels of halophilic archaea, was optimized for the quantification of genome copy numbers of bacteria. Slow-growing (doubling time 103 minutes) and fast-growing (doubling time 25 minutes) E. coli cultures were used as a positive control. The copy numbers of the origin and terminus region of the chromosome were determined and the results were in excellent agreement with published data. The approach was also used to determine the ploidy levels of Caulobacter crescentus (an alpha-proteobacterium) and Wolinella succinogenes (an epsilon-proteobacterium), both of which are monoploid. In contrast, Pseudomonas putida (a gamma-proteobacterium) contains 20 genome copies and is thus polyploid. A survey of the proteobacteria with experimentally-determined genome copy numbers revealed that only three to four of 11 species are monoploid and thus monoploidy is not typical for proteobacteria. The ploidy level is not conserved within the groups of proteobacteria, and there are no obvious correlations between the ploidy levels with other parameters like genome size, optimal growth temperature or mode of life.
Dual function of the NK cell receptor 2B4 (CD244) in the regulation of HCV-specific CD8+ T cells
(2011)
The outcome of viral infections is dependent on the function of CD8+ T cells which are tightly regulated by costimulatory molecules. The NK cell receptor 2B4 (CD244) is a transmembrane protein belonging to the Ig superfamily which can also be expressed by CD8+ T cells. The aim of this study was to analyze the role of 2B4 as an additional costimulatory receptor regulating CD8+ T cell function and in particular to investigate its implication for exhaustion of hepatitis C virus (HCV)-specific CD8+ T cells during persistent infection. We demonstrate that (i) 2B4 is expressed on virus-specific CD8+ T cells during acute and chronic hepatitis C, (ii) that 2B4 cross-linking can lead to both inhibition and activation of HCV-specific CD8+ T cell function, depending on expression levels of 2B4 and the intracellular adaptor molecule SAP and (iii) that 2B4 stimulation may counteract enhanced proliferation of HCV-specific CD8+ T cells induced by PD1 blockade. We suggest that 2B4 is another important molecule within the network of costimulatory/inhibitory receptors regulating CD8+ T cell function in acute and chronic hepatitis C and that 2B4 expression levels could also be a marker of CD8+ T cell dysfunction. Understanding in more detail how 2B4 exerts its differential effects could have implications for the development of novel immunotherapies of HCV infection aiming to achieve immune control.
DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.
Background: The Radical-Pair-Model postulates that the reception of magnetic compass directions in birds is based on spin-chemical reactions in specialized photopigments in the eye, with cryptochromes discussed as candidate molecules. But so far, the exact subcellular characterization of these molecules in the retina remained unknown. Methodology/Principal Findings: We here describe the localization of cryptochrome 1a (Cry1a) in the retina of European robins, Erithacus rubecula, and domestic chickens, Gallus gallus, two species that have been shown to use the magnetic field for compass orientation. In both species, Cry1a is present exclusively in the ultraviolet/violet (UV/V) cones that are distributed across the entire retina. Electron microscopy shows Cry1a in ordered bands along the membrane discs of the outer segment, and cell fractionation reveals Cry1a in the membrane fraction, suggesting the possibility that Cry1a is anchored along membranes. Conclusions/Significance: We provide first structural evidence that Cry1a occurs within a sensory structure arranged in a way that fulfils essential requirements of the Radical-Pair-Model. Our findings, identifying the UV/V-cones as probable magnetoreceptors, support the assumption that Cry1a is indeed the receptor molecule mediating information on magnetic directions, and thus provide the Radical-Pair-Model with a profound histological background.
Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8m delta) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na+/H+ antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8m delta. Nevertheless, nb8m delta still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.
Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.
The p53-family member p73 plays a role in various cellular signaling pathways during development and growth control and it can have tumor suppressor properties. Several isoforms of p73 exist with considerable differences in their function. Whereas the functions of the N-terminal isoforms (TA and delta Np73) and their opposing pro- and antiapoptotic roles have become evident, the functional differences of the distinct C-terminal splice forms of TAp73 have remained unclear. Here, we characterized the global genomic binding sites for TAp73alpha and TAp73beta by chromatin immunoprecipitation sequencing as well as the transcriptional responses by performing RNA sequencing. We identified a specific p73 consensus binding motif and found a strong enrichment of AP1 motifs in close proximity to binding sites for TAp73alpha. These AP1 motif-containing target genes are selectively upregulated by TAp73alpha, while their mRNA expression is repressed upon TAp73beta induction. We show that their expression is dependent on endogenous c-Jun and that recruitment of c-Jun to the respective AP1 sites was impaired upon TAp73beta expression, in part due to downregulation of c-Jun. Several of these AP1-site containing TAp73alpha-induced genes impinge on apoptosis induction, suggesting an underlying molecular mechanism for the observed functional differences between TAp73alpha and TAp73beta.
The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2´-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2´-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2´-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg2+ is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.
The interactions of changes in climate and biodiversity with societal actions, structures and processes are a priority topic within the international scientific debate – and thus, a relevant subject matter for BiKF’s work. This paper outlines a concept for transdisciplinary research within BiKF. It focuses on the analysis of social-ecological systems supporting society with biodiversity driven ecosystem services. Such research is considering different issues: defining sustainable societal adaptations to climate induced biodiversity changes; permitting adequate understanding of the social-ecological reproduction of ecosystem functions, including their conservation and restoration; analysing the societal values and socio-economic utilisation of ecosystem services. Gaining knowledge in these areas provides an improved basis for decision-making in biodiversity and resource management.
This study comprises a survey on ecology, morphology and taxonomy of parasitic fungi infecting Pteridophytes and Orchidaceae found by the author on several field trips to Western Panama as part of the project plant parasitic micro-fungi of Western Panama (ppMP). In Panama, approximately 9500 species of vascular plants are found. Of these, Orchidaceae are with ca. 1150 (ca. 12%) species by far the most speciose family. The Pteridophytes in Panama comprise ca. 940 species in 31 families. Most fungal pathogens on Orchidaceae in tropical regions were described from plants in culture or from material intercepted at borders by plant quarantine services and not from their natural habitats. Therefore, little is known about distribution and ecology of these pathogens in their natural range. The author determined and classified several hundred Orchidaceae-species and Pteridophytes at the sites selected in the context of the project. This work facilitated the identification of many host plants (at least to genus-level) even in sterile condition in the field. About 65 species of Pucciniales are known to infest Orchidaceae and ca. 38% of them are described from tropical America. All available types of Pucciniales on Orchidaceae in tropical America were studied and compared with 91 specimens of rust fungi on orchids collected by the author in Panama. Several hundred additional specimens housed in the BPI, almost all intercepted from plant quarantine services, were used for comparison. As result of this work, it is suggested to combine Uromyces stenorrhynchi Henn. to Sphenospora and, as this is the oldest epithet, to synonymize S. kevorkianii Linder, S. mera Cumm. and S. saphena Cumm. with it. Further, it could be demonstrated that Uredo aurantiaca Montemartini, U. cyrtopodii Syd. & P. Syd., U. epidendri Henn., U. guacae Mayor, U. gynandrearum Corda, U. lynchii (Berk.) Plowr., U. neopustulata Cumm. (≡U. pustulata Henn.), U. nigropuncta Henn., U. oncidii Henn., U. ornithidii F. Kern., Cif. & Thurst., and presumably U. scabies Cke., are anamorphs of this variable species. U. gynandrearum is the oldest anamorph-name for all these taxa. Therefore, it can be established that this rust infects more than 80 species of Orchidaceae in three subfamilies. In total, the anamorph of this species was collected by the author on 17 different species of Orchidaceae in Panama which, apart from one species, are all new hosts to science. The molecular data obtained by the author confirm this view, although more data, especially from material from the whole range of distribution of U. gynandrearum, are necessary. Puccinia spiranthicola Cumm. was found to be a synonym of P. cinnamomea Diet. & Holw. and was found by the author on three different Orchidaceae in two subfamilies. Uredo pleurothallidis Keissl. is now considered a synonym of U. wittmackiana Henn. and the latter as the anamorph of Puccinia oncidii Cumm. In the anamorph genus Uredo, a new species was found infecting at least five different species of Sobralia and Elleanthus (Sobraliinae) at different localities. Molecular data indicate it to be related to the currently polyphyletic Phakopsoraceae. For the rusts with suprastomatal sori on Orchidaceae, now separated from Hemileia and placed in the genus Desmosorus (nom. inval.), the current concept with only one taxon is rejected and the establishment of three subspecies is suggested. The complicated taxonomy is discussed and makes it necessary to validate the genus-name and make a new combination. Another Hemileia-anamorph species was found by the author and is considered to be new to science. This is the first species of this alliance in America on Orchidaceae. Molecular data obtained by the author confirm the separation of Desmosorus from Hemileia and the position of the new species. For rusts on Pteridophytes, a new species of Milesia, (teleomorph: Milesina) and a new anamorphic species of Uredinopsis was found, both on hosts hitherto not known. In Calidion, the presumable anamorph-genus of Uncol, the species C. cf. cenicafeae Salazar & Buriticá was found on several new hosts. Further, the teleomorph was found. Morphologically, this teleomorph did not agree with the description of Uncol by the author of the genus, although the anamorph characteristics left no doubt that it is Calidion. Apparently, the description of Uncol is inadequate, but cannot be improved, as the type is unavailable. Molecular data obtained by the author show this species to be closest to Desmosorus. For Uredo superficialis Speg., the anamorph of Desmella, nine new hosts in eight different fern families were found by the author and the collaborators of the ppMP-project. Ecological data indicate that this species includes different host specific races, which, however could not be distinguished morphologically. For all these rusts, a thorough discussion of the ecology in their habitats is given. In total, 21 LSU rDNA sequences from 6 different rust species on Orchidaceae and Pteridophytes were obtained and analyzed with the Maximum Parsimony and Minimum Evolution method. Here, the position of several groups could be confirmed, and some anamorphs could be assigned to different teleomorphic relationships. Within the Ascomycota and their anamorphs, several hitherto unknown species and species not known from these hosts or not known from Panama were found and analyzed. On Orchidaceae, the following fungi belonging to the Ascomycota are described, illustrated and discussed: In the Phyllachorales, a hitherto not known Phyllachora sp. was found on Oncidium warszewiczii Rchb. f. and was compared with the other species of this order currently known from Orchidaceae. In the Asterinaceae s. l. Lembosia cf. epidendri Meir. Silva & O. R. Pereia was found on Maxillaria crassifolia (Lindl.) Rchb. f., which is a new host and new host alliance for this fungus hitherto only known from Brazil. The fungus is described and compared with all species of Asterinaceae currently known on Orchidaceae. In the Meliolaceae, Meliola orchidacearum Cif. was found on Camaridium biolleyi (Schltr.) Schltr. and an Epidendrum sp. which are new hosts and new host alliances of this fungus which was hitherto only known from the Caribbean Islands. It is described, illustrated and compared with the type. In the Glomerellaceae, Glomerella cingulata and its anamorph Colletotrichum gloeosporioides were found on several hosts. The species is illustrated, described and compared with data from literature. In the anamorphic Mycosphaerellaceae, Pseudocercospora odontoglossii (Prill. & Delacr.) U. Braun, a species currently only known from culture, was found on the new host Pleurothallis imraei Lindl. It is illustrated, described and compared with data from literature. On ferns, the following other fungi are described, illustrated and discussed: A conspicuous undescribed form of Polycyclus was found by the author on Elaphoglossum ciliatum (C. Presl.) T. Moore (Dryopteridaceae) and Serpocaulon loriceum (L.) A. R. Sm. (Polypodiaceae). A conspectus of Parmulariaceae infecting ferns is given and demonstrated that Polycyclina should be synonymized under Polycyclus. Summing up, it can be assessed, especially for the Pucciniales, that the most speciose plant family in Panama carries remarkable few species of specific parasites, and that many of them seem to be distributed over a wide range of species which often are not closely related. One reason amongst others seems to be that parasites need a minimum density of host plants in a habitat to survive. As orchid species often occur with only few (and often small) individual plants at a given locality, the probability for a specific pathogen to infect a plant gets too low, hence high diversity by low abundance of hosts might be an impediment for specific pathogens. In this case, unspecific parasites, or such which are infecting larger alliances, are in advantage. Other reasons could be specific traits of orchids, like succulence and mycotrophy which might hamper fungal infections.
Background: The zinc finger transcription factor Egr-1 (Early growth response 1) is central to several growth factors and represents an important activator of target genes not only involved in physiological processes like embryogenesis and neonatal development, but also in a variety of pathophysiological processes, for example atherosclerosis or cancer. Current options to investigate its transcription and activation in vivo are end-point measurements that do not provide insights into dynamic changes in the living organism. Results: We developed a transgenic mouse (Egr-1-luc) in which the luciferase reporter gene is under the control of the murine Egr-1 promoter providing a versatile tool to study the time course of Egr-1 activation in vivo. In neonatal mice, bioluminescence imaging revealed a high Egr-1 promoter activity reaching basal levels three weeks after birth with activity at snout, ears and paws. Using a model of partial hepatectomy we could show that Egr-1 promoter activity and Egr-1 mRNA levels were increased in the regenerating liver. In a model of wound healing, we demonstrated that Egr-1 promoter activity was upregulated at the site of injury. Conclusion: Taken together, we have developed a transgenic mouse model that allows real time in vivo imaging of the Egr-1 promoter activity. The ability to monitor and quantify Egr-1 activity in the living organism may facilitate a better understanding of Egr-1 function in vivo. Additional File 1: BLI of adult Egr-1-luc mice with opened body cavity. Transgenic Egr-1-luc mice (one month old) received 6 mg luciferin in 100 μl PBS by intraperitoneal injection. Ten minutes thereafter the animal was killed by cervical dislocation, the body cavity opened immediately, skin from the ventral side partially removed and BLI measurement was carried out (10 min signal collection, setting 'high resolution'). A representative animal is shown with similar amplification setting as in Figure 2A.
Background: Factors and processes shaping the population structure and spatial distribution of genetic diversity across a species' distribution range are important in determining the range limits. We comprehensively analysed the influence of recurrent and historic factors and processes on the population genetic structure, mating system and the distribution of genetic variability of the pulmonate freshwater snail Radix balthica. This analysis was based on microsatellite variation and mitochondrial haplotypes using Generalised Linear Statistical Modelling in a Model Selection framework. Results: Populations of R. balthica were found throughout North-Western Europe with range margins marked either by dispersal barriers or the presence of other Radix taxa. Overall, the population structure was characterised by distance independent passive dispersal mainly along a Southwest-Northeast axis, the absence of isolation-by-distance together with rather isolated and genetically depauperated populations compared to the variation present in the entire species due to strong local drift. A recent, climate driven range expansion explained most of the variance in genetic variation, reducing at least temporarily the genetic variability in this area. Other factors such as geographic marginality and dispersal barriers play only a minor role. Conclusions: To our knowledge, such a population structure has rarely been reported before. It might nevertheless be typical for passively dispersed, patchily distributed taxa (e.g. freshwater invertebrates). The strong local drift implied in such a structure is expected to erode genetic variation at both neutral and coding loci and thus probably diminish evolutionary potential. This study shows that the analysis of multiple factors is crucial for the inference of the processes shaping the distribution of genetic variation throughout species ranges. Additional files Additional file 1: Distribution of Radix taxa. Spatial distribution of the Radix MOTU as defined in Pfenninger et al. 2006 plus an additional, newly discovered taxon. This map is the basis for the inference of the species range of R. balthica. Additional file 2: Sampling site table and spatial distribution of diversity indices, selfing estimates and inferred population bottlenecks for R. balthica. Table of sampling site code, geographical position in decimal degrees latitude and longitude, number of individuals analysed with microsatellites (Nnuc), expected heterozygosity (HE) and standard deviation across loci, mean rarefied number of alleles per microsatellite locus (A) and their standard deviation, number of individuals analysed for mitochondrial variation (Nmt), rarefied number of mitochondrial COI haplotypes (Hmt), number of individuals measured for body size (Nsize). Figures A1 - A3 show a graphical representation of the spatial distribution of He, Hmt and, s, respectively. Additional file 3: Assessment of environmental marginality. PCA (principle component analysis) on 35 climatic parameters for the period from 1960 - 2000 from publicly availableWorldClim data. Additional file 4: Inference of a recent climate driven range expansion in R. balthica. Analysis of the freshwater benthos long term monitoring data of the Swedish national monitoring databases at the Swedish University of Agricultural Sciences SLU with canonical correspondence analysis.
Background: The automation of objectively selecting amino acid residue ranges for structure superpositions is important for meaningful and consistent protein structure analyses. So far there is no widely-used standard for choosing these residue ranges for experimentally determined protein structures, where the manual selection of residue ranges or the use of suboptimal criteria remain commonplace. Results: We present an automated and objective method for finding amino acid residue ranges for the superposition and analysis of protein structures, in particular for structure bundles resulting from NMR structure calculations. The method is implemented in an algorithm, CYRANGE, that yields, without protein-specific parameter adjustment, appropriate residue ranges in most commonly occurring situations, including low-precision structure bundles, multi-domain proteins, symmetric multimers, and protein complexes. Residue ranges are chosen to comprise as many residues of a protein domain that increasing their number would lead to a steep rise in the RMSD value. Residue ranges are determined by first clustering residues into domains based on the distance variance matrix, and then refining for each domain the initial choice of residues by excluding residues one by one until the relative decrease of the RMSD value becomes insignificant. A penalty for the opening of gaps favours contiguous residue ranges in order to obtain a result that is as simple as possible, but not simpler. Results are given for a set of 37 proteins and compared with those of commonly used protein structure validation packages. We also provide residue ranges for 6351 NMR structures in the Protein Data Bank. Conclusions: The CYRANGE method is capable of automatically determining residue ranges for the superposition of protein structure bundles for a large variety of protein structures. The method correctly identifies ordered regions. Global structure superpositions based on the CYRANGE residue ranges allow a clear presentation of the structure, and unnecessary small gaps within the selected ranges are absent. In the majority of cases, the residue ranges from CYRANGE contain fewer gaps and cover considerably larger parts of the sequence than those from other methods without significantly increasing the RMSD values. CYRANGE thus provides an objective and automatic method for standardizing the choice of residue ranges for the superposition of protein structures. Additional files Additional file 1: Dependence of Q on the order parameter rank. The quantity Qi is plotted against the order parameter rank i for 9 different protein structure bundles. Additional file 2: Dependence of P on the clustering stage. The quantity Pi is plotted against the clustering stage i for 9 different protein structure bundles. Additional file 3: Dependence of CYRANGE results on the minimal cluster size parameter my. The sequence coverage (red) and RMSD (blue) of the residue ranges determined by CYRANGE were plotted as a function of my for 9 different protein structure bundles. The dotted vertical line indicates the default value, my = 8. Where CYRANGE found two domains, the RMSD values of the individual domains are shown in light and dark blue. Additional file 4: Dependence of CYRANGE results on the domain boundary extension parameter m. See Additional File 3 for details. Additional file 5: Dependence of CYRANGE results on the minimal gap width g. See Additional File 3 for details. Additional file 6: Dependence of CYRANGE results on the relative RMSD decrease parameter delta. See Additional File 3 for details. Additional file 7: Dependence of CYRANGE results on the absolute RMSD decrease parameter delta abs. See Additional File 3 for details. Additional file 8: Dependence of CYRANGE results on the gap penalty parameter gamma. See Additional File 3 for details. Additional file 9: Correlation between the sequence coverage from CYRANGE, FindCore and PSVS, and the GDT total score, GDT_TS. Each data point represents a protein shown in Figures 3 and 4. The coverage is the percentage of amino acid residues included in the residue ranges found by the different methods. The GDT_TS value is defined by GDT_TS = (P1 + P2 + P4 + P8)/4, where Pd is the fraction of residues that can be superimposed under a distance cutoff of d Å. Additional file 10: Correlation between the RMSD value for the residue ranges from CYRANGE, FindCore and PSVS, and the GDT total score, GDT_TS. Each data point represents one protein domain. See Additional File 9 for details.
Background: Natural history museums receive a rapidly growing number of requests for tissue samples from preserved specimens for DNA-based studies. Traditionally, dried vertebrate specimens were treated with arsenic because of its toxicity and insect-repellent effect. Arsenic has negative effects on in vivo DNA repair enzymes and consequently may inhibit PCR performance. In bird collections, foot pad samples are often requested since the feet were not regularly treated with arsenic and because they are assumed to provide substantial amounts of DNA. However, the actual influence of arsenic on DNA analyses has never been tested. Findings: PCR success of both foot pad and body skin samples was significantly lower in arsenic-treated samples. In general, foot pads performed better than body skin samples. Moreover, PCR success depends on collection date in which younger samples yielded better results. While the addition of arsenic solution to the PCR mixture had a clear negative effect on PCR performance after the threshold of 5.4 μg/μl, such high doses of arsenic are highly unlikely to occur in dried zoological specimens. Conclusions: While lower PCR success in older samples might be due to age effects and/or DNA damage through arsenic treatment, our results show no inhibiting effect on DNA polymerase. We assume that DNA degradation proceeds more rapidly in thin tissue layers with low cell numbers that are susceptible to external abiotic influences. In contrast, in thicker parts of a specimen, such as foot pads, the outermost horny skin may act as an additional barrier. Since foot pads often performed better than body skin samples, the intention to preserve morphologically important structures of a specimen still conflicts with the aim to obtain optimal PCR success. Thus, body skin samples from recently collected specimens should be considered as alternative sources of DNA.
Poster presentation from Twentieth Annual Computational Neuroscience Meeting: CNS*2011 Stockholm, Sweden. 23-28 July 2011. To truly appreciate the myriad of events which relate synaptic function and vesicle dynamics, simulations should be done in a spatially realistic environment. This holds true in particular in order to explain the rather astonishing motor patterns presented here which we observed within in vivo recordings which underlie peristaltic contractions at a well characterized synapse, the neuromuscular junction (NMJ) of the Drosophila larva. To this end, we have employed a reductionist approach and generated three dimensional models of single presynaptic boutons at the Drosophila larval NMJ. Vesicle dynamics are described by diffusion-like partial differential equations which are solved numerically on unstructured grids using the uG platform. In our model we varied parameters such as bouton-size, vesicle output probability (Po), stimulation frequency and number of synapses, to observe how altering these parameters effected bouton function. Hence we demonstrate that the morphologic and physiologic specialization maybe a convergent evolutionary adaptation to regulate the trade off between sustained, low output, and short term, high output, synaptic signals. There seems to be a biologically meaningful explanation for the co-existence of the two different bouton types as previously observed at the NMJ (characterized especially by the relation between size and Po),the assigning of two different tasks with respect to short- and long-time behaviour could allow for an optimized interplay of different synapse types. As a side product, we demonstrate how advanced methods from numerical mathematics could help in future to resolve also other difficult experimental neurobiological issues.
Poster presentation from Twentieth Annual Computational Neuroscience Meeting: CNS*2011 Stockholm, Sweden. 23-28 July 2011. Parallel multiunit recordings from V1 in anesthetized cat were collected during the presentation of random sequences of drifting sinusoidal gratings at 12 fixed orientations while gamma oscillations were present. In agreement with the seminal work [1], most units were orientation selective to varying degrees and synchronization was evident in spike train crosscorrelograms computed between units with similar preferred orientations, particularly during the presentation of optimal stimuli. Interestingly, a subset of units, which we refer to as synchronization hubs, were additionally found to synchronize with units having differing preferred orientations which was consistent with a previous study [2]. Moreover, oscillatory patterning in spike train autocorrelograms was also found to be strongest in units denoted as synchronization hubs, and synchronization hubs also tended to have narrower tuning curves relative to other units. We used simplified computational models of small networks of V1 neurons to demonstrate that neurons subject to a sufficiently strong level of inhibitory input can function as synchronization hubs. Neurons were endowed either with integrate-and-fire or conductance-based dynamics and each neuron received a combination of excitatory (AMPA) synaptic inputs that were Poisson-distributed and inhibitory (GABA) inputs that were coherent at a gamma-frequency range. If the strength of rhythmic inhibition was increased for a subset of neurons in the network, and excitation was increased simultaneously to maintain a fixed firing rate, then these neurons produced stronger oscillatory patterning in their discharge probabilities. The oscillations in turn synchronized these neurons with other neurons in the network. Importantly, the strength of synchronization increased with neurons of differing orientation preferences even though no direct synaptic coupling existed between the hubs and the other neurons. Enhanced levels of inhibition account for the emergence of synchronization hubs in the following way: Inhibitory inputs exhibiting a gamma rhythm determine a time window within which a cell is likely to discharge. Increased levels of inhibition narrow down this window further simultaneously leading to (i) even stronger oscillatory patterning of the neuron's activity and (ii) enhanced synchronization with other neurons. This enables synchronization even between cells with differing orientation preferences. Additionally, the same increased levels of inhibition may be responsible for the narrow tuning curves of hub neurons. In conclusion, synchronization hubs may be the cells that interact most strongly with the network of inhibitory interneurons during gamma oscillations in primary visual cortex.