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Die im Mittelhirn lokalisierten dopaminergen (DA) Neurone sind in einer Vielzahl von Hirnfunktionen involviert und werden aufgrund von anatomischen, molekularen sowie funktionellen Unterschieden in mehrere Subpopulationen aufgeteilt. DA Neurone, die in der Substantia nigra (SN) pars compacta lokalisiert sind, spielen durch ihre Projektion in das dorsale Striatum eine Rolle in der Steuerung der Willkürmotorik. Die Area tegmentalis ventralis (VTA) enthält DA Neurone, die in den präfrontalen Cortex, die basolateralen Amygdala sowie den Nucleus accumbens projizieren und in höheren kognitiven Funktionen, wie dem Arbeitsgedächtnis, der Motivation sowie belohnungsassoziierten Lernvorgängen involviert sind.
In dieser Arbeit wurden die differentiellen Eigenschaften des transienten A-Typ Kaliumstroms sowie dessen Funktion für die intrinsische elektrische Aktivität und die Integration von synaptischen Eingängen in Subpopulationen von DA Neuronen untersucht. Dieser spannungsgesteuerte Strom ist an der Kontrolle der Schrittmacheraktivität beteiligt, beeinflusst die Form und Dauer von Aktionspotentialen und moduliert die Erregbarkeit des somatodendritischen Kompartiments. Der A-Typ Kaliumkanal besteht in DA Neuronen aus einem Tetramer von porenbildenden KV4.3 α-Untereinheiten. Die Koexpression von akzessorischen β-Untereinheiten moduliert maßgeblich die biophysikalischen Parameter des A-Stroms, wie z. B. die Kinetik der Inaktivierung sowie die Spannungsabhängigkeit der Aktivierung und Inaktivierung. Zu diesen β-Untereinheiten gehören die cytoplasmatischen Kaliumkanal-interagierenden Proteine (KChIPs) sowie die transmembranären Dipeptidylpeptidase-ähnlichen Proteine (DPPLs). Während in DA SN Neuronen vor allem KChIP3 exprimiert wird und einen schnell inaktivierenden A-Strom gewährleistet, sind DA VTA Neurone durch die zusätzliche Expression der KChIP4a Splice-Variante charakterisiert, welche durch Inhibition der schnellen Inaktivierung in einem langsam inaktivierenden A-Strom resultiert. Die Bedeutung der differentiellen KChIP4a-Expression für DA Mittelhirnneurone wurde mit Hilfe von KChIP4-Knock-Out (KO)-Mäusen untersucht. Alle Versuche wurden in vitro an akuten Hirnschnitten adulter Wildtyp (WT)- und KChIP4-KO-Tiere durchgeführt und die DA neurochemische Identität sowie die Lage der gemessenen Zellen im Anschluss immunhistochemisch bestätigt. Die biophysikalischen Eigenschaften des A-Stroms wurden mit der Patch-Clamp Technik in der nucleated outside-out Konfiguration untersucht, welche optimale Bedingungen für Voltage-Clamp Experimente gewährleistet. Der A-Strom in DA VTA Neuronen aus KChIP4-KO-Tieren wies dabei eine siebenfach schnellere Inaktivierungskinetik als in vergleichbaren Neuronen aus WT-Tieren auf, während die Inaktivierungskinetik in DA SN Neuronen aus KChIP4-KO-Tieren lediglich um den Faktor zwei schneller war. Außerdem wurde festgestellt, dass selektiv in DA VTA Neuronen das halbmaximale Aktivierungspotential ebenfalls von der KChIP4-Expression abhängig war. Somit konnte gezeigt werden, dass die Expression von KChIP4 für die charakteristischen A-Strom-Eigenschaften von DA VTA Neuronen verantwortlich ist.
Die funktionelle Rolle des KChIP4-vermittelten langsamen A-Stroms wurde mit Hilfe von Current-Clamp Messungen in Ganzzellableitungen untersucht. Dabei wurde deutlich, dass die Expression von KChIP4 die Spontanaktivität von DA SN und VTA Neuronen nicht beeinflusst. Das für DA VTA Neuronen charakteristische verzögerte Wiedereintreten der Spontanaktivität nach einer Inhibition zeigte allerdings eine Abhängigkeit von der KChIP4-Expression, da der sog. rebound delay in DA VTA Neuronen aus KChIP4-KO-Tieren signifikant kürzer war, als in Zellen aus WT-Tieren. Dies konnte sowohl durch Strominjektionen, die in ihrer Kinetik GABAergen synaptischen Eingängen ähnelten, als auch nach direkter Aktivierung von GABA-Rezeptoren durch iontophoretische GABA-Applikation bestätigt werden. KChIP4 könnte somit einen internen Verzögerungsmechanismus nach einer transienten Inhibition von DA Neuronen gewährleisten, die z.B. bei Präsentation von aversiven Stimuli sowie beim Ausbleiben von erwarteten Belohnungen auftritt. Somit könnte die physiologische Relevanz des KChIP4-gesteuerten A-Stroms in der Integration von inhibitorischen synaptischen Eingängen im Kontext von belohnungsgesteuerten Lernprozessen liegen.
Zottelige Landschaftspfleger
(2013)
Glioblastoma is the most common and most aggressive type of brain tumor in adults. In contrast to epithelial cancers, glioblastomas do not metastasize. While the major treatment challenge in epithelial cancers is not the primary tumor but metastasis, glioblastoma patients die of the primary tumor.
However, there is a common theme which underlies the malignant properties of progressed epithelial cancers and glioblastoma: invasion from the primary tumor into the surrounding tissue. In the case of epithelial cancers this is the first and necessary step to metastasis, whereas invasion leads inevitably to tumor recurrence after resection in the case of glioblastoma, causing it to be incurable.
A cellular program which has been described in detail to promote the invasive phenotype in epithelial tumors, is the epithelial-mesenchymal-transition (EMT). Differentiated neural cells are not epithelial, thus, strictly speaking, EMT does not occur in glioblastoma. However, the traits acquired in the process of EMT, especially invasiveness and stemness, are highly relevant to glioblastoma. One of the key transcription factors known to induce EMT in epithelial cancers is ZEB1, which has been described only marginally in the central nervous system so far. Here, I investigate the expression and function of ZEB1 in glioblastoma and during human fetal neural development.
ZEB1 mRNA was significantly upregulated in all histological types of glioma, including glioblastoma, when compared to normal brain. There was no correlation between ZEB1 mRNA levels and tumor grade. Immunohistochemical staining of glioma samples demonstrated that ZEB1 was highly expressed in the great majority of tumor cells. In the developing human brain, intense staining for ZEB1 could be observed in the ventricular and subventricular zone, where stem- and progenitor cells reside. ZEB1 positive cells included cells stained with stem- and progenitor markers like PAX6, GFAP and Nestin. In contrast, ZEB1 was never found in early neuronal cells as identified by TUBB3 staining.
To gain insight into ZEB1 function I generated a human fetal neural stem cell line and a glioblastoma cell line with ZEB1 knockdown, which were compared with their respective control cell lines. First, I found that ZEB1 does not regulate the micro RNA 200 family in either cell line, which has been described as an essential ZEB1 target in epithelial cancers. Second, regulated target genes were identified with a genome wide microarray. The third approach was to directly identify genomic binding sites of ZEB1 by chromatin immunoprecipitation sequencing (ChIP-seq). All three approaches showed that the ZEB1 transcriptional program is surprisingly similar in the neural stem cell line and the glioblastoma cell line. In contrast, it bears only little resemblance to the program described in epithelial cancers.
The most interesting, previously unrecognized ZEB1 target gene identified in this study is integrin b1. It was regulated after ZEB1 knockdown detected by microarray analysis, and has a ZEB1 binding site in its promoter region detected by ChIP-seq. Finally, I addressed the question whether ZEB1 influences tumor growth and invasiveness in a glioblastoma model. After intracranial xenotransplantation in mice, ZEB1 knockdown glioblastoma cells formed significantly smaller and less invasive tumors than control glioblastoma cells.
This study demonstrates that ZEB1 is widely expressed in glioma and relevant for glioblastoma growth and invasion. In contrast to what is known about ZEB1 function in epithelial cancers, ZEB1 is not associated with glioma progression, but instead seems to be an early and necessary event in tumorigenesis. Also with regard to ZEB1 target genes, ZEB1 functions differently in glioblastoma than in epithelial cancers. The two most important ZEB1 targets in epithelial cancers are E-cadherin and the miR-200 family members. Both are not relevant to ZEB1 function in glioblastoma. Interestingly, while the ZEB1 transcriptional program is different from the one described in epithelial cancers, it is highly similar in glioblastoma cells and fetal neural stem cells. This suggests that an embryonic pathway restricted to stem- and progenitor cells during development is reactivated in glioblastoma.
Previously known ZEB1 target genes were tissue specific and therefore seemed unlikely to mediate ZEB1 function in the central nervous system. However, the newly identified ZEB1 target gene integrin b1 is well known to play pivotal roles in both glioblastoma tumorigenesis and invasion as well as in neural stem cells. Additionally, integrin b1 is widely expressed and seems a likely ZEB1 target in other organs than the brain.
Taken together, I demonstrate that ZEB1 is a new regulator of glioblastoma growth and invasion. The transcriptional program of ZEB1 differs from the one in epithelial cancers but is strikingly similar to the one in neural stem cells. The newly identified ZEB1 target gene integrin b1 is likely to mediate crucial ZEB1 functios. Thus, this study identifies ZEB1 as a yet unrecognized player in glioblastoma and neural development. Furthermore, it sets the stage for more research which will help to deepen our understanding of ZEB1 function in the central nervous system and beyond.
Wie andere Vögel auch, verfügen Hühner über zwei verschiedene Magnetfeldrezeptoren. In der vorliegenden Arbeit werden diese beiden Rezeptoren, vor allem unter dem Aspekt Verhaltensontogenie eingehender untersucht. Meine Ergebnisse werden durch histologische Untersuchungen gestützt. Ich untersuchte zwei Hühnerrassen, einen braunen und einen weißen Legehuhn Stamm. Mit der Standardmethode konnte ich die Befunde der Literatur bestätigen. Zur Untersuchung des Magnetkompasses im Auge, habe ich Hühner darauf trainiert einen roten Tischtennisball, auf den sie geprägt wurden, in einer bestimmten magnetischen Richtung zu suchen. Im unbelohnten“ Test ist das Magnetfeld um 90 Grad gedreht, so dass der magnetische Norden nun im geographischen Osten liegt. Die braunen Hühner benutzen den Magnetkompass zum Lösen der gestellten Aufgabe, die weißen Hühner wählen zufällig eine Richtung. Eine Veränderung der Trainingsmethode, ein Training im gedrehten Magnetfeld und eine „Bestrafung“, haben das Ergebnis verändert. Die weißen Hühner sind nun in der Lage, die magnetisch richtige Richtung zu finden, die braunen Hühner reagieren verängstigt und wählen nur zufällig eine Richtung. Beide Hühnerrassen können also - unter verschiedenen Voraussetzungen - einen magnetischen Kompass für die Orientierung benutzen.
Introduction: Gastropoda are guided by several sensory organs in the head region, referred to as cephalic sensory organs (CSOs). These CSOs are innervated by distinct nerves. This study proposes a unified terminology for the cerebral nerves and the categories of CSOs and then investigates the neuroanatomy and cellular innervation patterns of these cerebral nerves, in order to homologise them. The homologisation of the cerebral nerves in conjunction with other data, e.g. ontogenetic development or functional morphology, may then provide insights into the homology of the CSOs themselves.
Results: Nickel-lysine axonal tracing (“backfilling”) was used to stain the somata projecting into specific nerves in representatives of opisthobranch Gastropoda. Tracing patterns revealed the occurrence, size and relative position of somata and their axons and enabled these somata to be mapped to specific cell clusters. Assignment of cells to clusters followed a conservative approach based primarily on relative location of the cells. Each of the four investigated cerebral nerves could be uniquely identified due to a characteristic set of soma clusters projecting into the respective nerves via their axonal pathways.
Conclusions: As the described tracing patterns are highly conserved morphological characters, they can be used to homologise nerves within the investigated group of gastropods. The combination of adequate number of replicates and a comparative approach allows us to provide preliminary hypotheses on homologies for the cerebral nerves. Based on the hypotheses regarding cerebral nerve homology together with further data on ultrastructure and immunohistochemistry of CSOs published elsewhere, we can propose preliminary hypotheses regarding homology for the CSOs of the Opisthobranchia themselves.
In dieser Arbeit wurde die physiologische Funktion der Klasse I Methyltransferase Rrp8 bei der Ribosomen-Biogenese der Hefe Saccharomyces cerevisiae untersucht. Ziel war es, die Bedeutung des Proteins für die rRNA-Prozessierungsschritte besser zu verstehen und das Substratmolekül zu identifizieren, das durch die katalytische Aktivität von Rrp8p modifiziert wird.
In einer rrp8-ΔC Mutante, bei der die für die C-terminale Methyltransferase-Domäne codierende Sequenz deletiert vorlag, konnte eine leichte Mengenreduktion der 40S Untereinheit gefunden werden, was für eine Beteiligung von Rrp8p an der Biogenese der kleinen Untereinheit sprach. Unter Anwendung eines artifiziellen Tetrazyklin-Aptamer-Systems, das die Regulation der Expression eines spezifischen Gens erlaubt, wurde eine bereits vorher bekannte synthetische Interaktion mit der essentiellen 90SKomponente Nep1p bestätigt. Mit Hilfe dieses Expressionssystems konnte auch für eine reduzierte Expression von Nop14p, einem Interaktionspartner des Nep1-Proteins, eine synthetisch kranke Beziehung mit rrp8-ΔC festgestellt werden. Zusammen mit der Untersuchung des Sedimentationsverhaltens eines markierten Rrp8-Proteins und bekannten Daten aus der Literatur wiesen die genetischen Analysen darauf hin, dass Rrp8p neben dem Einfluss auf späte Reifungsschritte des 90S prä-Ribosoms auch für die frühen Reifungsschritte der 60S Untereinheit wichtig ist. Weitere Interaktionen mit Faktoren, die an der Translation beteiligt sind (TIF4631, DOM34) und die Messung der Translationsaktivität zeigten, dass der Ausfall von Rrp8p nicht nur die Biogenese verzögert, sondern gleichfalls die Funktionsfähigkeit des Ribosoms beeinflusst.
Die in dieser Arbeit durchgeführte phänotypische Analyse einer rrp8-ΔC tc-GAR1 Doppelmutante unterstützte die Vermutung, dass Rrp8p auch frühe Reifungsschritte der 60S Untereinheit beeinflusst. Mit einem in vitro Experiment konnte die Bindung von SAM an Rrp8p gezeigt werden und RP-HPLC Analysen der 25S rRNA verdeutlichten, dass Rrp8p neben dem Einfluss auf die Prozessierungsstelle A2 für die m1A645 Modifikation in Helix 25.1 verantwortlich ist. Die phänotypische Untersuchung einer von P. Kötter und S. Lamberth angefertigten rRNA Mutante (A645U) zeigte, dass die Sequenzveränderung innerhalb der Helix 25.1 der 25S rRNA, die zugleich zum Verlust der Modifikation führt, eine deutliche Auswirkung auf das Zellwachstum und auf das Polysomenprofil hat. Ähnliche Polysomenprofile wurden in den Mutanten rrp8-G209R und rrp8-G209A beobachtet, die ein punktmutiertes Rrp8-Protein exprimieren. Eine reduzierte SAM-Bindungsaktivität des mutierten Proteins führte ebenfalls zu einer reduzierten Menge an m1A645 modifizierter 25S rRNA. Eine im Unterschied zur rrp8-ΔC Mutante auftretende Reduktion der 60S Untereinheit in den Punktmutanten spricht für einen bisher noch unbekannten Einfluss von Rrp8p auf die Biogenese der 60S Untereinheit.
In Zusammenarbeit mit S. Sharma durchgeführte 2D-DIGE Experimente und quantitative Messungen von Transkriptmengen zeigten, dass im Vergleich zu einem Wildtyp-Stamm in einer rrp8-ΔC Mutante einige glykolytische Enzyme in geringerem Maße exprimiert werden, was in Zusammenhang mit einer in höheren Eukaryoten bekannten nukleolären Stressantwort gebracht werden kann. Dies verdeutlicht die komplexe Wechselwirkung zwischen der Ribosomenfunktion und dem Energiemetabolismus.
he Influence of trehalose-based glycolipids in the virulence of Mycobacterium tuberculosis (Mtb) is recognised; however, the actual role of these cell-wall glycolipids in latent infection is unknown. As an initial approach, we determined by two-dimensional thin-layer chromatography the sulfolipid (SL) and diacyltrehalose/polyacyltrehalose (DAT/PAT) profile of the cell wall of hypoxic Mtb. Then, qRT-PCR was extensively conducted to determine the transcription profile of genes involved in the biosynthesis of these glycolipids in non-replicating persistent 1 (NRP1) and anaerobiosis (NRP2) models of hypoxia (Wayne model), and murine models of chronic and progressive pulmonary tuberculosis. A diminished content of SL and increased amounts of glycolipids with chromatographic profile similar to DAT were detected in Mtb grown in the NRP2 stage. A striking decrease in the transcription of mmpL8 and mmpL10 transporter genes and increased transcription of the pks (polyketidesynthase) genes involved in SL and DAT biosynthesis were detected in both the NRP2 stage and the murine model of chronic infection. All genes were found to be up-regulated in the progressive disease. These results suggest that SL production is diminished during latent infection and the DAT/PAT precursors can be accumulated inside tubercle bacilli and are possibly used in reactivation processes.
Objective: Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink−/−) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency.
Methods: The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function, and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation.
Results: PINK1 deficiency results in loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink−/− larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalization of mitochondrial function, with resulting rescue of dopaminergic neurons in pink−/− larvae. There was also marked microglial activation in pink−/− larvae, but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis.
Interpretation: Pink1−/− zebrafish are the first vertebrate model of PINK1 deficiency with loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD. Ann Neurol 2013;74:837–847
Background: Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species.
Results: The literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identified (co-)orthologues in plants, which in combination yielded 148 and 143 orthologues in A. thaliana and S. lycopersicum, respectively. Interestingly, we traced 82% overlap in findings from both approaches though we did not find any orthologues for 27% of the factors by either procedure. In turn, 29% of the factors displayed the presence of more than one (co-)orthologue in tomato. Moreover, our analysis revealed that the genomic composition of the translocation machineries in the bryophyte Physcomitrella patens resemble more to higher plants than to single celled green algae. The monocots (Z. mays and O. sativa) follow more or less a similar conservation pattern for encoding the translocon components. In contrast, a diverse pattern was observed in different eudicots.
Conclusions: The orthologue search shows in most cases a clear conservation of components of the translocation pathways/machineries. Only the Get-dependent integration of tail-anchored proteins seems to be distinct. Further, the complexity of the translocation pathway in terms of existing orthologues seems to vary among plant species. This might be the consequence of palaeoploidisation during evolution in plants; lineage specific whole genome duplications in Arabidopsis thaliana and triplications in Solanum lycopersicum.
The TolC-like protein HgdD of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 is part of multiple three-component "AB-D" systems spanning the inner and outer membranes and is involved in secretion of various compounds, including lipids, metabolites, antibiotics, and proteins. Several components of HgdD-dependent tripartite transport systems have been identified, but the diversity of inner membrane energizing systems is still unknown. Here we identified six putative resistance-nodulation-cell division (RND) type factors. Four of them are expressed during late exponential and stationary growth phase under normal growth conditions, whereas the other two are induced upon incubation with erythromycin or ethidium bromide. The constitutively expressed RND component Alr4267 has an atypical predicted topology, and a mutant strain (I-alr4267) shows a reduction in the content of monogalactosyldiacylglycerol as well as an altered filament shape. An insertion mutant of the ethidium bromide-induced all7631 did not show any significant phenotypic alteration under the conditions tested. Mutants of the constitutively expressed all3143 and alr1656 exhibited a Fox(-) phenotype. The phenotype of the insertion mutant I-all3143 parallels that of the I-hgdD mutant with respect to antibiotic sensitivity, lipid profile, and ethidium efflux. In addition, expression of the RND genes all3143 and all3144 partially complements the capability of Escherichia coli ΔacrAB to transport ethidium. We postulate that the RND transporter All3143 and the predicted membrane fusion protein All3144, as homologs of E. coli AcrB and AcrA, respectively, are major players for antibiotic resistance in Anabaena sp. PCC 7120.
Cell-cell adhesion is an essential process during the development of multicellular organisms. It is based on various cellular junctions and ensures a tight contact between neighboring cells, enabling interactive exchanges necessary for morphological and functional differentiation and maintaining the homeostasis of healthy tissue organization. Two important types of cell-cell adhesions are the adherens junction (AJ) and the desmosome which link the actin cytoskeleton and intermediate filaments to cadherin-based adhesion sites. The core of these structures is composed of single-span transmembrane proteins of the cadherin superfamily which include, among other members, the classical cadherins, e.g. E-cadherin, as well as the desmosomal cadherins, e.g. desmoglein-3. The cytoplasmic domains of the desmosomal and classical cadherins enable interactions with proteins of the catenin family. Classical cadherins preferentially associate with β-catenin and p120-catenin, whereas desmosomal cadherins bind to γ-catenin and plakophilins. Intriguingly, γ-catenin, also known as plakoglobin, is so far the only protein known to be present both in the AJ and the desmosome.
In this study, we showed that the two homologous, membrane raft-associated proteins flotillin-1 and flotillin-2 associate with core proteins of the AJ and the desmosome in vitro and in vivo. In confluent human, non-malignant epithelial MCF10A cells and human skin cryosections, flotillin-2 colocalized with E-cadherin, desmoglein-3 and γ-catenin at cell-cell contact sites, whereas flotillin-1 showed barely any overlap with these proteins. In addition, we detected a colocalization of both flotillins with the actin-binding protein α-actinin in membrane ruffles in subconfluent and at cell-cell contact sites in confluent MCF10A cells as well as in human skin cryosections. The interaction with α-actinin was later shown to be flotillin-1 dependent by performing indirect GST pulldown experiments with purified α-actinin-1-GST in MCF10A cell lysates.
Since flotillin-2 strongly colocalized with cell-cell junctions, this suggested that flotillins might be found in complex with cell adhesion proteins. Thus, we performed coimmunoprecipitation experiments in murine skin lysates and various cell lines of epithelial origin, such as human breast cancer MCF7 cells, human keratinocyte HaCaT cells and primary mouse keratinocytes. These experiments demonstrated that flotillins, especially flotillin-2, coprecipitated with E-cadherin, desmosomal cadherins and γ-catenin in relation to the respective cell type and the maturation status of these cell-cell adhesion structures. However, since γ-catenin is so far the only protein known to be present in the AJ and the desmosome, we further assumed that the complex formation of flotillins with cell adhesion structures is mediated by γ-catenin. For this, we performed indirect GST pulldown experiments in MCF10A cell lysates with bacterially expressed, purified flotillin-1-GST, flotillin-2-GST and γ-catenin-GST and were able to verify the complex formation of adhesion proteins and flotillins in vitro. To further test if the interaction of γ-catenin and flotillins is a direct one, we used purified flotillin-1-GST or flotillin-2-GST and γ-catenin-MBP fusion proteins. Both flotillins directly interacted with γ-catenin in this in vitro assay. In addition, mapping of the interaction domains in γ-catenin by using GST fusion proteins carrying different parts of γ-catenin suggested that flotillins bind to a discontinuous γ-catenin binding domain which consists of a Major determinant around ARM domains 6-12, most likely with a major contribution of the ARM domain 7, and possibly including the NT part of γ-catenin.
To study the effect of flotillin depletion on cell-cell adhesion, we generated stable MCF10A cell lines in which flotillins were knocked down by means of lentiviral shRNAs. Staining of E-cadherin and γ-catenin in these cells showed that the localization at the cell-cell borders was significantly altered after flotillin-2 depletion, which pointed to a role for flotillin-2 in the formation of cell-cell adhesion structures in epithelial cells. Furthermore, isolation of detergent resistant membranes (DRMs) from these cells demonstrated that upon depletion of flotillin-2, a significant amount of E-cadherin and γ-catenin shifted into raft fractions. On the contrary, no change was detected in flotillin-1 knockdown cells. These observations point to a functional role of flotillin-2 in the regulation of raft association of cell-cell adhesion proteins. To gain more insight into the in vivo relevance of our findings, we next studied the function of flotillins in the skin of Flot2-/- knockout mice. Analysis of lysates prepared from the skin of one year old female animals revealed an increased expression of E-cadherin, desmoglein-1 and γ-catenin but not β-catenin, implicating that specific adhesion proteins are upregulated in flotillin-2 knockout skin.
Since flotillins are tightly associated with membrane microdomains we next studied the interaction of flotillin-2 with membrane cholesterol. Using the photoreactive cholesterol analog azocholestanol, we were able to show that flotillin-2 and cholesterol directly interacted. In addition, previous studies speculated that flotillin-2 interacts with cholesterol via two putative cholesterol recognition/interaction amino acid consensus (CRAC) motifs. Analysis of the flotillin-2 sequence revealed that flotillin-2 actually contains four putative CRAC motifs. However, using various flotillin-2 CRAC mutant GFP fusion proteins, we were able to show that none of the putative CRAC motifs is functional, which suggested that flotillin-2 interacts with membrane cholesterol, e.g., via posttranslational modifications, such as myristoylation and palmitoylation which were previously shown to be essential for membrane association of flotillin proteins.
Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes.
The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na(+)-translocating electron transport chain that couples electron transfer from reduced ferredoxin to NAD(+) with the generation of a primary electrochemical Na(+) potential across its cytoplasmic membrane. In previous assays in which Ti(3+) was used to reduce ferredoxin, Na(+) transport was observed, but not a Na(+) dependence of the electron transfer reaction. Here, we describe a new biological reduction system for ferredoxin in which ferredoxin is reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-reduced ferredoxin, NAD(+) reduction was highly specific and strictly dependent on ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na(+) dependence of this electron transfer reaction. The Km was 0.2 mm. Na(+) could be partly substituted by Li(+). Na(+) dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na(+) as a coupling ion. Electron transport from reduced ferredoxin to NAD(+) was coupled to electrogenic Na(+) transport, indicating the generation of ΔμNa(+). Vice versa, endergonic ferredoxin reduction with NADH as reductant was possible, but only in the presence of ΔμNa(+), and was accompanied by Na(+) efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes ferredoxin reduction at the expense of an electrochemical Na(+) gradient. The physiological significance of this finding is discussed.
Background: Ferredoxin:NAD+-oxidoreductases (Rnf) found in many bacteria are novel ion-translocating electron transport chains.
Results: A Na+ requirement for the reaction and its reversible coupling to the transmembrane Na+ gradient are demonstrated.
Conclusion: Na+ is the coupling ion. Rnf not only generates a Na+ potential but also uses it to drive the reverse reaction.
Significance: Evidence for a function of Rnf in ferredoxin reduction is provided.
Photorhabdus is a genus of Gram-negative entomopathogenic bacteria that also maintain a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus has an extensive secondary metabolism that is required for the interaction between the bacteria and the nematode. A major component of this secondary metabolism is a stilbene molecule, called ST. The first step in ST biosynthesis is the non-oxidative deamination of phenylalanine resulting in the production of cinnamic acid. This reaction is catalyzed by phenylalanine-ammonium lyase, an enzyme encoded by the stlA gene. In this study we show, using a stlA-gfp transcriptional fusion, that the expression of stlA is regulated by nutrient limitation through a regulatory network that involves at least 3 regulators. We show that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is absolutely required for stlA expression. We also show that stlA expression is modulated by σS and Lrp, regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria. This work is the first that describes pathway-specific regulation of secondary metabolism in Photorhabdus and, therefore, our study provides an initial insight into the complex regulatory network that controls secondary metabolism, and therefore mutualism, in this model organism.
Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. In eukaryotes a comprehensive collection of experimentally verified ribosome biogenesis factors (RBFs) exists only for Saccharomyces cerevisiae. Far less is known for other fungi, animals or plants, and insights are even more limited for archaea. Starting from 255 yeast RBFs, we integrated ortholog searches, domain architecture comparisons and, in part, manual curation to investigate the inventories of RBF candidates in 261 eukaryotes, 26 archaea and 57 bacteria. The resulting phylogenetic profiles reveal the evolutionary ancestry of the yeast pathway. The oldest core comprising 20 RBF lineages dates back to the last universal common ancestor, while the youngest 20 factors are confined to the Saccharomycotina. On this basis, we outline similarities and differences of ribosome biogenesis across contemporary species. Archaea, so far a rather uncharted domain, possess 38 well-supported RBF candidates of which some are known to form functional sub-complexes in yeast. This provides initial evidence that ribosome biogenesis in eukaryotes and archaea follows similar principles. Within eukaryotes, RBF repertoires vary considerably. A comparison of yeast and human reveals that lineage-specific adaptation via RBF exclusion and addition characterizes the evolution of this ancient pathway.
We report here the effects of temperature on the p1 neuromuscular system of the stomatogastric system of the lobster (Panulirus interruptus). Muscle force generation, in response to both the spontaneously rhythmic in vitro pyloric network neural activity and direct, controlled motor nerve stimulation, dramatically decreased as temperature increased, sufficiently that stomach movements would very unlikely be maintained at warm temperatures. However, animals fed in warm tanks showed statistically identical food digestion to those in cold tanks. Applying dopamine, a circulating hormone in crustacea, increased muscle force production at all temperatures and abolished neuromuscular system temperature dependence. Modulation may thus exist not only to increase the diversity of produced behaviors, but also to maintain individual behaviors when environmental conditions (such as temperature) vary.
The spider genus Eusparassus Simon, 1903 (Araneae: Sparassidae: Eusparassinae; stone huntsman spider) is revised worldwide to include 30 valid species distributed exclusively in Africa and Eurasia. The type species E. dufouri Simon, 1932 is redescribed and a neotype is designated from Portugal. An extended diagnosis for the genus is presented. Eight new species are described: Eusparassus arabicus Moradmand, 2013 (male, female) from Arabian Peninsula, E. educatus Moradmand, 2013 (male, female) from Namibia, E. reverentia Moradmand, 2013 (male, female) from Burkina Faso and Nigeria, E. jaegeri Moradmand, 2013 (male, female) from South Africa and Botswana, E. jocquei Moradmand, 2013 (male, female) from Zimbabwe, E. borakalalo Moradmand, 2013 (female) from South Africa, E. schoemanae Moradmand, 2013 (male, female) from South Africa and Namibia and E. mesopotamicus Moradmand and Jäger, 2012 (male and female) from Iraq, Iran and Turkey. 22 species are re-described six of them are transferred from the genus Olios Walckenaer, 1837. Six species-groups are proposed: the dufouri-group [8 species: E. dufouri, E. levantinus Urones, 2006, E. barbarus (Lucas, 1846), E. atlanticus Simon, 1909, E. syrticus Simon, 1909, E. oraniensis (Lucas, 1846), E. letourneuxi (Simon, 1874), E. fritschi (Koch, 1873); Iberian Peninsula to parts of north-western Africa], walckenaeri-group [3 species: E. walckenaeri (Audouin, 1826), E. laevatus (Simon, 1897), E. arabicus; eastern Mediterranean to Arabia and parts of north-eastern Africa], doriae-group [7 species: E. doriae (Simon, 1874), E. kronebergi Denis, 1958, E. maynardi (Pocock, 1901), E. potanini (Simon, 1895), E. fuscimanus Denis, 1958, E. oculatus (Kroneberg, 1846) and E. mesopotamicus; Middle East to Central and South Asia], vestigator-group (3 species: E. vestigator (Simon, 1897), E. reverentia, E. pearsoni (Pocock, 1901); central to eastern Africa and an isolated area in NW India], jaegeri-group [4 species: E. jaegeri, E. jocquei, E. borakalalo, E. schoemanae; southern and south-eastern Africa], tuckeri-group [2 species: E. tuckeri (Lawrence, 1927), E. educatus; south-western Africa). Two species, E. pontii Caporiacco, 1935 and E. xerxes (Pocock, 1901) cannot be placed in any of the above groups. Two species are transferred from Eusparassus to Olios: O. flavovittatus (Caporiacco, 1935) and O. quesitio Moradmand, 2013. 14 species are recognized as misplaced in Eusparassus, thus nearly half of the described species prior to this revision were placed mistakenly in this genus. Neotypes are designated for E. walckenaeri from Egypt, E. barbarus, E. oraniensis and E. letourneuxi (all three from Algeria) to establish their identity. The male and female of Cercetius perezi Simon, 1902, which was known only from the immature holotype, are described for the first time. It is recognized that the monotypic and little used generic name Cercetius Simon, 1902 — a species, which had been known only from the immature holotype — as a synonym of the widely used name Eusparassus. The case proposal 3596 (conservation of name Eusparassus) is under consideration by ICZN.
The first comprehensive molecular phylogeny of the family Sparassidae with focus on the genus Eusparassus is investigated using four molecular markers (mitochondrial COI and 16S; nuclear H3 and 28S). The monophyly of Eusparassus and the dufouri, walckenaeri and doriae species-groups are recovered with the latter two groups more closely related. The monophyly of the tuckeri-group is not supported and the position of E. jaegeri as the only available member of the jaegeri-group is not resolved within the Eusparassus clade. DNA samples of the vestigator-group were not accessible for this study. The origination of the genus Eusparassus around 70 million years ago (MA) is estimated according to molecular clock analyses. Using this recent result in combination with some biogeographic and geological data, the Namib Desert is proposed as the place of ancestral origin for Eusparassus and putative Eusparassinae genera.
Further analyses are done on the phylogenetic relationships of Sparassidae and its subfamilies. The Eusparassinae are not confirmed as monophyletic, with the two original genera Eusparassus and Pseudomicrommata in separate clades and only the latter clusters with most other assumed Eusparassinae, here termed the "African clade". Monophyly of the subfamilies Sparianthinae, Heteropodinae sensu stricto, Palystinae and Deleninae is recovered. The Sparianthinae are supported as the most basal clade, diverging considerably early (143 MA) from all other Sparassidae. The Sparassinae and genus Olios are found to be polyphyletic. The Sparassidae are confirmed as monophyletic and as most basal group within the RTA-clade. The divergence time of Sparassidae from the RTA-clade is estimated with 186 MA in the Jurassic. No affiliation of Sparassidae to other members of the "Laterigradae" (Philodromidae, Selenopidae and Thomisidae) is observed, thus the crab-like posture of this group was proposed a result of convergent evolution. Only the families Philodromidae and Selenopidae are found members of a supported clade. Including a considerable amount of RTA-clade representatives, the higher-level clade Dionycha is not but monophyly of the RTA-clade itself is supported.
Background: The rationale for gathering information from plants procuring nitrogen through symbiotic interactions controlled by a common genetic program for a sustainable biofuel production is the high energy demanding application of synthetic nitrogen fertilizers. We curated sequence information publicly available for the biofuel plant sugarcane, performed an analysis of the common SYM pathway known to control symbiosis in other plants, and provide results, sequences and literature links as an online database.
Methods: Sugarcane sequences and informations were downloaded from the nucEST database, cleaned and trimmed with seqclean, assembled with TGICL plus translating mapping method, and annotated. The annotation is based on BLAST searches against a local formatted plant Uniprot90 generated with CD-HIT for functional assignment, rpsBLAST to CDD database for conserved domain analysis, and BLAST search to sorghum's for Gene Ontology (GO) assignment. Gene expression was normalized according the Unigene standard, presented as ESTs/100 kb. Protein sequences known in the SYM pathway were used as queries to search the SymGRASS sequence database. Additionally, antimicrobial peptides described in the PhytAMP database served as queries to retrieve and generate expression profiles of these defense genes in the libraries compared to the libraries obtained under symbiotic interactions.
Results: We describe the SymGRASS, a database of sugarcane orthologous genes involved in arbuscular mycorrhiza (AM) and root nodule (RN) symbiosis. The database aggregates knowledge about sequences, tissues, organ, developmental stages and experimental conditions, and provides annotation and level of gene expression for sugarcane transcripts and SYM orthologous genes in sugarcane through a web interface. Several candidate genes were found for all nodes in the pathway, and interestingly a set of symbiosis specific genes was found.
Conclusions: The knowledge integrated in SymGRASS may guide studies on molecular, cellular and physiological mechanisms by which sugarcane controls the establishment and efficiency of endophytic associations. We believe that the candidate sequences for the SYM pathway together with the pool of exclusively expressed tentative consensus (TC) sequences are crucial for the design of molecular studies to unravel the mechanisms controlling the establishment of symbioses in sugarcane, ultimately serving as a basis for the improvement of grass crops.
Es ist wohl unumstritten, dass das Leben, wie wir es kennen, ohne die sauerstoffproduzierenden Organismen unserer Erde nicht möglich wäre. Zu ihnen gehören nicht nur die Landpflanzen, deren mannigfaltige Nutzung wichtiger Bestandteil unseres Alltags ist. Auch mikroskopisch kleine Algenarten leisten einen entscheidenden Beitrag zu den Stoffwechselkreisläufen dieser Welt. Unter ihnen befinden sich die Kieselalgen (Diatomeen), die mit einer Varietät von bis zu 10000 Spezies etwa 40 % der marinen Primärproduktion verantworten. Der Ursprung der heutigen zur oxygenen Photosynthese befähigten Eukaryoten geht auf Endosymbioseereignisse zurück, von denen aus sich diese Organismen ausgesprochen vielfältig entwickelt haben. Diese Vielfalt wird dabei nicht nur anhand ihrer äußeren Morphologie, sondern auch auf subzellulärer Ebene, deutlich. So zum Beispiel durch die unterschiedlichen Strukturen der Thyakoidmembranen, die sich in Kieselalgen wie Cyclotella meneghiniana in dreilagigen Bändern arrangieren. In Pflanzen wie Nicotiana tabacum (Tabak) hingegen bilden sie große, stapelartige Bereich aus, die zur räumlichen Separation der in den Thylakoiden eingebetteten Photosystemen beitragen. Auch die an die Photosysteme (PS) gebundenen Lichtsammelproteine (Lhcs) haben sich in Tabak und Cyclotella unterschiedlich entwickelt. Gemäß ihrem Namen zeichnen sie sich zwar allesamt durch die Sammlung und Weiterleitung der Lichtenergie an die Photosysteme aus, grenzen sich aber in Hinblick auf Proteingröße und Pigmentierung voneinander ab.
Die Lhcs der höheren Pflanzen werden entsprechend ihrer Zuordnung zu den Photosystemen in den aus zwei Heterodimeren bestehenden LHCI des PSI und die Lhcb-Antennenproteine des PSII unterschieden. Zu letzteren gehören der trimere Hauptantennenkomplex LHCII und die monomeren, minoren Antennenproteine. Die Lhcs binden die zur Lichtsammlung benötigten Pigmente, vor allem Chlorophyll a und Chlorophyll b, aber auch primäre Carotinoide wie Violaxanthin, Lutein und Neoxanthin, in unterschiedlichen Stöchiometrien. Es ist bereits bekannt, dass die Pigmentierung entscheidend zur Stabilität der Lichtsammelproteine beiträgt, wenngleich zum Teil auch eine gewisse Flexibilität in Bezug auf die Art der gebundenen Pigmente an den entsprechenden Bindestellen der Proteine besteht.
Im Rahmen dieser Arbeit liegt der Fokus auf der Fragestellung inwieweit die in der Regel nicht in Pflanzen vorkommenden Ketocarotinoide die Struktur und Funktion des LHCII aus einer Ketocarotinoide produzierenden N. tabacum - Transformante (bkt-Linie) beeinflussen und welche Auswirkungen sie auf dessen Photosyntheseapparat im Allgemeinen haben. Die bkt-Linie bildet dabei zum Teil auf Kosten ihrer primären Carotinoide sowohl das als antioxidativ und als anti-kanzerogen beschriebene Astaxanthin, als auch dessen Vorstufe Canthaxanthin und einige Derivate dieser Pigmente, die, nach vergleichenden HPLC-Analysen von Blättern und Thylakoidfraktionen, zu einem großen Teil mit der Thylakoidmembran assoziiert sind. Durch spektroskopische Untersuchungen konnte gezeigt werden, dass diese Ketocarotinoide in Hinblick auf die Energieweiterleitung zum Chlorophyll a nicht funktionell an den LHCII binden, ihre Produktion aber die Trimerisierung dieses Lichtsammelkomplexes in N. tabacum nachhaltig beeinträchtigt. Auch die Assemblierung der PSII-LHCII-Superkomplexe wird dadurch maßgeblich gestört. Elektronenmikroskopische Aufnahmen von Chloroplasten der bkt-Linie verdeutlichten zudem die Beeinträchtigung der Granathylakoid-Stapelung: Sie fällt ungeordneter aus als im Wildtyp, was durch den Mangel an intakten LHCII-Trimeren begründet sein kann.
In funktioneller Hinsicht stören die Ketocarotinoide die Energieweiterleitung innerhalb des PSII und bewirken die Reduktion der photoprotektorischen, nicht-photochemischen Fluoreszenzlöschung des Wirtsorganismus nachhaltig. Zeitgleich reduzieren sie durch einen abschirmenden Effekt auf Grund ihrer Assoziation mit der Thylakoidmembran und/oder durch einen eventuellen S1-S1-Energietransfer von Chl a auf die Ketocarotinoide aber auch die Menge der Lichtenergie, die über die Lhcs an die Photosysteme weitergeleitet wird. Dadurch kommt ihnen neben dem nachhaltig störenden Einfluss auf die Intaktheit des Photosyntheseapparats zugleich auch eine schützende Wirkung vor einem Übermaß an Lichtenergie zu.
Aus Cyclotella meneghiniana sind zwei Hauptantennenkomplexe bekannt: FCPa und FCPb. Im Gegensatz zu den Lhcs der Chl a/b-haltigen Organismen binden die Lichtsammelproteine der Diatomeen das Xanthophyll Fucoxanthin anstelle des Luteins, und Chlorophyll c anstelle des Chlorophyll b. Im Gegensatz zu der bereits sehr detailliert aufgeklärten Struktur des trimeren LHCII in höheren Pflanzen, existieren für den Aufbau des FCPb in C. meneghiniana bisher nur fundierte Modellvorschläge. Diese postulieren eine homotrimere Grundstruktur für den FCPb, die zu höheren Oligomeren assembliert.
In der vorliegenden Arbeit konnte anhand elektronenmikroskopischer Aufnahmen und der anschließenden Einzelpartikelanalyse nun erstmalig die Struktur des etwa 6-7 nm großen, trimeren FCPb gezeigt und die Richtigkeit der bisher postulierten Modellvorschläge in Hinblick auf die Struktur des Trimers bewiesen werden. Nach den hier dargelegten Erkenntnissen gleicht die Anordnung der Untereinheiten des FCPb-Trimers der des LHCII. Zudem ergibt sich aus dem Zusammenhang der hier erhobenen Daten und den in der Fachliteratur veröffentlichten Ergebnissen zum Thema FCPb ein klares Bild über die Anordnung der höheren Oligomere in Form von Nonameren. Auch diese Erkenntnisse unterstützen das ursprünglich von C. Büchel vorgeschlagene Modell für die oligomere Struktur des FCPb in C. meneghiniana.
The project focuses on the efficiency of combined technologies to reduce the release of micropollutants and bacteria into surface waters via sewage treatment plants of different size and via stormwater overflow basins of different types. As a model river in a highly populated catchment area, the river Schussen and, as a control, the river Argen, two tributaries of Lake Constance, Southern Germany, are under investigation in this project. The efficiency of the different cleaning technologies is monitored by a wide range of exposure and effect analyses including chemical and microbiological techniques as well as effect studies ranging from molecules to communities.