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Background The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. Results A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 uM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. Conclusions The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6% - 28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.
Expeditionen ins Pilzreich Panamas : Pionierarbeit in einer der artenreichsten Regionen unserer Erde
(2007)
Als Bindeglied zwischen Nord- und Südamerika ist Panama ein »Biodiversitäts-Hotspot« – es beherbergt eine außerordentlich hohe Artenvielfalt an Pflanzen, Tieren und Pilzen. Pilze übernehmen in tropischen Ökosystemen wichtige Aufgaben: Sie zersetzen totes organisches Material, helfen den Pflanzen bei der Aufnahme von Wasser und Mineralstoffen aus dem Boden, und sie leisten sogar als Parasiten einen Beitrag zum Erhalt einer großen Artenvielfalt. Aufgrund einzelner Stichproben wissen wir, dass die Anzahl der Pilzarten in den Tropen diejenige der Pflanzen um ein Vielfaches übertrifft. Doch während für Panama zirka 9500 verschiedene Arten von Gefäßpflanzen bekannt sind, zählt eine im Rahmen unserer Arbeit erstellte Checkliste der Pilze nur zirka 1800 Arten. Das zeigt, dass für die Erforschung der Pilze noch umfangreiche Pionierarbeit geleistet werden muss. Zwischen 2003 und 2006 geschah dies im Rahmen einer Universitätspartnerschaft der Universität Frankfurt mit der Universidad Autónoma de Chiriquí, die durch den Deutschen Akademischen Austauschdienst (DAAD) gefördert wurde. Im Zentrum eines Projekts der Deutschen Forschungsgemeinschaft (DFG) steht die Erforschung der Vielfalt und Ökologie pflanzenparasitischer Pilze. Des Weiteren untersucht unsere Arbeitsgruppe Pilze an Insekten sowie an menschlichen Haut- und Nagelläsionen.
Ahnenforschung unter sozialen Amöben : die morphologische Taxonomie muss umgeschrieben werden
(2007)
Seit fast 150 Jahren forschen Wissenschaftler aus aller Welt über den faszinierenden Wechsel zwischen Einzelligkeit und Vielzelligkeit im Lebenszyklus der »zellulären Schleimpilze«. Diese Forschung war bisher so erfolgreich, dass einem Vertreter der zellulären Schleimpilze, Dictyostelium discoideum, vom US-amerikanischen Gesundheitsministerium National Institutes of Health (NIH) ganz offiziell der Status eines Modellorganismus für biomedizinische Forschung verliehen wurde. Obwohl wir inzwischen glauben, viel über die »sozialen Amöben«, die sich bei Nahrungsmangel von Einzellern zu einem vielzelligen Verband zusammenlagern, gelernt zu haben, basiert unser Wissen doch fast ausschließlich auf Arbeiten mit der einen Art D. discoideum. Man kennt allerdings heute mehr als 100 Arten sozialer Amöben. Alle bilden multizelluläre Fruchtkörper aus, die aus Stielen und Sporenpaketen bestehen. Bisher ging man davon aus, dass die Spezies mit azellulären Stielen in ihren Fruchtkörpern phylogenetische Vorläufer der Vertreter mit zellulären Stielen sind, und dass die Vertreter mit verzweigten Fruchtkörpern näher mit sich selbst als mit den anderen sozialen Amöben verwandt sind. Diese Hypothesen wurden nun durch aktuelle molekulargenetische Analysen widerlegt.
Background Identification and evaluation of surface binding-pockets and occluded cavities are initial steps in protein structure-based drug design. Characterizing the active site's shape as well as the distribution of surrounding residues plays an important role for a variety of applications such as automated ligand docking or in situ modeling. Comparing the shape similarity of binding site geometries of related proteins provides further insights into the mechanisms of ligand binding. Results We present PocketPicker, an automated grid-based technique for the prediction of protein binding pockets that specifies the shape of a potential binding-site with regard to its buriedness. The method was applied to a representative set of protein-ligand complexes and their corresponding apo-protein structures to evaluate the quality of binding-site predictions. The performance of the pocket detection routine was compared to results achieved with the existing methods CAST, LIGSITE, LIGSITEcs, PASS and SURFNET. Success rates PocketPicker were comparable to those of LIGSITEcs and outperformed the other tools. We introduce a descriptor that translates the arrangement of grid points delineating a detected binding-site into a correlation vector. We show that this shape descriptor is suited for comparative analyses of similar binding-site geometry by examining induced-fit phenomena in aldose reductase. This new method uses information derived from calculations of the buriedness of potential binding-sites. Conclusions The pocket prediction routine of PocketPicker is a useful tool for identification of potential protein binding-pockets. It produces a convenient representation of binding-site shapes including an intuitive description of their accessibility. The shape-descriptor for automated classification of binding-site geometries can be used as an additional tool complementing elaborate manual inspections.
Background The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabdeltaC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E.coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.
High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.
Obwohl die reiche Artenvielfalt der westafrikanischen Savannenlandschaften erst in Ansätzen erforscht und dokumentiert ist, geht aus Beobachtungen der ansässigen Bevölkerung hervor, dass viele Pflanzenarten bedroht sind. Dies ist nicht nur ein ökologisches, sondern auch ein soziokulturelles Problem. So werden beispielsweise in Nord-Benin etwa 80 Prozent aller vorkommenden Pflanzen zu medizinischen Zwecken herangezogen und stellen damit die Basisgesundheitsversorgung besonders für die ländliche Bevölkerung dar. Neben der Verwendung der Pflanzen in der traditionellen Medizin kommt ihnen auch in der täglichen Ernährung, als Baumaterial und zur Herstellung von Kosmetika eine entscheidende Rolle zu. Das interdisziplinäre BIOTA-Projekt der Universitäten Frankfurt und Mainz, des Forschungsinstituts Senckenberg und der Universitäten Ouagadougou (Burkina Faso) und Abomey-Calavi (Benin) hat es sich zur Aufgabe gemacht, die biologische Artenvielfalt und das damit verbundene lokale Wissen zu erforschen, zu schützen und zu erhalten. Erste Erfolge konnten bereits durch die Anpflanzung besonders bedrohter Arten und die Einrichtung eines Medizinalpflanzengartens, gemeinsam mit lokalen Heilkundigen in Nord-Benin, erzielt werden.
Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.