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Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane β-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.
Moderately elevated levels of plasma plant sterols have been suspected to be causally involved in atherosclerosis. The aim of this study was to investigate whether plant sterols and other markers of sterol metabolism predicted all-cause and cardiovascular mortality in participants of the Ludwigshafen Risk and Cardiovascular health (LURIC) study. A total of 1,257 individuals who did not use statins and at baseline had a mean (± SD) age of 62.8 (± 11.0) years were included in the present analysis. Lathosterol, cholestanol, campesterol, and sitosterol were measured to estimate cholesterol synthesis and absorption. The mean (± SD) time of the follow-up for all-cause and cardiovascular mortality was 7.32 (± 2.3) years. All-cause (P = 0.001) and cardiovascular (P = 0.006) mortality were decreased in the highest versus the lowest lathosterol to cholesterol tertile. In contrast, subjects in the third cholestanol to cholesterol tertile had increased all-cause (P < 0.001) and cardiovascular mortality (P = 0.010) compared with individuals in the first tertile. The third campesterol to cholesterol tertile was associated with increased all-cause mortality (P = 0.025). Sitosterol to cholesterol tertiles were not significantly related to all-cause or cardiovascular mortality. The data suggest that high absorption and low synthesis of cholesterol predict increased all-cause and cardiovascular mortality in LURIC participants.
Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.
Peroxisome proliferator-activated receptor γ (PPARγ) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARγ expression. Considering that regulation of PPARγ expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARγ1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3′-untranslated regions (UTRs), we analyzed the impact of the PPARγ-3′-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARγ-3′-UTR, suggesting that PPARγ1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3′-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARγ1 mRNA decay, whereas miR-27b overexpression decreased PPARγ1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARγ1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARγ1 mRNA. Understanding molecular mechanisms decreasing PPARγ might help to better appreciate inflammatory diseases.
Bare incrusted soils are a degradation stage often encountered in the Sahel zone. Our study documents the success of restoration (= regreening) experiments using deep ploughing in an experimental site south of Gorom-Gorom in the Oudalan province of Burkina Faso. We used phytosociological relevés and maximum likelihood classifications of digital photography to analyze changes in vegetation. Plant cover in treated plots was found to be about 20 times higher than in control plots, mean species richness more than twice as high. Therefore, this promising restoration method should be tested also in other Sahelian regions. Our approach to combine phytosociological relevés and maximum likelihood classifications of digital photography proved to be very useful.
Background: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood.
Methodology/Principal Findings: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation.
Conclusions/Significance: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG.
Naturally, the floodplains of Central Asian rivers harbour riparian, so-called ‘Tugai’ forests, reeds with Phragmites australis, and shrub communities which form a mosaic depending on the variety of available ground water. In recent decades, these natural ecosystems have been strongly altered anthropogenically or even completely destroyed. In order to restore those ecosystems, knowledge on vegetation, ecosystem dynamics, and natural regeneration processes is essential. In our study, we present results of ecological investigations at the Tarim River. We gathered comprehensive data on soil, vegetation, forest stand age, tree vitality, river course dynamics, and land use and brought it to the landscape level. Thus, recommendations are derived for the maintenance of these floodplain ecosystems, in particular with regard to their biological diversity.
The Ucayali River originates in the high Andean Mountains near the city of Cusco in Peru. After about 1,600 km, it joins with the Marañón River. Both the Ucayali River and the Marañón River are sources of the Amazon. From 2001 to 2005, the Ministry of Transport and Communication of Peru funded a study to determine the navigability of the Ucayali River. In the process, an extensive data set was acquired including hydrological and sedimentological data as well as a comprehensive topographical survey of the riverbed. Since the Ucayali River has experienced no stream channel modification in the past, the available data provide information about the flow pattern for a natural stream and insight into a reference ecosystem. This paper offers the first analysis of the available data and a sediment transport calculation for the Ucayali River.
In recent decades, the number of streams on the Canary Islands has decreased dramatically due to the non-sustainable consumption of water for agriculture and tourism. Natural reaches of streams with an endemic macroinvertebrate fauna do, however, still exist in protected areas of Tenerife and La Gomera. Those reaches serve as a reference to develop an assessment method for streams on islands. This method takes into account common parameters such as water quality and hydromorphology, while emphasizing biodiversity and endemism. The latter concepts as they relate to stream conservation are important in both nature conservation and protection of species as many endemic aquatic organisms are endangered.
This study represents the first comprehensive biological and hydrochemical investigation of small coastal ponds in the saltmarsh dominated Tijuana Estuary, southern California (U.S.). Special attention is given to the brackish water biotopes.
Different salinities and considerable fluctuations in water level characterized these shallow ponds and restrict the biological settlement. Fluctuations of salinities ranged from brackish water to hyperhaline water conditions. Due to different salinity levels, the ponds vary in hydrochemistry, macroinvertebrate species composition and plant communities. The macroinvertebrate community of the brackish waters were dominated by Gastropoda, Odonata, and Coleoptera containing a mixture of freshwater/brackish water species and marine macroinvertebrates. Typical plants of the brackish habitat were Typha domingensis (Southern cattail), and Scirpus californicus (California bulrush) associated with Juncus acutus (Siny rush). These brackish habitats with a wide range of salinity fluctuations are sparsely colonized but represent a niche for typical highly adaptable species. Especially, it is a biotope for species with a wide range of salt tolerance. Therefore, endangered species occurred besides introduced or invasive species in the ponds of the Tijuana Estuary. This fact has to be taken into account in case of wetland restoration. Due to the freshwater influence, the restoration of brackish habitats focuses on the problem of invasive species.
As a part of the interdisciplinary research project ”Integration of nature protection goals with organic farming: an the example from the Hessian ”state domain” [Staatsdomäne] area Frankenhausen”, different restoration measures have been carried out within this site, 15 km north of Kassel. Since 1998, intensive conventional agricultural practices have been substituted with organic farming here. One intention of the agricultural restructuring was to realise nature protection goals in cooperation with sustainable organic agricultural production.
The hydrologic portion of the project addresses both the implementation of restoration measures in rivers and streams and their scientific monitoring. Starting in July 2007, several restoration measures were carried out in the hydrologic systems of the Jungfernbach and Esse streams within the Frankenhausen site. Both systems are formed by typical loess streams (catchment size about 9 km2) which had been heavily degraded for several hundred years by intensive agriculture. The most important restoration measures were removal of a piped section of a tributary of the Jungfernbach at Totenhof, restoration of biological passability by removal of weirs and substitution of narrow pipes under farm paths, relocation of a section of the Jungfernbach from the edge of the floodplain to its original location in the centre, widening of narrow sections and partial raising of the deepened stream bed by means of rough ramps (stone bars) and racks made of oak wood or iron.
These physical restoration measures were accompanied by a scientific monitoring programme comprising morphological, hydrochemical and biological (aquatic macrophytes, aquatic macroinvertebrates, fish and amphibians) aspects.
The aim of this study was to document the original ecological conditions, the restoration measures and the early ecological effects on the stream sections for the first six months following restoration as a basis for further ecological monitoring.
The restoration measures effected clear morphological changes in cross-section and passability. The chemical condition of the streams showed slight changes in some aspects following the restoration, e. g. a reduction of phosphorus, magnesium and potassium concentration. Other than macrophytic algae in the newly shaped sections, aquatic macrophytes did not develop over the winter season before the end of the monitoring phase in April 2008. Within the newly shaped stream sections of a small tributary and of the Jungfernbach, up to 14 aquatic macroinvertebrate taxa started to colonise the new habitats 6 months after restoration.
Fish fauna were very poorly represented in the streams and included only a few specimens of brown trout (Salmo trutta). This did not change markedly after restoration, possibly due to the isolation of the population caused by impassable weirs downstream of the investigation area.
The survival of the approximately 1,000 artificial ponds in the Pfälzerwald (Palatinate Forest) biosphere reserve is endangered as they continue to be abandoned, but a large number of them have conservation and historical value. An overall management concept is needed as the high costs for restoration and the requirements of the EU Water Framework Directive regarding river continuity will make it impossible to maintain all of the ponds. Most of the ponds are migration barriers for fish and aquatic invertebrates. The assessment methods presented here are based on readily available data for the evaluation of the ecological and cultural-historical importance of the ponds, their implications within the landscape, and their (often negative) impact on stream ecology. The assessment of the condition of the ponds’ manmade structures leads to conclusions about the urgency for action. The assessment classes are linked with recommendations for action. In the synopsis of all assessments, management concepts emerge for the individual ponds, and priority lists of ponds can be generated that point out where actions are preferential.
The use of water buffalos for landscape maintenance started ten years ago in Germany. Now, more than 2,100 buffalos are kept by about 90 breeders, and first results concerning their usefulness for landscape management are available. Buffalos are mainly used on particularly wet sites which cannot be grazed by cattle or other domestic animals. Although grazing of wetlands, river banks and water bodies is still controversial, early results from literature and our own research clearly indicate the beneficial impact of moderate grazing on such sites for birds, amphibians, vegetation and insects. This paper presents a short literature review and the first results of the BUBALUS project at Brandenburg University of Technology (BTU) and general experience from other projects.
Species-rich wet grasslands in floodplains are on focus of European nature conservation policy. However, since the seventies of the last century large areas with grasslands in floodplains have been meliorated, ploughed and used for intensive cropping in Germany. Therefore, restoration strategies for large-scale conversion of former arable land into species-rich grasslands and integration into a long-term sustainable land use regime are needed. Dealing with large areas in restoration projects causes high costs which often exceed the possibilities of NGO’s or other stakeholders. Aiming to develop and implement new cost-efficient strategies for restoration and long-term management of wetlands on former arable land local NGO’s and the Anhalt University of Applied Sciences started a co-operation within a project in a heavily degraded floodplain in the Elbe river valley. Up to now, more than 40 ha former arable land was successively bought and immediately grazed by large herbivores (Heck-cattle and Przewalski-horses). The local farmers apply a year-round grazing regime without additional feeding and low stocking density. Scientific evaluation of the project progress and experiments with different re-vegetation variants (natural recovery, hay transfer, seeding of commercial seed mixture) revealed the following results: (1) on former arable land immediate grazing with large herbivores without additional feeding is possible and leads to a successive development of typical grassland communities with low nutrient status, (2) integration of old pastures into the grazing system enhances colonization of native grassland species alongside animal tracks, (3) seeding of a commercial seed mixture impedes the colonization of native grassland species, (4) transfer of species-rich hay accelerates the colonization rate of several grassland species, and (5) highest cover of target species was found on regularly wet sites. Therefore, we conclude that grazing with large herbivores proved to be successful in converting former arable land into species-rich grasslands. Nevertheless, rising of the groundwater table is most important for further development of species-rich wet grasslands in the Wulfener Bruch.
The restoration of ecosystems has become a major challenge throughout the world in the 21st century (see comprehensive surveys from, e. g. Temperton et al. 2004, van Andel & Aronson 2006, Walker et al. 2007, and Zerbe & Wiegleb 2009). Due to non-sustainable land use and inefficient use of natural resources, many ecosystems have been degraded or completely destroyed. Consequently, the functioning of ecosystems has been severely affected and many ecosystem services have been lost.
While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.
FIAS Scientific Report 2009
(2010)
In this Annual Report we present some of the ongoing activities of FIAS and of the associated graduate
school, the “Frankfurt International Graduate School for Science” (FIGSS) in the year 2009. The main part of the Report consists of a collection of short reports describing the research projects of scientists working at or associated with FIAS.
The TATA Box Binding Protein (TBP) is a 20 kD protein that is essential and universally conserved in eucarya and archaea. Especially among archaea, organisms can be found that live below 0°C as well as organisms that grow above 100°C. The archaeal TBPs show a high sequence identity and a similar structure consisting of α-helices and β-sheets that are arranged in a saddle-shape 2-symmetric fold. In previous studies, we have characterized the thermal stability of thermophilic and mesophilic archaeal TBPs by infrared spectroscopy and showed the correlation between the transition temperature (Tm) and the optimal growth temperature (OGT) of the respective donor organism. In this study, a “new” mutant TBP has been constructed, produced, purified and analyzed for a deeper understanding of the molecular mechanisms of thermoadaptation. The β-sheet part of the mutant consists of the TBP from Methanothermobacter thermoautotrophicus (OGT 65°C, MtTBP65) whose α-helices have been exchanged by those of Methanosarcina mazei (OGT 37°C, MmTBP37). The Hybrid-TBP irreversibly aggregates after thermal unfolding just like MmTBP37 and MtTBP65, but the Tm lies between that of MmTBP37 and MtTBP65 indicating that the interaction between the α-helical and β-sheet part of the TBP is crucial for the thermal stability. The temperature stability is probably encoded in the variable α-helices that interact with the highly conserved and DNA binding β-sheets.
The estimation model PhytoCalc allows a non-destructive quantification of dry weight and nutrient pools of understorey plants in forests by using the relationship between species biomass, cover and mean shoot length. The model has been validated with independent samples in several German forest types and can be a useful tool in forest monitoring. However, in open areas within forests (e.g. clearcuts), the current model version underestimates biomass and produces unreliable nutrient pool estimations. Thus, tissue density, as approximated by leaf dry matter content (LDMC), is systematically higher under high light compared to low light conditions. We demonstrate that the ratio of LDMC under clearcut conditions to LDMC under forest conditions can be used to adjust the PhytoCalc model to clearcut conditions. We investigated the LDMC ratio of five exemplary species commonly occurring on clearcuts. Integrating the square of the ratio as a correction factor improved estimates of biomass to more than 70% fit between observations and predictions. Results also suggest this ratio can be used to correct nutrient concentrations modelled in PhytoCalc, which tend to be overestimated in clearcuts. As morphological groups of plant species exhibit significantly different ratios, we advise using group-specific correction factors for clearcut adjustments in the future.
The display of foreign polypeptides and proteins on the surface of viruses or cells provides an important tool for the engineering of biomolecules and the analysis of their interactions with binding partners. The most extensively used display platform is the coat protein of the filamentous bacteriophage (Smith, 1985). Phage display libraries have often been selected for polypeptides, e.g. single chain (sc) antibodies that bind to a protein of interest, but in vivo selection could only be demonstrated for peptides so far. An alternative display platform is the retrovirus murine leukemia virus (MLV). Here, polypeptides are displayed at the N-terminus of the viral envelope glycoprotein. Proof of principle for this platform was demonstrated for protease substrate libraries, which can be selected through coupling proteolytic activation with viral infectivity (Buchholz et al., 1998). Selection of the library CX4A on living cells resulted in viruses with more than three orders of magnitude improved spreading efficiency through tumor cells (Hartl et al., 2005). Also scAb libraries have recently been displayed and selected using retroviruses (Urban et al., 2005). The library scFvlibxMo displays the repertoire of phage display preselected sc antibodies for laminin-1 binding. The retrovirus based selection process resulted in laminin-specific sc antibodies with improved expression levels in mammalian cells.
This thesis describes the in vivo (i.e. in mouse tumor models) selection of the C-X4-A and scFvlibxMo for tumor homing upon systemic delivery.
For selection of the protease substrate library C-X4-A a subcutaneous tumor was induced in SCID mice followed by three systemic injections of the library. The selection process was monitored over a period of 34 days. After the incubation period mice were sacrificed and virus load in organs and tumor determined. PCR analysis after 34 days showed that virus from the library had preferentially infected the tumor. Sequence analysis showed the selection of protease substrates with the most prominent one with a frequency of over 65%. The four most prominent protease substrate variants where reconstituted into the original viral backbone for further investigation (C-SK-A, C-HI-A, C-HM-A and C-HS-A). Interestingly, these viruses exhibited a reduced spreading capacity in vitro on HT1080 cells as compared to the C-AK-A virus, which had previously been selected on HT1080 cells. When assayed for tumor homing, however, viruses C-HI-A and C-HS-A had clearly improved in comparison to C-AK-A. Tumor tissue had been infected at rates of over 55% while virus load of extratumoral organs was very low (infection rates <0.7 for C-HS-A and <0.02 for C-HI-A). Tumor targeting capacity had thus been improved over 10-fold by the in vivo selection of the C-X4-A library.
The experimental set up for the in vivo selection of the scFvlibxMo library was performed according to that of the C-X4-A library. Fingerprint analysis of the selected viruses that infected tumor tissue resulted in the identification of seven antibody variants showing unique CDR3 sequences. Two prominent clones (M49T-A and M49T-B) were cloned back into the MoMLV genome for further analysis of the reconstituted viruses. While variant B bound laminin-1 efficiently, variant A was unable to do so, although it was selected at highest frequency (76%). Both reconstituted viruses were equally well infectious and spread through HT1080rec1 cells at a similar efficiency as MoMLV. In an in vivo competition experiment the selected viruses clearly out-competed a laminin-1 binding reference virus L36xMo for tumor homing. To understand the molecular driving forces behind the in vivo selection process the epitope of the selected scFv M49T-A was identified using a phage peptide library approach. In silico analysis led to the identification of a small group of possible antigens, including tenascin, fibronectin and collagen.
The data described in this thesis demonstrate that the retrovirus display platform is capable of allowing the in vivo selection of protease substrates and scFvs. Notably, the replication competence of the system introduced an additional level of complexity to the library. The performed in vivo selections significantly enhanced tumor tropism. Selective infection of tumor cells combined with transfer of anti-tumoral genes is an attractive strategy for cancer therapy being in focus of current research. The viruses selected in this thesis build prime candidates for targeted retrovirus based tumor therapy.
Drug toxicity and viral resistance limit long-term efficacy of antiviral drug treatment for HIV
infection. Thus, alternative therapies need to be explored. Previously, group of “Prof. von Laer”
tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an
HIV entry inhibitory peptide (maC46). Gene-modified autologous T cells were infused into 10
HIV-infected patients with advanced disease and multidrug resistant virus during antiretroviral
combination therapy. T cell infusions were tolerated well with no severe side effects. A
significant increase of CD4 counts was observed post infusion. At the end of the one-year
follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified
cells could be detected in peripheral blood, lymph nodes and bone marrow throughout the oneyear
follow-up, whereby marking levels correlated with the cell dose. No significant changes of
viral load were observed during the first four months. Four of the seven patients that changed
their antiviral drug regimen thereafter responded with a significant decline in plasma viral load.
In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene
marking and may improve immune competence in HIV-infected patients with advanced disease
and multidrug resistant virus. However, the low level of gene marking and the lack of substantial
long-term in vivo accumulation of gene-protected cells observed in this trial clearly demonstrate
the requirement for new vectors with new strategy.
In this thesis self‐inactivating lentiviral vectors harboring internal promoters and RNA elements
were therefore evaluated for their potential use in a clinical gene‐therapy trial. The results from
this work provide the basis for the selection of a suitable candidate vector for extensive
preclinical testing. Apart from being capable of transducing non‐dividing cells, lentiviral vectors
incorporate a number of additional features that are of potential value for gene therapeutic
applications. These include a larger packaging capacity, higher titers than γ‐retroviral vectors
and, most importantly, a reduced risk of deregulating cellular genes due to its natural integration
profile. The use of internal promoters to drive expression of the therapeutic transgene maC46
should further improve the safety profile of these new‐generation vectors, while an additional
artificial splice acceptor (SA) into the 5‟UTR of the transgene over all elevate transgene
expression. The rationale for this is that hematopoietic stem and progenitor cells will be
Summary
98
protected from enhancer‐mediated transactivation effects and also from potential side effects due
to the aberrant expression of maC46 while at the same time the full clinical benefit for the
patients is maintained.
In order to find a suitable candidate for preclinical studies, two candidate therapeutic vectors
harboring different regulatory elements were selected based on results from pilot experiments.
The internal promoters used to drive expression of codon optimized maC46 were the PGK
promoter and MPSV promoter. This work focuses on the transgene expression levels in
lymphoid cells and antiviral activity. The issues of long term expression, propensity to
methylation mediated silencing of the promoters, and genotoxicity were also touched. In a first
step the performance of different vectors was evaluated in the human T cell lines. Based on
promising data from ex vivo human peripheral blood mononuclear cells, the vector carrying the
MPSV promoter along with intron were selected for in vivo transplantation experiments.
In summary, the ex vivo data suggested the long term survival of lentiviral gene modified cells,
along with maintained expression of introduced genes. It was observed that the expression of
these constructs depends strongly on the activation and differentiation status of the targeted T
cells. This regulation was not linked to any specific promotor. In vivo study shows that maC46
can be introduced into murine multiple hematopoietic lineages via lentiviral vector and expressed
at high levels in their mulilineage progeny, without altering the hematopoiesis. There was no
sign of any kind of hematopoietic or lymphoid malignancies. Although gene-modified
lymphocytes persisted in-vivo, the downregulation of transgene expression was consistent with
the ex-vivo observation. In contrast to that the T cells transplanted group showed delayed
engraftment of donor cells and there was no expression of C46 in blood and lymphatic organs. .
In conclusion, when considering HIV gene therapy focusing CD4+ T cells, potential problems of
T cell activation status as related to the desired clinical effect must be addressed. These results
might open the way for a gene therapy targeting mainly or exclusively activated T cells and
could be exploited for immunostimulatory as well as suppressive approaches.
This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 × C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM—ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2–21 alleles and polymorphic information content value 0.04–0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago.
EGFL7 regulates adult neural stem cell maintenance and differentiation by inhibition of Notch1
(2010)
In neurobiology the preexisting dogma on the unchangeability of the adult mammalian brain and its inability to give rise to new neurons has been challenged since the early nineties. Generally, it is now accepted that neurogenesis persists in adults. Progress in developmental and stem cell biology in recent years led to an increasing interest in regeneration-based treatment strategies for damaged tissue of the central nervous system. Thus, the enhancement of the endogenous potential of the brain to repair itself is potentially a feasible therapeutic strategy to treat various types of brain damage. Therefore, it is of great interest to understand the molecular mechanism that regulate adult neurogenesis. One of the prominent pathways suggested to be involved here is the Notch signaling cascade. Previously, it has been shown that various components of Notch signaling are expressed in the stem cell niche of the adult subventricular zone (SVZ) in vivo. Interestingly, a recent study demonstrated that the self-renewal potential of adult neural stem cells (NSCs) isolated from the SVZ depend on Notch signaling in vitro.
Recently, we identified a novel non-canonical Notch ligand termed epidermal growth factor-like domain 7 (EGFL7), which was originally described as a protein secreted by endothelial cells and functionally implicated in cellular responses of the vascular system such as cell migration and blood vessel formation. We were able to show that secreted EGFL7 binds to a region in Notch that is involved in ligand-mediated receptor activation, thus acting as an antagonist of Notch signaling.
The present study identifies neurons of the human and murine brain as a novel source of EGFL7, which suggests functions of EGFL7 in the neural system. Expression analyses by quantitative RT-PCR (qRT-PCR) revealed EGFL7 is down regulated in the adult SVZ, which suggests that endogenous EGFL7 may act as a Notch modulator of NSCs. We assessed the expression of Notch pathway components in adult NSCs isolated from the SVZ of adult mice and demonstrated that inhibition of Notch activity by the γ-secretase inhibitor DAPT reduced the self-renewal potential of NSCs. Accordingly, adenoviral-mediated expression of EGFL7 in vitro decreased Notch-specific signaling and reduced proliferation and self-renewal of NSCs. Conversely, activation of Notch by a constitutive active form of Notch (NICD) rescued the EGFL7-mediated reduction of NSC self-renewal verifying that this effect was directly linked to Notch signaling. Congruent to the reduced proliferation rate measured in vitro, induced expression of EGFL7 in vivo significantly reduced the number of Ki-67 positive cells within the SVZ upon cerebroventricular injection of EGFL7 adenovirus.
Expression analyses in the developing brain showed single EGFL7-positive cells within the marginal zone of the neocortex as measured by in situ hybridization. These cells might be Cajal-Retzius cells, specialized neurons, which specifically express Reelin, which is a protein of the extracellular matrix known to control neuronal migration and differentiation. Interstingly, we could show that Reelin and EGFL7 are expressed in a subtype of neurons of the adult mouse cortex. This implied an interaction of both proteins and was verified by co-immunoprecipitation assays, suggesting an additional role for EGFL7 in neuronal maintenance. QRT-PCR based expression analyses in vitro comparing differentiated and non-differentiated NSCs displayed an increase in EGFL7 expression during the differentiation process, which was paralled by reduced Notch signaling. NSCs differentiated on coverslips coated with EGFL7 differentiated into all three cell types - neurons, oligodendrocytes and astrocytes. EGFL7 favored the formation of neurons as compared to control comparable to the effect of the Notch-inhibitor DAPT. Furthermore, additional oligodendrocytes were formed. These cells displayed a mature morphology with distinct sprouts and branches in contrast to the small and round oligodendrocytes that formed on control coverslips, which resembled us of precursor cells. Neurons and oligodendrocytes were formed at the expense of astrocytes. Congruently to the effect observed in vitro, adenoviral-based expression of EGFL7 in the SVZ yielded a slight induction of neuronal differentiation in vivo. Taken together, these results show for the first time a previously unrecognized role for EGFL7 in the brain by modulation of the Notch pathway in adult NSCs, which changes the proliferation and differentiation potential of adult NSCs in vitro and in vivo.
We report on screening tests of 66 extracts obtained from 35 marine sponge species from the Caribbean Sea (Curaçao) and from eight species from the Great Barrier Reef (Lizard Island). Extracts were prepared in aqueous and organic solvents and were tested for hemolytic, hemagglutinating, antibacterial and anti-acetylcholinesterase (AChE) activities, as well as their ability to inhibit or activate cell protein phosphatase 1 (PP1). The most interesting activities were obtained from extracts of Ircinia felix, Pandaros acanthifolium, Topsentia ophiraphidites, Verongula rigida and Neofibularia nolitangere. Aqueous and organic extracts of I. felix and V. rigida showed strong antibacterial activity. Topsentia aqueous and some organic extracts were strongly hemolytic, as were all organic extracts from I. felix. The strongest hemolytic activity was observed in aqueous extracts from P. acanthifolium. Organic extracts of N. nolitangere and I. felix inhibited PP1. The aqueous extract from Myrmekioderma styx possessed the strongest hemagglutinating activity, whilst AChE inhibiting activity was found only in a few sponges and was generally weak, except in the methanolic extract of T. ophiraphidites.
Background: Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research.
Methodology/Principal Findings: Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region.
Conclusions/Significance: The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.
Activation of Notch1 signaling in neural progenitor cells (NPCs) induces self-renewal and inhibits neurogenesis. Upon neuronal differentiation, NPCs overcome this inhibition, express proneural genes to induce Notch ligands, and activate Notch1 in neighboring NPCs. The molecular mechanism that coordinates Notch1 inactivation with initiation of neurogenesis remains elusive. Here, we provide evidence that Prox1, a transcription repressor and downstream target of proneural genes, counteracts Notch1 signaling via direct suppression of Notch1 gene expression. By expression studies in the developing spinal cord of chick and mouse embryo, we showed that Prox1 is limited to neuronal precursors residing between the Notch1+ NPCs and post-mitotic neurons. Physiological levels of Prox1 in this tissue are sufficient to allow binding at Notch1 promoter and they are critical for proper Notch1 transcriptional regulation in vivo. Gain-of-function studies in the chick neural tube and mouse NPCs suggest that Prox1-mediated suppression of Notch1 relieves its inhibition on neurogenesis and allows NPCs to exit the cell cycle and differentiate. Moreover, loss-of-function in the chick neural tube shows that Prox1 is necessary for suppression of Notch1 outside the ventricular zone, inhibition of active Notch signaling, down-regulation of NPC markers, and completion of neuronal differentiation program. Together these data suggest that Prox1 inhibits Notch1 gene expression to control the balance between NPC self-renewal and neuronal differentiation.
Sucrose- and H+-dependent charge movements associated with the gating of sucrose transporter ZmSUT1
(2010)
Background: In contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly proton-driven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features.
Methodology/Principal Findings: To unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H+ co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H+ transport was associated with a decrease in membrane capacitance (Cm). In addition to sucrose Cm was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these Cm changes, presteady-state currents (Ipre) of ZmSUT1 transport were analyzed. Decay of Ipre could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to Ipre, was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed Cm changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependent-potassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I = Q/τ) was sufficient to predict ZmSUT1 transport-associated currents.
Conclusions: Taken together our results indicate that in the absence of sucrose, ‘trapped’ protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to Cm changes. Upon application of external sucrose, protons can pass the membrane turning presteady-state into transport currents.
Background: Leishmaniasis is a chronic disease that is found in various countries of the world. The aim of the current study was to investigate the impact of leishmaniasis on the world's research output. The present study assessed benchmarking of research output for the period between 1957 and 2006. Using large database analyses, research in the field of leishmaniasis was evaluated. Furthermore, cooperation between different countries was identified.
Results: The number of publications increased with time. Most publications came from Western countries such as the US, UK or Germany. Interestingly, countries like Brazil and India had a high research output. We found a substantial amount of cooperation between countries.
Conclusion: Although leishmaniasis is of limited geographic distribution it attracts a wide research interest. The central hub of research cooperation is the USA.
Tumor hypoxia and nutrient starvation are common phenomena in cancerous tissue. Cells that resist this hostile environment are selected for a more aggressive phenotype, usually accompanied by therapy resistance. The hypoxia inducible factors HIF-1a and HIF-2a play a key role in the adaptive homeostatic responses to these challenging conditions inducing a number of target genes that are involved in the regulation of a variety of cellular processes such as angiogenesis, proliferation, metabolism, self-renewal and cell death/cycle arrest. Thus, the HIF pathway encompasses opposing adaptive responses on tumor growthgrowth promoting abilities on the one hand and growth inhibiting on the other. A recent study in our lab uncovered that this switch between cell death and cell survival critically depends on HIF-2a protein levels. Since PHDs (HIF prolyl hydroxylases) are the main regulators of HIF protein abundance and hypoxia drives the malignant phenotype of tumors, we wanted to characterize HIF regulatory functions of PHDs under hypoxic conditions. Our intention was to reveal the importance of PHD contribution to the opposing functions of HIFs under hypoxia. Characterization of PHD1-4 mRNA and protein expression levels under normoxic and hypoxic conditions in glioblastoma cell lines led to the identification of PHD2 and PHD3 as hypoxia inducible PHD isoforms and highlighted their predominant function under hypoxia. Mechanistically, we demonstrated that HIF mediates the hypoxic induction of PHD2 and 3 within a negative feedback loop, promoting its own degradation during prolonged hypoxia. The functional impact of PHD2 and 3 abundance on cell viability under hypoxic conditions was analyzed by disrupting PHD2 and PHD3 function either through a siRNA mediated approach or by application of the PHD inhibitor DMOG. These experiments uncovered that PHD2 and 3 are protective under hypoxic conditions and that PHD inhibition expedites cell death. Combined HIF and PHD suppression under hypoxic conditions abrogated this increased susceptibility to cell death, clearly showing that PHD2 and 3 act in a negative feedback regulatory loop to limit the HIF response under prolonged hypoxia. With respect to possible future therapeutical applications we co-treated cells with a PHD inhibitor and pro-apoptotic agents staurosporine or TRAIL. Co-challenging tumor cells even potentiated the cell death response, indicating a more widespread protective function of PHD. Taken together PHD2 and 3 protect tumor cells from cell death induction, functioning in a negative feedback regulatory loop to constrain the HIF dependent cell death responses under hypoxia. Interestingly, however, when assessing the role of PHD2 and PHD3 in in vivo tumor growth using an intracranial tumor model, we identified an exclusive tumor suppressor function for PHD3. Loss of PHD3 function enhanced tumor growth whereas increased PHD3 expression diminished the tumor burden. The accelerated tumor growth following PHD3 loss could be attributed to a decrease in the induction of apoptosis and an increase in proliferation. Tumor cells are frequently exposed to temporary and spatial depletion of nutrients. Interestingly, PHD3 loss conferred a growth advantage under growth factor deprivation. The growth regulatory function of PHD3 was isoform specific, HIF independent and importantly, did not require the hydroxylase function of PHD3. Previous reports have uncovered a regulatory function of the PHD system in NF-kB signaling. However, our results demonstrated that NF- kB signaling remained unaffected by alteration in the PHD3 status of the cell. Additionally, the PHD3 tumor suppressor function proved to be independent of two putative PHD3 downstream effectors, ATF4 and KIF1Bb. Mechanistically, PHD3 suppression reduced EGFR internalization, enhancing the amount of EGFR expressed on the cell surface. We further showed that the impaired EGFR internalization during PHD3 loss resulted in receptor hyperactivation under stimulated and growth factor deprived conditions. Importantly, PHD3 physcially associated with the EGFR complex as evidenced by co-immunoprecpitation. Consequently, this extended EGFR activation in PHD3 deficient cells resulted in enhanced downstream activation of EGFR signaling and increased proliferation. Consistent with the interpretation that PHD3 loss is beneficial for tumor growth, we found PHD3 promoter methylation in glioblastoma cell lines, hinting at a epigenetic mechanism to finetune PHD3 expression on top of the hypoxic driven gene regulation. Finally, we demonstrated that PHD3 tumor suppressor function is not restricted to glioblastomas since PHD3 suppression in lung adenocarcinoma accelerated subcutaneous tumor growth. With these findings, we expand the knowledge of PHD3 action from its oxygen sensing role to a regulatory function in growth factor signaling. This clearly discriminates PHD3 from the other isoforms and supports the exclusive tumor suppressor function in glioblastoma. Taken together our results identify a complex role of PHD signaling in cancer and delineate HIF dependent and HIF independent functions of the PHD system. We think that the HIF dependent protective effect of PHD2 and 3 and the HIF independent PHD3 tumor suppressor function are not mutually exclusive, but might be activated according to the heterogeneous intra-tumoral conditions. However, PHD3 hydroxylase activity is dispensable for its HIFindependent tumor suppressor function in glioma. This uncouples PHD3 function from co-factor and co-substrate requirements and allows it to act over a broader physiological range, since its influence on cellular processes is not constrained by the availability of rate limiting factors. It might explain, why the enzymatic independent functions of PHD3 predominate in vivo. Thus, therapeutic modulation of the PHD system to inhibit tumor growth has to be based on these contrasting functions of the PHD system. However, their differential dependence on the hydroxylase activity may facilitate a therapeutic strategy to specifically inhibit or promote the protective versus suppressive functions of the PHD system.
Project I: The progression of rod and cone degeneration in retinally degenerate (rd) mice ultimately results in a complete loss of photoreceptors and blindness. The inner retinal neurons survive and several recent studies using genetically targeted, light activated channels have made these neurons intrinsically light sensitive. We crossbred a transgenic mouse line expressing channelrhodopsin2 (ChR2) under the control of the Thy1 promoter with the Pde6b(rd1) mouse, a model for retinal degeneration (rd1/rd1). Approximately 30-40% of the ganglion cells of the offspring expressed ChR2. Extracellular recordings from ChR2-expressing ganglion cells in degenerated retinas revealed their intrinsic light sensitivity which was approximately 7 log U less sensitive than the scotopic threshold and approximately 2 log U less sensitive than photopic responses of normal mice. All ChR2-expressing ganglion cells were excited at light ON. The visual performance of rd1/rd1 mice and ChR2 rd1/rd1 mice was compared. Behavioral tests showed that both mouse strains had a pupil light reflex and they were able to discriminate light fields from dark fields in the visual water task. Cortical activity maps were recorded with optical imaging. The ChR2rd1/rd1 mice did not show a better visual performance than rd1/rd1 mice. In both strains the residual vision was correlated with the density of cones surviving in the peripheral retina. The expression of ChR2 under the control of the Thy1 promoter in retinal ganglion cells does not rescue vision. Project II: Lentiviral vectors are becoming the vector of choice for transgene delivery into cells due to their ability to infect non- dividing cells and stably integrate the gene into the genome of the host. Two different viral vector systems, namely HIV-1 and SIV and three different viral vectors PLECYT, PHRCMVChR2 of HIV-1 family and PBjChR2 of SIV were used in this study. The efficiency of the vectors was analyzed by applying them onto the retinal explants in culture and checking the transgene expression. The transgene in the PLECYT lentiviral vector was driven by the EF1A promoter. Upon administration of 5.2 X 106 infectious units of PLECYT viral vector suspension onto the retinal explant resulted in the transduction of retinal ganglion cells. Very few other retinal neurons were found transduced. In the case of PHRCMVChR2, approximately 5 X 105 TU/ml of the vector was used and resulted in the transduction of different neuronal subtypes. Many amacrine cells, ganglion cells and Müller cells were found expressing the transgene. For PBjChR2, 5.6 X104 TU/ml was used which resulted in Müller cell- specific transduction. Very few or no other retinal neurons were found transduced. This study demonstrates the transduction efficiency of different viral vectors on the retinal neurons in vitro. An interesting observation on these viral vectors is their altered tropism. The glycoprotein of the virus is critical for determining their tropism and in this study, all the viral vectors generated were pseudotyped with VSVG, which confers a broad non-specific spectrum of infection. However, analyzing the transgene expression, the viral vectors differ from one another and show remarkable difference in their transduction pattern. To list a few factors that might possibly responsible for the drastic transduction difference exerted by the viral vectors include; 1. Promoters used to drive the transgene expression. 2. HIV or SIV component of the vector in combination with the promoter 3. Titre of the vector used and 4. Other factors like pH and serum used in the study. Therefore optimizing the viral vectors and generating high titers would increase the efficiency and cell-type specific expression of the transgene.
Fuer die schlechte Prognose von Glioblastompatienten mit einer ueberlebenszeit von 9-15 Monaten (Norden and Wen, 2006) ist vor allem die hohe Invasivitaet dieser Tumore verantwortlich. Nach operativer Entfernung des Haupttumors entstehen aus den verbleibenden invadierten Zellen sekundaere Tumore, die sich mitunter ueber weite Bereiche des Hirns verteilen. Des Weitern sind die hochinvasiven Tumorzellen oft resistent gegen Chemo- und Strahlentherapie (Drappatz et al., 2009; Lefranc et al., 2005). In Maustumormodellen und Pateinten konnte zudem gezeigt werden, dass die neuartige antiangiogenetische Therapie zwar das Tumorwachstum verringert, jedoch die Invasivitaet stark erhoeht. (Norden et al., 2008; Ebos et al., 2009; Paez-Ribes et al., 2009). Ueber die Mechanismen die diese hohen Invasivitaet induzieren, ist bislang nur sehr wenig bekannt. Die durch Reduktion von Blutgefaessen steigende Hypoxie des Tumors foerdert die Expression von Matrix-Metalloproteinasen (MMPs). Dies fuehrt zum Abbau der extrazelluaeren Matrix des umgebenden gesunden Gewebes und beguenstigt dadurch die Tumorzellinvasion (Indelicato et al., 2010; Miyazaki et al., 2008; Shyu et al., 2007). Die Umformung des Aktinzytoskeletts und damit die Mobilitaet von Zellen wird vorwiegend durch ein akkurates Zusammenspeil der Rho GTPasen Rac, Rho und Cdc42, kontrolliert (Ridley et al., 2003). Fuer die Organisation von Axonen im Nervensystem und fuer die Blut- und Lymphgefaessbildung wurde gezeigt, dass die Interaktion der Eph-Rezeptortyrosinkinasen und Ihrer Ephrin-Liganden Signalwege induziert, die in die Regulation dieses Zusammenspiels involviert sind (Egea and Klein, 2007; Makinen et al., 2005; Palmer et al., 2002; Sawamiphak et al., 2010). Des Weiteren zeigt die Analyse der Genloci von Eph-Rezeptoren und Ephrinen in verschieden Hirntumoren eine gehaeufte Deletionen des Ephrin-B2-Gens. Die Quantifizierung von Ephrin-B2 mRNA in diesen Tumoren hat ausserdem ergeben, dass mit zunehmender Malignitaet die Expression von Ephrin-B2 sinkt. Aus diesen Gruenden wurden die Untersuchungen in dieser Arbeit auf die Rolle von Ephrin-B2 anhaengigen Signalwegen in der Glioblastomzellinvasion konzentriert. In einem modifiziertem Boyden-Chamber-Assay konnte gezeigt werden, dass das Ephrin-B2 induzierte EphB4 forward signaling und EphB4 induzierte Ephrin-B2 reverse signaling die Invasivitaet der human Glioblastomzelllinien LN-229, G55 und SNB-19 reduziert. In einem Maustumormodel konnte weiterhin gezeigt werden, dass Ephrin-B2 Knock-Out (KO) Astrozytomzellen, im Vergleich zu Wild-Typ (WT) Zellen, Tumore mit einem groesseren Volumen und einer erhoehten Invasivitaet bilden. Da die Expressionslevel fuer die Ephrin-B2 bindenden Rezeptoren EphA4, EphB1 EphB3 und EphB6 auch im adulten Hirn hoch sind (Hafner et al., 2004), weisen diese in vitro und in vivo Ergebnisse auf eine Tumorsupressorfunktion von Ephrin-B2 hin, die durch repulsive Effekte des Ephrin-B2 reverse signaling vermittelte werden koennten. Dies geht mit Erkenntnissen ueber kolorektale Tumore einher (Batlle et al., 2005). Die in einem Sphaeroid-Invasionsassay mit einer EphB-Rezeptoren freien Umgebung beobachtete verminderte Invasion von Ephrin-B2 WT deutet auf eine zusaetzliche invasionsblockierende Rolle der Ephrin-B2-Eph-Rezeptor Interaktion zwischen benachbarten Tumorzellen hin, wie sie auch in Brusttumoren gefunden wurde (Noren et al., 2006). Es scheint als sei Tumorprogression und Invasion erst moeglich, nachdem die Expression von Ephrin-B2 vermindert wurde. Es konnte weiterhin gezeigt werden, dass in hypoxischen Glioblastomzellen die Ephrin-B2 Expression durch die direkte Bindung des den Transkriptionsfaktors ZEB2 an den Ephrin-B2 Promoter reprimiert wird. In einem Weiteren Maustumormodel konnte gezeigt werden, dass die Blockierung der ZEB2 Expression mittels shRNA und die damit einhergehenden Inhibition der hypoxie induzierten Ephrin-B2 Repression das Wachstum und die Invasivitaet von Glioblastomen verringert. Zusaetzlich wurde gezeigt, dass der Verlust von ZEB2 ausreicht, die durch antiangiogenetische Therapie induzierte stark erhoehte Invasivitaet zu vermeiden. Die in dieser Arbeit gewonnen Erkenntnisse fuehren zu folgendem Modelmechanismus. In kleinen normoxischen Tumoren koennen repulsive Effekte des Ephrin-B2 reverse signalings und EphB forward signalings zwischen Tumorzellen und Zellen des umgebenden Gewebes die Ausbreitung und Invasion des Tumors unterdruecken. Zusaetzlich koennte das Ephrin-B2 induzierte EphB forward signaling zwischen benachbarten Tumorzellen die Mobilitaet der Tumorzellen wie in Brusttumoren inhibieren. Beim Erreichen einer bestimmten Tumorgroesse tritt Hypoxie auf, wodurch HIF-1alpha stabilisiert wird. Dies fuehrt dann zur ZEB2 Expression und leitet die Repression von Ephrin-B2 ein, was wiederum zur erhoehten Tumorzellemobilitaet und im Zusammenspiel mit MMPs zu Invasion fuehren kann. Gleichzeitig werden durch den HIF-induzierten VEGF-Gradienten neue Blutgefaesse rekrutiert. Damit wird der hypoxie-induzierten Invasivitaet entgegengewirkt. Wird mittels antiangiogenetischer Behandlung versucht Tumorprogression entgegenzuwirken, resultiert daraus eine erneut gesteigerte Hypoxie, die dann durch die ZEB2 vermittelte Repression von Ephrin-B2 wieder eine erhoehte Invasivitaet induzieren kann. Das Blockieren der ZEB2 Expression kann dieser durch antiangiogenetischen Behandlung induzierten Invasivitaet entgegenwirken.
Background: ClC-7 is a ubiquitous transporter which is broadly expressed in mammalian tissues. It is implied in the pathogenesis of lysosomal storage disease and osteopetrosis. Because of its endosomal/lysosomal localization it is still poorly characterized. Methodology/Principal Findings: An electrophysiological characterization of rat ClC-7 using solid-supported membrane-based electrophysiology is presented. The measured currents show the characteristics of ClC-7 and confirm its function as a Cl−/H+-antiporter. We have used rat ClC-7 in CHO cells as a model system to investigate the functionality and cellular localization of the wt transporter and its variant G213R ClC-7 which is the analogue of human G215R ClC-7 responsible for autosomal dominant osteopetrosis type II. Our study shows that rat G213R ClC-7 is functional but has a localization defect in CHO cells which prevents it from being correctly targeted to the lysosomal membrane. The electrophysiological assay is tested as a tool for drug discovery. The assay is validated with a number of drug candidates. It is shown that ClC-7 is inhibited by DIDS, NPPB and NS5818 at micromolar concentrations. Conclusions/Significance: It is suggested that the scenario found in the CHO model system also applies to the human transporter and that mislocalization rather than impaired functionality of G215R ClC-7 is the primary cause of the related autosomal dominant osteopetrosis type II. Furthermore, the robust solid-supported membrane-based electrophysiological assay is proposed for rapid screening for potential ClC-7 inhibitors which are discussed for treatment of osteoporosis.
We established a protocol of the SuperSAGE technology combined with next-generation sequencing, coined “High-Throughput (HT-) SuperSAGE”. SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. In the present protocol, index (barcode) sequences are employed to discriminate tags from different samples. Such barcodes allow researchers to analyze digital tags from transcriptomes of many samples in a single sequencing run by simply pooling the libraries. Here, we demonstrated that HT-SuperSAGE provided highly sensitive, reproducible and accurate digital gene expression data. By increasing throughput for analysis in HT-SuperSAGE, various applications are foreseen and several examples are provided in the present study, including analyses of laser-microdissected cells, biological replicates and tag extraction using different anchoring enzymes.
Background: Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype. Methodology/Principal Findings: We found that Per2Brdm1 mutant mice as well as mice lacking Cry2-/- displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2-/- displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2-/- mutants despite the simultaneous inactivation of Per2. Conclusions/Significance: This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters.
Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells
(2010)
Background: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. Methodology/Principal Findings: The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. Conclusion/Significance: Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.
Background: The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings: We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal alpha-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.
Variants resistant to compounds specifically targeting HCV are observed in clinical trials. A multi-variant viral dynamic model was developed to quantify the evolution and in vivo fitness of variants in subjects dosed with monotherapy of an HCV protease inhibitor, telaprevir. Variant fitness was estimated using a model in which variants were selected by competition for shared limited replication space. Fitness was represented in the absence of telaprevir by different variant production rate constants and in the presence of telaprevir by additional antiviral blockage by telaprevir. Model parameters, including rate constants for viral production, clearance, and effective telaprevir concentration, were estimated from 1) plasma HCV RNA levels of subjects before, during, and after dosing, 2) post-dosing prevalence of plasma variants from subjects, and 3) sensitivity of variants to telaprevir in the HCV replicon. The model provided a good fit to plasma HCV RNA levels observed both during and after telaprevir dosing, as well as to variant prevalence observed after telaprevir dosing. After an initial sharp decline in HCV RNA levels during dosing with telaprevir, HCV RNA levels increased in some subjects. The model predicted this increase to be caused by pre-existing variants with sufficient fitness to expand once available replication space increased due to rapid clearance of wild-type (WT) virus. The average replicative fitness estimates in the absence of telaprevir ranged from 1% to 68% of WT fitness. Compared to the relative fitness method, the in vivo estimates from the viral dynamic model corresponded more closely to in vitro replicon data, as well as to qualitative behaviors observed in both on-dosing and long-term post-dosing clinical data. The modeling fitness estimates were robust in sensitivity analyses in which the restoration dynamics of replication space and assumptions of HCV mutation rates were varied.
The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free) expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate) mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent) mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated Pf value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.
Structured RNA regions are important gene control elements in prokaryotes and eukaryotes. Here, we show that the mRNA of a cyanobacterial heat shock gene contains a built-in thermosensor critical for photosynthetic activity under stress conditions. The exceptionally short 5´-untranslated region is comprised of a single hairpin with an internal asymmetric loop. It inhibits translation of the Synechocystis hsp17 transcript at normal growth conditions, permits translation initiation under stress conditions and shuts down Hsp17 production in the recovery phase. Point mutations that stabilized or destabilized the RNA structure deregulated reporter gene expression in vivo and ribosome binding in vitro. Introduction of such point mutations into the Synechocystis genome produced severe phenotypic defects. Reversible formation of the open and closed structure was beneficial for viability, integrity of the photosystem and oxygen evolution. Continuous production of Hsp17 was detrimental when the stress declined indicating that shutting-off heat shock protein production is an important, previously unrecognized function of RNA thermometers. We discovered a simple biosensor that strictly adjusts the cellular level of a molecular chaperone to the physiological need.
The plastids of cryptophytes, haptophytes, and heterokontophytes (stramenopiles) (together once known as chromists) are surrounded by four membranes, reflecting the origin of these plastids through secondary endosymbiosis. They share this trait with apicomplexans, which are alveolates, the plastids of which have been suggested to stem from the same secondary symbiotic event and therefore form a phylogenetic clade, the chromalveolates. The chromists are quantitatively the most important eukaryotic contributors to primary production in marine ecosystems. The mechanisms of protein import across their four plastid membranes are still poorly understood. Components of an endoplasmic reticulum-associated degradation (ERAD) machinery in cryptophytes, partially encoded by the reduced genome of the secondary symbiont (the nucleomorph), are implicated in protein transport across the second outermost plastid membrane. Here, we show that the haptophyte Emiliania huxleyi, like cryptophytes, stramenopiles, and apicomplexans, possesses a nuclear-encoded symbiont-specific ERAD machinery (SELMA, symbiont-specific ERAD-like machinery) in addition to the host ERAD system, with targeting signals that are able to direct green fluorescent protein or yellow fluorescent protein to the predicted cellular localization in transformed cells of the stramenopile Phaeodactylum tricornutum. Phylogenies of the duplicated ERAD factors reveal that all SELMA components trace back to a red algal origin. In contrast, the host copies of cryptophytes and haptophytes associate with the green lineage to the exclusion of stramenopiles and alveolates. Although all chromalveolates with four membrane-bound plastids possess the SELMA system, this has apparently not arisen in a single endosymbiotic event. Thus, our data do not support the chromalveolate hypothesis. Key words: Emiliania huxleyi, secondary endosymbiosis, chromalveolate, hypothesis, complex plastid, plastid protein import, algal evolution
Wastewater treatment plants (WWTPs) do not eliminate micropollutants completely and are thus important point sources for these substances. In particular, concerns about en-docrine disrupting compounds in WWTP effluents give rise to the implementation of advanced treatment steps for the elimination of trace organic contaminants. The present study investigated ozonation (O3) and activated carbon treatment (AC) at two WWTPs. For an ecotoxicological assessment at WWTP Regensdorf, conventionally treated wastewater, wastewater after ozonation, and ozonated wastewater after sand filtration were evaluated in parallel via the fish early life stage toxicity test (FELST) using rainbow trout (Oncorhynchus mykiss). Additionally, a comparative toxicity evalu-ation of ozonated and activated carbon treated effluents was performed at the pilot scale treatment plant in Neuss (WWTP Neuss). For this purpose, four invertebrate tests and one higher plant toxicity test were selected to assess potential biological effects on or-ganisms [Lemna minor growth inhibition test, chironomid toxicity test with Chironomus riparius, Lumbriculus variegatus toxicity test, comet assay with haemolymph of the zebra mussel (Dreissena polymorpha), reproduction test with Potamopyrgus antipo-darum]. All in vivo assays were performed on site at the treatment plants in flow-through test systems. Furthermore, the present study investigated the effects of ozona-tion and activated carbon treatment on endocrine activities [estrogenicity, anti-estrogenicity, androgenicity, anti-androgenicity, aryl-hydrocarbon receptor (AhR) agonistic activity] with yeast based bioassays using solid phase extracted water samples. To evaluate the removal of in vitro non-specific toxicity, a cytotoxicity assay using a rat cell line was applied. The FELST at WWTP Regensdorf revealed a considerable developmental retardation of test organisms exposed to ozonated WW. This was accompanied by a significant decrease in body weight and length compared to reference water, to the conventionally treated WW, and to the ozonated water after sand filtration. Hence sand filtration obvi-ously prevents from adverse ecotoxicological effects of ozonation. An additional test – starting with yolk-sac larvae – resulted in a significant reduction of vitellogenin levels in fish exposed to ozonated wastewater compared to fish reared in conventionally treat-ed wastewater. This demonstrates the effective removal of estrogenic activity by ozonation. At WWTP Neuss, the reproduction test with the mudsnail P. antipodarum exhibited a decreased reproductive output after advanced treatment compared to conventional treatment. This indicates an effective estrogenicity removal by ozonation and activated carbon treatment and is confirmed by results of the yeast estrogen screen with a reduc-tion of in vitro estrogenic activity by > 75%. The L. variegatus test revealed a signifi-cantly enhanced toxicity after ozonation compared to conventional treatment, whereas this effect was reduced following subsequent sand filtration. When ozonation was applied, a significantly increased genotoxicity was observed, detected with the comet assay using haemolymph of the zebra mussel. Again, this effect was removed by subsequent sand filtration to the level of conventional treatment. Activated carbon treatment even resulted in a significant reduction of genotoxicity. At both treatment plants, adverse effects after ozonation may have been a result of the formation of toxic oxidation by-products. However, sand filtration reduced toxication effects, indicating that these oxidation by-products are readily degradable or adsorbable. The results point out that, in any case, ozonation should not be applied without subsequent biologically active post treatment appropriate for oxidation by-products removal (e.g. sand filtration). However, only activated carbon achieved a toxicity reduction compared to the conventional treated wastewater. Thus, it cannot be excluded that po-tential beneficial effects due to ozonation might be masked by residual toxic oxidation by-products passing the sand filter or ozonation is not as effective in toxicity removal as PAC treatment. The yeast based assays with solid phase extracted samples revealed an effective endo-crine activity removal during ozonation and activated carbon filtration (estrogenicity: 77 – 99%, anti-androgenicity: 63 – 96%, AhR agonistic activity: 79 – 82%). The cyto-toxicity assay exhibited a 32% removal of non-specific toxicity after ozonation com-pared to conventional treatment. Ozonation in combination with sand filtration reduced cytotoxic effects by 49%, indicating that sand filtration contributes to the removal of toxicants. Activated carbon treatment was the most effective technology for cytotoxici-ty removal (61%). Sample evaporation reduced cytotoxic effects by 52% (after activated carbon treatment) to 73% (after ozonation), demonstrating that volatile substances contribute considerably to toxic effects, particularly after ozone treatment. These results confirm an effective removal or transformation of toxicants with receptor mediated mode of action and non-specific toxicants during both investigated treatment steps. However, due to the limited extractability, polar ozonation by-products were neglected for toxicity analysis, and hence non-specific toxicity after O3 is underestimated. In the long run, only on-site comparisons at WW receiving water bodies (e.g. communi-ty analysis of fish, macroinvertebrates, plants, microorganisms) – before and after up-grading WWTPs – allow drawing environmentally relevant conclusions regarding bene-fits and risks of advanced WW treatment methods. Conclusively, the benefits and possible negative impacts have to be carefully evaluated to prove that not more environmental impact will be induced than removed by advanced treatment technologies as each additional treatment requires considerable amounts of energy, resources, and infrastructure facilities. Accordingly, comprehensive sustainable approaches for pollution prevention and wastewater treatment (e.g. source control and source separation) are preferable compared to end-of-pipe treatment systems.
Employing NMR spectroscopy, it is not only possible to calculate the three dimensional structures of single proteins, but also to study dynamics and conformational changes of protein-complexes. In fact that is an important aspect, since the protein function depends on dynamics and interactions with other molecules. Therefore the study of protein-protein interactions is of highest importance for a better understanding of biological processes. Based on NMR methods, in this thesis we were able to determine protein-protein interactions within the enterobacterial Rcs signalling complex which is regulated via a phosphorelay. Originally identified as regulator of capsule synthesis, the Rcs phosphorelay is now considered to be implicated in stress response caused by disturbances in the peptidoglycan layer. Beyond that the Rcs system is involved in multiplex transcriptional networks including cell division, motility, biofilm formation and virulence. Because of such global nature and its extraordinary structural organisation involving membrane integrated sensor proteins (RcsC, RcsD), coactivators (RcsF, RcsA) and a transcription factor (RcsB), the Rcs system is one of the most remarkable phosphorelays in the family of enterobacteriacaea. During the complex phosphotransfer the histidine phosphotransferase (HPt) domain of the intermediary RcsD protein mediates the phosphotransfer between RcsC and RcsB, and probably modulates the phosphorylation state of the response regulator RcsB. Therefore the present work has been focused on the interface between RcsD and RcsB in more detail. In the first part of the thesis a new domain within the RcsD protein has been identified and structurally analysed by liquid NMR spectroscopy. RcsD is an inner membrane bound hybrid sensor like-kinase composed of a periplasmic sensor domain and a cytoplasmic portion. The cytoplasmic part contains the histidine like-kinase (HK) domain and the histidine phosphotransferase (HPt) domain. By analysis of the secondary structure in more detail, it was shown here that the two domains are intermitted by an additional 13.3 kDa domain. Corresponding to the position of the ABL (α−β−loop) domain of RcsC, located C-terminal to the RcsC-HK domain, the new identified domain was named RcsD-ABL. The central structural element of RcsD-ABL is a β-sheet composed of six strands with a β1−β2−β3−β4−β6−β5 topology and surrounded by two α-helices α1 and α2. In the second part of the thesis, RcsD-ABL is identified as a binding domain for the response regulator RcsB by NMR titration experiments. Such a binding domain for a response regulator has so far only been described for the histidine kinase CheA. In reportergene assays with β-galactosidase and ONPG as substrate it was shown that overexpression of RcsD-ABL in high amounts inhibited binding of RcsB to its target promoter. The β-galactosidase activity was reduced by 80 % with respect to cells carrying no plasmid encoding RcsD-ABL. The mapping of the binding interface was successfully achieved by chemical shift perturbations, a fast mapping protocol and selective labelling. It was shown that the interaction between RcsD-ABL and RcsB takes place via a binding interface comprising mainly the two α-helices of RcsD-ABL and the α-helices α7, α8 and α10 in the effector domain of RcsB. In the third part of the thesis, the interaction of RcsB with RcsD-ABL was related to that with RcsD-HPt. Using NMR titration experiments and ITC measurements, a comparison of the binding constants (Kd) of RcsB interacting either with the isolated RcsD-ABL (2 PM) or the isolated RcsDHPt domain (40 PM) revealed a higher affinity of RcsD-ABL to RcsB. A conjugate of RcsD-ABL-HPt interacting with RcsB decreased the Kd in the one-site fitting mode to 10 PM. However, the two-site fitting mode applied for RcsD-ABL-HPt/RcsB interaction resulted in a Kd (RcsD-ABL) of 2 PM and a Kd (RcsD-HPt) of 8 PM, indicating that RcsD-ABL enhances the binding of RcsD-HPt to RcsB. In the last part of the thesis, it was partly possible together with the data obtained from NMR titration experiments, PRE measurements and a HADDOCK protocol to develop a geometrical model for the interaction of RcsD with RcsB. In this model the receiver domain of RcsB interacts with the RcsD-HPt domain and the RcsB effector domain interacts with the RcsD-ABL domain. These results lead to surprising insights on the regulation of phosphorelays, since normally the effector domain binds to DNA. Here the effector domain is recognized by the newly identified RcsD-ABL domain. Prospectively, further investigations of phosphorylation affects and mutational studies will be of great interest.
With which political developments is BiKF confronted as a research centre as well as concerning its research and transfer efforts? Are there any hints for emerging research questions that meet practical needs? This paper gives an overview – as of June 2010 – on priority issues in the run-up to CBD’s COP-10, the 10th Conference of the Parties to the Convention on Biological Diversity (CBD), which will take place in Nagoya/Japan in October 2010. Highlighted discourse threads are: (1) the state of negotiations for an Access and Benefit Sharing (ABS) regime within CBD, (2) European and international preparations for renewing the political objectives for protecting biodiversity (Post-2010 Targets) and (3) the recent decision on an Intergovernmental Science-Policy Platform for Biodiversity and Ecosystem Services (IPBES). These three threads are selected against the background of an in depth analysis of the discourse field which was carried out in 2008/09 for BiKF. They show how the field progresses and which developments are worth being incorporated into BiKF’s further work. This Knowledge Flow Paper documents the talk given by the author during the second BiKF Retreat, 17–18 July 2010.
One of the key functions of blood vessels is to transport nutrients and oxygen to distant tissues and organs in the body. When blood supply is insufficient, new vessels form to meet the metabolic tissue demands and to re-establish cellular homeostasis. Expansion of the vascular network through sprouting angiogenesis requires the specification of ECs into leading (sprouting) tip and following (non-sprouting) stalk cells. Attracted by guidance cues tip cells dynamically extend and retract filopodia to navigate the nascent vessel sprout, whereas trailing stalk cells proliferate to form the extending vascular tube. All of these processes are under the control of environmental signals (e.g. hypoxia, metabolism) and numerous cytokines and peptide growth factors. The Dll4/Notch pathway coordinates several critical steps of angiogenic blood vessel growth. Even subtle alterations in Notch activity can profoundly influence endothelial cell behavior and blood vessel formation, yet little is known about the intrinsic regulation and dynamics of Notch signaling in endothelial cells. In addition, it remains an open question, how different growth factor signals impinging on sprouting ECs are coordinated with local environmental cues originating from nutrient-deprived, hypoxic tissue to achieve a balanced endothelial cell response. Acetylation of lysines is a critical posttranslational modification of histones, which acts as an important regulatory mechanism to control chromatin structure and gene transcription. In addition to histones, several non-histone proteins are targeted for acetylation reversible acetylation is emerging as a fundamental regulatory mechanism to control protein function, interaction and stability. Previous studies from our group identified the NAD+-dependent deacetylase SIRT1 as a key regulator of blood vessel growth controlling endothelial angiogenic responses. These studies revealed that SIRT1 is highly expressed in the vascular endothelium during blood vessel development, where it controls the angiogenic activity of endothelial cells. Moreover, in this work SIRT1 has been shown to control the activity of key regulators of cardiovascular homeostasis such as eNOS, Foxo1 and p53. The present study describes that SIRT1 antagonizes Notch signaling by deacetylating the Notch intracellular domain (NICD). We showed that loss of SIRT1 enhances DLL4-induced endothelial Notch responses as assessed by different luciferase responsive elements as well as transcriptional analysis of Notch endogenous target genes activation. Conversely, SIRT1 gain of function by overexpression of pharmacological activation decreases induction of Notch targets in response to DLL4 stimulation. We also showed that the NICD can be directly acetylated by PC AF and p300 and that SIRT1 promotes deacetylation of NICD. We have identified 14 lysines that are targeted for acetylation and their mutation abolishes the effects of SIRT1 of Notch responses. Furthermore, over-expression or activation of SIRT1 significantly reduces the levels of NICD protein. Moreover, SIRT1-mediated NICD degradation can be reversed by blockade of the proteasome suggesting a mechanism resulting from ubiquitin-mediated proteolysis. Indeed, we have shown that SIRT1 knockdown or pharmacological inhibition decreased NICD ubiquitination. We propose a novel molecular mechanism of modulation of the amplitude and duration of Notch responses in which acetylation increases NICD stability and therefore permanence at the promoters, while SIRT1, by inducing NICD degradation through its deacetylation, shortens Notch responses. In order to evaluate the physiological relevance of our findings we used different models in which the Notch functions during blood vessel formation have been extensively characterized. First, retinal angiogenesis in mice lacking SIRT1 activity shows decreased branching and reduced endothelial proliferation, similar to what happens after Notch gain of function mutations. ECs from these mice exhibit increased expression of Notch target genes. Second, these results were reproducible during intersomitic vessel growth in sirt1-deficient zebrafish. In both models, the defects could be partially rescued by inhibition of Notch activation. Third, we used an in vitro model of vessel sprouting from differentiating embryonic bodies in response to VEGF in a collagen matrix. Our results showed that Sirt1-deficient cells shows impaired sprouting which correlated with increased NICD levels. In addition, when in competition with wild-type cells in this assay, Sirt1-deficient cells are more prone to occupy the stalk cell position. Taken together, our study identifies reversible acetylation of NICD as a novel molecular mechanism to adapt the dynamics of Notch signaling and suggest that SIRT1 acts as a rheostat to fine-tune endothelial Notch responses. The NAD+-dependent feature of SIRT1 activity possibly links endothelial Notch responses to environmental cues and metabolic changes during nutrient deprivation in ischemic environments or upon other cellular stresses.
Der L-Carnitin/gamma-Butyrobetain Antiporter CaiT ist ein Mitglied der Betain/Carnitin/Cholin Transporter (BCCT) Familie. Sekundärtransporter der BCCT Familie transportieren Substrate, die eine positiv-geladene quartäre Ammoniumgruppe besitzen. CaiT besteht aus 504 Amiosäuren und besitzt ein moleculares Gewicht von etwa 56 kDa. In Enterobakterien wie Escherichia coli, Proteus mirabilis und Salmonella typhimurium wird die Expression des caiTABCDE Operons unter anaeroben Bedingungen induziert. Unter diesen Bedinungen ist CaiT der Haupttransporter des Betain-Derivates L-Carnitin. In Enterobakterien wird L-Carnitin unter anaeroben Bedingungen aufgenommen und dehydratisiert wobei Crotonobetain ensteht. Crotonobetain wird anschließend zum Endprodukt gamma-Butyrobetain reduziert. Gamma-Butyrobetain ist das Gegensubstrat, das aus der Zelle hinaustransportiert wird, wenn L-Carnitin in die Zelle aufgenommen wird. Der Austauschmechanismus von LCarnitin gegen gamma-Butyrobetain geschieht ohne das Vorhandensein eines elektrochemischen Gradients, d.h. CaiT ist sowohl H+- als auch Na+-unabhängig. Ein Ziel dieser Arbeit war es die drei-dimensionale (3D) Struktur von CaiT mittels Röntgenstrukturanalyse zu lösen. Weiterhin sollten mit Hilfe der 3D-Struktur und funktionellen Studien detailiertere Erkenntnisse über den kationenunabhängigen Antiportmechanismus von CaiT ermittelt werden. Im Rahmen dieser Arbeit wurden die 3D-Röntgenkristallstrukturen von drei CaiT-Homologen der Enterobakterien P. mirabilis (PmCaiT), E. coli (EcCaiT) und S. typhimurium (StCaiT) mittels molekularem Ersatz (engl.: molecular replacement, MR) mit einem Alanin-Model des CaiT verwandten Na+/Glycinbetain Symporters BetP gelöst. PmCaiT konnte mit einer Auflösung von 2.3 Å gelöst werden. Das Protein kristallisierte in der Kristallraumgruppe H3, mit drei Molekülen in der asymmetrischen Einheit (engl.: asymmetric unit, AU). Die drei PmCaiT-Moleküle ordneten sich innerhalb der AU um eine kristallographische dreifach Symmetrieachse an. EcCaiT wurde mittels MR mit einem Alanin-Model von PmCaiT bei einer Auflösung von 3.5 Å gelöst. EcCaiT kristallisierte in der Kristallraumgruppe P32, ebenfalls mit drei Molekülen in der AU, jedoch ohne kristallographische Symmetry. Während der Verfeinerung des EcCaiT-Models wurde eine strenge dreifache nichtkristallographische Symmetry (engl.: non-crystallographic symmetry, NCS) angewandt. StCaiT, das ebenfalls mittels MR mit einem Alanin-Model von PmCaiT, aber bei einer Auflösung von 4.0 Å gelöst wurde, kristallisierte in der Kristallraumgruppe P65, ebenfalls mit drei StCaiT-Molekülen in der AU, ohne kristallographische Symmetry. Bei der Verfeinerung des StCaiT-Modells wurde wie bei EcCaiT eine strenge NCS angewandt. Da die Auflösung von 4.0 Å bei StCaiT zu niedrig ist um detailierte moleculare Erkenntnisse zu gewinnen, wurden Protein- sowie Substratinteraktionen nur an den Strukturen von PmCaiT und EcCaiT analysiert. Alle drei CaiT-Homologe weisen jedoch einen ähnlichen strukturellen Aufbau auf. In der Röntgenkristallstruktur bildet CaiT ein symmetrisches Trimer, das über ionische und polare Wechselwirkungen zwischen den Protomeren stabilisiert wird. Der trimere Oligomerisierungszustand von CaiT in Detergenzlösung sowie in zweidimensionalen Lipidmembrankristallen wurde bereits in früheren Arbeiten gezeigt. Jedes der drei CaiT-Protomere besteht aus zwölf Transmembranhelices (TMH), die N- und C-terminalen Domänen des Proteins befinden sich auf der cytoplasmatischen Seite. Zehn der TMH bilden zwei invertierte Wiederholungseinheiten aus jeweils fünf TMH. Die erste Einheit besteht aus den TMH 3 – 7, die invertierte zweite Einheit besteht aus den TMH 8 – 12. Beide Wiederholungseinheiten sind strukturell nahezu identisch und lassen sich fast vollständig übereinanderlegen, jedoch weisen die Aminosäuren der beiden Einheiten keine signifikante Sequenzidentität auf. Die ersten beiden Helices der Wiederholungseinheiten, die TMH 3 – 4 und die TMH 8 – 9, bilden ein antiparalleles vier-Helix-Bündel, in dem in CaiT zwei Substratbindestellen lokalisiert sind. Eine derartige Transporterarchitektur wurde erstmals in der Struktur des Na+/Alanin Symporters LeuTAa des thermophilen Bakteriums Aquifex aeolicus gezeigt. Bislang wurden, inklusive CaiT, sieben Sekundärtransporterstrukturen gelöst, die diese LeuT-Transporterarchitektur aufweisen. Ungewöhnlich dabei ist, dass diese sieben Sekundärtransporter fünf verschiedenen Transporterfamilien angehören und eine Verwandschaft auf Basis der Aminosäuren nicht zu finden ist. Da jedoch die tertiäre Struktur dieser Tansporter konserviert ist, kann davon ausgegangen werden, dass sie alle von einem Urprotein entstanden sind, welches zunächst aus fünf TMH bestanden haben muss. Im Laufe der Evolution hat sich das Urgen des Urproteins zunächst dupliziert und die weitere Evolution hat zwar die Aminosäuresequenz verändert und den Umweltbedingungen angepasst, jedoch ist die tertiäre Struktur erhalten geblieben. Da sich die tertiäre Struktur der sieben Sekundärtransporter so stark ähnelt, ist zu vermuten, dass auch der Transportmechanismus ähnlich, jedoch nicht identisch ist. Nach dem strukturellen Aufbau der Transporter, der Lage der Substratbindestellen in den jeweiligen Transportern und der Tatsache, dass es sich bei diesen Proteinen um Membranproteine handelt, wurde ein Transportmechanismus aufgestellt, in dem die Bindestelle des zu transportierende Substrats alternierend zu beiden Seiten der Membran zugänglich ist, ohne jedoch jemals den Substratweg innerhalb des Proteins vollständig zu öffnen. Dieser Mechanismus wurde als “alternating access mechanism” beschrieben. Anhand der unterschiedlichen Zustände, in denen einige der Transporter kristallisierten, kann abgeleitet werden, welche Konformationsänderungen erforderlich sind um das Substrat von einer Seiter der Membran auf die andere zu transportieren. Bisher kristallisierten einzelne der sechs Transporter in der nach außen gerichteten offenen Form, der nach außen gerichteten Form, in der die Substratbindestelle jedoch nicht mehr zugänglich ist, in einer Form, die keine Öffnungspräferenz der Substratbindestelle zu einer Seite der Membran hat und in der nach innen gerichteten Form, in der die Substratbindestelle jedoch nicht geöffnet ist. CaiT kristallisierte in der noch fehlenden Konformation, der nach innen gerichteten Form, in der die Substratbindestelle zugänglich ist. Mit dieser noch fehlenend Konformation kann der Transportzyklus des “alternating access mechanism” vollständig beschrieben werden. Alle drei CaiT-Homologe kristallisierten in der nach innen gerichteten, offenen Konformation. Im Gegensatz zur EcCaiT-Struktur kristallisierte PmCaiT in der substratungebundenen Form. In der StCaiT-Struktur konnte aufgrund der niedrigen Auflösung kein Substrat nachgewiesen werden. In der EcCaiT-Struktur sind zwei gamma-Butyrobetain-Moleküle gebunden. Das erste Molekül wurde in der zentralen Substratbindestelle, der sogenannten Tryptophan-Box bestehend aus vier Tryptophanen, im Zentrum des Protein lokalisiert. Das zweite gamma-Butyrobetain-Molekül wurde in einer Vertiefung an der extrazellulären Proteinoberfläche gefunden. Beide Substrate werden hauptsächlich über Kation-Pi-Interaktionen zwischen der positiv geladenen quatären Ammoniumgruppe des Substrats und des Pi-Elektronensystems der Tryptophane in den jeweiligen Bindestellen gebunden. Eine besondere Eigenschaft von CaiT ist der H+- bzw. Na+-unabhängige Substrattransport. Die CaiT-Struktur erklärt warum kein zusätzliches Kation benötigt wird um Substrat zu binden oder zu transportieren. In der EcCaiT-Struktur ist eine wichtige polare nicht-bindende Interaktion zwischen der Carboxylgruppe des gamma-Butyrobetains und dem Schwefelatom eines Methionins in der zentrale Bindestelle zu erkennen. Dieses Methionin ist konserviert in den prokaryotischen CaiTs und in den Na+-unabhängigen eukaryotischen L-Carnitin Transportern (OCTN), jedoch ist es nicht konserviert im Na+-abhängigen verwandten Glycinbetain Transporter BetP. In BetP ist diese Position des Methionins durch ein Valin ersetzt. Die Mutation des Methionins in CaiT zu Valin ermöglicht zwar immernoch die H+- bzw. Na+-unabhängige Bindung des Substrates durch die Tryptophan-Box, jedoch ist der Substrattransport nahezu vollständig zerstört. Eine derart wichtige Substratkoordinierende Funktion des Schwefelatoms eines Methionins wurde bisher nicht beschrieben. Eine weitere Stelle, die in H+- bzw. Na+-abhängigen Transporter mit H+ bzw. Na+ besetzt ist, ist in CaiT von einem positiv geladenen Arginin eingenommen. Eine positive Ladung an dieser Stelle stabilisiert den Bereich im Protein in der Nähe der zentralen Substratbindestelle. Die Mutation des Arginins zu Glutamat in CaiT erzielt eine vollständige Inaktivierung des Substrattansports. Durch Zugabe von Na+ im Transportansatz kann die Substrattransportaktivität der Glutamat-Mutante jedoch teilweise zurückerlangt werden. Diese eben beschriebenen Aminosäurereste in den beiden Stellen des Proteins erklären die Kationenunabhängigkeit von CaiT. Die Aktivierung des Antiportmechanismus in CaiT wurde mit Hilfe von Bindungsstudien an rekonstituiertem Protein ermittelt. Diese Messungen ergaben für das Wildtypprotein ein sigmoidales Substratbindungsverhalten, was auf ein positiv-kooperatives Bindungsverhalten hindeutet. Die beiden Substratbindestellen im Protein sowie die beiden unterschiedlichen Substrate, L-Carnitin und gamma-Butyrobetain, lassen auf einen heterotropen positiv-kooperativen Bindungs- und einen allosterisch regulierten Transportmechanismus schließen. Bei diesem Mechanismus erhöht die Bindung eines Substrats in der regulatorischen Bindestelle durch induzierte Konformationsänderungen die Affinität eines anderen Substrats in einer weiteren Substratbindestelle. Die regulatorische Bindestelle in CaiT befindet sich an der extrazellulären Proteinoberfläche. Eine Schwächung der Substrataffinität in dieser Bindestelle durch Einführung einer Mutation, verstärkt das sigmoidale Substratbindungsverhalten und hat einen negativen Einfluss auf den Substrattransport. Durch die in dieser Arbeit gelösten 3D-Röntgenkristallstrukturen der zwei CaiT-Homologen, PmCaiT und EcCaiT, sowie den durchgeführten funktionellen Studien sowohl an Wildtypprotein wie auch an Mutanten konnte ein L-Carnitin/gamma-Butyrobetain Antiport-Mechanismus für CaiT vorzuschlagen werden.
This work investigated the applicability of global pairwise sequence alignment to the detection of functional analogues in virtual screening. This variant of sequence comparison was developed for the identification of homologue proteins based on amino acid or nucleotide sequences. Because of the significant differences between biopolymers and small molecules several aspects of this approach for sequence comparison had to be adapted. All proposed concepts were implemented as the ‘Pharmacophore Alignment Search Tool’ (PhAST) and evaluated in retrospective experiments on the COBRA dataset in version 6.1. The aim to identify functional analogues raised the necessity for identification and classification of functional properties in molecular structures. This was realized by fragment-based atom-typing, where one out of nine functional properties was assigned to each non-hydrogen atom in a structure. These properties were pre-assigned to atoms in the fragments. Whenever a fragment matched a substructure in a molecule, the assigned properties were transferred from fragment atoms to structure atoms. Each functional property was represented by exactly one symbol. Unlike amino acid or nucleotide sequences, small drug-like molecules contain branches and cycles. This was a major obstacle in the application of sequence alignment to virtual screening, since this technique can only be applied to linear sequences of symbols. The best linearization technique was shown to be Minimum Volume Embedding. To the best of knowledge, this work represents the first application of dimensionality reduction to graph linearization. Sequence alignment relies on a scoring system that rates symbol equivalences (matches) and differences (mismatches) based on functional properties that correspond to rated symbols. Existing scoring schemes are applicable only to amino acids and nucleotides. In this work, scoring schemes for functional properties in drug-like molecules were developed based on property frequencies and isofunctionality judged from chemical experience, pairwise sequence alignments, pairwise kernel-based assignments and stochastic optimization. The scoring system based on property frequencies and isofunctionality proved to be the most powerful (measured in enrichment capability). All developed scoring systems performed superior compared to simple scoring approaches that rate matches and mismatches uniformly. The frameworks proposed for score calculations can be used to guide modifications to the atom-typing in promising directions. The scoring system was further modified to allow for emphasis on particular symbols in a sequence. It was proven that the application of weights to symbols that correspond to key interaction points important to receptor-ligand-interaction significantly improves screening capabilities of PhAST. It was demonstrated that the systematic application of weights to all sequence positions in retrospective experiments can be used for pharmacophore elucidation. A scoring system based on structural instead of functional similarity was investigated and found to be suitable for similarity searches in shape-constrained datasets. Three methods for similarity assessment based on alignments were evaluated: Sequence identity, alignment score and significance. PhAST achieved significantly higher enrichment with alignment scores compared to sequence identity. p-values as significance estimates were calculated in a combination of Marcov Chain Monte Carlo Simulation and Importance Sampling. p-values were adapted to library size in a Bonferroni correction, yielding E-values. A significance threshold of an E-value of 1*10-5 was proposed for the application in prospective screenings. PhAST was compared to state-of-the-art methods for virtual screening. The unweighted version was shown to exhibit comparable enrichment capabilities. Compound rankings obtained with PhAST were proven to be complementary to those of other methods. The application to three-dimensional instead of two-dimensional molecular representations resulted in altered compound rankings without increased enrichment. PhAST was employed in two prospective applications. A screening for non-nucleoside analogue inhibitors of bacterial thymidin kinase yielded a hit with a distinct structural framework but only weak activity. The search for drugs not member of the NSAID (non-steroidal anti-inflammatory drug) class as modulators of gamma-secretase resulted in a potent modulator with clear structural distiction from the reference compound. The calculation of significance estimates, emphasizing on key interactions, the pharmacophore elucidation capabilities and the unique compound rannkings set PhAST apart from other screening techniques.
Apoptotic cell (AC)-derived factors alter the physiology of macrophages (M Phi s) towards a regulatory phenotype that is characterized by enhanced production of anti-inflammatory mediators, an attenuated pro-inflammatory cytokine profile and reduced nitric oxide (NO) formation. Impaired NO production in response to ACs or AC-conditioned medium (CM) is facilitated by arginase II (ARG II) expression, which competes with inducible NO synthase for L-arginine. In this study, I investigated the signaling pathway that allowed CM to upregulate ARG II in M Phi s. A sphingolipid, further identified as sphingosine-1-phosphate (S1P), was required but authentic S1P alone only produced small effects. S1P acted synergistically with a so far unidentified factor to elicit high ARG II expression. S1P signaled through S1P receptor 2 (S1P2), since the S1P2-antagonist JTE013 and siRNA knock-down of S1P2 prevented ARG II upregulation. Further, inhibition and knock-down of extracellular signal-regulated kinase 5 (ERK5) attenuated CM-mediated ARG II protein induction. Exploring ERK5-dependent transcriptional regulation, promoter deletion and luciferase reporter analysis of the murine ARG II promoter (mpARG II) suggested the involvement of cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB). This was confirmed by EMSA analysis and decoyoligonucleotides scavenging CREB, thereby preventing it from activating target genes and thus, blocking ARG II expression. I concluded that AC-derived S1P binds to S1P2 and acts synergistically with other factors to activate ERK5 and concomitantly CREB. This signaling cascade shapes an anti-inflammatory M Phi phenotype by ARG II induction. Further investigations of ERK5-dependent CREB activation suggested an indirect mechanism implying that ERK5 inhibited phosphodiesterase 4 (PDE4) and thus, prevented hydrolysis of cAMP. Since S1P-dependent ERK5 activation presumably inhibited PDE4, subsequent cAMP accumulation led to enhanced PKA activity and CREB-mediated transcription. The unidentified factor(s) besides S1P probably provoked the required elevation of cAMP production in M Phi s. Indeed, pharmacological inhibition of cAMP-producing adenylyl cyclase with SQ22536 as well as cAMP-dependent protein kinase A (PKA) with KT5720 suggested cAMP to be involved in CM-mediated ARG II up-regulation. Furthermore, forskolin-dependent activation of adenyly cyclase and simultaneous rolipram-mediated inhibition of PDE4 mimicked CM-induced ARG II expression. Considering these findings, I propose that one or several unidentified factors in CM provoke cAMP production in M Phi s. In parallel, AC-derived S1P activates ERK5, which inhibits PDE4-dependent cAMP hydrolysis, further raising intracellular cAMP levels. Thus, unrestricted continuous cAMP signaling via PKA/CREB, results in a time-dependent and sustained ARG II induction.
The eukaryotic glyoxalase system consists of two enzymatic components, glyoxalase I (lactoylglutathionelyase) and glyoxalase II (hydroxyacylglutathione hydrolase). These enzymes are dedicated to the removal of toxic alpha-oxoaldehydes like methylglyoxal (MG). MG is formed as a by-product of glycolysis and MG toxicity results from its damaging capability leading to modifications of proteins, lipids and nucleic acids. An efficient removal of MG appears to be essential to ensure cellular functionality and viability. Here we study the effects of the genetic modulation of genes encoding the components of the glyoxalase system in the filamentous ascomycete and aging model Podospora anserina. Overexpression of PaGlo1 leads to a lifespan reduction on glucose rich medium, probably due to depletion of reduced glutathione. Deletion of PaGlo1 leads to hypersensitivity against MG added to the growth medium. A beneficial effect on lifespan is observed when both PaGlo1 and PaGlo2 are overexpressed and the corresponding strains are grown on media containing increased glucose concentrations. Notably, the double mutant has a ‘healthy’ phenotype without physiological impairments. Moreover, PaGlo1/PaGlo2_OEx strains are not long-lived on media containing standard glucose concentrations suggesting a tight correlation between the efficiency and capacity to remove MG within the cell, the level of available glucose and lifespan. Overall, our results identify the up-regulation of both components of the glyoxalase system as an effective intervention to increase lifespan in P. anserina. Key words: Podospora anserina, aging, lifespan, glycation, glucose, methylglyoxal, advanced glycation end products
In the adult mammalian central nervous system, two defined neurogenic regions retain the capacity to generate new neurons throughout adulthood, namely the subependymal zone (SEZ) at the lateral ventricles and the subgranular layer of the hippocampus (SGL). Adult neurogenesis consists of a whole set of events including proliferation, fate specification, migration, survival and finally synaptic integration of newly born neurons. Each of these events is controlled by the interplay of numerous factors. In this study two signalling systems were analysed with regard to their functional role in adult neurogenesis in vivo, namely the purinergic system and the growth factor EGF. Neither short- nor long-term application of the P2Y receptor agonists UTP and ADPβS and the P2Y receptor antagonist suramin into the lateral ventricle of adult mice altered cell responses as compared to vehicle controls in vivo. In contrast, analysis of the expansion rates of cultured neural stem cells (NSCs) from knockout mice revealed a strong increase in the number of NSCs from NTPDase2-/- mice, whereas cell numbers of NSCs from P2Y1-/- and P2Y2-/- mice were significantly reduced in comparison to wildtype levels. Notably, in vivo proliferation rates were potently elevated in the SGL and the SEZ of NTPDase2-deficient mice. However, in vivo proliferation in both neurogenic niches of the single receptor knockout mice P2Y1-/- and P2Y2-/- and P2Y1-/- P2Y2-/-double-knockout mice did not differ significantly from the wildtype. In mice lacking the P2Y2 receptor the survival of newly born neurons in the hippocampal granule cell layer was significantly increased. These data provide the first line of evidence that purinergic signalling is involved in the control of neural stem cells behaviour not only in vitro but also in vivo. In order to further characterise the role of epidermal growth factor (EGF) in adult neurogenesis, transit amplifying precursors (TAPs) and type B astrocytes were identified as EGF-responsive cell populations following ventricular EGF injection, whereas ependymal cells, neuroblasts and NG2-positive cells did not or only to a minor extent respond to EGF injection. These EGF-responsive cell populations were found on both, the septal as well as striatal lateral ventricle walls. Long-term ventricular EGF infusion for 6d, 1. increased cell proliferation of both ventricle walls revealing a gradient along the rostro-caudal axis, 2. altered the balance between neuronal and macroglial cell fates to generate oligodendrocyte precursors and 3. lead to an entire remodelling of the classical architecture of the SEZ.